Cell Biology and Genetics Notes PDF

UBT 008: Cell Biology and Genetics Sections Topics Module I Cell Structure and Functions MST [5 weeks] Module II...

UBT 008: Cell Biology and Genetics Sections Topics Module I Cell Structure and Functions MST [5 weeks] Module II Cell Division Module III Genetics EST [5 weeks] Module IV Linkage and Recombination Course Objectives: 1. To imparts knowledge of structural and functional aspects of cells as units of living systems. 2. To understand functions of various organelles and transport of information and matter across cell membrane. 3. To understand Mendelian Laws of Inheritance and their significance in genetic diseases. Course Learning Outcomes: 1. Acquire knowledge about the organizational and functional aspects of cell and organelles. 2. Learn about interaction of cells with outside environment through exchange of information and transport of molecules. 3. Learn about classical genetics and transmission of characters from one generation to the next which will make foundation for advanced genetics. 4. Develop innovative research ideas for curing genetic disorders in human. 06-09-2024 Cell Biology : Basics 1 Books to be Referred for Cell Biology and Genetics 1. Bruce Alberts et al., Essential Cell Biology. Garland Science (Taylor and Francis) 2. Veer Bala Rastogi, Cell Biology (MedTech Science Press) 3. Geoffrey M. Cooper., The Cell: A molecular Approach 4. H. Lodish et al., Molecular Cell Biology (4th Edition), WH Freeman 5. Gardner, Simmons and Snustad, Principle of Genetics by John Wiley and Sons 6. Eddon John Gardner, Principle of Genetics (Wiley, 8th Edition) 7. Peter J. Russel. Genetics: A Molecular Approach (Pearson Education India) Course Instructor: Dr. Jyotsana Mehta Email Id: [email protected] Contact No. 8708290308 06-09-2024 Cell Biology : Basics 2 Module I: Cell Structure and Functions 1. Cell Biology Basics 1.1. Living Organisms : Unicellular and Multicellular Organisms 1.2. Cell: Definitions; Basic Features; Hierarchy 1.3. Cell Theory and Cell Doctrine 1.4. Types of Cells: Based on Size, Shape and Morphology 06-09-2024 Cell Biology : Basics 3  1.1. Living Organisms: Unicellular and Multicellular Organisms  Organism can be broadly defined as a molecule assembly functioning in totality and exhibits properties of life.  Monomeric unit of their existence is called Cell.  Basic parameter of an organism is its life span. Some organisms live for one day, while some plants and fungi can live thousands of years.  Basically made up of complex system of macromolecules.  Macromolecules like carbohydrates, proteins, nucleic acid and lipids play important role in day to day function of the organism. 06-09-2024 Cell Biology : Basics 4  1.1. Living Organisms: Unicellular and Multicellular Organisms Unicellular Organisms Unicellular Multi-cellular  Known as a single-celled organism (Most primitive form of organisms).  Main groups of unicellular organisms are bacteria, archaea, protozoa, algae and fungi.  Fall into two general categories: prokaryotic and eukaryotic organisms (based on cellular organization)  Oldest form of life, possibly existing 3.8 billion years ago.  Mostly these organisms are microscopic (cannot be seen by naked eyes) and categorised as microorganisms.  Examples are: Bacteria like Escherichia coli, Mycobacteria, Bacillus sp. etc. Protozoans like Amoeba, Paramecium etc. Algae like Chlorella sp, Chlamydomonas, Diatoms, Euglenophyta, Dinoflagellates etc; Fungi like yeast. 06-09-2024 Cell Biology : Basics 5  1.1. Living Organisms: Unicellular and Multicellular Organisms Multi-cellular Organisms  Consist of many cells specialized to do different functions for maintaining the complexity of the organism.  Most bacteria are unicellular, but some bacterial species are multicellular like Myxobacteria. Some species of cyanobacteria are also multicellular like Spirogyra etc.  Most eukaryotic organism are multicellular. Multicellular organisms have well developed body structure and also have specific organs for specific function.  Most well developed plants and animals are multicellular. All animals are eukaryotic in nature and most of them are multicellular. In plants highly evolved types like angiosperms and gymnosperms are multicellular. Examples of unicellular plants are Chlamydomonas ( green algae), chlorella (single-celled green algae), etc. Acetabularia is the largest unicellular green algae. It reaches to the size 0.5 to 10 cm in length.  Cell divisions is essential for three major functions in multicellular organisms: Growth, Development, and Repair.  Two types of divisions are present: Mitosis (Vegetative Divisions) and Meiosis (Reductive Cell division). 06-09-2024 Cell Biology : Basics 6  Differences between Unicellular and Multicellular Organisms Unicellular Organisms Multicellular Organisms  Body is made up of single cell  Body is made up of numerous cells  Division of labour may be at cellular, tissue,  Division of labour is at the organelle level. It organ and organ system level. It gives high gives a low level of operational efficiency. degree of operational efficiency.  Different cells are specialized to perform  A single cell carries out all the life processes different functions.  Only outer cells are specialized to face the  The cell body is exposed to the environment environment. Inner cells are devoted to other on all sides. functions. 06-09-2024 Cell Biology : Basics 7 Contd………  Unicellular Organisms Multicellular Organisms  Injury or death of some cells does not affect  An injury to the cells can cause death of the the whole organism as the same can be organism. replaced by new one.  Cell body cannot attain a large size  A multicellular body can attain a large size because of the limit imposed by surface increasing the number of small cells. area to volume ratio.  Lifespan is long due to limited load of work  Lifespan is short due to heavy load of work. for each cell type.  Certain specialized cells lose power of  Power of division is not lost. division.  The capacity of regeneration decreases with  Capacity of regeneration present. increasing specialization. 06-09-2024 Cell Biology : Basics 8  Common Features of Unicellular and Multi-cellular Organisms All organisms must accomplish the same functions:  uptake and processing of nutrients  excretion of wastes  response to environmental stimuli  reproduction among others 06-09-2024 Cell Biology : Basics 9  1.2. Cell: Definitions; Basic Features; Hierarchy What is a Cell?  Cell is a basic structural, functional and biological unit of all living organisms (Unicellular and multicellular).  Term originated from Latin Word “Cellula” or “Cellus” meaning small room or little space and was discovered by Robert Hook in 1665 while studying cork under microscope. Robert Hook Microscope Cells of Cork  It is a self-replicating structures that are capable of responding to changes in the environment.  Often called building block of life.  Study of cell is called cell biology. 06-09-2024 Cell Biology : Basics 10  1.2. Cell Theory  Robert Hook published findings about Cells in his book entitled Micrographia in which he gave 60 ‘observations of various objects under an optical microscope’.  One observation was from very thin slices of bottle cork (tiny empty boxes/hexagonal dimensions). Hooke did not know their real structure or function. He had thought that cells were actually empty cell walls of plant tissues.  With microscopes of low magnification at that time, Hooke was unable to see internal components of the cells he was observing. So, he thought cells were dead and his observations gave no indication of the nucleus and other organelles found in most living cells.  Antonie van Leeuwenhoek is another scientist who saw cells soon after Hooke did. He made use of a microscope containing improved lenses that could magnify objects almost 300 fold. Under these microscopes, Leeuwenhoek found motile objects and he stated that motility is a quality of life therefore these were living organisms. Over time, he wrote many more papers in which described many specific forms of microorganisms like bacteria and protozoa. He called these tiny creatures “animalcules.”  He also gave accurate description of red blood cells, sperm cells, fertilization process and thus, ended the theory of spontaneous generation. 06-09-2024 Cell Biology : Basics 11  1.2. Cell: Definitions; Basic Features; Hierarchy  The cells in animal tissues were observed after plants because the tissues were so fragile and susceptible to tearing, it was difficult for such thin slices to be prepared for studying.  Biologists believed that there was a fundamental unit to life, but were unsure what this was. It would not be until over a hundred years later that this fundamental unit was connected to cellular structure and existence of cells in animals or plants.  