Ultra structure of cell wall

For B.Sc Biotechnology Students

1. Ultra Structure of Cell Wall and Plasma Membrane By DR. KRISHNA Assistant Professor in Biotechnology Tumkur University, Tumakuru

2. Introduction: The cell wall - rigid, semi-permeable protective layer in some cell types. Cell wall is positioned next to the cell membrane (plasma membrane) in most plant cells , fungi , bacteria , algae , and some archaea . Animal cells however, do not have a cell wall. The cell wall conducts many important functions in a cell: 1. Protection, 2. Structure, 3. Support. Cell wall composition varies depending on the organism. In plants - mainly of strong fibers of the carbohydrate polymer cellulose . Cellulose is the major component of cotton fiber and wood and is used in paper production.

3. Plant Cell Wall Structure

4. There are at least 8000 to 15,000 glucose monomers per cellulose molecule and are 0.25 to 0.5 µm long. The molecules are flat and ribbon like, and lie parallel to each other. Hydrogen bonding occurs between the molecules, thus crystallizing and producing aggregates. These aggregates are called microfibril . Each microfibril contains 40 to 70 chains, which lie side by side, and these can be seen in Electron micrographs. The spaces between the microfibrils are filled up with lignin, cutin , pectic substances, hemicellulose, water etc. Thus, the microfibril gains considerable strength.

5. In primary cell wall , the orientation of microfibril is transverse to the long axis, and during growth the arrangement may be longitudinal. The orientation in secondary wall may differ from primary wall. Tracheids and fibres show three layers in their secondary wall the outer layer (S 1 ), the central layer (S 2 ) and the inner layer (S 3 ), among which the central (S 2 ) is the thickest. The S 1 and S 3 layers lie adjacent to primary wall and cell lumen respectively. These layers S 1 , S 2 and S 3 may be distinguished by their respective orientation of cellulose microfibrils . In S 1 and S 3 , the microfibrils are in the form of a lax helix and in S 2 , it is a steep one

6. Primary (growing) plant cell wall : M ajor carbohydrates are cellulose , hemicellulose and pectin . cellulose microfibrils are linked via hemicellulosic tethers to form the cellulose-hemicellulose network, which is embedded in the pectin matrix. C ommon hemicellulose in the primary cell wall is xyloglucan O uter part of the primary cell wall of the plant epidermis is usually impregnated with cutin and wax Secondary cell walls –range of compounds that modify their mechanical properties and permeability. The major polymers that make up wood (largely secondary cell walls) include: cellulose, 35-50% xylan , 20-35%, a type of hemicellulose lignin , 10-25%, a complex phenolic polymer that penetrates the spaces in the cell wall between cellulose, hemicellulose and pectin components, driving out water and strengthening the wall.

7. Layers of Cell wall The primary cell wall , generally a thin, flexible and extensible layer formed while the cell is growing. The secondary cell wall , a thick layer formed inside the primary cell wall after the cell is fully grown. It is not found in all cell types. Some cells, such as the conducting cells in xylem , possess a secondary wall containing lignin , which strengthens and waterproofs the wall. The middle lamella , a layer rich in pectins . This outermost layer forms the interface between adjacent plant cells and glues them together.

8. Plant Cell Wall Function A major role of the cell wall is to form a framework for the cell to prevent over expansion. Cellulose fibers , structural proteins , and other polysaccharides help to maintain the shape and form of the cell. Additional functions of the cell wall include: Support - the cell wall provides mechanical strength and support. It also controls the direction of cell growth.​ Withstand turgor pressure - turgor pressure is the force exerted against the cell wall as the contents of the cell push the plasma membrane against the cell wall. This pressure helps a plant to remain rigid and erect, but can also cause a cell to rupture.​ Regulate growth - sends signals for the cell to enter the cell cycle in order to divide and grow. Regulate diffusion - the cell wall is porous allowing some substances, including proteins , to pass into the cell while keeping other substances out.​ Communication - cells communicate with one another via plasmodesmata (pores or channels between plant cell walls that allow molecules and communication signals to pass between individual plant cells).​ Protection - provides a barrier to protect against plant viruses and other pathogens. It also helps to prevent water loss.​ Storage - stores carbohydrates for use in plant growth, especially in seeds.

