BIO102 : Cell Biology lecture slides.pdf

Cell Biology

1. Course Contents, Bio 102
Cell Theory, Cell-the building blocks of life, structural components of cells and
their function, Prokaryotic cell and Eukaryotic cell -Structure and function
(Overview, Cell Wall, Cell membrane, Cytoplasm and components therein,
Cytoskeleton and cell motility).
Structure and functions of cell organelles: Mitochondria, Chloroplasts, Golgi,
ER and lysosomes; Cell division; Approaches to study cellular processes-
microscopy, biochemical and biophysical assays with specific examples, Cell
Division.
Suggested Readings
Molecular Biology of the Cell: Bruce Alberts, Alexander Johnson, Julian Lewis,
Martin Raff, Keith Roberts, Peter Walter; New York: Garland Science; 5th
edition ;2008
Essential Microbiology: Stuart Hogg; John Wiley & Sons; 2005
NCERT basic biology books

2. *What are the major organelles
and their functions?
*How to cell divide?
*How do small and large
molecules pass through the
membrane barrier?
*How do cells communicate with
each other across their
respective plasma membranes?
*How are extracellular signals
recognized & processed by the
cell?
Questions that we will be addressing:

3. Average composition of a cell
•The interactions between water and the other
constituents of cells are factors in biological
chemistry
•Because of their polar nature, water molecules can
form hydrogen bonds with each other and with other
polar molecules, as well as interacting with
positively or negatively charged ions. As a result of
these interactions, ions and polar molecules are
readily soluble in water (hydrophilic).
•In contrast, nonpolar molecules, which cannot
interact with water, are poorly soluble in an aqueous
environment (hydrophobic)
Three major types of Noncovalent Interactions
help bring molecules together in cells:
•Ionic bonds
•Hydrogen bonds
•Van der Waals interactions

4. Differences between living and non living things
Living Non-Living

5. Cell Theory:
•In 1838~1839, Schleiden and
Schwann elaborated that:
•All organism (either animals or plants)
composed of cells.
•The cell is the fundamental structural and
functional unit of biological systems.
•Cells arise only from preexisting cells.
Theplasma membraneencloses the cell, defines its boundaries, and maintains the essential differences between
thecytosoland the extracellular environment. Inside eucaryoticcells, the membranes of theendoplasmic
reticulum, Golgi apparatus, mitochondria, and othermembrane-enclosed organelles maintain the characteristic
differences between the contents of eachorganelleand the cytosol. Ion gradients across membranes, established
by the activities of specialized membrane proteins, can be used to synthesize ATP, to drive the transmembrane
movement of selected solutes, and transmit electrical signals. In all cells, theplasma membranealso contains
proteins that act as sensors of external signals, allowing the cell to change its behavior in response to
environmental cues; theseproteinsensors, orreceptors,transfer information.
The cell is the fundamental structural
and functional unit of biological systems

6. Urey-Miller’s experiment (1953):
abiogeneticsynthesis of large amountof variety of
organic compounds from a mixture of gases
Possiblesynthesisoforganic
compoundsthatledtotheappearance
oflivingorganisms.Intheir
experiment,amixtureofgaseswas
allowedtocirculateoverelectric
dischargefromantungstenelectrode.
Asmallflaskwaskeptboilingand
thesteamemanatingfromitwas
madetomixwiththemixtureof
gases(ammonia,methaneand
hydrogen)inthelargechamberthat
wasconnectedtotheboilingwater.
Experiment was conducted
continuouslyforaweekandthe
liquidwasanalysed.Glycine,alani
ne,betaalanineandasparticacid
wereidentified.
Later in similar experiments, formation of all types of amino
acids, and nitrogen bases were noticed.

7. The three kingdoms of organisms
The biological universe consists of two types of
cells, prokaryotic cells,which lack a
definednucleusand have a simplified internal
organization, andeukaryotic cells,which have a
more complicated internal structure including a
defined,membrane-limitednucleus. Detailed
analysis of theDNAfrom a variety of prokaryotic
organisms in recent years has revealed two
distinct types: bacteria (often called “true”
bacteria oreubacteria) andarchaea(also
calledarchaebacteriaorarchaeans).
Thearchaeaare in some respects more similar to
eukaryotic organisms than to the true bacteria.

8. Nucleus:
-contains DNA genome
-site of DNA and RNA synthesis
Cytoplasm:
-comprises ~50% of cell volume
-site of protein synthesis and intermediary
metabolism
Endoplasmic Reticulum (ER):
-made up of smooth and rough ER
-site of entry into the secretion pathway
-major site of lipid biosynthesis
-comprises > 50% of total cell membrane
Mitochondria:
-responsible for most of the ATP production in
eukaryotic cells
-enclosed within a double membrane
Endosomes:
-a series of compartments that serve as an
intermediate destination for endocytosed material
Ribosomes:
Golgi Complex:
-made up of stacks of disc-like compartments
-an intermediate compartment of the secretion
pathway
Lysosomes:
-contains a variety of digestive or degradative
enzymes
-responsible for degradation of cellular
macromolecules, endocytosed material and
defunct organelles (autophagy)
Peroxisomes:
-vesicular compartments that carry out a variety
of oxidative reactions
Cell Compartments

9. Possible origin of
the ER and
nucleus
Possible origins of
the mitochondria

10. Ineukaryoticcells,DNAispackagedintheformofchromatin
Epigenetic mechanisms are affected by
intracellular and extracellular factors:
?Development
?Environmental chemicals
?Drugs/Pharmaceuticals
?Diet, physical activity
?Aging and so on…………
Epigenetic changes to the chromatin
1. Chemical: covalent post translational
modifications of histone proteins and DNA
methylation
2. Physical: Chromatin remodellingand exchange
of histone variants
3. Irreversible: Proteolytic clipping of histone tails
3 billion base pairs of DNA in each human cell fits into a 6
microns space. If you stretch the DNA of one cell, it would
beabout 2m longand all the DNA of all cells of a human,
would be about twice the diameter of the Solar System.

11. Histoneproteinsareposttranslationallymodifiedto
regulatetheDNAdependentprocesses;
DNAReplication,Transcription,DNArepairandrecombination
Post Translational
Modifications
•Acetylation
•Methylation
•Phosphorylation
•Ubiquitination
•Clipping of histone tails
•…………….
Histone Code
Combinations of certain PTMs acts as Code to specifically regulate a biological process

12. Important differences between
eukaryotic and prokaryotic cells

13. •Cell wall is an important structure of a bacteria. It give
shape, rigidity and support to the cell.
•On the basis of cell wall composition, bacteria are
classified into two major group ie. Gram Positive and
gram negative.
Bacterial Cell wall: Structure, Composition and Types
-
-
-
-
-

14. Peptidoglycan:
•Peptidoglycan (also known as murein) is porous cross linked polymer which is
responsible for strength of cell wall.
•Peptidoglycan is composed of three components.
•Glycan backbone
•Tetra-peptideside chain ( chain of 4 amino acids)linked to NAM
•Peptide cross linkage
•Glycan backbone is the repeated unit of N-acetyl muramic acid (NAM) and N-
acetyl glycosamine(NAG) linked by -glycosidicbond.
•The glycan backbone are cross linked by tetra-peptide linkage. The tetra-
peptide are only found in NAM.
•More than 100 peptidoglycan are known with the diversity focused on the
chemistry of peptide cross linkage and interbridge.
•Although the peptidoglycan chemistry vary from organism to organism the
glycan backbone ieNAG-NAM is same in all species of bacteria.

15. Endotoxin was considered to be a toxin retained
inside the bacterial cell as in contrast to the exotoxin that
is released into the surroundings by live bacteria,
although subsequent work showed that endotoxin can be
released by live bacteria as well. Today endotoxin is
commonly used as a synonym for lipopolysaccharides
(LPS).
LPS is the major component of the outer membrane of
Gram-negative bacteria. Gram-negative bacteria do not
retain crystal violet as they carry a thin cell wall which is
made of a single layer of peptidoglycan surrounded by
the outer membrane which invariably contains LPS.
During Gram-negative sepsis, endotoxin binds to host
macrophages and induces release of pro-inflammatory
cytokines and excessive inflammation can lead to
multiple organ failure and death.
Macrophages(to eat) are a type ofwhite blood cell of
theimmune systemthat engulfs and digests pathogens,
such ascancercells,microbes, cellular debris, and
foreign substances. They are found in all tissues. The
process is calledphagocytosis, which acts to defend the
host against infection and injury.

16. When the stain combines
with bacteria in a sample,
the bacteria will either stay
purple or turn pink or red.
If the bacteria stays purple,
they are Gram-positive. If
the bacteria turns pink or
red, they are Gram-negative
When the bacteria is stained with primary stain Crystal Violet and fixed by the mordant, some of the bacteria
are able to retain the primary stain and some are decolorized by alcohol. The cell walls of gram positive
bacteria have a thick layer of protein-sugar complexes called peptidoglycan and lipid content is
low.Decolorizing the cell causes this thick cell wall to dehydrate and shrink, which closes the pores in the cell
wall and prevents the stain from exiting the cell.
In case of gram negative bacteria, cell wall also takes up the CV-Iodine complex but due to the thin layer of
peptidoglycan and thick outer layer which is formed of lipids, CV-Iodine complex gets washed off. When they
are exposed to alcohol,decolorizer dissolves the lipids in the cell walls, which allows the crystal violet-iodine
complex to leach out of the cells. Then when again stained with safranin, they take the stain and appears red
in color.

17. Lipopolysaccharides(LPS) are largemoleculesconsisting of alipidand
apolysaccharidecomposed of O-antigen, outer core and inner core joined by
acovalent bond; they are found in theouter membraneofGram-negative bacteria

18. Sepsisis a life-threatening illness caused
by your body's response to an infection.
Your immune system protects you from
many illnesses and infections, but it's also
possible for it to go into overdrive in
response to an infection

20. Biological membranes have a common general structure:
The lipidandproteinmolecules, held together mainly by noncovalent interactions.
Cell membranes are dynamic, fluid structures, and most of their molecules are able to move about in the plane of
themembrane.
The lipid molecules are arranged as a continuous double layer about 5nmthick.
Thislipid bilayerserves as a relatively impermeable barrier to the passage of most water-soluble molecules. Protein
molecules that span the lipid bilayer mediate nearly all of the other functions of the membrane, transporting specific
molecules across it, for example, or catalyzing membrane-associated reactions, such as ATP synthesis.
In theplasma membrane, some proteins serve as structural links that connect thecytoskeletonthrough the lipid
bilayer to either theextracellular matrixor an adjacent cell, while others serve as receptors to detect and transduce
chemical signals in the cell's environment.
It is estimated that about 30% of the proteins that are encoded in an animal cell'sgenomeare membrane proteins.
(A)Anelectronmicrographof
aplasma membrane(of a
humanred blood cell) seen in
crosssection.
(B and C) These drawings show two-
dimensional and three-dimensional
views of a cell membrane.

21. Bacterial plasma membranes consist of one major type of phospholipid and contain no cholesterol.
Their mechanical stability is enhanced by an overlying cell wall. In archaea, lipids usually contain 20-
25 carbon long prenylchains instead of fatty acids
Approximate Lipid Compositions of Different Cell Membranes

22. •Eachlipidsincellmembranesareamphipathicinnature;hasahydrophilicheadanda
hydrophobictail.Phospholipidsarethemostabundantcellularlipids.
•Lipid molecules constitute about 50% of the mass of most animal cell membranes
(1000000000 lipid molecules in the plasma membrane of a animal cell).
•Inbacteriaandyeastwhichhavetoadapttovaryingtemperatures,thelengthand
degreeofsaturationofthehydrocarbontailsinthebilayerareadjustedconstantlyto
maintainthemembranewithconsistentfluidity.Athighertemperature,cellmakes
lipidswithtailsthatarelongerandlessnumberofdoublebonds.
•Thefatsproducedbyplantsaregenerallyunsaturatedandthereforeremainsliquidat
RTbutanimalfatssuchasbuttergenerallycontainsaturatedandthusremainssolidat
RT.Toproducemargarine,vegetablesoilsarehydrogenatedwhichremovesdouble
bondsmakingoilmoresolidandbutterlikeatRT.
•Thefluidityoflipidbilayerdependsuponitscomposition,mainlythenatureof
hydrocarbontails.Thelength(variesfrom14to24carbons)andthenumberofdouble
bonds.Withnodoublebonds(saturatedwithhydrogenatoms)areknownassaturated.
Doublebond(unsaturated)intailscreatesasmallkinkintailwhichmakestails
difficulttopackagainsteachother.Duetothisreason,tailswithunsaturated
hydrocarbonsaremorefluid.
Cholesterol
Lipid composition influences fluidity of membranes
Inanimalcells,cholesteroltendsto
stiffencellmembranes

23. Phospholipids and glycolipids are distributed asymmetrically in the lipid bilayer of animal cell plasma membrane
Phosphatidylcholine and
sphingomyelin in the non-cytosolic
(outer) monolayer
Phosphatidylserine (negatively
charged) and
Phosphatidylethanolamine are
found in cytosolic (inner)
monolayer
Glycolipids are exclusively found
in the noncytosolicmonolayer of
the membrane
Glycolipids
Phospholipids
glycolipids are drawn with hexagonal polar head groups
(blue). Cholesterol (not shown) is thought to be distributed
about equally in both monolayers
Four major
phospholipids in
mammalian
plasma
membranes
Phosphoglycerides, sphingolipids, and sterols are major lipids in cell membranes.
Charges difference between two halves of the bilayer?

24. Other phospholipids, such as the inositol phospholipids
•Present in smaller quantities but are functionally very important
•Inositol phospholipids have a crucial role in cell signalling, endocytosis etc.

25. Lipid rafts are transient domains within a bilayer that form as a
result of the physical properties of some lipid molecules
Particular
proteins can be
concentrated
within lipid rafts
Lipid rafts are involved in several cellular functions, such as pathogen
recognition, cell signaling
Lipid rafts in pathogen sensing and cytokine release: Lipid rafts play an
important role in pathogen sensing by recruiting pathogen sensing receptors
such as toll like receptors when cells are stimulated with bacterial cell wall
components such as LPS
Lipid rafts influencemembrane fluidityandmembrane proteintrafficking,
thereby regulatingneurotransmissionand receptor trafficking

26. The asymmetry of the lipid bilayer is functionally important
•The lipid compositions of the two monolayers in many membranes are very different. For example in the
membranes of human RBC, phosphatidyl choline and sphingomyelin are located in the outer monolayer whereas
phosphatidylserine and phosphatidylethanolamine on inner monolayer
•Because the negatively charged phosphatidylserine are located in inner monolayer, significant charge difference
exist between two monolayers
•Membrane bound phospholipid translocators generate and maintain lipid asymmetry
•Lipid asymmetry is functionally important, especially in converting extracellular signals into intracellular ones
•Many cytosolic proteins binds to specific lipid head groups in the cytosolic monolayer of the lipid bilayer. The
enzyme PKC is activated in response to extracellular signals. It binds to cytosolic face where PS is concentrated
and requires this negatively charged PS for its activity.
•In some cases various lipid kinases can add phosphate groups at distinct positions in the inositol ring of
phosphatidylinositol which creates a binding site that recruits specific proteins from the cytosol to the membrane.
Example, a PI 3 kinase which is activated upon extracellular signal which helps to recruit specific intracellular
signalling proteins to the cytosolic face of the plasma membrane.
•In some cases, membrane phospholipases are activated by extracellular signal which cleave specific phospholipid
molecule in the membrane which acts as short-lived intracellular mediators. Example, phospholipases C cleaves an
inositol phospholipid in cytosolic monolayer into two fragments. One fragment, remain in the membrane which
helps to activate the PKC and other one is released in the cytosol and stimulates release of calcium ion from ER.
•Animals exploit phospholipid asymmetry to distinguish between live and dead cells. During apoptosis, PS rapidly
translocate to the outer monolayer. PS signals macrophages to phagocytose the dead cell and digest it.

27. (A) Extracellular signals can activate PI3-kinase, which phosphorylates inositol phospholipids in the plasma
membrane. Various intracellular signaling molecules then bind to these phosphorylated lipids and are thus
recruited to the membrane, where they can interact and help relay the signal into the cell.
(B) Other extracellular signals activate phospholipases that cleave phospholipids. The lipid fragments then act
as signaling molecules to relay the signal into the cell.
(C) Illustration of the sites where different classes of phospholipases cleave phospholipids. As indicated,
phospholipases A1 and A2 cleave ester bonds, whereas phospholipases C and D cleave at phosphoesterbonds
Some functions of membrane phospholipids in cell signaling

28. Apoptotic cells are rapidly engulfed and removed by phagocytes after
displaying cell surface eat-me signals
Among many phospholipids, only phosphatidylserine (PS) is known to act as an eat-me signal on apoptotic cells
Externalized PS is a well-established eat-me signal for the phagocytic clearance of apoptotic cells

29. Galactocerebrosideiscalledaneutralglycolipidbecausethesugarthatformsitsheadgroupisuncharged.(B)
Agangliosidealwayscontainsoneormorenegativelychargedsialicacidresidues(alsocalledN-
acetylneuraminicacid,orNANA)
Glycolipids Are Found on the Surface of All Plasma Membranes
Glycolipids probably occur in all animal cell plasma
membranes, where they generally constitute about 5% of
thelipidmolecules in the outer monolayer
These molecules are found exclusively in the
noncytosolicmonolayer of thelipid bilayer, where they
are thought to partition preferentially into lipid rafts.
More than 40 different gangliosides have been
identified. They are most abundant in theplasma
membraneof nerve cells,
They are also found in much smaller quantities in
other cell types.