Henri Dutrochet was the first person to state that “the cell is the fundamental element of organization”, he also claimed that cells were not just a structural unit, but also a physiological unit.  In 1804, Karl Rudolphi and J.H.F. Link were awarded the prize for "solving the problem of the nature of cells", meaning they were the first to prove that cells had independent cell walls. Prior to that, it was though that cells shared cell wall and fluid passed between them.  Later in 1838 concept of cell theory came into existence. 06-09-2024 Cell Biology : Basics 12  1.2. Cell – Important Features  Grow  Repair and Maintain  Reproduce  Undergo change  Move  Respond  Grow old and die  Why basic unit of life?? Smallest of biological structure that perform all basic activities of life 06-09-2024 Cell Biology : Basics 13  1.2. Cellular Hierarchy Organism (Human) Organ-system (Respiratory system) Organ (Lung) Tissue (Epithelial tissue, interstitial connective tissue, and lymphoid tissue Cells Cell (Monocyte) 06-09-2024 Cell Biology : Basics 14  1.3. Cell Theory  In biology, cell theory is a scientific theory which describes the properties of cells.  Cell theory is the foundation of biology and is the most widely accepted explanation regarding the structural and functional aspects of cells.  With continual improvements made to microscopes over time, magnification technology advanced enough to discover cells in the 17th century.  After the discovery of cells, many debates started about properties, role and function of cells. Eventually in 1838-1839, Cell theory was formulated by Matthias Schleiden and Theodor Schwann. Other scientists like Rudolf Virchow also contributed to the theory. 06-09-2024 Cell Biology : Basics 15  1.3. Cell Theory Schleiden suggested:  Cells or result of cells contribute to structural part of a plant.  Cells are made by a crystallization process either within other cells or from the outside. Schwann suggested:  Like plants, structurally animals are also composed of cells or a group of cells/products of cells. The following are the basic principles of the cell theory: 1. All living organisms are composed of one or more cells. 2. The cell is the most basic unit of life. 06-09-2024 Cell Biology : Basics 16  1.3. Essential Features of Cell Theory Following are the essential features of cell theory: 1. Cells are fundamental units of structure and function in all living organisms. 2. Cells are physiological units of living organisms. 3. Cell is the smallest unit of life. All activities of living organisms are the outcome of the activities of its constituent cells. 06-09-2024 Cell Biology : Basics 17 Significance of Cell Theory  Concept of cell theory emphasizes the structural and functional relationship among the diverse living forms from bacteria to man.  All cells irrespective of their function and position have a nucleus embedded in the cytoplasm and bounded by cell membrane (unity in their structural plan).  Same metabolic processes occur in all the cells primitive or specialized (unity of function).  This implies that all the living things have originated from the same primitive ancestral types that originated billion years ago. Exceptions to the Cell Theory  Viruses are the exception to cell theory because they are made up of proteins and one of the nucleic acid (DNA or RNA), lack protoplasm.  Bacteria lack well-organised nucleus. Nuclear membrane, nucleolus and nucleoplasm are also absent. Nucleic acid (DNA) alone forms the chromosome and lies in direct contact with cytoplasm. Basic proteins associated with nucleic acid are absent in bacteria.  Coenocytic hyphae of Rhizopus and cells of Vaucheria are multinucleate. 06-09-2024 Cell Biology : Basics 18  1.3. Objections to Cell Theory 06-09-2024 Cell Biology : Basics 19  1.3. Cell Principle or Cell Doctrine  Such objections necessitated modification of Cell theory.  The modified form of cell theory is described as “Cell Principle or Cell doctrine” Events that led to the development of Cell Principle or Cell Doctrine  In 1858, a German biologist Rudolph Virchow found that all living cells arise from pre-existing cells (“omnis cellula e cellula”)  In 1866, Ernst Haeckel suggested that nucleus might store and transmit the hereditary information.  The improvement made in the field of microscopes and techniques for the study of cells, enabled scientists to collect enormous information on the structural and functional organisation of cells.  As a result of these developments, the cell theory had to be modified and cell principle was formulated. 06-09-2024 Cell Biology : Basics 20  1.3. Important Features of Cell Principle 06-09-2024 Cell Biology : Basics 21  1.3. Important Features of Cell Principle 06-09-2024 Cell Biology : Basics 22  1.3. Cell Theory versus Cell Principle Cell principle is better than cell theory because: 1. It is applicable to all the cells present in living things like plants, animals and micro-organisms 2. Cell principle incorporates all the modern findings related to a cell 06-09-2024 Cell Biology : Basics 23  1.4. Diversity in Cell Size and Shape  Diversity exists in the cells as far as size, shape and number is concerned.  Most cells are microscopic, visible only under the high power of microscope.  Micrometre is usually used to measure the cell-size. Majority of cells are 5-15 micrometer in size [RBCs are 5-8 micrometer].  Nerve cells are the longest (1-2 m in humans).  Egg of Ostrich is about 175 mm x 135 mm.  In human body, cell size ranges between 3-4 micron (Leukocytes) to over 90-100 cm (nerve cells).  The nucleo-cytoplasmic ratio and surface area are the two important factors that restrict the cell size. 06-09-2024 Cell Biology : Basics 24  1.4. Diversity in Cell Size and Shape  The shape of the cells is related to their functions.  Some blood cells and Amoeba change their shape whereas others have constant shape.  The cells may be spherical, oval, rounded or elongated, cuboidal, cylindrical, tubular, polygonal, plate-like, discoidal or irregular. The cell size and shape is influenced by : 1. Surface area to volume ratio 2. Nucleocytoplasmic ratio 3. Rate of cellular activity 4. Cell association 06-09-2024 Cell Biology : Basics 25  1.4. Diversity in Cell Size and Shape 06-09-2024 Cell Biology : Basics 26  1.4. Diversity in Cell Size and Shape: Influencing Factors 1. Surface Area to Volume Ratio  The interior content of the cell is separated from the external environment by the cell membrane. This distinction, however, does not imply isolation.  The membrane allows a variety of chemicals (nutrients) to flow through it. These nutrients are required for the cell’s functions to perform.  A cell’s entire surface area is just enough to hold its internal contents. Any increase in surface area might cause a massive rise in cell volume, which throws the balance off.  As a result, the ratio of surface area to interior volume determines the specific form and size of a cell. 06-09-2024 Cell Biology : Basics 27  1.4. Diversity in Cell Size and Shape: Influencing Factors 2. Nucleo-Cytoplasmic Ratio  The coordinated activity of the cell’s many components allows it to operate. The coordination between the nucleus and the cytoplasm is the most crucial.  The nucleus generates chemicals that enter the cytoplasm and regulate its activities. A cell’s cytoplasmic region is just large enough for a nucleus to regulate.  The nucleus will be unable to control its activity if the cytoplasmic region becomes too large.  As a result, a cell’s nucleo-cytoplasmic ratio never exceeds one-seventh to one-twelfth of its total size.  In certain circumstances, the presence of many nuclei in a big cell overcomes this issue. 06-09-2024 Cell Biology : Basics 28  1.4. Diversity in Cell Size and Shape: Influencing Factors 3. Rate of Cellular Activity  Despite the fact that all cells in a live organism are vital, some are metabolically more active than others.  The more active cells are generally smaller, with a higher surface-volume ratio and nucleo-cytoplasmic ratio than the bigger cells, allowing them to function more actively. 4. Cell Association  In multicellular forms that exhibit some rigidity, cell-to-cell attachment is critical.  The degree of attachment has a significant impact on the structure of the cell and its functional properties. All four variables are clearly connected, with the surface-volume ratio and nucleo-cytoplasmic ratio playing the most significant roles in determining the cell’s size, shape, and characteristics. 06-09-2024 Cell Biology : Basics 29  1.4. Types of Cells based on Morphology Cells are generally three types: 1. Prokaryotic cells: Relative simple cells with no membrane bound organelles like Golgi complex, Mitochondria, Chloroplast or Lysosomes. The hereditary material is highly coiled circular chromosome lying naked in the cytoplasm. Example: Bacterial cells 2. Eukaryotic cells: These cells contain a true nucleus. Hereditary material that is DNA is associated with basic proteins inside nucleus separated from cytoplasm by nuclear envelop. Membrane bound organelles are present like plant and animal cells. 