9. The Cell Wall of Bacteria cell wall in prokaryotic bacteria is composed of peptidoglycan . Peptidoglycan is a polymer composed of double-sugars and amino acids ( protein subunits). This molecule gives the cell wall rigidity and helps to give bacteria shape. Peptidoglycan molecules form sheets which enclose and protect the bacterial plasma membrane . The cell wall in gram-positive bacteria contains several layers of peptidoglycan. These stacked layers increase the thickness of the cell wall. In gram-negative bacteria , the cell wall is not as thick because it contains a much lower percentage of peptidoglycan . The gram-negative bacterial cell wall also contains an outer layer of lipopolysaccharides (LPS). The LPS layer surrounds the peptidoglycan layer and acts as an endotoxin (poison) in pathogenic bacteria (disease causing bacteria). The LPS layer also protects gram-negative bacteria against certain antibiotics, such as penicillins .

10. Cell Wall Structure of Bacteria:

11. Types of cell wall 1. Gram positive cell wall Cell wall composition of gram positive bacteria. Peptidoglycan Lipid Teichoic acid 2. Gram negative cell wall Cell wall composition of gram negative bacteria Peptidoglycan Outermembrane : Lipid Protein Lipopolysaccharide (LPS)

12. Composition of cell wall : 1. Peptidoglycan: Peptidoglycan is porous cross linked polymer which is responsible for strength of cell wall. Peptidoglycan is composed of three components. Glycan backbone Tetra-peptide side 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 β- glycosidic bond. 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 ie NAG-NAM is same in all species of bacteria.

13. The aminoacids found in tetra-peptide are- L-alanine: 1 st position in both gm+ve and gm- ve bacteria D-glutamic acid: 2 nd position D- aminopimelic acid/ L-lysine: 3 rd position (variation occurs) D-alanine : 4 th position

14. Peptide cross linkage in Gram positive and Gram negative bacteria: In gram negative bacteria, peptide cross linkage occur between  Diaminopamilic acid (3rd position) of one glycan back bone and D-alanine of adjacent  glycan back bone. In gram positive bacteria, peptide cross linkage occur by peptide interbridge . The type and number of aminoacids in interbridge vary among bacterial species.

15. 2. Teichoic acid: Teichoic acid is water soluble polymer of glycerol or ribitol phosphate. It is present in gram positive bacteria. It constitutes about 50% of dry weight of cell wall. It is the major surface antigen of gram positive bacteria. 3. Outer membrane It is an additional layer present in gram negative bacteria. It is composed of lipid bilayer, protein and lipopolysaccharide (LPS) layer Function of outer membrane: Structure component of gram- ve cell wall LPS is an endotoxin produced by gram – ve bacteria Lipid-A is antigenic Figure: Outer membrane in cell wall of gram negative bacteria

16. 4. LPS LPS is attached to outer membrane by hydrophobic bond. LPS is synthesized in cytoplasmic membrane and transported to outer membrane. LPS is composed of lipid-A and polysaccharide. Lipid-A: it is phosphorylated glucosamine disaccharide. Polysaccharide: it consists of core-polysaccharide and O-polysaccharide.   Gram positive bacteria Micrococcus Staphylococcus Streptococcus Leuconostoc Gram negative bacteria E. coli Salmonella Klebsiella Shigella Pseudomonas

17. Cell Membrane: The term was originally used by Nageli and Cramer (1855) for the membranous covering of the protoplast. The same was named plasmalemma by Plowe (1931). Plasmalemma or plasma membrane was discovered by Schwann (1838). Membranes also occur inside the cytoplasm of eukaryotic cells as covering of several cell organelles like nucleus, mitochondria, plastids, lysosomes, golgi bodies, peroxisomes, etc.

18. Models and Ultrastructure of Plasma Membrane The top four historical models of Plasma Membrane. The models are : 1. Lipid and Lipid Bilayer Models 2. Unit Membrane Model (Protein-Lipid Bilayer-Protein) 3. Fluid Mosaic Model 4. Dannelli Model.     1. Lipid and Lipid Bilayer Model: This model to explain the structure of plasma membrane was given by Overton, Gorion and Grendel . Previously only indirect information was available to explain the structure of plasma membrane. In 1902, Overton observed that substances soluble in lipid could selectively pass through the membranes. On this basis he stated that plasma membrane is composed of a thin layer of lipid. Subsequently, Gorter and Grendel in 1926 observed that the extracted from erythrocyte membranes was twice the amount expected if a single layer was present throughout the surface area of these cells. On this basis they stated that plasma membrane is made up of double layer of lipid molecules. These models of Gorter and Grendel could not explain the proper structure of plasma membrane but they put the foundation of future models of membrane structure.