30. •The glycolipids help to protect the membrane from the
harsh conditions (such as low pH and degradative
enzymes). Charged glycolipids, such as gangliosides, may
be important for their electrical effects: their presence will
alter the electrical field across the membrane and the
concentrations of ions.
•Glycolipids may also play a role in electrical insulation in
the myelin membrane.
•They are also thought to function in cell-recognition
processes in which membrane-boundcarbohydrate-
binding proteins(lectins)bind to thesugargroups on both
glycolipids and glycoproteins in the process of cell-cell
adhesion
•The ganglioside, for example, acts as a cell-surface
receptor for the bacterial toxin that causes the diarrhea of
cholera..
•The evidence that glycolipids can help cells to bind to the
extracellular matrix, as well as to other cells.
the surface that the cell presents to the
exterior consists largely of carbohydrate,
which forms a glycocalyx or cell coat
Glycolipids Are Found on the Surface of All Plasma Membranes

31. Bilayer behave like a two-dimensional fluid
in which the individual lipid molecules are
able to move in their own monolayer. Lipid
molecules do not move spontaneously from
one monolayer to the other.
Phospholipid
mobility. The types
of movement
possible for
phospholipid
molecules in a lipid
bilayer.
Membrane phospholipids move within the lipid bilayer
The double bonds make it more difficult to pack the
chains together and therefore make the lipid bilayer
more difficult to freeze
Chains of unsaturated lipids are more spread apart,
lipid bilayers containing them are thinner than bilayers
containing saturated lipids
Influence of cis-double bonds in hydrocarbon chains

32. Many membrane proteins are able to move laterally
within the membrane (lateral diffusion). The first
direct evidence that some plasma membrane
proteins are mobile in the plane of the membrane
was provided in 1970 by an experiment in which
mouse cells were artificially fused with human cells
to produce hybrid cells (heterocaryons). Two
differently labeled antibodies were used to
distinguish selected mouse and human plasma
membrane proteins. Although at first the mouse and
human proteins were confined to their own halves of
the newly formed heterocaryon, the two sets of
proteins diffused and mixed over the entire cell
surface within half an hour or so.
Experiment demonstrating the mixing of plasma membrane
proteins on mouse-human hybrid cells.

33. Most trans-membrane proteins are thought to extend across the bilayer
as a single a helix (1) or as multiple a helices (2); some of these "single-
pass" and "multipass" proteins have a covalently attached fatty acid
chain inserted in the cytoplasmic monolayer (1). Other membrane
proteins are attached to the bilayer solely by a covalently attached lipid -
either a fatty acid chain or prenylgroup -in the cytoplasmic monolayer
(3) or, less often, via an oligosaccharide, to a minor phospholipid,
phosphatidylinositol, in the noncytoplasmicmonolayer (4). Finally,
many proteins are attached to the membrane only by noncovalent
interactions with other membrane proteins (5) and (6).
Six ways in which membrane proteins associate with the lipid bilayer
The great majority of transmembrane
proteins are glycosylated. Like
glycolipids, the sugar residues are added
in the lumen of the endoplasmic
reticulum and Golgi apparatus and for
this reason the oligosaccharide chains are
always present on the noncytoplasmic
side of the membrane. A further
asymmetry arises as a result of the
reducing environment of the cytosol,
which prevents the formation of
intrachain (and interchain) disulfide (S -
S) bonds between cysteine residues on
the cytosolic side of membranes. These
bonds do form on the noncytosolicside,
where they can play an important part in
stabilizing either the folded structure of
the polypeptide chain or its association
with other polypeptide chains.

34. Like membrane lipids, membrane proteins do not tumble (flip-flop)
across the bilayer, but they do rotate about an axis perpendicular to
the plane of the bilayer (rotational diffusion). In addition, many
membrane proteins are able to move laterally within the membrane
(lateral diffusion). The first direct evidence that some plasma
membrane proteins are mobile in the plane of the membrane was
provided in 1970 by an experiment in which mouse cells were
artificially fused with human cells to produce hybrid cells
(heterocaryons). Two differently labeled antibodies were used to
distinguish selected mouse and human plasma membrane proteins.
Although at first the mouse and human proteins were confined to their
own halves of the newly formed heterocaryon, the two sets of proteins
diffused and mixed over the entire cell surface within half an hour or
so.
Experiment demonstrating the mixing of plasma membrane proteins
on mouse-human hybrid cells.

35. Ionophores Can Be Used as Tools to Increase the
Permeability of Membranes to Specific Ions
•Ionophores are small hydrophobic molecules that dissolve in lipid bilayers and increase permeability to specific inorganic ions.
•They are mostly synthesized by microorganisms.
•Used by cell biologists as tools to increase the ion permeability of membranes in studies on synthetic bilayers, cells, or cell organelles.
•There are two classes of ionophores -mobile ion carriers and channel formers, operate by shielding the charge of the transported ion
to penetrate the hydrophobic interior of the lipid bilayer.
•Ionophores are not coupled to energy sources, they permit net movement of ions only down their electrochemical gradients.
•Valinomycin is an example of a mobile ion carrier, transports K+ down its electrochemical gradient by picking up K+ on one side of
the membrane, diffusing across the bilayer, and releasing K+ on the other side.
•The ionophore A23187 is another example of a mobile ion carrier, but it transports divalent cations such as Ca2+ and Mg2+. It
normally acts as an ion-exchange shuttle. When cells are exposed to A23187, Ca2+ enters the cytosol. Accordingly, this ionophoreis
widely used in cell biology to increase the concentration of free Ca2+ in the cytosol, thereby mimicking certain cell signaling
mechanisms.
•Gramicidin A is an example of a channel-forming ionophore. As a linear peptide of only 15 amino acid residues, all with hydrophobic
side chains. Two gramicidin molecules are thought to come together end to end across the bilayer to form a transmembrane channel
which selectively allows monovalent cations to flow down their electrochemical gradients. With a large electrochemical gradient,
gramicidin A can transport about 20,000 cations per open channel each millisecond, which is 1000 times more ions than can be
transported by a single mobile carrier molecule in the same time. Gramicidin is made by certain bacteria, perhaps to kill other
microorganisms by collapsing the H+, Na+, and K+ gradients that are essential for cell survival, and it has been useful as anantibiotic.
A23187
Valinomycin
Gramicidin

36. The use of mild detergents for solubilizing, purifying, and
reconstituting functional membrane protein systems. In this
example functional Na+-K+ ATPase molecules are purified
and incorporated into phospholipid vesicles. The Na +-K+
ATPase is an ion pump that is present in the plasma
membrane of most animal cells; it uses the energy of ATP
hydrolysis to pump Na+ out of the cell and K+ in,
Mild detergents helps to purify membrane proteins

37. Membrane Transport Proteins
-Multiple pass transmembrane proteins
-Exhibit solute specificity
-Two major classes:
1) Transporter proteins
-Also called carriers or permeases
-Bind to solute and undergo a series of
conformation changes that result in the
transfer of bound solute across bilayer
2) Channel proteins
-Form a hydrophilic pore; do not bind
solute
-Faster rate of transfer
There are two major classes of membrane transport proteins;
transporters and channels
Bind the specific
solute and undergo
a series of
conformational
changes to
transport the
bound solute
across the plasma
membrane
Channels form aqueous pores
that extend across the lipid
bilayer, transport occurs on a
faster rate than transporters,
Although water can diffuse
across lipid bilayers but all
cell contain specific channel
proteins (water channels or
aquaporins) that increases the
permeability of these
membranes to water

38. 1) Passive Transport
-Also called "Facilitated Diffusion"
-Solutes are transferred passively across
membrane
-Mediated by both carrier & channel proteins
2) Active Transport
-Transfer solutes againsttheir electrochemical gradient
-Transport is coupled to an expenditure of energy; e.g.
ATP hydrolysis
-Mediated by carrier proteins
All channels and many
transporters allow
solutes to cross the
membrane only
passively known as
passive transport
Cells also require
transport proteins that
actively pump certain
solutes across the
plasma membrane but
against the
electrochemical
gradients, known as
active transport
Two major types of transport mechanisms

39. Types of Carrier Proteins
Two types of coupled transport reactions, symport and antiport.
Symporterstransfer second solute in the same direction as the first.
Antiporterstransfer second solute in the opposite direction as the first.
Uniporters
Transport a single solute
from one side of the
membrane to another.
Coupled Transporters
Transfer of one solute
depends on the simul-
taneoustransport of
a second solute.

40. •The concentration of K+ is typically 10 to 20 times higher inside cells than outside,
whereas the reverse is true of Na+, maintained by a Na+-K+ pump that is found in the
plasma membrane of all animal cells.
•Functions as an antiporter; Na
+
is pumped out and K
+
is pumped in.
•The Na+ gradient produced by the pump regulates cell volume through its osmotic
effects and is also exploited to drive transport of sugars and amino acids into the cell.
•Almost one-third of the energy requirement of a typical animal cell is consumed in
fueling this pump.
•Na
+
gradient is used to regulate cell volume and to drive the transport of sugars and
amino acids into the cell.
•Almost 33% of the energy required by a typical animal cell is used to drive this pump.
•ATP hydrolysis is required for Na
+
/K
+
pump action.
•Pump is electrogenic, as it pumps 3 Na
+
out for every 2 K
+
pumped in.
•A major advance in understanding that Na+-K+ pump came in 1957, ouabain an
inhibitor, inhibits the ATPase.
•The Na+-K+ATPasehas been purified and found to consist of a large, multipass,
transmembrane catalytic subunit (about 1000 amino acids long) and an associated
smaller, single-pass glycoprotein. The larger subunit has binding sites for Na+ and ATP
on its cytoplasmic surface and a binding site for K+ on its external surface, and is
reversibly phosphorylated and dephosphorylated during the pumping cycle.
•A functional Na+-K+ pump can be reconstituted from the purified complex: the ATPase
is solubilized in detergent, purified, and mixed with appropriate phospholipids. When
the detergent is removed, membrane vesicles are formed that pump Na+ and K+ in
opposite directions in the presence of ATP.
The Na+-K+ ATPase. This carrier protein actively
pumps Na+ out of and K+ into a cell against their
electrochemical gradients. For every molecule of
ATP hydrolyzed inside the cell, three Na+ are
pumped out and two K+ are pumped in. The specific
pump inhibitor ouabain and K+ compete for the
same site on the external side of the ATPase.
The Plasma
Membrane contains
Na+-K+ Pump, an
ATPase

41. •The concentration of K+ is typically 10 to 20 times higher inside cells
than outside, whereas the reverse is true of Na+, maintained by a Na+-
K+ pump that is found in the plasma membrane of all animal cells.
•Functions as an antiporter; Na
+
is pumped out and K
+
is pumped in.
•The Na+ gradient produced by the pump regulates cell volume through
its osmotic effects and is also exploited to drive transport of sugars and
amino acids into the cell.
•Almost 33% of the energy required by a typical animal cell is used to
drive this pump.
•ATP hydrolysis is required for Na
+
/K
+
pump action.
•Pump is electrogenic *, as it pumps 3 Na
+
out for every 2 K
+
pumped in.
•ouabain an inhibitor, inhibits the ATPase.
•The Na+-K+ATPasehas been purified and found to consist of a large,
multipass, transmembrane catalytic subunit (about 1000 amino acids
long) and an associated smaller, single-pass glycoprotein. The larger
subunit has binding sites for Na+ and ATP on its cytoplasmic surface
and a binding site for K+ on its external surface, and is reversibly
phosphorylated and dephosphorylated during the pumping cycle.
•A functional Na+-K+ pump can be reconstituted from the purified
complex: the ATPase is solubilized in detergent, purified, and mixed
with appropriate phospholipids. When the detergent is removed,
membrane vesicles are formed that pump Na+ and K+ in opposite
directions in the presence of ATP.
The Na+-K+ ATPase. This carrier protein actively pumps Na+
out of and K+ into a cell against their electrochemical
gradients. For every molecule of ATP hydrolyzed inside the
cell, three Na+ are pumped out and two K+ are pumped in.
The specific pump inhibitor ouabain and K+ compete for the
same site on the external side of the ATPase.
The Plasma Membrane contains
Na+-K+ Pump, an ATPase
*producing a change in the electrical potential of a cell

42. •Lower doses, can be used to treat hypotension
•It is a plant derived toxic substance that was
traditionally used as an arrow poison in eastern
Africa for both hunting and warfare
•Ouabain can be found in the roots, stems, leaves,
and seeds of theAcokanthera
schimperiandStrophanthusgratusplants, both of
which are native to eastern Africa
Acokantheraschimperi
Strophanthusgratusplant.
Ouabain isa cardiac glycoside

43. The binding of Na+ (1) and the
subsequent phosphorylation by ATP of
the cytoplasmic face of the ATPase (2)
induce the protein to undergo a
conformational change that transfers the
Na+ across the membrane and releases it
on the outside (3). Then the binding of
K+ on the extracellular surface (4) and
the subsequent dephosphorylation (5)
return the protein to its original
conformation, which transfers the K+
across the membrane and releases it into
the cytosol (6).
A schematic model of the pumping cycle of the Na+ -K+ ATPase
Although for simplicity only one Na+-and one K+-binding site are shown, in the real pump there are thought
to be three Na+-and two K+-binding sites. Moreover, although the ATPase is shown as alternating between
two conformational states, there is evidence that it goes through a more complex series of conformational
changes during the actual pumping cycle.

44. The Na+ -K+ ATPase Is Required to Maintain Osmotic Balance and
Stabilize Cell Volume
The Na+-K+ ATPase does have a direct role in regulating cell volume: it controls the solute concentration
inside the cell, thereby regulating the osmotic forces that can make a cell swell or shrink.
There are, of course, other ways for a cell to cope with its osmotic problems. Plant cells and many
bacteria are prevented from bursting by the semirigid cell wall that surrounds their plasma membrane; in
amoebae the excess water that flows in osmotically is collected in contractile vacuoles, which periodically
discharge their contents to the exterior. But for most animal cells, the Na+-K+ ATPase is crucial.

45. Cell Response to Osmotic Pressures
Cells generally contain higher concentrations of biomolecules
and ions than their surroundings, so osmotic pressure tends to
drive water into cells. If not somehow counterbalanced can
cause bursting of the cell (osmotic lysis).
Several mechanisms have evolved to prevent this catastrophe.
In bacteria and plants, the plasma membrane is surrounded by
a nonexpandablecell wall of sufficient rigidity and strength to
resist osmotic pressure and prevent osmotic lysis. Certain
freshwater protists that live in a highly hypotonic medium
have an organelle (contractile vacuole) that pumps water out of
the cell. In multicellular animals, blood plasma and interstitial
fluid (the extracellular fluid of tissues) are maintained at an
osmolarity close to that of the cytosol. The high concentration
of albumin and other proteins in blood plasma contributes to
its osmolarity. Cells also actively pump out ions such as Na-
into the interstitial fluid to stay in osmotic balance with their
surroundings.
Osmosis, water movement across a semipermeable
membrane driven by differences in osmotic
pressure, is an important factor in the life of most
cells. Plasma membranes are more permeable to
water than to most other small molecules, ions, and
macromolecules. This permeability is due partly to
simple diffusion of water through the lipid bilayer
and partly to protein channels (aquaporins)
membrane that selectively permit the passage of
water.

46. The contractile vacuole is a specialized type ofvacuolethat regulates the
quantity ofwaterinside acell. Infreshwaterenvironments, theconcentrationofsolutes
ishypotonic, lesser outside than inside the cell. Under these conditions,osmosiscauses
water to accumulate in the cell from the external environment. The contractile vacuole
acts as part of a protective mechanism that prevents the cell from absorbing too much
water.
The contractile vacuole, as its name suggests, expels water out of the cell by
contracting. The water gathering and contraction water expulsion of the contractile
vacuole are periodical.
Water always flows first from outside the cell into thecytoplasm, and is only then
moved from the cytoplasm into the contractile vacuole for expulsion.
The best understood contractile vacuoles belong to the
protistsParamecium,Amoeba,DictyosteliumandTrypanosoma
Osmosis also has consequences for laboratory protocols. Mitochondria, chloroplasts,
and lysosomes, for example, are bounded by semipermeable membranes. In isolating
these organelles from broken cells, biochemists must perform the fractionations in
isotonic solutions. Buffers used in cellular fractionations commonly contain sufficient
concentrations (about 0.2 M) of sucrose or some other inert solute to protect the
organelles from osmotic lysis.

