Intercellular connections and molecular motors

1. Intercellular Connections and Molecular Motors

2. Learning Objectives Cell Adhesion Molecules Intercellular Connections. Brief description of each type and their function. Molecular motors. What are they and what they do?

3. Cell Adhesion Molecules (CAMs) Important cell surface proteins molecules promoting cell–cell and cell–matrix interactions. I mportant for many normal biological processes -embryonic cell migration, immune system functions, wound healing. Involved in intracellular signaling pathways (primarily for cell death/survival, secretion etc.)

4. Cell Adhesion Molecules (CAMs) Express 3 major domains : The extracellular domain allows one CAM to bind to another on an adjacent cell . The transmembrane domain links the CAM to the plasma membrane through hydrophobic forces. The cytoplasmic domain is directly connected to the cytoskeleton by linker proteins .

5. Cell Adhesion Molecules (CAMs) Interactions between CAMs can be mediated by : Binding of an adhesion molecule on one cell to the same adhesion molecule on a second cell Cadherin - cadherin An adhesion molecule on one cell type binds to a different type of cell adhesion molecule on a second cell Selectins – mucins The linker molecule in most cases is Laminin , a family of large cross shaped molecules with multiple receptor domains.

6. Principal classes of cell-adhesion molecules Identified by using specific monoclonal antibodies ( mAbs ). genes encoding these molecules has shown that they are structurally different from each other. These cell adhesion molecules can be divided into 4 major families The cadherin superfamily T he selectins T he immunoglobulin superfamily and The integrins

7. The Cadherin superfamily Cadherins are the most prevalent CAMs in vertebrates. 125 kD transmembrane glycoproteins - mediate intercellular adhesion in epithelial and endothelial cells by Ca2 + dependent homophilic adhesion. Primarily link epithelial and muscle cells to their neighbors Form desmosomes and adherens junctions Play critical role during development ( cell sorting). Do not interact with extracellular matrix.

8. The Cadherin superfamily C ontain a short transmembrane domain and a relatively long extracellular domain containing four cadherin repeats (EC1-EC4), each of which contains calcium binding sequences Cadherins interact with specific cytoplasmic proteins, e.g., catenins ( α , β and γ ), as a means of being linked to the actin cytoskeleton. The binding of cadherins to the catenins is crucial for cadherin function.

9. The Cadherin superfamily E-cadherin is thought to be important during embryonic development, and is also involved in generating and maintaining epithelial layers in adult tissues. The loss of E cadherin expression has been linked to the invasive behavior of tumour cells

10. The Cadherin superfamily Ca binds in the hinge regions between cadherin domains, and prevent the flexing. Without Ca the molecule is floppy and adhesion fails

11. The Selectins Involved in heterophilic cell-cell interactions. Family of Ca+2 dependent carbohydrate binding proteins, mediate the initial attachment of leukocytes to the endothelium on the blood vessel wall during the rolling step of leukocyte extravasation in inflammation. Selectins recognize fucosylated carbohydrate ligands , especially structures containing Sialyl -Lewis x ( sLex ) and Sialyl -Lewis a ( sLea ), which are heavily expressed on neutrophils and monocytes

12. The Selectins Structural features of selectins include: NH2-terminal C-type Ca2+ dependent lectin like binding domain, which determines the ability of each selectin to bind to specific carbohydrate lingands . an epidermal growth factor-like region. a number of repeat sequences. a membrane-spanning region and a short cytoplasmic region

13. The Selectins Selectin family L eukocyte-expressed L- selectin (CD62L) E ndothelial-expressed E- selectin (CD62E) P- selectin (CD62P ) which is expressed by both platelets and endothelial cells

14. The Selectins Recently elevated levels of L- selectin have been observed in the serum of patients with AIDS and leukemia (1 ) E selectin has been found to regulate adhesion of human colon cancer cells to the endothelium by binding to sLea and sLex carbohydrate ligands (2)

15. Immunoglobulin Superfamily Molecules Have a series of globular Ig -like domains, formed by disulfide bonds . Mediate Ca -independent cell adhesion . P rimarily homophilic cell-cell adhesion but also some heterophilic. Activate intracellular signaling pathways. Play critical role during morphogenesis and differentiation of muscle, glial and nerve cells In neurons promote the formation of myelin In vascular endothelial cells leukocyte adhesion and extravasation.

16. Immunoglobulin Superfamily Molecules C onsists of more than 25 molecules. Important ones being: Intracellular adhesion molecule 1(ICAM1 ; CD54) I ntercellular adhesion molecule 2 ( ICAM2 ), V ascular cell adhesion molecule1 ( VCAM1 ; CD106), Platelet endothelial cell adhesion molecule 1 (PECAM 1 ; CD31) and the mucosal addressin cell adhesion molecule 1 ( MAdCAM1 ).