19. 2. Unit Membrane Model (Protein-Lipid Bilayer-Protein ): This is also known as unit membrane model. This model was proposed by Davson , Daniell and Robertson . When surface tension measurements made on the membranes, it suggests the presence of proteins. After the existence of proteins the initial lipid bilayer model proposed by Gorter and Grendel was modified. It was suggested that surface tension of cells is much lower than what one would expect if only lipids were involved. It may also be observed that if protein is added to model lipid water system, surface tension is lowered. This suggested indirectly the presence of proteins. On this basis Davson and Danielli proposed that plasma membrane contained a lipid bilayer with protein on both surfaces.

20. Initially they supposed that proteins existed as covalently bonded globular structures bound to the polar ends of lipids. Subsequently they developed the model in which the protein appears to be smeared over the hydrophilic ends of the lipid bilayer. This model makes its popularity for a long time. With the availability of electron microscope later, fine structure of plasma membrane could be studied. Definite plasma membrane of 6 nm to 10 nm (10nm = 100 Å; 1 nm = 10 _6 mm) thickness was observed on surface of all cells, and plasma membranes of two adjacent cells were found to be separated by a space, 1-15nm wide . It was also observed that the plasma membrane of most of the cells appeared to be three layered. Two outer dense layers were about 2.0nm thick and the middle layer about 3.5nm . The early ideas of Gorter and Grendel and those of Davson and Danielli were first formalized by Robertson in 1959 in the form of his unit membrane concept.

21. This concept of unit membrane with three layers (two protein layers and one lipid bilayer) only supported the concept proposed earlier by Davson and Danielli . In this unit membrane the less dense middle layer corresponded to hydrocarbon chains of lipids . Thickness of unit membrane (10nm) was found to be greater in plasma membrane than in intracellular membranes of endoplasmic reticulum or golgi complex.

22. 3. Fluid Mosaic Model: To explain the structure plasma membrane various models have been put forward from time to time. But none was universally accepted. In this relation Gorter and Grendel, Davson and Danielli , etc. proposed model for plasma membrane after that Fluid Mosaic Model for plasma membrane was proposed which was universally accepted. It was proposed by Singer and Nicholson (1912). This model postulates that lipid and integrated proteins are disposed in a sort of mosaic pattern and all the biological membranes have a quasi-fluid structure where both lipid and protein components are able to perform transitional movement within lipid bilayer . In this model, lipid molecules may exhibit intra-molecular movement or may rotate about their axis or may display flip-flop movement including transfer from one side of bilayer to the other. Thus this concept implies that main components of the membrane, i.e., lipids, proteins and oligosaccharides are held together by means of non-covalent interactions as suggested by Gitler (1972). A term amphipathy was coined by Hartley (1936) to the molecules having both hydrophilic and hydrophobic groups. Thus lipids and integrated proteins are amphipatic in nature.

23. 4. Dannelli Model : ( i ) Lipid and intrinsic proteins are present in a mosaic arrangement and (ii) Biological membranes are semifluid so that lipids as well as intrinsic proteins are able to make movements within the bilayer. This concept of fluidity implies that lipids, proteins and oligosaccharides are held in their positions by means of non-covalent interactions. On this basis, it can be stated that components can be dispersed by solvents or detergents without breaking any bonds. Intrinsic proteins are also intercalated to greater or lesser extent into a continuous lipid bilayer. Such proteins may be in contact with an aqueous solvent on both sides of the membrane.

24. Sandwich Model (Lamellar Models) of Plasma Membrane Denielli and Davson have proposed that in the plasma membrane a double layer lipid molecule is sandwiched between the two different layers of protein molecules. The lipid layers are found internally and the protein molecules are found externally. The inner ends of lipid molecules are non-polar and hydrophobic where as outer ends are polar and hydrophilic . James Danielli and Hugh Davson in 1935

25. Unit Membrane Model of Plasma Membrane Robertson in the year 1953 proposed the unit membrane model consisting of three layers of the plasma membrane and it is complimentary to the sandwich model. According to this there are two outer layers of protein molecules each with 20 angstroms thick, embracing a central layer of lipid molecules of about 35 angstroms thick for a total thickness of 75 angstroms . Robertson Model: J. David Robertson (1959) modified the model of Danielli and Davson by proposing that the lipid bilayer is covered on the two surfaces by extended or (3-protein molecules)