47. Na
+
gradient to drives the glucose uptake
Na
+
and glucose bind cooperatively
•Intestine and kidney cells contains a variety of symporters that are driven by Sodium ion
gradient across the plasma membrane.
•The sodium ion driven symporters are specific to import of a small group of sugars or
amino acids into the cells.
•Sodium tends to move into the cells down its electrochemical gradient, these small
molecules (amino acids and sugars) are dragged into the cell with it.

48. Comparison of ion concentrations
inside and outside a typical
mammalian cell
Inside Outside
Lipid bilayers are highly impermeable to ion
•Given the time any molecule can diffuse through protein free lipid bilayer down its concentration gradient
•Rate of diffusion varies

49. An asymmetric distribution of carrier proteins underlies
transcellular transport in epithelial cells
The transcellular transport of glucose across an
intestinal epithelial cells depends on the asymmetrical
distribution of transport proteins in the cell's plasma
membrane.
The process shown results in the transport of glucose
from the gut lumen to the extracellular fluid (from
where it passes into the blood). Glucose is pumped
into the cell through the apical domain of the
membrane by a Na+-powered glucose symport,and
glucose passes out of the cell (down its concentration
gradient) by facilitated diffusion mediated by a
different glucose carrier protein in the basal and
lateral membrane domains. The Na+ gradient
driving the glucose symport is maintained by the
Na+-K+ ATPase in the basal and lateral plasma
membrane domains, which keeps the internal
concentration of Na+ low .
Transcellulartransportinvolvesthe
transportationofsolutesbyacellthrougha
cell.Oneclassicexampleisthemovement
ofglucosefromtheintestinal
lumentoextracellularfluidbyepithelialcells.

50. •Proteins are the end products of most information pathways. A typical cell
requires thousands of different proteins at any given moment. These must be
synthesized in response to the cell’s current needs, transported (targeted) to
their appropriate cellular locations, and degraded when no longer needed
•Many of the fundamental components and mechanisms used by the protein
biosynthetic machinery are well conserved from bacteria to higher eukaryotes
•The synthesis of proteins is the most complex biosynthetic process
•Eukaryotic protein synthesis requires more than 70 different ribosomal
proteins; 20 or more enzymes to activate the amino acid precursors; a dozen
or more auxiliary enzymes and other protein factors for the initiation,
elongation, and termination of polypeptides; perhaps 100 additional enzymes
for the final processing of different proteins; and 40 or more kinds of transfer
and ribosomal RNAs. Overall, almost 300 different macromolecules
cooperate to synthesize polypeptides
•Protein synthesis can account for up to 90% of the chemical energy used by a
cell for all biosynthetic reactions.
•The 15,000 ribosomes, 100,000 molecules of protein synthesis–related
protein factors and enzymes, and 200,000 tRNA molecules in a typical
bacterial cell can account for more than 35% of the cell’s dry weight
•A polypeptide of 100 residues is synthesized in an Escherichia coli cell (at 37
°C) in about 5 seconds.
Electron micrograph of a portion of a
pancreatic cell, showing ribosomes attached to
the outer (cytosolic) face of the endoplasmic
reticulum (ER). The ribosomes are the
numerous small dots bordering the parallel
layers of membranes
Ribosome

51. Composition of ribosomes in prokaryotic and eukaryotic cells

52. Three major advances set the stage for our present
knowledge of protein biosynthesis
First,in the early 1950s, Paul Zamecnikand his colleagues designed a set of experiments to investigate where in the cell proteins
are synthesized. They injected radioactive amino acids into rats and, at different time intervals after the injection, removed the
liver, homogenized it, fractionated the homogenate by centrifugation, and examined the subcellular fractions for the presenceof
radioactive protein. When hours or days were allowed to elapse after injection of the labeled amino acids, all the subcellular
fractions contained labeled proteins. However, when only minutes had elapsed, labeled protein appeared only in a fraction
containing small ribonucleoprotein particles. These particles, visible in animal tissues by electron microscopy, were therefore
identified as the site of protein synthesis from amino acids, and later were named ribosomes.
Secondkey advance was made by MahlonHoagland and Zamecnikwhen they found that amino acids were “activated” for
protein synthesis when incubated with ATP and the cytosolic fraction of liver cells. The amino acids became attached to a heat-
stable soluble RNA of the type that had been discovered and characterized by Robert Holley, and later called transfer RNA
(tRNA), to form aminoacyl-tRNAs. The enzymes that catalyze this process are the aminoacyl-tRNA synthetases.
Thirdadvance was how the genetic information encoded in the 4-letter language of nucleic acids could be translated into the 20-
letter language of proteins. The tRNA adaptor, the same molecule that activates the amino acid for peptide bond formation, also
“translates” the nucleotide sequence of an mRNA into the amino acid sequence of a polypeptide. The overall process of mRNA-
guided protein synthesis is often referred to simply as translation.

53. Ribosomes are macromolecular
machines, found within all living
cells, that perform biological
protein synthesis. Ribosomes link
amino acids together in the order
specified by the codons of
messenger RNA molecules to
form polypeptide chains
Steps of protein biosynthesis

54. •Peroxisomes are found in all eucaryoticcells
•They contain oxidative enzymes, such as catalase and urate oxidase, at
high concentrations and are visible under electron microscope because
of the presence of a crystalloid core, largely composed of urate oxidase
•They are surrounded by only a single membrane, and they do not
contain DNA or ribosomes.
•Peroxisomes are thought to acquire their proteins from the cytosol.
•Because peroxisomes have no genome, all of their proteins must be
imported.
•Peroxisomes are self-replicating membrane-bounded organelles that
exist without genomes of their own
•Like the mitochondrion, the peroxisome is a major site of oxygen
utilization. Peroxisomes are ancient organelle that carried out all of the
oxygen metabolism in the primitive ancestors of eucaryoticcells. Later
development of mitochondria rendered the peroxisome largely
obsolete.
•The oxidative reactions carried out by peroxisomes in present-day
cells would therefore be those that have important functions not taken
over by mitochondria.
Peroxisomes
Three peroxisomes from rat liver
are shown. Two contain dense
regions, which are paracrystalline
arrays of the enzyme urate
oxidase
-
-
-
-
-
-
·

55. •Peroxisomes are so called because they usually contain one or more enzymes that use molecular
oxygen to remove hydrogen atoms from specific organic substrates (R) in an oxidative reaction that
produces hydrogen peroxide (H2O2):
•Catalaseutilizes the H2O2 generated by other enzymes in the organelle to oxidize a variety of other
substrates -including phenols, formic acid, formaldehyde, and alcohol -by the "peroxidative"
reaction.
•This type of oxidative reaction is particularly important in liver and kidney cells, whose peroxisomes
detoxify various toxic molecules that enter the bloodstream.
•In addition, when excess H2O2 accumulates in the cell,
catalase converts it to H2O (2H2O2 2H2O + O2).

56. •In animal cells,cholesteroland dolichol are
synthesized in peroxisomes as well as in theER.
•In the liver, peroxisomes are also involved in the
synthesis of bile acids, which are derived from
cholesterol.
•In addition, peroxisomes containenzymesrequired for
the synthesis ofplasmalogens—a family
ofphospholipidsin which one of the hydrocarbon
chains is joined to glycerol by an ether bond rather
than an ester bond.
•Plasmalogens are important membrane components in
some tissues, particularly heart and brain, although
they are absent in others.
In addition to providing a compartment for oxidation reactions,
peroxisomes are involved in lipid biosynthesis.
The plasmalogen shown
is analogous to
phosphatidylcholine.
However, one of the fatty
acid chains is joined to
glycerol by an ether,
rather than an ester,
bond.

57. •A major function of the oxidative reactions carried out
in peroxisomes is the breakdown of fatty acid
molecules by a process called beta oxidation, the alkyl
chains of fatty acids are shortened sequentially by
blocks of two carbon atoms at a time that are converted
to acetyl CoA and exported from the peroxisomes to the
cytosol for reuse in biosynthetic reactions.
•Beta oxidation in mammalian cells occurs both in
mitochondria and peroxisomes.
The oxidation of a fatty acid is accompanied by the production of
hydrogen peroxide (H
2O
2) from oxygen. The hydrogen peroxide is
decomposed bycatalase
Beta oxidation of fatty acids

58. •In yeast and plant cells, however, this essential reaction
(beta oxidation) is exclusively found in peroxisomes.
•Peroxisomes also can adapt to changing conditions.
Yeast cells grown on sugar, for example, have small
peroxisomes. But when some yeasts are grown on
methanol, they develop large peroxisomes that oxidize
methanol; and when grown on fatty acids, they develop
large peroxisomes that break down fatty acids to acetyl
CoA by beta oxidation.
There are two different types of peroxisomes in plants. One type is present in leaves, where it catalyzes
process is called photorespiration because it uses up O2 and liberates CO2.
The other type of peroxisome is present in germinating seeds, where it plays an essential role in converting the
fatty acids stored in seed lipids into the sugars needed for the growth of the young plant. Because this
conversion of fats to sugars is accomplished by a series of reactions known as the glyoxylate cycle, these
peroxisomes are also called glyoxysomes. In the glyoxylate cycle two molecules of acetyl CoA produced by
fatty acid breakdown in the peroxisome are used to make succinic acid, which leaves the peroxisome and is
converted into glucose. The glyoxylate cycle does not occur in animal cells, and animals are thus unable to
convert the fatty acids in fats into carbohydrates.
Two types of peroxisomes in plants

59. In plants theglyoxylatecycle occurs in
specialperoxisomeswhich are
calledglyoxysomes. This cycle allows
seeds to use lipids as a source of energy
to form the shoot duringgermination.
The seed cannot produce biomass using
photosynthesis because of lack of an
organ to perform this function. The
lipid stores of germinating seeds are
used for the formation of the
carbohydrates that fuel the growth and
development of the organism.

60. Photorespiration in brief
Ribulose bisphosphate carboxylase or rubisco,
sometimes catalyzes the addition of O
2instead of CO
2,
producing one molecule of 3-phosphoglycerate and one
molecule of phosphoglycolate
Photorespiration(also known as theoxidative
photosyntheticcarbon cycle orC
2cycle) refers to a process inplant
metabolismwhere theenzymeRuBisCOoxygenatesRuBP, wasting
some of the energy produced by photosynthesis
Factors which influence this include the atmospheric abundance of the
two gases, the supply of the gases to the site of fixation (i.e. in land
plants: whether thestomataare open or closed), the length of the liquid
phase (how far these gases have to diffuse through water in order to
reach the reaction site). For example, when the stomata are closed to
prevent water loss duringdrought: this limits the CO
2supply, whileO
production within the leaf will continue

61. Lysosome and vacuole
a typical animal cell
a typical plant cell

62. Lysosomesare membrane-enclosed organelles that contain an array
of50 different degradative enzymescapable of breaking down all types of
biological polymers; proteins, nucleic acids, carbohydrates, andlipids.
Lysosomes function as the digestive system of the cell, serving both to
degrade material taken up from outside the cell and to digest obsolete
components of the cell itself.
Mutations in the genes that encode these enzymes are responsible for more
than 30 different human genetic diseases, which are calledlysosomal storage
diseasesbecause undegraded material accumulates within the lysosomes of
affected individuals.
For example, Gaucher’s disease (the most common of these disorders) results
from amutationin thegenethat encodes a lysosomal enzyme required for the
breakdown of glycolipids.
I-cell disease, which is caused by a deficiency in the enzyme that catalyzes
the first step in the tagging of lysosomal enzymes with mannose-6-phosphate
in theGolgi apparatus.
Three pathways to degradation in lysosomes
Lysosomes contain a variety of acid
hydrolases that are active at the acidic pH
maintained within thelysosome, but not at
the neutral pH of the cytosol. The acidic
internal pH of lysosomes results from the
action of a proton pump in the lysosomal
membrane, which imports protons from the
cytosol coupled to ATP hydrolysis.
Most of the lysosomal membrane proteins
are unusually highly glycosylated, which
helps to protect them from the lysosomal
proteases in the lumen.
Lysosomes Are the Sites of
Intracellular Digestion

63. Lysosomes were initially discovered by the
biochemical fractionation of cell extracts;
only later were they seen clearly in
theelectron microscope. Although
extraordinarily diverse in shape and size,
they can be identified as members of a
single family of organelles by staining them
with specific antibodies.
Theseelectronmicrographs show two
sections of a cell stained to reveal the
location ofacidphosphatase, a
markerenzymefor lysosomes. The
largermembrane-enclosed organelles,
containing dense precipitates of lead
phosphate, are lysosomes. Their diverse
morphology reflects variations in the
amount and nature of the material they
are digesting. The precipitates are
produced when tissue fixed
withglutaraldehyde(to fix the enzyme
in place) is incubated with a
phosphatasesubstratein the presence of
lead ions. Two small vesicles thought to
be carrying acid hydrolases from the
Golgi apparatus are indicated byred
arrowsin the top panel.

64. Transport of newly
synthesized lysosomal
hydrolases to lysosomes
The precursors of lysosomal hydrolases are covalently modified by the addition ofmannose 6-phosphate (M6P)groups in thecisGolgi network. They then
become segregated from all other types of proteins in thetransGolgi network because adaptinsin theclathrincoat bind the M6P receptors, which, in turn, bind
the modified lysosomal hydrolases. The clathrin-coated vesicles produced bud off from thetransGolgi network and fuse with late endosomes. At the lowpHof
the lateendosome, the hydrolases dissociate from the M6P receptors, and the empty receptors are recycled to the Golgi apparatus for further rounds of transport.
It is not known which type of coat mediatesvesiclebudding in the M6Preceptorrecycling pathway. In the late endosomes, the phosphate is removed from the
mannose sugars attached to the hydrolases, further ensuring that the hydrolases do not return to the Golgi apparatus with thereceptor
The M6P Receptor Shuttles Between Specific Membranes

65. TheM6Preceptorproteinbinds its specificoligosaccharideatpH6.5–6.7 in thetransGolgi
network and releases it at pH 6, which is the pH in the interior of late endosomes. Thus, in the late
endosomes, the lysosomal hydrolases dissociate from the M6P receptor.
As the pH drops further during endosomal maturation, the hydrolases begin to digest the
endocytosed material delivered from early endosomes. Having released their bound enzymes, the
M6P receptors are retrieved into transport vesicles that bud from late endosomes; the receptors are
then returned to themembraneof thetransGolgi network for reuse.
Transport in either direction requires signal peptides in the cytoplasmic tail of the M6P receptor that
specify transport of this protein to the lateendosomeor back to the Golgi apparatus.
Not all of the hydrolase molecules that are tagged withM6Pfor delivery to lysosomes get to their
proper destination. Some escape the normal packaging process in thetransGolgi network and are
transported “by default” to the cell surface, where they are secreted into the extracellular fluid.
Some M6P receptors, however, also take a detour to theplasma membrane, where they recapture
the escaped lysosomal hydrolases and return them byreceptor-mediated endocytosisto lysosomes
via early and late endosomes. As lysosomal hydrolases require an acidic milieu to work, they can do
little harm in the extracellular fluids, which usually has a neutralpH.
The structure of mannose
6-phosphate on a
lysosomal enzyme

66. •Most plant and fungal cells contain one or several very large, fluid-filled vesicles called vacuoles. A vacuole is surrounded by a membrane called thetonoplast. They
typically occupy cell volume in the range of 30% -90%. Vacuoles are related to lysosomes of animal cells, containing a variety of hydrolytic enzymes, but their
functions are remarkably diverse.
•The organelle has no basic shape or size; its structure varies according to the requirements of the cell
•The plant vacuole can act as a storage organelle for nutrients and waste products, as a degradative compartment, as an economical way of increasing cell size, and as a
controller of turgor pressure (the osmotic pressure that pushes outward on the cell wall and protects the plant from wilting. Different vacuoles with distinct functions
such as digestion and storage are often present in the same cell
•The vacuole is important as a homeostatic device, enabling plant cells to withstand wide variations in their environment. When the pH in the environment drops due to
the flux of H+ into the cytosol is balanced by increased transport of H+ into the vacuole so as to keep the pH in the cytosol constant. Similarly, many plant cells
maintain constant turgor pressure by changing the osmotic pressure of the cytosol and vacuole by the controlled breakdown andre-synthesis of polymers such as
polyphosphate in the vacuole and by altering rates of transport of sugars, amino acids, and other metabolites across the plasma membrane and the vacuolar membrane.
•Substances stored in plant vacuoles in different species range from rubber to opium to the flavoring of garlic. Often, the stored products have a metabolic function.
Proteins, for example, can be preserved for years in the vacuoles of the storage cells of many seeds, such as those of peas and beans. When the seeds germinate, the
proteins are hydrolyzed and the mobilized amino acids provide a food supply for the developing embryo. Anthocyanin pigments thatare stored in vacuoles color the
petals of many flowers to attract pollinating insects, while noxious molecules that are released from vacuoles when a plant is eaten or damaged provide a defense
against predators.
Vacuoles
-
- -
-
-
-
-

67. Plant and Fungal Vacuoles Are Remarkably Versatile Lysosomes
The plant cell vacuole. This electron micrograph of cells in
tobacco leaf shows that the cytosol consist of a large
vacuole surrounded by a thin layer known as tonoplast.
Chloroplasts, pressed against the cell wall
A large increase in cell volume can be achieved without increasing the
volume of the cytosol. Localized weakening of the cell wall orients a
turgor-driven cell enlargement that accompanies the uptake of water into an
expanding vacuole. The cytosol is eventually confined to a thin peripheral
layer that is connected to the nuclear region by strands of cytosol, which
are stabilized by bundles of actin filaments.