3. Mesokaryotic cells: In these cells nuclear membrane is present around the nucleus but DNA is not associated with histones. These cells are more advance than prokaryotes and less advance than eukaryotes. Example: Dinoflagellates, Marine algae 06-09-2024 Cell Biology : Basics 30 1.4. Structure of Eukaryote and Prokaryote Cells Dougherty (1957) has divided cells into two types – prokaryotic cells and eukaryotic cells 06-09-2024 Cell Biology : Basics 31  Criteria Prokaryotes Eukaryotes Differences Between Eukaryotic and Type of Cell Always unicellular Unicellular and multicellular Cell Size Ranges in size from 0.1 μm – 5.0 μm in Size ranges from 10 μm – 100 μm in diameter diameter Cell Wall Usually present, chemically complex in nature When present chemically simple Prokaryotic Cells Nucleus Absent, Instead have a nucleoid region in the Present cell Ribosomes Present; Smaller in size and circular in shape Present; Comparatively larger in size and linear in shape DNA arrangement Circular in nature Linear in nature Membrane bound Absent Present organelles Plasmids Present Rarely found in eukaryotes Lysosome Absent Present Cell division/Reproduction Binary fission/Asexual Sexual and asexual Examples Archaea and Bacteria Plant and Animal Cells 06-09-2024 Cell Biology : Basics 32  1.4. Essential Features of Mesokaryotic Cells  The cells are medium-sized and the size of the cell is in between prokaryotic cells and eukaryotic cells.  They do not have a cell wall but instead have a pellicle or theca.  The cell membrane is present.  The nucleus is well-organized and surrounded by the membrane.  Membrane-bound cell organelles (like mitochondria, plastids, endoplasmic reticulum) are present in the cytoplasm.  The ribosomes in the cytoplasm are 80s type.  The histone protein is completely absent in the cell.  The chromosome is present and is formed with the help of acidic proteins.  Cell division occurs through the method of amitosis.  Flagella are present and consist of filaments. Dodge has first used the  The presence of chloroplast in the cell contains photosynthetic pigments. term mesokaryotic in 1966  Respiratory enzymes are present in the cytoplasm or mitochondria. 06-09-2024 Cell Biology : Basics 33 Module I Cell Morphology: Structure and Functions External Cellular Components Morphology and Function of Plant Cell Wall  Plant Cell Wall Structure  Structure of Plasmodesmata  Plant Cell Wall Functions 11-09-2024 Cell Morphology: External Cellular Components 1 The plant cell wall is multi-layered and consists of up to three sections. From the outermost layer of the cell wall, these layers are identified as the 1). Middle Lamella; 2). Primary Cell Wall, and 3). Secondary cell wall. While all plant cells have a middle lamella and primary cell wall, not all have a secondary cell wall. 11-09-2024 Cell Morphology: External Cellular Components 2  Plant Cell Wall Structure  Middle Lamella: This outer cell wall layer contains polysaccharides called pectins (Calcium pectate). Pectins aid in cell adhesion by helping the cell walls of adjacent cells to bind to one another.  Primary Cell Wall: This layer is formed between the middle lamella and plasma membrane in growing plant cells. It is primarily composed of cellulose microfibrils contained within a gel-like matrix of hemicellulose fibers and pectin polysaccharides. The primary cell wall provides the strength and flexibility needed to allow for cell growth. [Composition: 25% cellulose + 25% hemicellulose + 35% pectin + 1-8% proteins approximately]  Secondary Cell Wall: This layer is formed between the primary cell wall and plasma membrane in some plant cells. Once the primary cell wall has stopped dividing and growing, it may thicken to form a secondary cell wall. This rigid layer strengthens and supports the cell. In addition to cellulose and hemicellulose, some secondary cell walls contain lignin. Lignin strengthens the cell wall and aids in water conductivity in plant vascular tissue cells. 11-09-2024 Cell Morphology: External Cellular Components 3   The primary cell and middle lamella never occur in the form of a continuous layer, but many minute apertures through the cells of a tissue maintain cytoplasmic relation with each other. Such cytoplasmic junctions or bridges between the adjacent cells are known as plasmodesmata. Plasmodesmata  Plasmodesmata are mostly found in plant cells. In animal cells, similar structures are presently called gap junctions.  Plasmodesmata permit to pass a molecule directly from one cell to another and are important in cellular communication.  They are essential for plant life because they serve as a channel for conveying water, fluids and transport of metabolites during developmental and defense signaling.  They permit the passage of molecules weighing less than 800 Da.  Transport through the plasmodesmata is also found under complex regulation which may involve Ca2+ and protein phosphorylation.  The plasmodesmata (singular, plasmodesma) were first reported by Strasburger in 1901.  The word plasmodesma is derived from the Latin word ‘plasmo’ meaning fluid and the Greek ‘desma’ meaning bond 11-09-2024 Cell Morphology: External Cellular Components 4  Plasmodesmata  The number of plasmodesmata may vary from one cell to another. For example, the number of plasmodesmata may vary from 1- 15 or greater per square micrometer of the wall surface. A typical plant cell will have plasmodesmata of 1-10 per µm2.  Callose, a plant polysaccharide, appears to serve as structural and functional element of plasmodesmata.  Plasmodesmata looks like H-shaped, and twinned structures.  Usually, young tissue has simple plasmodesmata and complex Types of Plasmodesmata: plasmodesmata developing later, after cell expansion. Primary Plasmodesmta is formed by trapping a portion of the endoplasmic reticulum cross the  A plasmodesma measures about 50 to 60 nm in diameter and it is middle lamella during the formation of the a roughly cylindrical membrane-lined channel. primary cell wall in the newly divided plant cells. Secondary Plasmodesmata: The lining that develops de-novo. Enzyme cytokinin in plants helps in its development. 11-09-2024 Cell Morphology: External Cellular Components 5  They are assembled in three compartments: Plasmodesmata Structur 1. Plasma Membrane Lining  Plasmodesmata have their own plasma membrane lining called e plasmalemma, which is the extension from the membrane of the cell.  Its structure is similar to having the phospholipid bilayer. 2. The Cytoplasmic Sleeve  A fluid-filled space surrounded by the plasmalemma is called a cytoplasmic sleeve and is a continuous extension of the cytosol.  Proteinaceous spike-like projections that are regularly positioned within the cytoplasmic sleeve are thought to create nanochannels of varying size.  The cytoplasmic sleeve helps to transport the ions and molecules through plasmodesmata via diffusion. 3. Desmotubule  The desmotubule runs from cell to cell through the center of plasmodesmata in most cases.  The desmotubule is a dense rod or narrower cylindrical structure, that is connected to the smooth endoplasmic reticulum of adjacent cells. 11-09-2024 Cell Morphology: External Cellular Components 6  Plasmodesmata Structur e 3. Desmotubule  Desmotubules are derived from the smooth endoplasmic reticulum of the connected cells (appressed ER).  It was initially called axial component.  Diameter of desmotubule is about 15 nm.  The annulus of the cytosol is present between the outside-inside of the desmotubule and cylindrical plasma membrane respectively.  The space between desmotubules and plasma membrane contains 8-10 microchannels.  They are used to transport lipid molecules. 11-09-2024 Cell Morphology: External Cellular Components 7  Plant Cell Wall Functions A major role of the cell wall is to form a framework for the cell. 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: The cell wall 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: The cell wall provides a barrier to protect against plant viruses and other pathogens. It also helps to prevent water loss.  Storage: The cell wall stores carbohydrates for use in plant growth, especially in seeds. 11-09-2024 Cell Morphology: External Cellular Components 8 Cell Morphology: Structure and Functions Morphology and Function of Bacterial Cell Wall  Bacterial Cell Wall Structure  Chemical Composition of Bacterial Cell Wall  Cell Wall Chemical Composition: Gram Positive Bacteria  Cell Wall Chemical Composition: Gram Negative Bacteria  Gram Staining as Identification of Bacterial Cell Wall 11-09-2024 Morphology and Function of Bacterial Cell Wall 1  Bacterial Cell Wall Structure  Unlike in plant cells, the cell wall in prokaryotic bacteria is composed of peptidoglycan. This molecule is unique to bacterial cell wall composition.  