17. Immunoglobulin Superfamily Molecules leukocyte endothelial cell adhesion, endothelial cell-endothelial cell, and leukocyte-leukocyte adhesion

18. The integrins Cell adhesion receptors responsible for the cell extracellular matrix adhesion Important signal transduction receptors for regulation of cell growth Present in membranes of all cells except erythrocytes. C omposed of heterodimers consisting of two non-covalently associated subunits, α and β , both of which are necessary for adhesive binding.

19. The integrins Fifteen different α and eight different β subunits give rise to over twently different heterodimeric combinations at cell surfaces. Bind epithelial and muscle cells to laminin in the basal lamina Allow platelets to stick to exposed collagen in a damaged blood vessel Allow fibroblasts and white blood cells to adhere to fibronectin and collagen as they move

20. Intercellular Connections . OCCLUDING JUNCTIONS Tight Junctions ( Zona Occludens ) ANCHORING JUNCTIONS Actin filament attachment sites Cell- cell junctions ( Zonula Adherens ) Cell-matrix junction (Focal Adhesions) Intermediate filament attachment sites Cell-cell junction (Desmosomes) Cell-matrix junction ( Hemidesmosomes ) CHANNEL FORMING JUNCTIONS Gap junctions SIGNAL RELAYING JUNCTIONS Chemical synapse

21. Tight Junctions Also known as Zona Occludens . Surround the apical margins of the cells in epithelia such as the intestinal mucosa, the walls of the renal tubules, and the choroid plexus. Made up of ridges—half from one cell and half from the other—which adhere so strongly at cell junctions that they almost obliterate the space between the cells. P ermit the passage of some ions and solute in between adjacent cells ( paracellular pathway) and the degree of this “ leakiness” varies , depending in part on the protein makeup of the tight junction .

22. Tight Junctions B asic architectural principle - transmembrane proteins are linked to a cytoplasmic plaque that is formed by a network of scaffolding and adaptor proteins, signalling components and actin-binding cytoskeleton

23. Tight Junctions TRANSMEMBRANE TIGHT JUNCTION PROTEINS : Tight Junctions contain two principal types of Transmembrane protein components – tetraspan and single-span transmembrane proteins. The tetraspan proteins are: occludin and the claudins have both the N- and C-termini in the cytosol. form the paracellular permeability barrier and determine the capacity and the selectivity of the paracellular diffusion pathway. T he single-span transmembrane proteins are the junctional adhesion molecules (JAMs),

24. Functions of Tight Junctions Paracellular permeability: allow the passive selective diffusion of ions and small hydrophilic molecules through the paracellular pathway across epithelia and endothelia. the claudin composition of TJs is a major determinant of the permeability properties of a tissue. Occludin regulates the paracellular diffusion of small hydrophilic molecules, and regulates the transepithelial migration of neutrophils. The passage of solute depends upon its size and charge.

25. Functions of Tight Junctions Cell proliferation, polarity and differentiation: Several studies have linked TJs to the regulation of cell proliferation and cell polarity. Occludin suppresses oncogenic Raf-1 signalling (Wang et al., 2005) and interacts with ZONAB, thereby regulating gene expression, cell proliferation and epithelial morphogenesis (Matter and Balda , 2007; Sourisseau et al .,2006) Occludin has also been linked to the regulation of various subcellular signalling pathways, such as MAP-kinase-dependent pathways.

26. Disease of Tight Junctions

27. Gap Junction Gap junctions are clusters of intercellular channels that allow direct diffusion of ions and small molecules between adjacent cells . At gap junctions, the intercellular space narrows from 25 nm to 3 nm. gap junctions were first discovered in myocardium and nerve because of their properties of electrical transmission between adjacent cells ( Weidmann 1952 ; Furshpan and Potter 1957 ).

28. Gap Junction The intercellular channels are formed by head-to-head docking of hexameric assemblies ( connexons ) of tetraspan integral membrane proteins, the connexins ( Cx ) ( Goodenough et al. 1996).

29. Gap Junction Electron microscopy of gap junctions joining adjacent hepatocytes in the mouse. The gap junction (GJ ) is seen as an area of close plasma membrane apposition

30. Function of Gap Junction The diameter of the connexon channel is normally about 2 nm, which permits the passage of ions, sugars, amino acids, and other solutes with molecular weights up to about 1000 Dalton. F unction as suppressors of somatic cell mutations - loss of a critical metabolic enzyme or ion channel in one cell compensated by its neighbours. Are particularly important in cardiac muscle: the signal to contract is passed efficiently through gap junctions, allowing the heart muscle cells to contract in tandem. A gap junction located in neurons referred to as an electrical synapse are important in neurotransmitter release

31. Disease associated with Gap Junctions 20 different genes code for connexins in humans, and mutations in these genes can lead to diseases that are highly selective in terms of the tissues involved. In humans, mutations in Cx32 underlie X-linked Charcot-Marie-Tooth syndrome, a common peripheral demyelination neuropathy. mutations in Cx47 result in a central demyelinating condition. disorders of the skin and the auditory system accompany mutations in Cx31 andCx30. Familial cataracts are commonly associated with mutations in either Cx46 or Cx50.