68. Free and
membrane-
bound ribosomes

69. The Endoplasmic Reticulum
•All eucaryoticcells have anendoplasmic reticulum(ER)
Itsmembranetypically constitutes more than half of the total
membrane of an average animal cell
•The ER is organized into a netlike branching tubules extending
throughout thecytosol
•The tubules are all thought to interconnect, so that the ER
membrane forms a continuous sheet enclosing a single internal
space
•This highly convoluted space is called theER lumen
•The ER membrane separates theER lumenfrom the cytosol,
•TheERhas a central role inlipidandproteinbiosynthesis
•Itsmembraneis the site of production of all the transmembrane
proteins and lipids for most of the cell's organelles, including
the ER itself, the Golgi apparatus, lysosomes, endosomes,
secretory vesicles, and theplasma membrane.
•The ER membrane makes a major contribution to mitochondrial
and peroxisomal membranes
•In addition, almost all of the proteins that will be secreted to the
cell exterior; plus those destined for thelumenof the ER, Golgi
apparatus, or lysosome, are initially delivered to theER lumen.
Electronmicrographof the roughER
Electronmicrographof the smoothER

70. Rough and Smooth Regions of ER Can Be Separated by Centrifugation

71. How are proteins directed to their final cellular destinations?
A Signal-Recognition Particle (SRP) Directs ER Signal Sequences to a Specific Receptor in the Rough ER Membrane
A short sequence of amino acids called a signal sequence (SS) recognized
by SRP marks them for translocation into the lumen of the ER. SS directs
a protein to its appropriate location in the cell and, for many proteins, is
removed during transport or after the protein has reached its final
destination.
Hundreds of such SS have been determined.
The carboxyl terminus of the SS contains a cleavage site which is cleaved
by a protease to remove the sequence after the protein is imported into the
ER. SS vary in length from 13 to 36 amino acid residues
The targeting pathway begins with initiation of protein synthesis on free ribosomes. The signal sequence (SS) appears early in the synthetic process,
because it is at the amino terminus, synthesized first.
As SS emerges from the ribosome, the SS and the ribosome itself are bound by the large signal recognition particle (SRP); SRPthen binds GTP and
halts elongation of the polypeptide when it is about 70 amino acids long.
The GTP-bound SRP now directs the ribosome (still bound to the mRNA) and the incomplete polypeptide to GTP-bound SRP receptors in the cytosolic
face of the ER; the nascent polypeptide is delivered to a peptide translocation complex in the ER, which may interact directly with the ribosome.
SRP dissociates from the ribosome, accompanied by hydrolysis of GTP in both SRP and the SRP receptor.
Elongation of the polypeptide now resumes, with the ATP-driven translocation complex feeding the growing polypeptide into the ERlumen until the
complete protein has been synthesized.
The signal sequence is removed by a signal peptidase within the ER lumen.
The ribosome dissociates and is recycled.

72. Glycosylation Takes Place in the Lumen of the Endoplasmic Reticulum and in the
Golgi Complex, Plays a Key Role in Protein Targeting, Protein Glycosylation
TheN-linked glycosylation
begins in the ER and continues
in the Golgi complex, whereas
theO-linked glycosylation
takes place exclusively in the
Golgi complex.
The 14-sugar-residue precursor
attached to this dolichol
phosphate intermediate is then
transferred as a bloc to a
specific asparagine residue of
the growing polypeptide chain.
Most Proteins Synthesized in the Rough ER Are Glycosylated by the Addition
of a Common N -linked Oligosaccharide

73. TheN-linked glycosylation begins in
the ER and continues in the Golgi
complex, whereas theO-linked
glycosylation takes place exclusively
in the Golgi complex.

74. The Golgi complex is the sorting center in the targeting of proteins to
lysosomes, secretory vesicles, and the plasma membrane. The cis face of the
Golgi complex receives vesicles from the ER, and the trans face sends a
different set of vesicles to target sites. Vesicles also transfer proteins from
one compartment of the Golgi complex to another
Transport Vesicles Carry Proteins from the Endoplasmic Reticulum to the Golgi
Complex for Further Glycosylation and Sorting
•Oligosaccharides Are Used as Tags to Mark the
State of Protein Folding
•Improperly Folded Proteins Are Exported from
the ER and Degraded in the Cytosol
-
-
--

75. The Golgi complex is the sorting center in the targeting of proteins to
lysosomes, secretory vesicles, and the plasma membrane. The cis face of the
Golgi complex receives vesicles from the ER, and the trans face sends a
different set of vesicles to target sites. Vesicles also transfer proteins from
one compartment of the Golgi complex to another
Transport Vesicles Carry Proteins from the Endoplasmic Reticulum to the Golgi
Complex for Further Glycosylation and Sorting
•Oligosaccharides Are Used as Tags to Mark the
State of Protein Folding
•Improperly Folded Proteins Are Exported from
the ER and Degraded in the Cytosol
-

76. Only Proteins That Are Properly Folded
and Assembled Can Leave the ER
•To exit from theER, proteins must be properly
folded and, if they are subunits of
multimericproteincomplexes, they may need to be
completely assembled. Those that are misfolded or
incompletely assembled are retained in the ER,
where they are bound to chaperone proteins
•Such failed proteins are eventually transported
back into thecytosolwhere they are degraded by
proteasomes
•This quality-control step is important, as misfolded
or misassembled proteins could potentially
interfere with the functions of normal proteins if
they were transported onward
Misfolded Proteins in the ER Activate
an Unfolded Protein Response
By this novel intracellular signaling pathway, the accumulation of
misfolded proteins in theER lumensignals to thenucleusto
activate the transcription of genes that encode proteins that help the
cell to cope with the abundance of misfolded proteins in the ER.

78. Similarities between
mitochondria and chloroplast
During thedevelopmentof the flagellum of the sperm tail, microtubules wind
helically around theaxoneme, where they are thought to help localize the
mitochondria in the tail; these microtubules then disappear, and the mitochondria
fuse with one another to create the structure shown

79. •Theoutermembranecontainsmanycopiesofa
transportproteincalledporin,whichformslargeaqueouschannels
throughthelipidbilayer
•Theinnermembraneishighlyspecialized.Itslipidbilayercontainsa
highproportionofthe“double”phospholipidcardiolipin,whichhas
fourfattyacidsratherthantwo.
•Innermembranealsocontainsavarietyoftransportproteinsthat
makeitselectivelypermeabletothosesmallmoleculesthatare
metabolizedorrequiredbythemanymitochondrialenzymes
concentratedinthematrix.
The matrix enzymes include those that metabolize pyruvate and fatty acids to produceacetyl CoAand those that oxidize acetyl CoA in
thecitricacidcycle.The principal end-products of this oxidation are CO
2, which is released from the cell as waste, and NADH, which is the main
source of electrons for transport along therespiratory chainor electron-transport chainin mitochondria. The enzymes of therespiratory chainare
embedded in the inner mitochondrial membrane, essential for the process ofoxidative phosphorylation,which generates most of the animal cell's ATP.
Theinner membraneis usually highly convoluted, forming infoldings, known ascristae, that project into the matrix. The cristae increase the area of the
inner membrane. The number of cristae is three times greater in the mitochondrion of acardiac musclecell than in the mitochondrion of a liver cell,
presumably because of the greater demand for ATP in heart cells.

80. Biochemical
fractionation of
purified
mitochondria
into separate
components

81. High-Energy Electrons Are Generated via the Citric Acid Cycle
Without mitochondria eukaryotic organisms would be dependent on the
glycolysisfor all of their ATP production. Whenglucoseis converted to
pyruvate by glycolysis, less than 10% of the total free energy from the glucose
is released. In the mitochondria, themetabolismof sugars is completed, about
30 molecules of ATP are produced for eachmoleculeof glucose oxidized. By
contrast, only 2 molecules of ATP are produced per glucose molecule by
glycolysis alone.
Mitochondria can use both pyruvate and fatty acids as fuel. Pyruvate comes
fromglucoseand other sugars, whereas fatty acids come from fats. Both of
these fuel molecules are transported across the inner
mitochondrialmembraneand then converted to metabolic intermediateacetyl
CoAby enzymes located in the mitochondrial matrix.
The acetyl groups in acetyl CoA are then oxidized in the matrix via thecitric
acid cycleand converts the carbon atoms in acetyl CoA to CO
2. The cycle
generates high-energy electrons, carried by thecarriermolecules NADH and
FADH
2. These high-energy electrons are then transferred to the inner
mitochondrial membrane, where they enter theelectron-transport chain. The
loss of electrons from NADH and FADH
2regenerates the NAD
+
and FAD that
is needed for continued oxidativemetabolism.
Pyruvate and fatty acids enter the mitochondrionand are broken down toacetyl CoA. The acetyl
CoA is then metabolized by the citricacidcycle, which reduces NAD
+
to NADH (and FAD to
FADH
2). In the process ofoxidative phosphorylation, high-energy electrons from NADH (and
FADH
2) are then passed along theelectron-transport chainin theinner membraneto oxygen (O
2).
This electron transport generates aprotongradient across the inner membrane, which is used to
drive the production of ATP byATP synthase. The NADH generated byglycolysisin
thecytosolalso passes electrons to therespiratory chain. Since NADH cannot pass across the
inner mitochondrialmembrane, theelectrontransfer from cytosolic NADH must be accomplished
indirectly by means of one of several “shuttle” systems.

82. Thechemiosmotic hypothesiswas
proposed by Peter Mitchell in 1978 which
suggests that the action ofATPsynthase is
coupled with that of a proton gradient.
According to this hypothesis protons are
pumped across the inner mitochondrial
membrane as electrons move through the
electron transfer chain. It is the action of the
proton gradient that causes a proton motive
force that allowsATPsynthase to
phosphorylate ADP and inorganic phosphate
toATP.

83. In some specializedfatcells, mitochondrialrespirationis normally uncoupled from ATP synthesis. In
these cells, known as brown fat cells, most of the energy of oxidation is dissipated as heat rather than
being converted into ATP. The inner membranes of the large mitochondria in these cells contain a
special transportproteinthat allows protons to move down theirelectrochemical gradient, by-
passingATP synthase. As a result, the cells oxidize their fat stores at a rapid rate and produce more heat
than ATP. Tissues containing brown fat serve as “heating pads,” helping to revive hibernating animals
and to protect sensitive areas of newborn human babies from the cold
H
+
Ionophores Uncouple Electron
Transport from ATP Synthesis:
several substances; such as 2,4-
dinitrophenol have been known to act
asuncoupling
agents,uncouplingelectrontransport from
ATP synthesis. The addition of these low-
molecular-weight organic compounds to
cells stops ATP synthesis by mitochondria
without blocking their uptake of oxygen.
In the presence of an uncoupling agent,
electron transport andH
+
pumping
continue at a rapid rate, but no H
+
gradient
is generated.
Thermogenesis: the dissipation of energy through the production of
heatand occurs in specialisedtissues including brown adipose tissue

84. Protein import by mitochondria. The amino-terminal signal peptide of
the precursor protein is recognized by receptors that reside in the
outer membrane. The protein is thought to be translocated across both
mitochondrial membranes at or near special contact sites, driven first
by the electrochemical gradient across the inner membrane and then
by ATP hydrolysis. The signal peptide is cleaved off by a signal
peptidase in the matrix to form the mature protein; the free signal
peptide is rapidly degraded.
•Although both organelles (mitochondria and chloroplast) contain
their own DNA, ribosomes, and other machinery for protein
synthesis, most of their proteins are encoded by nuclear genome
and imported from the cytosol
•Proteins imported into the mitochondrial matrix are usually
taken up from the cytosol
•These mitochondrial precursor proteins almost always have a
signal peptide (20-80 residues long) at their amino terminus that
is removed after import by a protease (the signal peptidase) in
the mitochondrial matrix.
•As a first step in mitochondrial import, the mitochondrial
precursor proteins have to bind to receptor proteins that reside in
the mitochondrial outer membrane and recognize the
mitochondrial signal peptides. The next step is the translocation
process itself.
Protein import by mitochondria

85. Protein import into the mitochondrial matrix requires hsp70 proteins
on both sides of the mitochondrial double membrane. After the
initial insertion of the signal peptide and of adjacent portions of the
polypeptide chain, the unfolded chain slides in a channel that spans
both membranes. Bound cytosolic hsp70 is released from the protein
in a step that depends on ATP hydrolysis; concomitantly,
mitochondrial hsp70 binds to regions of the polypeptide chain as
they become exposed in the matrix, thereby pulling the protein into
the interior of the mitochondrion.
Transport of mitochondrial precursor proteins across the two mitochondrial
membranes at a contact site is guided by members of the chaperone family of
proteins
It is now known that cytosolic chaperone proteins (called chaperonins)
belonging to the hsp70 family, helping to ensure the correct folding of cytosolic
proteins, play an essential part in protein import into mitochondria by binding
the precursor in its unfolded state during translocation. The release of newly
synthesized polypeptides from the hsp70 family of chaperone proteins requires
ATP hydrolysis.
The essential role of the chaperone proteins in translocation across internal
cellular membranes was first indicated by genetic studies in yeasts. When the
genes encoding certain members of the hsp70 family of chaperone proteins are
inactivated, mitochondrial precursor proteins fails to import into mitochondria
and accumulate in the cytosol instead. It is thought that newly synthesized
precursor proteins, as they are released from polyribosomes in the cytosol, bind
to hsp70 proteins, which prevent the precursor proteins from aggregating or
folding up spontaneously before they bind to the protein translocator in the
target membrane. The energy liberated by the hydrolysis of ATP is used to
release the bound hsp70 proteins as the translocated protein is passed across the
membrane.
Proteins Are Imported into the
Mitochondrial Matrix in an Unfolded State

86. The Nucleus

87. The nuclear envelope. The
double-membrane envelope is
penetrated by nuclear pores and
is continuous with the
endoplasmic reticulum. Pore
composed of more than 100
different proteins, arranged in a
octagonal symmetry
The nuclear envelope consists of an inner and an
outer nuclear membrane. The outer membrane is
continuous with the ER membrane, and the space
between it and the inner membrane is continuous
with the ER lumen.
RNA molecules, which are made in the nucleus, and
ribosomal subunits, which are assembled there, are
exported to the cytosol, while all of the proteins that
function in the nucleus are synthesized in the cytosol
and are then imported.
The extensive traffic of materials between nucleus
and cytosol occurs through nuclear pores that
provide a direct passageway across the nuclear
envelope.
Proteins containing nuclear localization signals are
actively transported inward through the pores, while
RNA molecules and newly made ribosomal subunits
are actively transported outward through the pores.
The nuclear envelope: Nuclear Pores Perforate the Nuclear Envelope

88. The many proteins that function in the nucleus -including
histones, DNA and RNA polymerases, gene regulatory
proteins, and RNA-processing proteins -are selectively
imported into the nuclear compartment from the cytosol
where they are made.
At the same time, tRNAs and mRNAs are synthesized in the
nuclear compartment and then exported to the cytosol.
Like the import process, the export process is selective;
mRNAs, for example, are exported only after they have been
properly modified by RNA-processing reactions in the
nucleus.
In some cases the transport process is much more complex:
ribosomal proteins, for instance, are made in the cytosol,
imported into the nucleus -where they assemble with newly
made ribosomal RNA into particles -and then exported again
to the cytosol as part of a ribosomal subunit; each of these
steps involves selective transport across the nuclear envelope
Bidirectional traffic occurs continuously between the
cytosol and the nucleus

89. The 45S rRNA transcript is packaged
in a large ribonucleoprotein particle
containing many ribosomal proteins
imported from the cytoplasm. While
this particle remains in the nucleolus,
selected pieces are discarded as it is
processed into immature large and
small ribosomal subunits. These two
subunits are thought to attain their
final functional form only as each is
individually transported through the
nuclear pores into the cytoplasm.
The Nucleolus is a Ribosome-producing Machine: The packaging
of ribosome occurs in the nucleolus

90. Breakdown and re-formation of the nuclear envelope during mitosis. The
phosphorylation of the lamins is thought to help trigger the disassembly
of the nuclear lamina, which in turn causes the nuclear envelope to break
up into vesicles. Dephosphorylation of the lamins is thought to help
reverse the process.
•Each pore complex contains one or more open aqueous
channels through which water-soluble molecules that
are smaller than a certain size can passively diffuse
•Because the nuclear localization signals are not
removed, nuclear proteins can be imported repeatedly,
as is required each time the nucleus reassembles
following mitosis.
•The transport of nuclear proteins and RNA molecules
through the pores can be regulated by transport
machinery in the nuclear pore complexes
•In general, the more active the nucleus is in
transcription, the greater the number of pore complexes
its envelope contains
•The nuclear envelope of a typical mammalian cell
contains 3000 to 4000 pore complexes. If the cell is
synthesizing DNA, it needs to import about 10 6 histone
molecules from the cytosol every 3 minutes in order to
package newly made DNA into chromatin, which
means, on average, each pore complex needs to
transport about 100 histone molecules per minute.
•If the cell is growing rapidly, each pore complex also
needs to transport about 6 newly assembled large and
small ribosomal subunits per minute from the nucleus.