Peptidoglycan is a polymer composed of 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 Gram positive bacteria: 20-80 nm thick peptidoglycan layer peptidoglycan layer and acts as an endotoxin (poison) in pathogenic Gram negative bacteria: 2-7 nm thick bacteria (disease causing bacteria). The LPS layer also protects peptidoglycan layer gram-negative bacteria against certain antibiotics, such as penicillin. 11-09-2024 Morphology and Function of Bacterial Cell Wall 2  Chemical Composition of Bacterial Cell Wall  Cell wall is composed of two polymers, one consisting of saccharide subunits and the other consisting of amino acid subunits. Thus a bacterial cell wall is glycopeptide which is also known as peptidoglycan.  The saccharide component of the cell wall has alternating The four amino acids that compose the tetra peptide repeating units of are: L-alanine, D-glutamine, L-lysine and D-alanine in Gram positive bacteria and actinomycetes. i). NAM (N-Acetyl Muramic acid) In Gram negative bacteria and myxobacteria, L-lysine ii). NAG (N-Acetyl Glucosamine) is replaced by diaminopimelic acid (DAP). The inclusion of both L and D amino acids in the structure  Both NAG and NAM form the back bone of the cell wall provides protection from digesting effect of proteases. structure.  The alternating units of NAG and NAM are linked together by a glycosidic bond (β-1, 4 linkages). This linkage gives stability and strength to the cell wall. The NAG and NAM chains are cross- linked to one another by tetra/penta peptides that extend off the NAM unit forming a lattice-like structure. 11-09-2024 Morphology and Function of Bacterial Cell Wall 3  Cell Wall Chemical Composition: Gram Positive Bacteria  Gram-positive cell wall is thick measuring about 20-80 nm and more homogenous compared to gram-negative cell wall.  This cell wall consists of large amount of peptidoglycan arranged in several layers. Peptidoglycan in gram-positive cell wall constitutes about 40-80% of the dry weight.  Gram positive bacterial cell wall consists of teichoic acid, lipoteichoic acid, and teichuronic acid (glycerol or ribitol phosphate and carbohydrates). Play role in adjusting to adverse conditions, pathogenicity, and ion transport, respectively. 11-09-2024 Morphology and Function of Bacterial Cell Wall 4  Cell Wall Chemical Composition: Gram Positive Bacteria Teichoic Acids 1. Discovered by Armstrong and team in 1958. 2. The term teichoic acid encompasses a diverse family of cell surface glycol-polymers containing phosphodiester-linked polyol repeat units. Teichoic acids are made up of fibres of glycerol phosphate (glycerol teichoic acid) or ribitol phosphate (ribitol teichoic acid). Teichoic acids are located in the outer layer of Gram-positive bacteria (such as Staphylococci, Streptococci, Lactobacilli, and Bacillus spp). Types of Teichoic Acids 1. Lipoteichoic acids : Teichoic acids that are covalently linked to the Functions of Teichoic Acids lipid in the cytoplasmic membrane. 1. Wall teichoic acids help in 2. Wall teichoic acids: Teichoic acids that are covalently attached to maintaining shape of cell in bacteria 2. Wall teichoic acid helps in transfer of muramic acid in the wall peptidoglycan. ions across the cell 3. Lipoteichoic acid induces septic shock 11-09-2024 Morphology and Function of Bacterial Cell Wall 5  Cell Wall Chemical Composition: Gram Negative Bacteria  The cell wall of Gram negative bacteria is thin and is composed of peptidoglycan.  The cell envelope has 3 layers including, a unique outer membrane, a thin peptidoglycan layer (2-7 nm), and the cytoplasmic membrane.  The outer membrane (OM) is a characteristic feature of Gram-negative bacteria, and in fact Gram-positive bacteria lack this structure. The OM is a lipid bilayer intercalated with proteins, superficially resembling the plasma membrane; it contains phospholipids confined to its inner leaflet, while the outer leaflet contains glycolipids, mainly lipopolysaccharide (LPS).  LPS acts as endotoxin (toxins released by the cell during infections) and function as receptors and blocks immune response.  The porin proteins (β barrel structures; oval shape laterally ∼30–35Å and ∼50 Å in height) are present in the upper layer of a cell wall, functions by regulating the entry and exit of the molecules within the cell. 11-09-2024 Morphology and Function of Bacterial Cell Wall 6  Cell Wall Component of Gram Negative Bacteria : Lipopolysaccharide Structure of LPS LPS is the most abundant antigen on the cell surface of most Gram-negative bacteria, contributing up to 80% of the outer membrane of E. coli and Salmonella. LPS increases the negative charge of the cell membrane and helps stabilize the overall membrane structure. It is of crucial importance to many Gram-negative bacteria, which die if the genes coding for it are mutated or removed. 11-09-2024 Morphology and Function of Bacterial Cell Wall 7 O-antigen Repetitive glycan polymer attached to the core oligosaccharide comprises the outermost domain of the LPS molecule. The composition varies from strain to strain; over 160 different O antigen structures produced by different E. coli strains. The presence or absence of O chains determines whether the LPS is considered "rough" or "smooth". O antigen is exposed on the very outer surface of the bacterial cell, and is a target for recognition by host antibodies. Core Contains an oligosaccharide component that attaches directly to lipid A and commonly contains sugars such as heptose and 3-Deoxy-D-manno-oct-2-ulosonic acid (also known as KDO, keto-deoxyoctulosonate). The LPS cores of many bacteria also contain non-carbohydrate components, such as phosphate, amino acids, and ethanolamine substituents. Lipid A A phosphorylated glucosamine disaccharide decorated with multiple fatty acids. Responsible for much of the toxicity of Gram-negative bacteria. When bacterial cells are lysed by the immune system, fragments of membrane containing lipid A are released into the circulation, causing fever, diarrhea, and possible fatal endotoxic shock (also called septic shock). The Lipid A moiety is a very conserved component of the LPS. 11-09-2024 Morphology and Function of Bacterial Cell Wall 8  Specific Functions of Gram Negative Bacterial Cell Wall  Porins form passive pores that do not bind their substrates; they generally form water-filled pores, through which relatively small solutes diffuse, driven by their concentration gradient. For nutrients that are present at low concentrations in the extracellular environment, passive diffusion is no longer efficient and transport occurs via substrate-specific and active transporters. For example: Outer membrane proteins (OMP) C and A porins in E. coli exclude the movement of hydrophobic compounds and does not allow transport of hydrophilic molecules more than 700 kDa.  Outer membrane (OM) of gram-negative bacteria forms barrier to lysozyme and other toxic microbial agents.  Inside animal host cells, OM may impede colonization of gram-negative bacteria by serum components and phagocytic cells.  OM forms different antigenic strains of bacterial pathogens.  OM is indicative of pathogenesis of bacteria and responsible for endotoxicity 11-09-2024 Morphology and Function of Bacterial Cell Wall 9  Bacterial Gram Staining Protocol as Identification of Bacterial Cell Wall  The Gram stain is fundamental to the phenotypic characterization of bacteria. The staining procedure differentiates organisms of the domain Bacteria according to cell wall structure. Gram-positive cells have a thick peptidoglycan layer and stain blue to purple. Gram-negative cells have a thin peptidoglycan layer and stain red to pink.  The Gram stain was first used in 1884 by Hans Christian Gram. Gram devised his method that used Crystal Violet (Gentian Violet) as the primary stain, an iodine solution as a mordant followed by treatment with ethanol as a decolorizer.  Example: Organisms that stained blue/violet with Gram’s stain are gram positive bacteria and include Streptococcus pneumoniae (found in the lungs of those with pneumonia) and Streptococcus pyogenes (from patients with Scarlet fever) while those that decolorize are gram negative bacteria such as the Salmonella typhi that is associated with Typhoid fever. 11-09-2024 Morphology and Function of Bacterial Cell Wall 10  Bacterial Gram Staining Protocol as Identification of Bacterial Cell Wall  The Gram stain, the most widely used staining procedure in bacteriology, is a complex and differential staining procedure. Through a series of staining and decolorization steps, organisms in the Domain Bacteria are differentiated according to cell wall composition.  