32. Desmosomes Also known as macula adherens is a cell structure specialized for cell-to-cell adhesion. Are molecular complexes of cell adhesion proteins and linking proteins that attach the cell surface adhesion proteins to intracellular keratin cytoskeletal filaments. The cell adhesion proteins of the desmosome, desmoglein and desmocollin , are members of the cadherin family. On the cytoplasmic side of the plasma membrane, there are two dense structures called the Outer Dense Plaque (ODP) and the Inner Dense Plaque (IDP). The Outer Dense Plaque is where the cytoplasmic domains of the cadherins attach to desmoplakin via plakoglobin and plakophillin . The Inner Dense Plaque is where desmoplakin attaches to the intermediate filaments of the cell.

33. Desmosomes

34. Hemidesmosomes Hemidesmosomes look like half-desmosomes that attach cells to the underlying basal lamina. Rather than using desmogleins , hemidesmosomes use desmopenetrin cell adhesion proteins,which are members of Integrin family. The integrin molecule attach to one of many multi-adhesive proteins such as laminin , resident within the extracellular matrix, thereby forming one of many potential adhesions between cell and matrix.

35. Molecular Motors Molecular motors composed of motor proteins. These proteins bind to a polarized cytoskeletal filament and use the energy derived from repeated cycles of ATP hydrolysis to move steadily along it Power movements of subcellular components Create local forces leading to cell shape changes Muscle contraction Power cell movements

36. Molecular Motors Dozens of different motor proteins coexist in every eucaryotic cell. They differ in the type of filament they bind to (either actin or microtubules), the direction in which they move along the filament, and the “cargo” they carry. There are three super families of molecular motors: kinesin , dynein, and myosin.

37. Molecular Motors, Kinesin The conventional form of kinesin is a doubleheaded molecule that tends to move its cargo toward the “+” ends of microtubules. Dimer of two heavy chains Each heavy chain complexes with a light chain Three domains Two globular head domains Long central coiled-coil stalk Two small globular tail domains (contain light chains

38. Molecular Motors, Kinesin Kinesin accomplishes transport by "walking" along a microtubule. Two mechanisms have been proposed to account for this movement. In the "hand-over-hand" mechanism, the kinesin heads step past one another, alternating the lead position . One head binds to the microtubule and then bends its neck while the other head swings forward and binds, producing almost continuous movement In the "inchworm" mechanism, one kinesin head always leads, moving forward a step before the trailing head catches up .

39. Molecular Motors, Dyenin Dynein transports various cellular cargo by "walking" along cytoskeletal microtubules towards the minus-end of the microtubule. C omposed of two or three heavy chains (that include the motor domain) and a large and variable number of associated light chains. Dyneins can be divided into two groups: cytoplasmic dyneins and axonemal dyneins , which are also called ciliary or flagellar dyneins .

40. Molecular Motors, Dyenin Cytoplasmic dyneins are found in all eucaryotic cells - important for vesicle trafficking, and for localization of the Golgi apparatus near the center of the cell. Axonemal dyneins , are highly specialized for the rapid and efficient sliding movements of microtubules that drive the beating of cilia and flagella. Dyneins are the largest of the known molecular motors, and they are also among the fastest: axonemal dyneins can move microtubules in a test tube at the remarkable rate of 14 μm /sec

41. Molecular Motors, Myosin Myosins comprise a family of ATP-dependent motor proteins and are best known for their role in muscle contraction and their involvement in a wide range of other eukaryotic motility processes. Most myosin molecules are composed of a head, neck, and tail domain. The head domain binds the filamentous actin, and uses ATP hydrolysis to generate force and to "walk" along the filament towards the barbed (+) end (with the exception of myosin VI, which moves towards the pointed (-) end). the neck domain acts as a lever arm for transducing force generated by the catalytic motor domain. The tail domain generally mediates interaction with cargo molecules and/or other myosin subunits. In some cases, the tail domain may play a role in regulating motor activity

42. Molecular Motors, Myosin 18 different families (identified by genetic analysis) Have different functions Myosin II powers muscle contraction and cytokinesis Myosins I transport of endocytic vesicles Myosin V phagocytosis and transport of cellular elements Myosins VI and VII – transport endocytic vesicles into the cell. Found in the inner ear and mutations in the gene coding for myosin VII cause deafness in mice and humans.

43. Molecular Motors, Myosin A myosin II molecule is composed of two heavy chains (each about 2000 amino acids long (green) and four light chains (blue ). the long coiled-coil tail bundles itself with the tails of other myosin molecules forming bipolar “thick filaments” that have several hundred myosin heads, oriented in opposite directions at the two ends.

44. Molecular Motors, Myosin How does the myosin move ? Cyclic attachment and detachment of myosin head from actin filament, each coupled to hydrolysis of one ATP ATP binding to myosin opens the cleft and disrupts actin binding Release of actin from myosin head ATP hydrolysis - bending of the head to the new position (generation of movement) After ATP hydrolysis the cleft closes on the next actin molecule

45. ………………Thank You Any Queries?