91. Types of chromosomes:
Chromosomesare thread-like structures located
inside the nucleus of animal and plant cells.
Eachchromosomeis made of protein and a
single molecule of deoxyribonucleic acid (DNA).
Human chromosomes are of twotypesautosomes
and sex chromosomes

92. Centromereandtelomereare two regions found on a
chromosome.Centromereis approximately located in the middle of a
chromosome andtelomereis located at the two ends of each sister chromatid. The
main difference betweencentromereandtelomereis their location on a
chromosome and role during the cell's life.
The centromereis a chromosomal region where the kinetochore is formed, which
is the attachment point of spindle fibers. Thus, it is responsible for the correct
chromosome segregation during cell division.
Telomeresprotect chromosome ends against enzymatic degradation, DNA
damage and fusions. Research results show that the identity and functions of these
chromosomal regions are epigenetically determined.
Telomeres and centromeres are both usually described as highly condensed
heterochromatin regions. However, the epigenetic nature of centromeres and
telomeres is unique, as epigenetic modifications characteristic of both eu-and
heterochromatin have been found in these areas. They are composed of tandem
arrays of repeated DNA sequences with a specific heterochromatic organization
A kinetochoreis a disc-shaped protein structure
associated with duplicated chromatids in
eukaryotic cells where the spindle fibers attach
during cell division to pull sister chromatids apart.
ThePCMserves as a platform for protein
complexes that regulate organelle trafficking,
protein degradation and spindle assembly.
Centromeres and telomeres are parts of each linear chromosome
and necessary for their proper functioning
The length of telomeric repeats is dynamically regulated and can be affected by changes in the telomere chromatin structure.
When telomeres are not properly controlled, the resulting chromosomal alterations can induce genomic instability and
ultimately the development of human diseases, such as cancer. Therefore, proper establishment, regulation, and maintenance of
the telomere chromatin structure are required for cell homeostasis.

93. Telomeraseis the enzyme responsible for maintenance of the length of telomeres by
addition of guanine-rich repetitive sequences.
Telomeraseis a ribonucleoprotein (RNP) enzyme comprised of two essential core subunits:
thetelomeraseRNA (TER) andtelomerasereverse transcriptase protein (TERT)
The structure of telomere

94. Chromosomal DNA is
packaged inside
microscopic nuclei with
the help of histones.
These are positively-
charged proteins that
strongly adhere to
negatively-charged DNA
and form complexes
called nucleosomes.
Each nuclesome is
composed of DNA
wound 1.65 times
around eight histone
proteins. Nucleosomes
fold up to form a 30-
nanometer chromatin
fiber, which forms loops
averaging 300
nanometers in length.
The 300 nm fibers are
compressed and folded
to produce a 250 nm-
wide fiber, which is
tightly coiled into the
chromatid of a
chromosome.
Chromosomes are composed of DNA tightly -wound around histones

95. Micrococcalnucleasedigestschromatin
innuclei,multiplesof200bpofDNA
bandsingelelectrophoresis.
Nucleosome is the fundamental unit of all Chromatin
Evidencetosupportthat
nucleosomeisthebasicunitof
chromatin
•MNasedigestion
•Electronmicroscopy
•Reconstitutionofnucleosome
Digestion of
Nuclei by
MNase
Arthur Kornberg Roger Kornberg
Nobel Prize in 1959 Nobel Prize in 2006
Synthesis of DNA Synthesis of RNA
Micrococcal nuclease preferentially digests the naked DNA between nucleosomes

96. Awestern blotis a laboratory method used to detect specific protein molecules including
epigenetic changes from among a mixture of proteins, associated with a particular tissue or cell type.

98. The cytoskeleton
A cell in culture has been fixed and stained with Coomassie blue, a
general stain for proteins. The variety of filamentous structures extends
throughout the cell.
Two extra methyl groups
Slightly greenish
Slightly reddish

99. Three basic types of cytoskeleton

100. Function of the Cytoskeleton
The cytoskeleton has several functions.
•First, it gives the cell shape. This is especially
important in cells without cell walls, such as animal
cells.
•It can also give the cell movement. The microfilaments
and microtubules can disassemble, reassemble, and
contract, allowing cells to crawl and migrate, and help
form structures like cilia and flagella that allow cell
movement.
•The cytoskeleton organizes the cell and keeps the cell’s
organelles in place, but it also aids in the movement of
organelles throughout the cell. For example,
duringendocytosiswhen a cell engulfs a molecule,
microfilaments pull thevesiclecontaining the engulfed
particles into the cell.
•Similarly, the cytoskeleton helps move chromosomes
during cell division.

101. •Microtubules are formed
fromproteinsubunits oftubulin.
Thetubulinsubunitis itself
aheterodimerformed from two closely
related globular proteins called.-
tubulinand-tubulin,tightly bound
together by noncovalent bonds.
•(DFK.RUmonomerhas abinding sitefor
onemoleculeof GTP. The hydrolysis of
GTP has an important effect
onmicrotubuledynamics.
The structure of a microtubule and its subunit
(A) Thesubunitof eachprotofilamentis atubulinheterodimerIRUPHGIURPDYHU\WLJKWO\OLQNHGSDLURI.-DQG-tubulin monomers. The GTPmoleculeLQWKH.-
tubulinmonomeris so tightly bound that it can be considered an integral part of theprotein7KH*73PROHFXOHLQWKH-tubulin monomer, however, is less tightly bound and has
an important role in filament dynamics. Both nucleotides are shown inred%2QHWXEXOLQVXEXQLW.-KHWHURGLPHUDQGRQHSURWRILODPHQWDUHVKRZQVFKHPDWLFDOO\(DFK
protofilament consists of many adjacent subunits with the same orientation. (C) Themicrotubuleis a stiff hollow tube formed from 13 protofilaments aligned in parallel. (D) A
short segment of a microtubule viewed in anelectron microscope. (E) Electronmicrographof a crosssectionof a microtubule showing a ring of 13 distinct protofilaments.
Themicrotubuleis a stiff hollow tube formed
from 13 protofilaments aligned in parallel

102. Ciliary beating is an extensively studied form of cellular movement.
Cilia are tiny hairlikeappendages about 0.25 µm in diameter with a bundle of microtubules at their core
They extend from the surface of many kinds of cells and are found in most animal species, many protozoa, and
some lower plants.
The primary function of cilia is to move fluid over the surface of the cell or to propel single cells through a
fluid.
Protozoa, for example, use cilia both to collect food particles and for locomotion. On the epithelial cells lining
the human respiratory tract, huge numbers of cilia sweep layers of mucus, together with trapped particles of
dust and dead cells, up toward the mouth, where they are swallowed and eliminated.
The flagella of bacteria are different from the cilia and flagella of eucaryoticcells. The protein of Bacterial
flagella is flagellin
The movement of a cilium or a flagellum is produced by the bending of its core, which is called the axoneme.
The axoneme is composed entirely of microtubules and their associated proteins.
The microtubules are arranged in a particular pattern: nine special doublet microtubules are arranged in a ring
around a pair of single microtubules.
This "9 + 2" array is characteristic of almost all forms of cilia and flagella. While each member of the pair of
single microtubules (the central pair) is a complete microtubule, each of the outer doublets is composed of one
complete and one partial microtubule fused together so that they share a common tubule wall.
In transverse sections each complete microtubule appears to be formed from a ring of 13 subunits, while the
incomplete tubule of the outer doublet is formed from only 11.
DyneinDrives the Movements of Cilia and Flagella.
The microtubules of an axoneme are associated with numerous proteins, which project at regular positions
along the length of the microtubules. Some serve as cross-links that hold the bundle of microtubules together.
Others generate the force that drives the bending motion
The most important of these accessory proteins is ciliary dynein, whose heads interact with adjacent
microtubules to generate a sliding force between the microtubules. Dynein has a motor domain, which
hydrolyzes ATP to move along a microtubule, and a tail region that carries a cargo, which in this case is an
adjacent microtubule. The tail of ciliary dynein binds only to the A tubule and not to the B tubule, which has a
slightly different structure. The resulting asymmetry in the arrangement of the dynein molecules is required to
prevent a fruitless tug-of-war between neighboring microtubules.

103. Microtubules are polar structures: one end (the plus end) is capable of rapid growth, while the other end (the minus end)
tends to lose subunits if not stabilized.
In most cells, the minus ends of microtubules are stabilized by embedding them in a structure called the centrosome, and
the rapidly growing ends are then free to add tubulin molecules.
The centrosome generally lies next to the nucleus, near the center of the cell. At any one time, several hundred
microtubules are growing outward from a centrosome, with some extending for many microns, so that their plus end is
at the edge of the cell. Each of these microtubules is a highly dynamic structure that can shorten as well as lengthen:
after growing outward for many minutes by adding subunits, its plus end may undergo a sudden transition that causes it
to lose subunits, so that the microtubule shrinks rapidly inward and may disappear.
A centrosome with attached
microtubules. As indicated, the
minus end of each microtubule
is embedded in the centrosome
matrix ( light green) that
surrounds a pair of structures
called centrioles. By nucleating
the growth of new
microtubules, this matrix helps
to determine the number of
microtubules in a cell.
The array of
microtubules
anchored in a
centrosome is
continually
changing, as new
microtubules grow (
red arrows) and old
microtubules shrink
( blue arrows).
Microtubules are polar and dynamic, emanate from the Centrosome
Centrosomes Are the Primary Site of Nucleation of Microtubules in Animal Cells

104. •The centrosome is the major microtubule-organizing center in almost all animal cells. In
interphase it is located to one side of the nucleus, close to the nuclear envelope. Centrosome
consist of a pair of cylindrical structures called centrioles arranged at right angles to each
other.
•The centrosome duplicates and splits into two equal parts during interphase
•The each daughter cell inherits one centrosome, containing two structures called centrioles.
•The centrosome replicates during theS phaseof the cell cycle.
•During theprocess of cell division calledmitosis, the centrosomes migrate to opposite poles
of the cell.
•Upon division, each daughter cell receives one centrosome.
•Aberrant numbers of centrosomes in a cell have been associated withcancer.
•The protein composition of the centrosome matrix is only partly known.
•However, it contains a number of centrosome-specific proteins, including a special minor
form of tubulin to help nucleate microtubules.
•In cell biology, thecentrosomeis an organelle that serves as the main microtubule
organizing center (MTOC) of the animal cell, as well as a regulator of cell-cycle
progression.
•The centriolesare surrounded by a dense, highly structuredmass ofproteintermed
thepericentriolar material(PCM).
•The PCM contains proteins responsible formicrotubule nucleationand anchoring
•Centrosomes are associated with thenuclear membraneduring theprophasestage of the cell
cycle. Duringmitosis, the nuclear membrane breaks down, and the centrosome-
nucleatedmicrotubulescan interact with thechromosomesto build themitotic spindle.
•Themitotic spindleis a highly dynamic molecular machine composed of tubulin, motors,
and other molecules. It assembles around the chromosomes and distributes the duplicated
genome to the daughter cells duringmitosis.
The centrosome

105. •Many of the microtubule arrays in cells are labile and required for their
function. Examples is the mitotic spindle, which forms at the onset of mitosis.
•The mitotic spindle is the target of a variety of specific antimitotic drugs that
act by interfering with the exchange of tubulin subunits between the
microtubules and the free tubulin pool.
•Colchicine, an alkaloid extracted from the meadow saffron that has been used
medicinally in the treatment of gout since ancient times.
•Each molecule of colchicine binds tightly to one tubulin molecule and prevents
its polymerization, but it cannot bind to tubulin when it is polymerized into a
microtubule. The exposure of a dividing cell to colchicine, or to the closely
related drug colcemid, causes the rapid disappearance of the mitotic spindle
•Antimitotic drugs, such as vinblastine and vincristine (whose effects are
similar to those of colcemid), are widely used in the treatment of cancer.
•The drug taxol, extracted from the bark of yew trees, has the opposite effect. It
binds tightly to microtubules and stabilizes them, and when added to cells, it
causes much of the free tubulin to assemble into microtubules. The
stabilization of microtubules by taxolarrests dividing cells in mitosis. Taxol is
also widely used as an anticancer drug.
Microtubules Are Highly Labile Structures, Sensitive to Specific Antimitotic Drugs
The mitotic spindle isa structure that forms during cell division
and separates duplicated chromosomes.
E
-
-
-
-
-
=
-
-
-
-
-
-
-

106. (A)Schematic diagram of a cell showing the typical arrangement of microtubules (green), endoplasmic reticulum (blue), and Golgi apparatus
(yellow). The nucleus is shown in brown and the centrosome in light green.
(B)Cell stained with antibodies to endoplasmic reticulum (upper panel) or to microtubules (lower panel). Motor proteins pull theendoplasmic
reticulum along microtubules, stretching it like a net from its attachments to the nuclear envelope.
(C)Cell stained with antibodies to the Golgi apparatus (upper panel) or to microtubules (lower panel). In this case motor proteins move the Golgi
apparatus inward to its position near the centro-some.
The placement of
organelles by
microtubules

107. both types of microtubule motor proteins exist in many forms,
each of which is thought to transport a different cargo.
•Microtubule-dependent motor proteins play an important part in
positioning membrane-bounded organelles within a eucaryoticcell.
The membrane tubules of the endoplasmic reticulum (ER), for
example, align with microtubules and extend almost to the edge of the
cell, whereas the Golgi apparatus is located near the centrosome.
•When cells are treated with a drug that depolymerizes microtubules,
both of these organelles change their location: the ER collapses to the
center of the cell, while the Golgi apparatus fragments into small
vesicles that disperse throughout the cytoplasm. When the drug is
removed, the organelles return to their original positions, dragged by
motor proteins moving along the re-formed microtubules.
•The normal position of each of these organelles is thought to be
determined by a receptor protein on the cytosolic surface of its
membrane that binds a specific microtubule-dependent motor -a
kinesin for the ER and a dynein for the Golgi apparatus.
The position of the cell organelles is determined by Microtubule motor proteins;
the kinesins, which generally move toward the plus end of a microtubule (away from the centrosome), and the dyneins,
which move toward the minus end (toward the centrosome).

108. Motor Proteins Use the Microtubule Network as a Scaffold
to Position Membrane-bounded Organelles
?If a living vertebrate cell is observed in a light microscope, its cytoplasm is seen to be in continual motion.
?Mitochondria and smaller membrane-bounded organelles change their positions by periodic movements, which are
directional.
?These and other intracellular movements in eucaryoticcells are generated by motor proteins, which bind to either an
actin filament or a microtubule and use the energy derived from repeated cycles of ATP hydrolysis to move steadily
along it.
?Dozens of different motor proteins have now been identified. They differ in the type of filament they bind to, the
direction in which they move along the filament, and the "cargo" they carry.
?The first motor protein to be discovered was myosin, a protein that moves along actin filaments and abundant in
skeletal muscle.
?Another family of motor proteins are the kinesins, which generally move toward the plus end of a microtubule
(away from the centrosome), and the dyneins, which move toward the minus end (toward the centrosome).
?Microtubule-dependent motor proteins play an important part in positioning membrane-bounded organelles within a
eucaryoticcell.
?The endoplasmic reticulum (ER), for example, align with microtubules and extend almost to the edge of the cell,
whereas the Golgi apparatus is located near the centrosome.
?When cells are treated with a drug that depolymerizes microtubules, both of these organelles change their location:
the ER collapses to the center of the cell, while the Golgi apparatus fragments into small vesicles that disperse
throughout the cytoplasm. When the drug is removed, the organelles return to their original positions, dragged by
motor proteins moving along the re-formed microtubules. Thus the normal position of each of these organelles is
thought to be determined by a receptor protein that binds a specific microtubule-dependent motor -a kinesin for the
ER and a dynein for the Golgi apparatus
-
autorograle-etrograde
-
--
-

109. Large flat pigment cells in fish
contains many pigment granules,
which can be dark brown, yellow,
red, or iridescent, depending on the
species of fish, are attached to
microtubules and can either
aggregate in the center of the cell or
disperse throughout the cytoplasm.
The movement of the pigment
granules occurs along the
microtubules and can be controlled
by the fish to change its skin color.
Fish pigment cells. These giant cells, which are responsible for changes in skin coloration in several species
of fish, contain large pigment granules (brown), which can change their location in the cell in response to a
neuronal or hormonal stimulus.
(A) Schematic view of a pigment cell, showing the dispersal and aggregation of pigment granules, which
occur along microtubules.
(B) Scanning electron micrograph of a pigment cell following a brief exposure to detergent. The plasma
membrane and soluble contents of the cytoplasm have been removed, exposing the array of microtubules
and associated pigment granules.
(C and D) Bright-field images of the same cell in a scale of an African cichlid fish, showing its pigment
granules either dispersed throughout the cytoplasm or aggregated in the center of the cell. (E) An
immunofluorescence picture of another cell from the same fish stained with antibodies to tubulin, showing
large bundles of parallel microtubules extending from the centrosome to the periphery of the cell.
The Microtubule Network Can Find the Center of the Cell