Gram-positive bacteria have cell walls that contain thick layers of peptidoglycan (80% of cell wall). These stain purple. Gram-negative bacteria have walls with thin layers of peptidoglycan (10% of wall), and high lipid content. These stain pink. This staining procedure is not used for Archeae or Eukaryotes as both lack peptidoglycan. The performance of the Gram Stain on any sample requires four basic steps that include applying a i). primary stain (crystal violet) to a heat-fixed smear, followed by ii). the addition of a mordant (Gram’s Iodine), iii). rapid decolorization with alcohol, acetone, or a mixture of alcohol and acetone and lastly, iv). counterstaining with safranin. Morphology and Function of Bacterial Cell Wall 11 11-09-2024  Structure and Function of Plasma Membrane Content 1. Plasma Membrane Basics 2. Timeline of Models to Study the Structure and Function of Plasma Membrane 3. Structure of Plasma Membrane 4. Lipids Present in Plasma Membrane 5. Function of Lipids in Plasma Membrane 6. Plasma Membrane Proteins 7. Biological Importance of Plasma Membrane Proteins 8. Fluid Mosaic Model describing Structural Aspects of Plasma Membrane 9. Plasma Membrane Fluidity 10. Biotechniques used to Study the Structural Features of Plasma Memebrane  The plasma membrane is also termed as a Cell Membrane Plasma Membrane Basics or Cytoplasmic Membrane.  The plasma membrane is present both in plant and animal cells (dynamic fluid structure that forms external boundary of cells)  It functions as the selectively permeable membrane, by permitting the entry of selective materials in and out of the cell according to the requirement.  In an animal cell, the cell membrane functions by providing shape and protects the inner contents of the cell.  Based on the structure of the plasma membrane, it is regarded as the fluid mosaic model. According to the fluid mosaic model, the plasma membranes are subcellular structures (quasi-fluid), made of a lipid bilayer in which the protein molecules are embedded. 14-09-2024 Cell Morphology: External Cellular Components 2 Timeline of Models to Study the Structure and Function of Plasma Membrane Name of Model Model Diagram Scientist Year Lipid Nature Thin layer of Lipids Overton 1890 Lipid Monolayer Langmuir 1905 Lipid layer with Non polar polar and non polar ends Polar Lipid Bilayer E. Gorter & F. Grendel 1926 Timeline of Models to Study the Structure and Function Name of the Model Model Diagram Scientist Year Lipid Bilayer plus Protein Sheets Davson and Danielli Lipid 1935 bilayer is coated with thin sheets of proteins Unit Membrane Robertson 1960 All cellular membrane share a of Plasma Membrane common underlying structure - Unit Timeline of Models to Study the Structure and Function of Plasma Membrane Name of Model Model Diagram Scientist Year Fluid Mosaic Model Singer and 1972 Nicholson  Structure of Cell Membrane  The fluid mosaic model was proposed by S.J. Components Location Singer and Garth L. Nicolson. This model explains the structure of the plasma membrane Phospholipid The main fabric of plasma membrane of animal cells as a mosaic of components such Cholesterol Between phospholipids and phospholipid as phospholipids, proteins, cholesterol, and bilayers carbohydrates. Integral proteins Embedded within phospholipid layers  These components give a fluid character to the Peripheral proteins Inner or outer surface of the phospholipid membranes. bilayer Carbohydrates Attached to proteins/lipids on outside  Each phospholipid has a hydrophilic head [5-10%] membrane layers (Forms cell pointing outside and a hydrophobic tail coat/glycocalyx) [Protects cell from [Amphipathic molecule] forming the inside of mechanical/chemical damage, specific the bilayer. transient, cell-cell adhesion]  Cholesterol and proteins are embedded in the Irrespective of the cell type, all Plasma membrane have similar components but their composition might vary. bilayer that gives the membrane a mosaic look.  Human RBCs contain 43% lipid and 49% proteins. Each component has a specific function to  Mouse liver cells contains 54% lipids and 46% proteins. perform. 14-09-2024 Cell Morphology: External Cellular Components 6 The cell membrane is primarily made up of : 1. Phospholipids 2. Proteins [Fatty acid tails]  Polar head group are in contact with intra/extra cellular aqueous phase  Non polar tails face each other (hydrophobic interior of membrane) 14-09-2024 Cell Morphology: External Cellular Components 7  Lipids of Plasma Membrane  Plasma membrane contains 3 classes of lipids 1. Phospholipids (Examples: Phosphophatidyl choline; Phosphophatidyl serine; Phosphophatidyl ethanolamine; Sphingomyelin)- Cell growth and division 2. Glycolipids (Cerebroside; Gangliosides)- Cell recognition and adhesion 3. Sterols (Cholesterol; Stigmasterol)- Cell growth and viability, and regulate the fluidity, permeability  Certain lipids (Cholesterol and Sphingo-phospholipids) are organized as aggregates in plasma membrane in the form of lipid rafts. These lipid rafts are membrane microdomains enriched with cholesterol and glycosphingolipids along with proteins.  Function of Lipid Rafts : a. Signal transduction; b. Cholesterol trafficking; c. Endocytosis  Types of Lipid Rafts: Caveolar [Flask shaped invagination having caveolin protein found in brain, micro-vessels of the nervous system, endothelial cells, astrocytes, oligodendrocytes, Schwann cells, dorsal root ganglia and hippocampal neurons] and Non-caveolar [Planar lipid rafts found in continuation with plasma membrane and contains flotillin proteins and are found in neurons]  Lipid Aggregates to interact with water occur in three forms a). Fatty acid side chain forms small spherical micellar structure (< 20 nm) b). Two lipid monolayer forms sheets c). Liposome: Closed (sealing solvent) filled vesicle 14-09-2024 Cell Morphology: External Cellular Components 8  Figure: Non-caveolar and caveolar lipid rafts (Martinez-Outschoorn et al., 2015) Figure: Lipid Aggregates  Function of Lipids in Plasma Membrane  Lipids are used for energy storage. These function primarily as anhydrous reservoirs for the efficient storage of caloric reserves.  The matrix of cellular membranes is formed by polar lipids, which consist of a hydrophobic and a hydrophilic portion. The inclination of the hydrophobic moieties to self-associate , and the tendency of the hydrophilic moieties to interact with aqueous environments and with each other, is the physical basis of the formation and stability of membranes.  This fundamental principle of amphipathic lipids is a chemical property that enabled the first cells to segregate their internal constituents from the external environment. This same principle is recapitulated within the cell to produce discrete organelles. This compartmentalization enables segregation of specific chemical reactions for the purposes of increased biochemical efficiency and restricted dissemination of reaction products.  In addition to the barrier function, lipids provide membranes with the potential for budding, fission and fusion, characteristics that are essential for cell division, biological reproduction and intracellular membrane trafficking.  Lipids also allow particular proteins in membranes to aggregate, and others to disperse. 14-09-2024 Cell Morphology: External Cellular Components 10  Plasma Membrane Proteins  Membrane Proteins are responsible for most of the dynamic processes in Plasma Membrane.  Most membrane proteins are classified as a). Peripheral [Extrinsic] or b). Integral [Intrinsic] Membrane proteins are among the most important proteins biologically because they allow the cells to communicate with their environments, they determine whether the immune system recognizes the cell as foreign or not, they are the targets of most, and perhaps all, pharmaceuticals, they control cell adhesion to form tissues, and they control important metabolic processes, including salt balance, energy production and transmission, and photosynthesis. In short, they are important in across medicine and agriculture. 14-09-2024 Cell Morphology: External Cellular Components 11  Plasma Membrane Proteins 1. Peripheral Proteins  These proteins are bound by electrostatic or hydrogen bonds to outer layer of membrane with no interaction with the hydrophobic core of lipid bilayer.  They are indirectly bound to the integral membrane protein (protein-protein interaction) or directly interact with the polar lipid head groups (protein-lipid interaction).  Most polar proteins are soluble in aqueous solution.  These proteins on plasma membrane are released from the membrane by mild extraction procedure like exposure to high ionic strength solution or extremely high/low pH (without disrupting the lipid bilayer)  Examples: Spectrin and ankyrin on RBCs membrane. Spectrin is a flexible rod-like protein that helps maintain the structure of the cell membrane and cell shape. It also helps with cell adhesion, cell spreading, and the cell cycle. Ankyrin is and adaptor protein that links membrane proteins to the cytoskeleton. It also helps resist shear stress and provides anchoring systems. 