110. Theactinsubunitis a single
globularpolypeptidechain and is thus
amonomerrather than a dimer. Liketubulin,
each actin subunit has abinding sitefor
anucleotide, but for actin the nucleotide is ATP
(or ADP) rather than GTP (or GDP).
The actin filament can be considered to consist
of two parallel protofilaments that twist around
each other in a right-handed helix.
Actin filaments are relatively flexible compared
with the hollow cylindrical microtubules.
The structures of an actin monomer and actin filament
(A) Theactinmonomerhas anucleotide(either ATP or ADP) bound in a deep cleft in the center of themolecule. (B) Arrangement of monomers in
a filament. Although the filament is often described as a single helix of monomers, it can also be thought of as consisting of two protofilaments,
held together by lateral contacts, which wind around each other as two parallel strands of a helix. All the subunits within the filament have the
same orientation. (C) Electron micrographs of negatively stained actin filaments.
Microfilaments

111. Examples of
different actin
structural
organizations

112. Endocytosis
In eucaryoticcells, internal membrane system that allows them to take up
macromolecules by a process called endocytosis and deliver them to digestive enzymes
that are stored intracellularly in lysosomes
The routes that lead inward to lysosomes from the cell surface start with the process of
endocytosis, by which cells take up macromolecules, particulate substances, and, in
specialized cases, even other cells.
Material to be ingested is progressively enclosed by a small portion of the plasma
membrane, which first invaginates and then pinches off to form an intracellular vesicle
containing the ingested substance or particle. There are two main types of endocytosis on
the basis of the size of the endocytic vesicles formed: pinocytosis ("cellular drinking"),
which involves the ingestion of fluid and solutes via small vesicles (less than 150 nm in
diameter), and phagocytosis ("cellular eating"), which involves the ingestion of large
particles, such as microorganisms or cell debris, via large vesicles called phagosomes,
generally > 250 nm in diameter. Although most eucaryoticcells are continually ingesting
fluid and solutes by pinocytosis, large particles are ingested mainly by specialized
phagocytic cells.
Phagocytosis by a macrophage.
Scanning electron micrograph
of a mouse macrophage
phagocytosing two chemically
altered red blood cells. The red
arrows point to edges of thin
processes (pseudopods) of the
macrophage that are extending
as collars to engulf the red cells.
Phagocytosis by a neutrophil. Electron micrograph
of a neutrophil phagocytosing a bacterium

113. 1.Antibody produced by
immune system targets the
bacterium (Opsinization)
2.WBC binds antibody bound
(AB) bacterium with its FC
receptors and internalization
starts.
3.Actin filaments together with
other proteins power the
engulfment
4.Lysosomes fuse with the
phagosome and digest the
particle
Phagocytosis involves branched actin filaments

114. Specialized Phagocytic Cells Can Ingest Large Particles: professional
phagocytes -macrophages and neutrophils
•Phagocytosis is a special form of endocytosis in which large particles such as microorganisms and cell debris are ingested
via large endocytic vesicles called phagosomes.
•In protozoa phagocytosis is a form of feeding: large particles taken up into phagosomes end up in lysosomes, and utilized
as food.
•Phagocytosisis important in most animals for purposes other than nutrition, carried out by specialized cells known as
professional phagocytes.
•In mammals there are two classes of white blood cells that act as professional phagocytes -macrophages and
neutrophils. These two types of cells defend us against infection by ingesting invading microorganisms. Macrophages also
play an important part in scavenging senescent and damaged cells and cellular debris.
•The phagosomes fuse with lysosomes, and the ingested material is degraded.
•Some of the internalized plasma membrane components are retrieved from the phagosome by transport vesicles and
returned to the plasma membrane. In order to be phagocytosed, particles must first bind to the surface of the phagocyte.
Phagocytes have a variety of specialized surface receptors. Unlike pinocytosis, which is a constitutive process that occurs
continuously, phagocytosis is a triggered process that requires that activated receptors transmit signals to the cell interior to
initiate the response.

115. Clathrin-coated Pits Can Serve as a Concentrating Device for Internalizing
Specific Extracellular Macromolecules
In most animal cells, clathrin-coated pits and vesicles
provide an efficient pathway for taking up specific
macromolecules from the extracellular fluid, a
process called receptor-mediated endocytosis. The
macromolecules bind to complementary cell-surface
receptors (which are transmembrane proteins),
accumulate in coated pits, and enter the cell as
receptor-macromolecule complexes in clathrin-coated
vesicles. A particularly well-understood and
physiologically important example is the process
whereby mammalian cells take up cholesterol.
Receptor-mediated endocytosis of LDL. LDL dissociates from its receptors in the acidic environment of the endosome. LDL ends up in lysosomes, where it
is degraded to release free cholesterol. In contrast, the LDL receptor proteins are returned to the plasma membrane via transport vesicles that bud off from
the tubular region of the endosome, as shown. For simplicity, only one LDL receptor is shown entering the cell and returning to the plasma membrane.
Whether it is occupied or not, an LDL receptor typically makes one round trip into the cell and back to the plasma membrane every 10 minutes, making a
total of several hundred trips in its 20-hour life-span.

116. Cells Import Cholesterol by Receptor-mediated Endocytosis
Many animal cells take up cholesterol through receptor-mediated endocytosis.
If the uptake is blocked, cholesterol accumulates in the blood and can contribute to the formation in blood vessel walls of atherosclerotic plaques -
the deposits of lipid and fibrous tissue that cause strokes and heart attacks by blocking blood flow.
Most cholesterol is transported in the blood bound to protein in the form of particles known as low-density lipoproteins, or LDL. When a cell needs
cholesterol for membrane synthesis, it makes transmembrane receptor proteins for LDL and inserts them into its plasma membrane.
Once in the plasma membrane, the LDL receptors diffuse until they associate with clathrin-coated pits.
Since coated pits constantly pinch off to form coated vesicles, any LDL particles bound to LDL receptors in the coated pits are rapidly internalized
in coated vesicles. After shedding their clathrincoats, these vesicles deliver their contents to early endosomes, which are located near the cell
periphery. Once in the endosomal compartment, the LDL moves inward and is delivered via late endosomes to lysosomes to free cholesterol, which
thereby becomes available to the cell for new membrane synthesis.
If too much free cholesterol accumulates in a cell, it shuts off both the cell's own cholesterol synthesis and the synthesis of LDL receptor proteins,
so that the cell ceases either to make or to take up cholesterol.
This regulated pathway for the uptake of cholesterol is disrupted in individuals who inherit defective genes encoding LDL receptor proteins and
whose cells, consequently, are deficient in the capacity to take up LDL from the blood.
The resulting high levels of blood cholesterol predispose these individuals to develop atherosclerosis prematurely, and most dieat an early age of
heart attacks resulting from coronary artery disease. More than 25 different receptors are known to participate in receptor-mediated endocytosis of
different types of molecules, and they all apparently utilize the same clathrin-coated-pit pathway.

117. Pinocytic Vesicles Form from Coated Pits in the Plasma Membrane
Virtually all eucaryoticcells continually ingest bits of their plasma membrane
in the form of small pinocytic (endocytic) vesicles that are later returned to the
cell surface.
Fibroblasts endocytose at a somewhat lower rate, whereas some amoebae
ingest their plasma membrane even more rapidly.
The endocytosis begins at specialized regions of the plasma membrane called
clathrin-coated pits, which typically occupy about 2% of the total plasma
membrane area. Clathrinwith other proteins, forms a characteristic basket or
cage. Within a minute or so they invaginate into the cell and pinch off to form
clathrin-coated vesicles. It has been estimated that about 2500 clathrin-coated
vesicles leave the plasma membrane of a cultured fibroblast every minute.
Since extracellular fluid is trapped in clathrin-coated pits as they invaginate to
form coated vesicles, substances dissolved in the extracellular fluid are
internalized -a process called fluid-phase endocytosis.

118. The cell in (A) has not been stimulated. The cell in (B) has been
activated to secrete its stored histamine by a soluble extracellular
ligand.
Histamine-containing secretory vesicles are dark, while those that
have released their histamine are light. The material remaining in
the spent vesicles consists of a network of proteoglycans to which
the stored histamine was bound.
Once a secretory vesicle has fused with the plasma membrane, the
secretory vesicle membrane often serves as a target to which other
secretory vesicles fuse. Thus the cell in (B) contains several large
cavities lined by the fused membranes of many spent secretory
vesicles, which are now in continuity with the plasma membrane.
Electron micrographs of exocytosis in rat mast cells.

119. Neutrophil extracellular traps (NETs) are composed of
nuclear components (DNA and histones) and are decorated
by proteins from primary granules (such as
myeloperoxidase and neutrophil elastase), secondary
granules (such as lactoferrin and pentraxin 3), and tertiary
granules. NETs have been shown to trap microorganisms.
Initially, the neutrophils become rounded with uniformly
condensed chromatin, occurs because of PAD-4-mediated
citrullination of histones, and then undergo nuclear
envelope breakdown so DNA-containing vesicles
eventually fuse with the plasma membrane, and NETs are
released to trap microorganism.NETs can cause
development of autoimmune disorders trough molecules
present in the NET or the degradation products of NETs by
DNAseI can act as auto-antigen or because excessive
formation of degradation failure of NETs.

120. Dynamic surface extensions containing actin filaments are a common
feature of animal cells, especially when the cells are moving or changing
shape.
The large, free-living cells of Amoeba proteus, for example, produce
pseudopodia with which they walk over surfaces. Many cells in vertebrate
tissues are also capable of migration over surfaces, especially when put into
tissue culture.
The leading edge of a crawling fibroblast regularly extends a thin, sheetlike
process known as a lamellipodium, which contains a dense meshwork of
actin filaments.
Many cells also extend thin, stiff protrusions called microspikes, which are
about 0.1 µ wide and 5 to 10 µ long and contain a loose bundle of about 20
actin filaments.
The growing tip of a developing nerve cell axon extends even longer
microspikes, called filopodia, which can be up to 50 µ long.
The actin-dependent extension and firm attachment of a lamellipodium at
the leading edge stretches the actin cortex. The cortical tension then
draws the body of the cell forward to relax some of the tension. New
focal contracts are made and old ones are disassembled as the cell crawls
forward. The same cycle can be repeated over and over again, moving the
cell forward in a stepwise fashion. The newly polymerized cortical actin
is shown in red
Many Cells Extend Dynamic Actin-containing Microspikesand
Lamellipodia from Their Leading Edge

121. •Protein translocation systems
•UPR
•Glycosylation
•Nucleolus

122. Overview of translocation process :
A B
Cotranslational(A)andtheposttranslationaltransportmechanism(B).
Duringco-translationaltransport,theSRPbindstoahydrophobicsignalsequence
locatedattheNterminusofthenascentprecursorpolypeptideandtotheribosome.
Subsequently,theSRPreceptorguidestheribosomenascentchaincomplextothe
polypeptideconductingchannel,Sec61.FollowingGTPhydrolysis,SRPdissociates
fromtheribosomeandtheSRPreceptorinducingaresumptionofproteinsynthesis
andthenascentpolypeptidechaininsertsintotheSec61channel.
Thetransportofprecursorproteinsintoandacrosstheendoplasmic
reticulum(ER)membranerepresentsahighlyconservedprocessin
eukaryoticcells.
Basically,thisprocesscanbedividedintotwomajorstepsasfollows:
(i)thetargetingofnascentandnewlysynthesizedprecursor
polypeptidestotheERmembraneandinsertionintothepolypeptide
conductingchannel;and
(ii)thereleaseofthetransmembraneproteinfromthechannelintothe
phospholipidbilayerortranslocationintotheERlumen
•MembraneproteinsdiffuselaterallyfromtheSec61complexinto
thebilayer.Alternatively,ERluminalchaperoneproteinssuchas
BiP/Grp78canfunctionas‘molecularratchets’alsoknownas
chaperoneshelpsunidirectionaltransportofthenascentprotein
throughtheSec61channelintotheERlumen.
•Sec63mediatetheirdirectinteractionbetweenchaperonesand
theprecursorpolypeptidesintransit
•AstheactivityofERluminalBiPdependsonATPhydrolysis,the
nucleotide-exchangefactorsSil1andGRP170guaranteea
replacementofADPwithATP.Duringoraftertheprecursor
proteintranslocationiscompleted,thesignalsequenceiscleaved
offbythesignalpeptidasecomplex.

123. Theposttranslationaltransport:
TomaintainaproteinstructurecompatiblewithtranslocationacrosstheERmembrane,cytosolicHsp40andHsp70chaperones
preventextensiveproteinfoldingatthisstageandkeepthesignalsequencefreeforinteractionwithreceptorsattheERsurface.
Dependingonstructuralcharacteristicsofthesynthesizedprotein,forexample,thechainlengthandtheextentoffolding,SRPas
wellasSec62canberequiredforanefficienttargetingtotheSec61translocon.Thesubsequentstepsofproteintransportare
comparablewiththeco-translationaltransport.
•Sec61complexisaheterotrimericchannelforproteinconductionusedbybothco-andpost-translationaltranslocationpathways.
•Inyeast,thetransloconiscomposedofthreeproteins,Sbh1,Sec61andSss1.
•ThecorestructureoftheSec61p,approximately53kDaprotein,comprisesof10transmembranehelices.
•ThesehelicesareseparatedbyloopsoutofwhichL6playsaroleintranslocationofproteinswhileL8containsahighlyconserved
residuewhichispositivelychargedandprovidesamajorribosomebindingsite.
•Sbh1,thebetasubunitoftranslocon,isasmallproteinofapproximately8.7kDa.
•Sss1isanessentialsmallproteinofthecomplexwhichisanintegralmembraneproteinwithitsaminoterminalexposedtothecytosol.
Yeast translocon:

124. Glycophospholipid-AnchoredSurfaceprotein(Gas1),aispost-translationallymodifiedthroughthe
attachmentofaglycolipidmoietycalledGlycosylphosphatidylinositol(GPI)attheirC-terminalendtoanchor
proteintotheplasmamembrane’souterleafletofthelipidbilayer.
CarboxypeptidaseY(CPY)issynthesizedintheformofpreproenzyme(ppCPY).Thepreproenzymeentersin
theERlumenthroughpost-translationaltranslocationwherethesignalsequenceiscleavedresultingin
proCPYafterwhichtheenzymegetsfoldedthroughdisulphidebondformationandglycosylationtogivethe
p1CPYform;theenzymeisthentransportedtoGolgiwheretheoutermannoseresiduesareaddedgivingrise
top2CPY.Finally,insidethevacuole,proteinaseBcleavesthep2CPYtoformthematureCPY(mCPY).
•Copperinhibitsthetranslocationofpost-translationallytranslocatingsecretoryproteins,Gas1andCPY.
•InthepresenceofcopperbothGas1andCPYaccumulateinthecytosolicformasshowninthefiguresA
(Gas1)andB(CPY,indicatedbyaccumulationofpreproCPY).
Post-translationally
translocating proteins in
yeast, Gas1 and CPY
A
150 kDa
75 kDa
100 kDa
Cu (mM) - 1
.TBP
precursor
mature
I
75 kDa
75 kDa
Cu (mM)
Tm(g/mL)
-- 1--
-- 1 -0.75
.TBP
ppCPY(in Cytosol)
proCPY(In ER)
p2CPY
proCPY
p2CPY
ppCPY
B

125. The mycolactone–bortezomib
combination rapidly killed patient-
derived MM cells ex vivo, but not
normal mononuclear cells.
EMBO Molecular Medicine 14: e14740 | 2022
ex vivo: experimentation or measurements done in
or on tissue from an organism in an external
environment with minimal alteration of natural
conditions
Multiple myeloma, cancerous plasma cells
(WBC) accumulate in the bone marrow and crowd
out healthy blood cells.
Mononuclear cells refer to blood cells that have a
single, round nucleus, such as lymphocytes and
monocytes. When isolated from circulating blood,
they are called peripheral blood mononuclear cells
(PBMC)

126. •Proteins are usually imported into mitochondria from the cytosol within seconds
or minutes after they are released from the ribosome
•In contrast to translocation into ER, mitochondrial proteins are first fully
synthesized in cytosol and then translocated into mitochondria by a post
translational mechanism
•One or more signal sequences direct the all mitochondrial proteins into the sub-
compartments
•Positively charged amino acid residues are present on the one side and
uncharged on the other side of signal sequence
•Specific receptor proteins recognize this charged configuration in the signal
•Import proteins transiently spans both the membranes
•Multisubunitprotein complexes known as protein translocators mediate the protein translocation across mitochondrial membranes. TOM complexes
transfer proteins across outer membrane and two TIM complexes (TIM23 and TIM22) across the inner membrane. Some components ofthese
translocators consist of receptors for mitochondrial proteins and others act as translocation channels.
•Mitochondrial proteins are imported as unfolded polypeptide chains.
•Studies on isolated cell free mitochondria with reconstituted translocation systems in test tube using radiolabelled mitochondrial precursor proteins
suggest that proteins do not fold into their native structures, they remain in unfolded state in the cytosol through interactions with HSP70 family protein
chaperones
•As a first step, Import receptors of TOM complex bind with the signal sequence
•Subsequently signal sequence of the unfolded protein is fed into the translocation channel
Mitochondrial protein translocators