14-09-2024 Cell Morphology: External Cellular Components 12  Plasma Membrane Proteins 2. Transmembrane Proteins  Proteins held in lipid bilayer very tightly, which cannot be released via mild extraction process are called integral proteins or transmembrane proteins (one or more segments are embedded in phospholipid bilayer).  Transmembrane proteins are non-polar amino-acid residues with hydrophobic side chains which interacts with the fatty acyl groups of membrane phospholipids, thus anchoring the proteins to the membrane.  They are characterized to contain 21-26 hydrophobic residues coiled into an α-helical structure spanning the lipid bilayer.  They may be single pass (monotopic) or multi-pass (polytopic).  Examples:  Glycophorin is a major single pass transmembrane protein with 131 amino acid residues in the RBCs plasma membrane, carry sugar molecules and help determine blood groups.  Band 3 protein/chloride-bicarbonate exchanger, 95 kDa multi-pass membrane protein for the exchange of chloride and bicarbonate. 14-09-2024 Cell Morphology: External Cellular Components 13  Characteristic Features of Plasma Membrane Proteins Property Peripheral Proteins Integral Proteins Treatment Mild Treatment: extreme pH change, Hydrophobic bond breaking agents exposure to ionic solvents – Detergents, organic solvents Association with lipids Usually soluble, free of lipids Usually associates with lipids when when solubilized solubilized (lipid bilayer also gets disrupted) Solubility after Soluble and molecularly disperses in Usually insoluble or aggregated in dissociation from neutral aqueous buffer neutral aqueous buffer. Membrane Examples Spectrin, Ankyrin – RBC Membrane Glycophorin, Band 3 protein 14-09-2024 Cell Morphology: External Cellular Components 14  Classification of Membrane Proteins According to Function Membrane proteins are classified as 1. Transport Proteins i.e. Carrier or Channel Proteins 2. Catalytic Proteins i.e. F0–F1 ATP synthase in IM of mitochondria 3. Structural Proteins  Carrier Proteins are classified as i). uniporters or ii). Co-transporters (anti-porters/symporters). The transport can be either active/passive. Example: Glucose transporter/ Chloride-bicarbonate exchange protein  Channel Proteins transport solutes down the concentration gradient.  Channel Proteins work based on specific signals. The major signals which cause ion channels to open are 1. Voltage gated channels 2. Ligand gated channels Example: Aquaporins and Ionophores Channels – Form open pores through the membrane allowing the free passage of any molecule of the appropriate size. Carriers – Selectively binds and transports specific molecules such as glucose rather then forming open channels, carriers protein acts like enzymes so as to facilitates the passage of specific molecules across the membrane. 14-09-2024 Cell Morphology: External Cellular Components 15  Biological Importance of Membrane Proteins  Membrane proteins are interesting scientifically because of their key roles in controlling the processes of life.  Receptors are membrane proteins that bind to chemicals (e.g., drugs) outside of the cell, and this binding process causes a chemical response on the inside of cells. For example, morphine binds to the opioid receptor in the membranes of brain cells, and the interiors of the cells respond by reducing nerve transmission.  Ion-channels are membrane proteins that allow transport of chemical species into and out of cells. Nerve transmission, for example, is caused by a difference in ionic strengths across the membrane and this is mediated by ion channels. Ion-channels are also targets for drug discovery, and new analytical technology developed for research on receptors will benefit research on ion-channels.  Drug companies want to identify receptors (plasma membrane proteins), study what binds to them (new drug candidates), and understand what the cell and organism do in response to the binding. 14-09-2024 Cell Morphology: External Cellular Components 16  Plasma Membrane Structure 14-09-2024 Cell Morphology: External Cellular Components 17  Fluid Mosaic Model describing Structural Aspects of Plasma Membrane  In 1972, Jonathan Singer and Garth Nicolson proposed the fluid mosaic model of membrane structure, which is now generally accepted as the basic paradigm for the organization of all biological membranes.  In this model, membranes are viewed as two-dimensional fluids in which proteins are inserted into lipid bilayers. Fluid Mosaic Model Postulates that 1. Lipids and integral protein are disposed in a kind of mosaic arrangement. 2. The biological membrane are Quasi-fluid structures in which both lipids and integral proteins are able to perform translational movements within the bilayer.  The concept of fluidity implies that the main components of the membrane are held by means of non – covalent interaction and the cohesive forces between lipids and proteins are hydrophobic in character.  In fluid mosaic model the integral proteins of the membrane are intercalated into a continuous lipid layer. Integral proteins are amphipathic, with polar regions protruding from the surface and non-polar regions embedded in the hydrophobic interior of the membrane 14-09-2024 Cell Morphology: External Cellular Components 18  Plasma Membrane Fluidity  Membrane fluidity refers to the fact that lipids have considerable freedom of lateral or transverse movements. Phospholipids molecules are capable of 3 kinds of movements: 1. Rotation along its long axis; 2. Lateral diffusion by exchanging places with neighboring molecules in the same monolayer; & 3. Transverse diffusion or “flip – flop” from one monolayer to another. For transverse diffusion to occur lipid head group which is polar must be charged and must move into hydrophobic interior of the bilayer. This movement requires energy in the form of ATP.  Rapid movements in the membranes is due to the presence of enzymes called phopsholipid translocators namely Flippases and Floppases, that catalyze the transverse diffusion of phospholipid molecules from one monolayer to another.  Flippases move the phospholipids from the outer monolayer to the cytoplasmic surface of plasma membrane.  Floppases move the phospholipids from the cytoplasmic surface of plasma membrane to the outer monolayer. 14-09-2024 Cell Morphology: External Cellular Components 19  Plasma Membrane Fluidity/Motion of Lipids  Lipids are not rigid/static structure.  In lipid bilayer, lipid molecules rotate freely around the long axis (rotational motion).  Lipids can also diffuse laterally around each leaflet.  The ability of lipids to traverse laterally and along the axis in lipid bilayer indicates that it can act as fluid.  The flip-flop of lipids might happen in several hours to days, and depends on the length of lipid molecule as well as its degree of unsaturation.  Membrane fluidity depends on two factors: 1. Temperature 2. Lipid Composition 14-09-2024 Cell Morphology: External Cellular Components 20  Plasma Membrane Fluidity/Motion of Lipids Effect of temperature on membrane fluidity  Increase in temperature increases the membrane fluidity (free-flowing, less viscous).  Decrease in temperature results in the membrane forming gel like structure The process of change in temperature and fluidity of plasma membrane is called Phase Transition. Temperature at which the transition occurs is called Transition Temperature. Effect of nature of fatty acids on membrane fluidity  Lipids with short/unsaturated fatty acyl chains undergo phase transition at lower temperature than lipids with long or saturated fatty acids. Short chains have less surface area, so the tendency of van der wall interaction is less, therefore can assume fluidic nature much easily.  Unsaturated fatty acids have kinks, thus tends to adopt a more random fluid state and form less Van der Wall interaction with other lipids. Thus, increased proportion of unsaturated to saturated fatty acids in the membrane increases fluidity of bilayer with reduced temperature. 14-09-2024 Cell Morphology: External Cellular Components 21  Plasma Membrane Fluidity/Motion of Lipids Effect of nature of fatty acids on membrane fluidity  Cholesterol is a major determinant of membrane fluidity.  At high temperature, cholesterol interferes with the movement of phospholipid fatty acid chains, making the membrane less fluid.  At low temperature, cholesterol interferes with the hydrophobic fatty acid chains and prevents the membrane from freezing. 14-09-2024 Cell Morphology: External Cellular Components 22  Bio-techniques used to Visualize/study Plasma Membrane 1. Hydropathy Plots: [Identify the number of alpha-helical protein residues in integral proteins] a) These plots is a means of representing hydrophobic regions and hydrophilic regions along the length of a transmembrane proteins (amino-acid sequence of protein). b) The plot has the amino-acid residues on the X-axis and degree of hydrophobicity and hydrophilicity on Y-axis. 2. Fluorescence Recovery after Photobleaching [FRAP]: The rapid lateral movement of membrane lipids can be visualized by the means of Fluorescence Microscopy through the use of Fluorescence Recovery after Photobleaching [FRAP]. 3. Freeze-fracture Technique: Freezing, fracturing and then subjected to vacuum (direct passage of ice to vapour) gives 3 dimensional view of the cell membrane with transmission electron microscope. 14-09-2024 Cell Morphology: External Cellular Components 23  Cell Organelles: Part I Cytoplasm and Cell Nucleus Contents 1. Cytoplasm Basics 2. Theories on Physical Nature of Cytoplasm 3. Structural Features and Composition of Cytoplasm 4. Parts and Function of Cytoplasm: Cytosol 5. Parts and Function of Cytoplasm: Cell Organelles 6. Parts and Function of Cytoplasm: Cytoplasmic Inclusions 7. Overall Functions of Cytoplasm 8. Cytoplasmic Inclusions 9. Cell Organelles: Definition and Function 10. Nucleus 11. Parts and Function of Nucleus 11.1. Nuclear Envelope and Nuclear Pores 11.2. Nuclear Lamina and Nucleoplasm 11.3. Nucleolus 11.4. Chromatin and Chromosome  Cytoplasm Basics  The cytoplasm is a rich, semifluid material present in cells of organisms that are closed off by the cell membrane. It contains various cytoplasmic components, such as the 1). cytosol, 2). cytoplasmic organelles and 3). Inclusion bodies.  The cytoplasm is found within the cell. In an eukaryotic cell — such as an animal cell and a plant cell, the cytoplasm is between the cell membrane and the nuclear envelope. As for a prokaryotic cell such as a bacterial cell, lacking a well-defined nucleus, the cytoplasm contains everything that is inside the cell, surrounded by a cell membrane.  The cytoplasm is defined as the cellular component inside the cell between the cell membrane and the nuclear envelope of the nucleus.  All cells have cytoplasm. However, the size of the cytoplasm may vary from one cell to another.  The watery component (fluid segment) of the cytoplasm is called cytosol. The cytosol is made up of mostly water with a few other dissolved salts and ions. 14-09-2024 Cell Morphology 2  Theories on Physical Nature of Cytoplasm  Sol-Gel Form: Cytoplasm is known to behave similarly to that of a sol-gel. A sol-gel is a mixture of molecules that sometimes act like a liquid (sol) and other times acts like a solid (gel) integrated network.  As a Glass: Cytoplasm sometimes have glass-like behavior as well. This is when the cytoplasm acts as though it is approaching the glass transition as a glass-forming liquid. This comes off the theory that sometimes that cytoplasm may contain many solid components and hence the cytosol needs to act as glass and hold the solid components together so that they do not move excessively. This behavior still allows however for the movement of organelles and other inclusions across the cytoplasm and membrane if needed. This ability of the cytoplasm to somewhat “freeze” everything in place actually becomes very handy as a self-defense mechanism. This frozen stature would prevent harmful physical effects to the cell while still allowing cellular activities to take place whenever the goes back to a more fluid state.  Non-Brownian Motion of Cytoplasmic Components: Constituents of cytoplasm move as a separate entity. These are theorized to be channeled by motor proteins which assist with this non-Brownian motion within the cells versus actually having random forces causing the movements. 14-09-2024 Cell Morphology 3  Structural Features and Composition of Cytoplasm 14-09-2024 Cell Morphology 4  Parts and Function of Cell Cytoplasm: Cytosol  The cytosol is the part of the cytoplasm that is the liquid-like portion. It is mostly made up of water, dissolved minerals, and cytoskeleton filaments.  It, however, does not contain any organelles but holds them within the cell as part of the entire cytoplasm.  It consists of water, organic molecules, and dissolved ions. The highest percentage of cytosol component is water, i.e. about 70%.  The typical ions in the mammalian cytosol are potassium, magnesium, calcium, sodium, chloride, bicarbonate, amino acids in proteins.  The cytosol serves as the site where many chemical reactions take place. In prokaryotes, it is where most metabolic reactions take place (others occur in the cell membrane). In eukaryotes, it is where the organelles and other cytoplasmic structures are suspended. Since the cytosol contains dissolved ions, it plays a role in osmoregulation and cell signaling. It is also involved in generating action potentials in cells, such as nerve, and muscle cells. 14-09-2024 Cell Morphology 5  Parts and Function of Cell Cytoplasm: Cell Organelles  Organelles are specialized structures within cells that carry out specific tasks for the cell. The term “organelles” is based on the organs, as the organs in animals and humans work similarly in carrying out a specific task for the body.  In eukaryotic cells, the nucleus, for instance, is the organelle that contains the genetic material, and therefore it controls cellular activities such as metabolism, growth, and reproduction by regulating gene expression.  Chloroplasts are plastids containing green pigments essential for photosynthesis.  Mitochondria are the organelles that synthesize energy for multifarious metabolic processes.  The endoplasmic reticulum occurs as an interconnected network of flattened sacs or tubules involved in lipid synthesis, carbohydrate metabolism, drug detoxification, and attachment of receptors on cell membrane proteins. It is also involved in intracellular transport, such as the transport of the products (of the rough endoplasmic reticulum) to other cell parts like the Golgi apparatus.  Golgi apparatus is made up of membrane-bound stacks. It is involved in glycosylation, packaging of molecules for secretion, transporting of lipids within the cell, and giving rise to lysosomes. Other cytoplasmic structures found in the cytoplasm are vacuoles and ribosomes.  Ribosomes, the site of protein synthesis, are comprised of protein and RNA. Some ribosomes are unbound whereas the others are attached to the endoplasmic reticulum. 14-09-2024 Cell Morphology 6  Parts and Function of Cell Cytoplasm: Cytoplasmic Inclusions  Cytoplasmic inclusions are part of the cytosol but are not membrane-bound so they are not considered organelles. Instead, they are suspended in the cytosol as small, insoluble particles.  Cytoplasmic inclusions depend on the type of cell they are in. Cytoplasmic inclusions called lipid droplets are used by both plant and animals cells to store lipids like fatty acids. Lipid droplets are made of both lipids and proteins so that they don’t dissolve into the cytosol. Cytoplasmic inclusions in plant cells stores excess glucose during photosynthesis in plants. 14-09-2024 Cell Morphology 7  Overall Functions of Cytoplasm The cytoplasm (of both eukaryotes and prokaryotes) is where the functions for cell expansion, growth, and metabolism are carried out. Numerous chemical reactions including cellular metabolism take part in the cytoplasm as it acts as a bridge between the cell membrane and most organelles. The cytoplasm also has many other functions including: 1. Support and Structure  To aid with cell structure and turbidity. It helps the cells to maintain their shape which is important for the arrangement of cells.  To keep organelles in place. The cytoplasm keeps the membrane-bound organelles in place within the cell and prevents them from making unnecessary movements.  The cytosol – part of the cytoplasm – fills up the empty spaces in the cell that are not covered by the organelles. 14-09-2024 Cell Morphology 8  Overall Functions of Cytoplasm 2. Protection  To protect the cell and its components from damage. The cytoplasm in helping keep cell shape but also being able to keep organelles in place which act as a major part in the cell’s defense strategies.  Often the cytoplasm acts as a shock-absorber when the cell is attacked and cushions that blow to the cell. 3. Storage  It contains materials such as storage units and enzymes that are essential for many metabolic activities.  In plant cells, they include important storage units that are used to store excess glucose made during photosynthesis. 4. Transport  Through the process of cytoplasmic streaming, the cytoplasm assists with the transport of organelles and cytoplasmic inclusions all over the cell.  The cytoplasm also transports waste material out of the cells. If there was no cytoplasm in the cell, the cell would not be able to function. It would be flat and have no shape. In addition, the organelles would not be able to be suspended in the cell either. 