127. •In principle, protein could reach mitochondrial metrixspace by crossing two membranes at once or one at a time
•The experiments suggest that proteins can pass through both the membranes at once to enter the metrixspace
•TOM complex first transport the signal sequence across the outer membrane into the intermembrane space where
it binds to TIM complex and then enters the matrix space or inserts into inner membrane
Proteins reach mitochondrial metrixspace by crossing two membranes at once or one at a time

128. •TOM complex initially transports into intermembrane space and helps to insert proteins into outer membrane
and folded properly by an additional translocator, SAM complex in the outer membrane
•TIM23 complex imports proteins into metrixspace and then helps to insert them into inner membrane
•TIM22 complex mediates insertion of a subclass of proteins into inner membrane that transports metabolites
including ADP, ATP and phosphate in and out of mitochondria
•OXA complex, mediates insertion of those mitochondrial inner membrane proteins which are synthesized
within the mitochondria

129. ATP hydrolysis and membrane potential drives protein import into metrixspace
Energy is required for chaperon proteins, to release the newly synthesized proteins from the HSP70 family.
The requirement of HSP70 and ATP in the cytosol can be bypassed if the precursor protein is artificially unfolded before adding
to the purified mitochondria
Once signal sequence passed through TOM complex and bind to the TIM translocation channel complex, requires membrane
potential (electrochemical H+ gradient) across the inner membrane.
This membrane potential not only helps to drive the ATP synthesis but also drives the translocation of positively charged signal
sequence through TIM complex by electrophoresis
Mitochondrial Hsp70 bound to the metrixside of TIM23 complex acts as a motor to pull the precursor protein into metrixspace
and releases the bound protein by ATP hydrolysis
Another chaperone protein Hsp60 helps the unfolded polypeptide chain to fold by binding and release by ATP hydrolysis

130. Transport into inner mitochondrial membrane and
intermembrane space occurs via several routes
First route; A hydrophobic
signal sequence located after
N-terminal signal acts as
Stop transfer sequence
preventing further
translocation across the inner
membrane, TOM complex
pulls the remaining protein
into intermembrane space
Another route; proteins are initially imported into matrix space by TIM23 complex. A matrix signal
peptidase then removes the N-terminal signal sequence, exposing the hydrophobic sequence which
guides proteins to OXA complex which inserts into inner membrane
-
-
-
-
-
-

131. •S. cerevisiae Ire1 is activated by ER stress.
•Upon activation, Ire1 undergoes trans-
autophosphorylation and oligomerization.
•HAC1 mRNA is spliced by activated Ire1 through its
RNase domain.
•Upon translation, transcription factor Hac1 up-
regulates UPR target genes to restore homoeostasis.
Cancer cells exploit unfolded protein response (UPR) to meet increased demands for protein and
lipid production needed for rapid proliferation and adapt to an oxygen-and nutrient-deprived
environment. The UPR is a pro-survival mechanism triggered by accumulation of unfolded or
misfolded proteins in the endoplasmic reticulum (ER), a condition referred to as ER stress. IRE1,
PERK and ATF6 are three ER anchored transmembrane receptors. Upon induction of ER stress,
they signal in a coordinated fashion to re-establish ER homoeostasis essential for cell survival.
Role for the UPR in the establishment and progression of several cancers

132. The roles of the UPR in organ physiology (blue) or pathological
conditions (pink) affecting the same tissues
Selected IRE1r-binding partners
(Nat Rev Mol Cell Biol. 2020, 421–438

133. The oligosaccharide shown in this figure
is transferred to the asparagine as an
intact unit in a reaction catalyzed by a
membrane-bound oligosaccharyl
transferase enzyme. There is one copy of
this enzyme associated with each protein
translocator in the ER membrane
Theoligosaccharideisassembledsugarby
sugarontothecarrierlipiddolichol.Dolicholis
longandveryhydrophobic:sothattheattached
oligosaccharideisfirmlyanchoredinthe
membrane.Thishigh-energybondactivatesthe
oligosaccharideforitstransferfromthelipidto
anasparaginesidechainofanascent
polypeptideontheluminalsideoftherough
ER.Thesynthesisoftheoligosaccharidestarts
onthecytosolicsideoftheERmembraneand
continuesontheluminalfaceafterthe(Man)
5(GlcNAc)2lipidintermediateisflipped
acrossthebilayer.Allofthesubsequent
glycosyltransferreactionsontheluminalside
oftheER.GlcNAc=Nacetylglucosamine;
Man=mannose;Glc=glucose.
Synthesis of the lipid-linked precursor oligosaccharide in the rough ER membrane

134. Processing of oligosaccharide in the Golgi

135. Congenital
disorders of
glycosylation

136. Electron micrograph of a thin section of a nucleolus in a human
fibroblast, showing its three distinct zones. (A) View of entire
nucleus. (B) High-power view of the nucleolus
These light micrographs of human
fibroblasts grown in culture show
various stages of nucleolar fusion.
The Nucleolus Is a Ribosome-producing Machine

137. •The nucleolus is the site where ribosomal RNA (rRNA) molecules are processed from a
larger precursor RNA and assembled into ribosomes by the binding of ribosomal proteins
•The continuous transcription of multiple gene copies by RNA polymerase I ensures an
adequate supply of the rRNAs, immediately packaged with ribosomal proteins to form
ribosomes. The packaging occurs in the nucleolus. The nucleolus contains large loops of
DNA emanating from several chromosomes, each of which contains a cluster of rRNA
genes. Each such gene cluster is known as a nucleolar organizer region.
•Nucleolus is the site where other RNAs are also produced and other RNA-protein
complexes are assembled. Example; U6 snRNP (contains RNA molecule and 7 proteins)
functions in preRNAsplicing.
•Teomeraseand SRP are also believed to be assembled in Nucleolus.
•tRNAs genes are also clustered in Nucleolus. They are transcribed and are also processed
in Nucleolus.
The function of the nucleolus in ribosome synthesis. The 45S
rRNA transcript is packaged in a large ribonucleoprotein particle
containing many ribosomal proteins imported from the
cytoplasm. While this particle remains in the nucleolus, selected
pieces are discarded as it is processed into immature large and
small ribosomal subunits. These two subunits are thought to attain
their final functional form only as each is individually transported
through the nuclear pores into the cytoplasm.
Nucleolus are factory, many different non-coding RNAs are
transcribed, processed and assembled with proteins to form
large variety of ribinucleoproteincomplexes

138. •18S, 5.8S and 28S by transcribed by RNA Pol I as a single large
transcript which is chemically modified and cleaved to produce these
RNAs. About 150 guide RNAs are needed for modification and
introduces conformational change that exposes site for cleavage by
nucleases
•Guide RNAs belong to a class of RNA known as small nucleolar RNAs
(snoRNAs) because these guide RNAs perform their function in
Nucleolus. Many snoRNAs encode ribosomal proteins and transcribed
by RNA Pol II
•5S rRNA are transcribed by RNA Pol III but does not require chemical
modifications
•Nucleus contains many subnuclear highly dynamic structures; Cajal
bodies, interchromatin granule clusters (speckles). Difficult to study
subnuclear structures as they are highly dynamic in structure and
function
Schematic view of subnuclear structures

139. •Changes in the appearance of
the nucleolus in a human cell
during the cell cycle.
•Only the cell nucleus is
represented in this diagram.
•In most eucaryoticcells the
nuclear membrane breaks
down during mitosis, as
indicated by the dashed
circles.

140. •The nucleolus is the site where ribosomal RNA (rRNA) molecules are processed from a
larger precursor RNA and assembled into ribosomes by the binding of ribosomal proteins
•The continuous transcription of multiple gene copies by RNA polymerase I ensures an
adequate supply of the rRNAs, immediately packaged with ribosomal proteins to form
ribosomes. The packaging occurs in the nucleolus. The nucleolus contains large loops of
DNA emanating from several chromosomes, each of which contains a cluster of rRNA
genes. Each such gene cluster is known as a nucleolar organizer region.
•Nucleolus is the site where other RNAs are also produced and other RNA-protein
complexes are assembled. Example; U6 snRNP (contains RNA molecule and 7 proteins)
functions in preRNAsplicing.
•Teomeraseand SRP are also believed to be assembled in Nucleolus.
•tRNAs genes are also clustered in Nucleolus. They are transcribed and are also processed
in Nucleolus.
The function of the nucleolus in ribosome synthesis. The 45S
rRNA transcript is packaged in a large ribonucleoprotein particle
containing many ribosomal proteins imported from the
cytoplasm. While this particle remains in the nucleolus, selected
pieces are discarded as it is processed into immature large and
small ribosomal subunits. These two subunits are thought to attain
their final functional form only as each is individually transported
through the nuclear pores into the cytoplasm.
Nucleolus are factory, many different non-coding RNAs are
transcribed, processed and assembled with proteins to form
large variety of ribinucleoproteincomplexes

141. Modification and subsequent cleavage of a large 45S rRNA
transcript forms 18S rRNA, 5.8 S rRNA and 28S rRNA

142. •Intermediate filaments are made up of smaller subunits that are themselves
elongated and fibrous, whereasactinfilaments and microtubules are made of
subunits that are compact and globular—actin subunitsfor actin
filaments,tubulinsubunitsfor microtubules.
•Many biological polymers—includingDNA,RNA, and proteins—are held
together by covalent linkages between their subunits. In contrast, the three
types of cytoskeletal “polymers” are held together by weak noncovalent
interactions, which means that their assembly and disassembly can occur
rapidly, without covalent bonds being formed or broken.
Intermediate filaments

143. The domain organization of intermediate filament protein monomers
Most intermediate filament proteins share a similar rod domain that is usually about 310 amino acids long
and forms an extended a helix. The amino-terminal and carboxyl-terminal domains are non-a-helical and
vary greatly in size and sequence in different intermediate filaments.

144. (A)pairswithanidenticalmonomertoformadimer
(B)Theconservedcentralroddomainsofthedimers
arealignedinparallelandwoundtogetherintoa
coiledcoil.
(C)Twodimersthenlineupsidebysidetoform
anantiparalleltetrameroffourpolypeptidechains.
(D)Withineachtetramer,thetwodimersareoffset
withrespecttooneanother,therebyallowingitto
associatewithanothertetramer.
(E)Inthefinal10-nmrope-likefilament,tetramersare
packedtogetherinahelicalarray,whichhas16
dimers.Anelectronmicrographofintermediate
filamentsareshownontheupperleft.
A model of intermediate filament assembly

145. Major Types of Intermediate Filament Proteins
in Vertebrate Cells
IntermediateFilamentsImpartMechanicalStabilitytoAnimalCells
Differentfamiliesofintermediatefilamentsareexpressedindifferentcelltypes.Themost
diverseintermediatefilamentfamilyisthatofthekeratins:thereareabout20foundin
differenttypesofhumanepithelialcells,andabout10arespecifictohairandnails.
EverykeratinfilamentismadeupofanequalmixtureoftypeI(acidic)andtypeII
(neutral/basic)keratinchains;theseformheterodimers,twoofwhichthenjointoformthe
fundamentaltetramericsubunit.Cross-linkedkeratinnetworksheldtogetherbydisulfide
bonds,formingtoughcoveringsforanimals,asintheouterlayerofskinandinhair,nails,
claws,andscales.
Epithelial cells area type of cell that lines the
surfaces of your body. They are found on your skin,
blood vessels, urinary tract, and organs

146. •Abnormalitiesinneurofilamentscausesdiseasessuchas
amyotrophiclateralsclerosis(ALS).
•PhysicistStephenHawking,diedduetoALSresultsfrom
progressivelossofmotorneurons,whichinturnleadstomuscle
atrophy,paralysis,andeventualdeath.
•ALSandothertypesofmotorneurondiseasearecharacterized
bytheaccumulationandabnormalassemblyofneurofilaments.
•OverexpressionofNF-LorNF-Hintransgenicmiceresultinthe
developmentofaconditionsimilartoALS.
Abnormalities in neurofilaments causes neurodegenerative disease
Degenerative nerve diseases include:
•Alzheimer's disease.
•Amyotrophic lateral sclerosis.
•Friedreich ataxia.
•Huntington's disease.
•Lewy body disease.
•Parkinson's disease.
•Spinal muscular atrophy.
-
-
-
-
-
(ALS)
-
-
-
-
-

147. Neurofilaments aggregation in neurodegeneration

148. How to study effect
of mutations in
genes that causes
neurodegeneration?

149. Intermediate Filaments Provide Mechanical Stability to Animal Cells
•Themajorfunctionofcytoplasmicintermediate
filamentsistoresistmechanicalstress.
•Inthehumangeneticdiseaseepidermolysis
bullosasimplex,mutationsinkeratingenesthat
arenormallyexpressedinthebasalcelllayerof
theepidermisdisruptthekeratinfilamentnetwork
inthesecells,makingthemverysensitiveto
mechanicalinjury:agentlesqueezecancausethe
mutantbasalcellstorupture,andtheskinin
affectedindividualsisblistered.Asimilar
conditioncanbeproducedintransgenicmicethat
expressmutantkeratinsofthistype
Blistering of the skin caused by a
mutant keratin gene. A mutant
gene encoding a truncated keratin
protein (lacking both the amino-
and carboxyl-terminal domains)
was expressed in a transgenic
mouse. Light micrographs of
normal (A) and mutant (B) skin
show that the blistering results
from the rupturing of cells in the
basal layer of the mutant
epidermis
Note: epidermis isthe
outermost layer of skin
on your body
Aplasmidencoding a mutantkeratinwas microinjected into one
pronucleus of a fertilized egg. Microinjected embryos were then
transferred to a foster mother, and some of the offspring were found to
have incorporated the mutant keratingeneinto their genome.
Expression of the mutant gene in these transgenic mice disrupted the
keratincytoskeletonof cells of the epidermis, resulting in severe skin
blistering due to cell lysis following mild mechanical stress.
Experimental demonstration of keratin function

150. The diversity in keratins is clinically useful in
the diagnosis of epithelial cancers
(carcinomas), as the particular set of keratins
expressed gives an indication of theepithelial
tissuein which the cancer originated and thus
can help to guide the choice of treatment.
There are many members of keratins. The 1-19
members of keratins are separated into type I
(or acidic, K9–K19) and type II (or basic to
neutral, K1–8)
keratins areextensively used as
diagnostic tumor markers Keratin expression in human cancer

151. An immuno-fluorescence micrograph of glial filaments in cultured
astrocytes. The bundles of intermediate filaments (green) are stained
with antibodies to glial fibrillary acidic protein. Nuclei are stained with
a blue DNA-binding dye.
(A)Schematic drawing showing the nuclear lamina.
The lamina is associated with both the
chromatin and the inner nuclear membrane.
(B)Electron micrograph of a portion of the nuclear
lamina in a frog oocyte.
Nuclear lamina
(IF: Lamins)

152. The major proteins of muscles are Myosin and actin
•Myosin (thick filaments): 6 subunits, two heavy chains each with 220 Kdamolecular
weight, and 4 light chains each with 20 Kda
•Actin (thin filaments) with 42 Kda
•Myosin and actin undergo transient interactions and slide past each other to cause
contraction
•Together myosin and actin constitute more than 80% of the protein mass of the muscle
Muscle contraction

153. •Myosin has two heavy chains, C-terminus is a
extended coiled tail structure and N-terminus has
globular domains (heads) where ATP is hydrolyzed
•Two light chains are associated with each myosin head
•Cleavage with trypsin and papain separates the myosin
heads (S1 and S2 fragments)
•S1 fragment is the motor domain
•Muscle cells consist of thick (myosin) and thin (actin)
filaments
Structure of myosin
How to analyse the structure of myosin by electrophoresis?

154. •The fibers are made up of many myofibrils surrounded by the
membranous sarcoplasmic reticulum.
•The organization of thick and thin filaments in a myofibril
gives it a striated appearance.
•By electron microscope alternating regions of high and low
electron density called A band and I bands were revealed
which consist of thick and thin filaments
•Bisecting I band is a thin structure known as Z disc (alpha-
Actinin, Desmin, Vimentin and Nebulin) and bisecting A
band is a M line or M disc (paramyosin, C-protein and M-
protein)
•M and Z discs regulated the length of sarcomere
•When muscle contracts, the I bands narrow and the Z disks
move closer together, as seen in electron micrographs of (b)
relaxed and (c) contracted muscle
Muscles consists of bundles of fibersand each fiberis a single multinucleated cell formed from
many cells fused together and each fibercontains about 1000 myofibrils (diameter, 2 micrometer)

155. •In muscle cells, molecules of myosin
aggregate to form structures called
thick filaments.
•The second major muscle protein, actin, is
abundant in almost all eukaryotic cells.
•In muscle, molecules of monomeric actin,
called G-actin, associate to form a long
polymer called F-actin (filamentous) also
known as thin filament consists of other
proteins, troponin and tropomyosin.
•Each actin monomer in the thin
filament can bind tightly and
specifically to one myosin head group.

156. The regulation: is mediated by a complex of two proteins, tropomyosin and troponin.
Tropomyosin binds to the thin filament, blocking the attachment sites for the myosin head
groups. Troponin is a Ca 2+ -binding protein. A nerve impulse causes release of Ca 2+ ions
from the sarcoplasmic reticulum. The released Ca 2+ binds to troponin and causes a
conformational change in the tropomyosin-troponin complexes, exposing the myosin-binding
sites on the thin filaments. Contraction follows

157. Thick filaments are created by the association
of many myosin molecules.
(a) Muscle contraction occurs by the sliding of
the thick and thin filaments past each other so
that the Z disks in neighboring I bands draw
closer together. (b) The thick and thin
filaments are interleaved such that each thick
filament is surrounded by six thin filaments.
Muscle contraction

158. Molecular mechanism of muscle contraction. Conformational changes in the myosin head
that are coupled to stages in the ATP hydrolytic cycle cause myosin to successively
dissociate from one actin subunit, then associate with another farther along the actin
filament. In this way, the myosin heads slide along the thin filaments

159. Myosin Thick Filaments Slide along Actin Thin Filaments The interaction between actin and myosin, like that between all proteinsand
ligands, involves weak bonds.
When ATP is not bound to myosin, a face on the myosin head group binds tightly to actin. When ATP binds to myosin and is hydrolyzed to
ADP and phosphate, a coordinated and cyclic series of conformational changes occurs in which myosin releases the F-actin subunitand binds
another subunit farther along the thin filament. The cycle has four major steps:
In step 1 , ATP binds to myosin and a cleft in the myosin molecule opens, disrupting the actin-myosin interaction so that the bound actin is
released.
ATP is then hydrolyzed in step 2 , causing a conformational change in the protein to a “high-energy” state that moves the myosinhead and
changes its orientation in relation to the actin thin filament. Myosin then binds weakly to an F-actin subunit closer to the Z disk than the one
just released.
As the phosphate product of ATP hydrolysis is released from myosin in step 3 , another conformational change occurs in which themyosin
cleft closes, strengthening the myosin-actin binding.
This is followed quickly by step 4 , a “power stroke” during which the conformation of the myosin head returns to the original resting state, its
orientation relative to the bound actin changing so as to pull the tail of the myosin toward the Z disk. ADP is then releasedtocomplete the
cycle. Each cycle generates about 3 to 4 pN(piconewtons) of force and moves the thick filament 5 to 10 nm relative to the thin filament.
Because there are many myosin heads in a thick filament, at any given moment some (probably 1% to 3%) are bound to thin filaments. This
prevents thick filaments from slipping backward when an individual myosin head releases the actin subunit to which it was bound.The thick
filament thus actively slides forward past the adjacent thin filaments. This process, coordinated among the many sarcomeres in amuscle fiber,
brings about muscle contraction

160. Cell cycle and regulation

161. Prophase, the nuclear envelope breaks down, microtubules form the mitotic spindle apparatus,
and chromosomes condense.
Metaphase, attachment of chromosomes to microtubules via their kinetochores.
Anaphase, motor proteins and the shortening of spindle microtubules pull the sister chromatids
toward opposite spindle poles. After chromosome movement to the spindle poles,
chromosomes decondense and Cells reassemble nuclear membranes around the daughter-cell
nuclei and undergo cytokinesis
Overview of the Cell Cycle and Its Control
•The eukaryotic cell cycle is divided into four phases: G1 (the
period between mitosis and the initiation of nuclear DNA
replication), S (the period of nuclear DNA replication), G2 (the
period between the completion of nuclear DNA replication and
mitosis), and M (mitosis).
•Cells commit to a new cell division at a specific point in G1
known as START or the restriction point.
•Cyclin-CDK complexes, composed of a regulatory cyclin
subunit and a catalytic cyclin-dependent kinase (CDK) subunit,
drive the progression of a cell through the cell cycle.
•Surveillance mechanisms, called checkpoint pathways,
guarantee that each cell cycle step is completed correctly
before the next one is initiated
Overview of cell cycle

162. •Cells reproduce by duplicating their contents and then
dividing in two.
•This cell-division cycle is the fundamental means by which
all living things are propagated.
•In unicellular species, such as bacteria and yeasts, each cell
division produces an additional organism.
•In multicellular species many rounds of cell division are
required to make a new individual, and cell division is
needed in the adult body, too, to replace cells that are lost by
wear and tear or by programmed cell death.
•Thus an adult human must manufacture many millions of new
cells each second, and if all cell division is halted -for
example, by a large dose of ionizing radiation -the individual
will die within a few days.
Overview of the cell cycle

163. Cytokinesis:
•When chromosome segregation is completed, the cytoplasm and
organelles are distributed between the two future daughter cells.
This process is called cytokinesis. The division of the cell is
brought about by a contractile ring made of actin and the actin
motor myosin. During cytokinesis, the ring contracts in a manner
similar to muscle contraction, pulling the membrane inward and
eventually closing the connection between the two daughter cells.
Cytokinesis must be coordinated with other cell cycle events in
space and time.
•In animal cells, the contractile ring forms during anaphase.
•The major signal for cytokinesis is the inactivation of mitotic
CDKs. Cells expressing mitotic cyclins progress through anaphase
but do not undergo cytokinesis.
•The cyclin-dependent kinases and ubiquitin-mediated protein
degradation are at the center of its control.

164. There are multiple ways to study cell cycle?
1.Light microscopyprovides some estimate of cell cycle progression. For example, light microscopy allows a researcher to determine whether cultured mammalian
cells are in interphase (G1, S phase, and G2) or in mitosis. Mammalian tissue culture cells are flat and adhere to their growth surface during interphase, but round
up and form spherical structures as they undergo mitosis.
2.Fluorescence microscopy by using specific cell cycle markers -proteins that are present only in certain cell cycle stages—allows for a more accurate
determination of cell cycle stage.
3.Flow cytometry by measuring DNA content of a cell population. Cells are treated with a DNA-binding fluorescent dye, and the amount of dye that is incorporated
into the DNA of cells can then be quantitatively assessed using a flow cytometer. By their DNA content, the percentages of cellsin G1, S phase, and G2 or mitosis
can be assessed. Cells in G1 have half as much DNA as cells in G2 or mitosis. Cells undergoing DNA synthesis in S phase have an intermediate amount of DNA.
4.This cell cycle arrest is accomplished by restricting nutrients or by adding anti-growth factors, which cause cells to arrest in G1. In budding yeast, forexample,
cells treated with a mating pheromone arrest in G1. When the pheromone is removed from the cells, the cells exit G1 and progressthrough the cell cycle in a
synchronous manner.
5.In mammalian cells, removal of growth factors by removing serum from the culture medium (serum starvation) arrests cells in G0. Re-addition of serum allows
cells to re-enter the cell cycle.
6.Other methods involve blocking a certain cell cycle step with chemicals. Hydroxyurea inhibits DNA replication, leading to arrest in S phase.On removal of the
drug, cells will resume DNA synthesis. Nocodazole disrupts the mitotic spindle and halts the cell cycle in mitosis. Once the drug is removed, cells will resume
progression through mitosis.
7.In budding and fission yeasts, the conditional cdcmutants are powerful tool for creating synchronous cultures. Temperature-sensitive cdcmutants, when
incubated at the non-permissive temperature, arrest in a particular cell cycle stage because they are defective in a certain keycell cycle protein. Returning cells to
the permissive temperature allows them to continue with the cell division cycle in a synchronous fashion.

165. •The cell-cycle control system is a cyclically operating biochemical
device constructed from a set of interacting proteins that induce and
coordinate the essential processes that duplicate and divide the cell's
contents.
•The control system is regulated by brakes that can stop the cycle at
specific checkpoints. They allow the cell-cycle control system to be
regulated by signals from the environment. The environmental
controls generally act on the control system at one or other of two
major checkpointsin its cycle -one in G1, just before entry into S
phase; the other in G2, at the entry to mitosis.
•In a continuously cycling cell the G1 checkpoint is the point where
the cell-cycle control system triggers a process that will initiate S
phase, and the G2 checkpoint is where it triggers a process that will
initiate M phase
A Central Control System Triggers the Essential Processes of the Cell Cycle

166. •The cell-cycle control system is based on two key families of
proteins.
•The first is the family of cyclin-dependent protein kinases (Cdkfor
short), which induce downstream processes by phosphorylating
selected proteins on serinesand threonines.
•The second is a family of specialized activating proteins, called
cyclins, that bind to Cdkmolecules and control their ability to
phosphorylate appropriate target proteins.
•The cyclic assembly, activation, and disassembly of cyclin-Cdk
complexes are the pivotal events driving the cell cycle. Cyclins are
so called because they undergo a cycle of synthesis and
degradation in each division cycle of the cell.
•There are two main classes of cyclins: mitotic cyclins, which bind
to Cdkmolecules during G2 and are required for entry into mitosis,
and G1 cyclins, which bind to Cdkmolecules during G1 and are
required for entry into S phase
The core of the cell-cycle
control system. Cdkis
thought to associate
successively with different
cyclins to trigger the
different downstream
processes of the cycle. Cdk
activity is terminated by
cyclin degradation
Two key components of the cell-cycle control
system. A complex of cyclin with Cdkacts as a
protein kinase to trigger downstream processes.
Without cyclin, Cdkis inactive.
The Cell-Cycle Control System depends on cyclically
activated cyclin-dependent protein kinases (Cdks)

167. •The most abundant Cdkregulators are Cyclins, undergo a cycle
of synthesis and degradation in each cell cycle. But the level of
CDK proteins remain constant
•There are four clyclins; G1/S-clyclins, S-cyclins, M-cyclins and
G1-cyclins (controls the activities of G1/S-clyclins
•In, yeast only one CDK which binds all the cyclins
•In vertebrate cells, there are four CDKs; two interacts with G1-
cyclins, one with G1/S and S-cyclins and one with M-cyclins
•Cyclin binding to CDK causes activation of the CDK enzyme
•The full activation of cyclin-CDK complex occurs when a
separate enzyme, Cdk-activating kinase (CAK) phosphorylates
an amino acid near the active site which causes a small
conformational change which further increases the activity of
CDK, allowing the kinase to phosphorylate its target proteins to
induce cell cycle specific events
The cell cycle control system depends upon cyclically
activated Cyclin-dependent protein kinases (CDKs)
Structural basis of Cdkactivation: binding of cyclin causes T-loop to move
out of the active site causes partial activation of Cdk. Phosphorylation of Cdk
by CAK at a Thrresidue on T-loop further activates the enzyme by changing
the shape of the T-loop improving the ability of the enzyme to bind its protein
substrates (this information is based on structure of human Cdk2)

168. The control of cell cycle not only depends upon protein
phosphorylation but by protein degradation.
•The key regulator of the metaphase to anaphase is the
Anaphase-promoting complex or cyclosome(APC/C), a
member of the ubiquitin ligase family of enzymes.
•APC/C which is activated by Cdc20 (in anaphase) and Cdh1
(from late mitosis through G1) catalyzesthe ubiquitylation and
destruction of two major proteins. First is Securin, which holds
sister chromatid pairs. APC/C with the help of E1 and E2
transfers multiple ubiquitin molecules onto the target protein.
The polyubiquitylated target is then degraded by proteasome.
•The S-and M-cyclins are second major targets of APC/C.
Destroying Cyclins inactivates the Cdks, resulting into
dephosphorylation of many proteins by various phosphatases
which are present in anaphase cell
•SCF: Ubiquitin ligase, targeting phosphorylated target protein
(CKI) for degradation
The control of proteolysis by APC/C and SCF

169. Rbprotein (pRb) is responsible for a major G1 checkpoint, blockingS-phaseentry and cell growth.
Theretinoblastoma protein(pRb) is dysfunctional in several majorcancers.
When the cell is ready to divide, pRbisphosphorylated, inactivating it, and the cell cycle is allowed to
progress. It is also a recruiter of severalchromatin remodelingenzymes such asmethylasesandacetylases.
The Rbprotein, a tumor suppressor, negatively regulates the
cell cycle and tumor progression
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170. Summary
of cell cycle
stages

171. •Abnormal cell growth
•Types of DNA damage and
repair mechanisms
•Meiosis

172. Introduction about cancerous growth
•Cancer occurs due to malfunction in normal proliferation rates. Most or all cases of cancer result from genetic
damage that is often caused by tumor-promoting chemicals, hormones, and sometimes viruses.
•Mutations in three broad classes of genes cause cancer.
•1. Protooncogenesnormally promote cell growth; mutations change them into oncogeneswhose products are
excessively active in growth promotion.
•2. Tumor-suppressor genes (p53, Rbetc.), mutations that inactivate these allow inappropriate cell division.
•3. genome maintenance genes, required for genome’s integrity, when inactivated, cause the deregulation of
cell growth and cause cancer. Many of the genes in these three classes encode proteins that help regulate cell
proliferation (i.e., entry into and progression through the cell cycle) or cell death by apoptosis; others encode
proteins that participate in repairing damaged DNA.
•The cancer-forming process, oncogenesis or tumorigenesis, is an interplay between genetics and the
environment.
•Most cancers arise by cancer-causing chemicals, carcinogens that alter genes (errors in replication and
repair).
•The cancerous cells also acquire other properties such as the ability to stimulate the growth of vasculature to
obtain oxygen. In some cases, cells from the primary tumor migrate to new sites, where they form secondary
tumors, a process termed metastasis.
•It may take many years for a cell to accumulate the multiple mutations to form a tumor
•Tumorscanbebenign(noncancerous)
ormalignant(cancerous).Benign
tumorstendtogrowslowlyanddonot
spread.Malignanttumorscangrow
rapidly,invadeanddestroynearby
normaltissues,andspreadthroughout
thebody.
•Disruptionofepigeneticprocessescan
leadtoalteredgenefunctionand
malignantcellulartransformation
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173. •Carcinomaisatypeofcancerthatstartsincellsthatmakeuptheskinorthetissueliningorgans,suchastheliverorkidneys.Likeother
typesofcancer,carcinomasareabnormalcellsthatdividewithoutcontrol.Theyareabletospreadtootherpartsofthebody,butnotalways.
•Sarcomas:Atypeofcancerthatbeginsinboneorinthesofttissuesofthebody,includingcartilage,fat,muscle,bloodvessels,fibroustissue,or
otherconnectiveorsupportivetissue.Differenttypesofsarcomaarebasedonwherethecancerforms.
Pathways of genome maintenance
•Genomemaintenancegenes:Genesinvolvedinactivities
suchasDNA repair,celldivisioncyclecontrol,and
checkpointsignalingpathwayspreservegenomeintegrity
andpreventdisease.Defectsinthesepathwayscausebirth
defects,neurodegeneration,prematureaging,andcancer.
•Acarcinogenisasubstance,organismoragentcapableof
causingcancer.Carcinogensmayoccurnaturallyinthe
environment(suchasultravioletraysinsunlightandcertain
viruses)ormaybegeneratedbyhumans(suchasautomobile
exhaustfumesandcigarettesmoke)

174. Types of DNA damage
and repair mechanisms

175. Mitosis Meiosis

176. Meiosis. Pre-meiotic cells have two copies of each chromosome (2n), one derived from the paternal parent and one from the maternal parent. For simplicity, the
paternal and maternal homologs of only one chromosome are diagrammed.
Step 1: Chromosomes replicate during S phase before the first meiotic division, giving a 4n chromosomal complement. Cohesincomplexes link the sister
chromatids composing each replicated chromosome.
Step 2: The replicated homologs pair and undergo homologous recombination. At metaphase I, both chromatids of one chromosome associate with microtubules.
Step 3: During anaphase of meiosis I, the homologous chromosomes, each consisting of two chromatids, are pulled to opposite spindle poles.
Step 4: Cytokinesis yields two daughter cells (now 2n), which enter meiosis II without undergoing DNA replication. At metaphase of meiosis II, the sister
chromatids associate with spindle microtubules from opposite spindle poles, as they do in mitosis. Steps 5 and 6 : Segregation of sister chromatids to opposite
spindle poles during anaphase of meiosis II, followed by cytokinesis, generates haploid gametes (1n) containing one copy of eachchromosome.

177. Meiosis
The meiotic division comprises one cycle of chromosome replication followed by two cycles of cell division to produce
haploid germ cells from a diploid pre-meiotic cell.
During meiosis I, homologous chromosomes are segregated; during meiosis II, sister chromatids separate.
During prophase of meiosis I, homologous chromosomes undergo recombination. At least one recombination event occurs
between the chromatids of each pair of homologous chromosomes.
Chiasmata and cohesinsdistal to them are responsible for holding the homologous chromosomes together during prophase
and metaphase of meiosis I.
At the onset of anaphase of meiosis I, cohesinson chromosome arms are phosphorylated and, as a result, cleaved by
separaseand sister chromatids remain linked to each other during segregation in meiosis I.
Cleavage of centromeric cohesinsduring anaphase of meiosis II allows individual chromatids to segregate into germ cells.
Meiotic cohesinsfacilitate the attachment of sister kinetochores to microtubules emanating from the same pole during
meiosis I.
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