14-09-2024 Cell Morphology 9  Cytoplasmic Streaming Cytoplasmic streaming occurs when the cytoplasm itself moves within the cell membrane in a plant or animal cell. The movement is made mainly as a transportation system in the cell to get the necessary components to the places they need to be. For instance, to move organelles around for metabolic activities or to get proteins and nutrients to the nucleus and other organelles. Cytoplasmic streaming is not completely understood in the way it works. However, it is believed that with a mixture of motor proteins and adenosine triphosphate (ATP) the process occurs and is able to transport molecules all over the cell 14-09-2024 Cell Morphology 10  Cell Organelles: Definition and Functions  The cellular components in a cell are enclosed in structures/compartments called cell organelles.  These cell organelles include both membrane and non-membrane bound organelles, present within the cells and are distinct in their structures and functions.  They coordinate and function efficiently for the normal functioning of the cell. A few of them function by providing shape and support, whereas some are involved in the locomotion and reproduction of a cell.  There are various organelles present within the cell and are classified into three categories based on the presence or absence of membrane.  Organelles without membrane: Ribosomes, and Cytoskeleton are non-membrane-bound cell organelles. They are present both in the prokaryotic cell and the eukaryotic cell.  Single membrane-bound organelles: Vacuole, Lysosome, Golgi Apparatus, Endoplasmic Reticulum are single membrane-bound organelles present only in a eukaryotic cell.  Double membrane-bound organelles: Nucleus, Mitochondria and Chloroplast are double membrane-bound organelles present only in a eukaryotic cell. 14-09-2024 Cell Morphology 11 14-09-2024 Cell Morphology 12 Nucleus  The nucleus is a double membrane-bound organelle located centrally only in a eukaryotic cell, enclosing the DNA, the genetic material. It is the most important and defining feature of all higher organisms, including plant and animal cells, whose main function is to control and coordinate the functioning of the entire cell.  The word ‘nucleus’ (plural: nuclei) is derived from the Latin word ‘nucleus‘, meaning ‘kernel’ or ‘seed’. Structure and Characteristics  The largest and most prominent organelle in the cell, the nucleus, accounts for almost 10% of the volume of the entire cell.  In mammalian cells, the average diameter of the nucleus is approximately 6 µm in size.  Mostly the shape of the nucleus is found to be either spherical or oblong.  Eukaryotes usually contain a single nucleus, however erythrocytes and platelets are without a nucleus and osteoclasts of bones have many of them. 14-09-2024 Cell Morphology 13 Nuclear Envelope and Nuclear Pores Parts and Function of Nucleus  Surrounding the nucleus, the nuclear envelope is made of a phospholipid bilayer, similar to cell membranes, and contains tiny openings called nuclear pores over them.  The two membranes are often referred to as the inner and outer nuclear membranes with a fluid-filled region called perinuclear space in between.  The perinuclear space has a thickness of 20 to 40 nm. The outer membrane is attached to ribosomes and is continuous with the cell’s endoplasmic reticulum, a system that helps to package, transport, and export substances outside the cell.  Nuclear pores enable the selective transport of water soluble molecules through the nuclear membrane are themselves composed of a large number of proteins (several hundred in mammals). Although many larger biomolecules, including proteins and RNA, are unable to passively diffuse through these pores they may be actively transported in conjunction with importin/karyopherin proteins and the RanGTP family of small GTPases.  Functions  Nuclear envelope separates the nuclear content from the cytoplasm an is selectively permeable in nature.  Nuclear pores regulate the flow of molecules into and out of the nucleus. 14-09-2024 Cell Morphology 14  Parts and Function of Nucleus Nuclear Lamina  They are meshwork of protein filaments organized in a net-like fashion that line below the inner nuclear membrane.  The proteins that make up the nuclear lamina are known as lamins, which are intermediate filament proteins.  Functions Nucleoplasm  Also  Supports theas known nuclear envelope,itmaintaining karyoplasm, the overall is found inside shape and the nucleus, structure and of the nucleus. is a gelatinous substance similar to the cytoplasm, being composed mainly of water with dissolved salts, enzymes, and suspended organic molecules.  Functions  Protects the nuclear content by providing a cushion around the nucleolus and the chromosome.  Supports the nucleus to hold its shape.  Provides a medium through which enzymes and fragments of genetic materials (DNA or RNA), can be transported throughout the nucleus. 14-09-2024 Cell Morphology 15  Parts and Function of Nucleus Nucleolus  Nucleolus was first described by F. Fontana is a non-membrane bound dynamic body which disappears in the late prophase and reappears in the telophase stage of cell division.  Each nucleolus is produced by a Nucleolar-Organizing Region (NOR) of a chromosome which is termed as nuclear organizing chromosome.  All the eukaryotic cell contain at least one such chromosome. Its number may be one or more (several hundred per nucleus), but 1 to 4 being the most common.  The number of nucleoli per nucleus also differs.  The yeast cell contains one relatively large nucleolus with respect to its nuclear volume.  At the other extreme , Xenopus oocytes contain over 1000 nucleoli per nucleus.  It is a site of transcription of ribosomal RNA and assembly of ribosome.  It is responsible for synthesizing a large nascent precursor ribosomal RNA (pre-rRNA). The concomitant assembly of these RNAs with incoming ribosomal proteins generates small and large ribosomal subunits that then pass into the cytoplasm. 14-09-2024 Cell Morphology 16  Parts and Function of Nucleus Nucleolus Structure : It consists of three major regions.  Fibrillar centres : Containing rRNA genes in the form of partly condensed chromatin.  Dense Fibrillar component: Surrounds the fibrillar center, which contains RNA molecules in the process of transcription.  Granular regions: Contains mature ribosomal precursor particles (pre-ribosome assembly). Nucleolar-Organizing Regions (NORs)  The nucleolus organizing region or nucleolar organizer is a region of chromosome around which the nucleolus forms.  In the human, the five chromosomes contains the NORs, which can be identified as secondary constriction on the metaphase chromosomes.  The area of chromosome distal to a nucleolar organizer is called satellite. A chromosome with a secondary constriction usually associated with a nucleolus is called SAT-chromosome. 14-09-2024 Cell Morphology 17  Parts and Function of Nucleus Chromatin and Chromosome  A chromatin is an organized structure of DNA and protein that is found in the nucleus of eukaryotic cells. It contains a single dsDNA in coiled and condensed form.  Chromatin and chromosomes are basically the same thing. The difference is that chromatin is less condensed and extended DNA while chromosomes are highly condensed and extended DNA.  The word chromosome comes from the Greek word “chroma” which means colour and “soma” which means body due their property of being very strongly stained by particular dyes.  The extent of chromatin condensation varies during the life cycle of the cell.  In non-dividing as well as interphase stages of the cell, most of the chromatin remain relatively decondensed. The light- staining, less condensed portions of the chromatin is termed euchromatin. The region is transcriptionally active and contains most of the transcribing genes.  The darkly stained and highly condensed region of the chromatin is termed as heterochromatin. Because of its condensed state, this heterochromatin is generally believed to be transcriptionally silent. 14-09-2024 Cell Morphology 18 Centromere Parts and Function of Nucleus  The constricted region of linear chromosomes is known as the centromere.  It is usually located exactly in the center of the chromosome and in some cases is located almost at the chromosome's end.  The regions on the either side of the centromere are referred to as the chromosome's arms.  The centromeres serve both as the sites of association of sister chromatids and as the attachment sites for microtubules of the mitotic spindle. Telomere  Telomeres are specialized structures, which cap the ends of eukaryotic chromosomes.  It consists of a long array of short, tandemly repeated sequences.  Sequencing of telomeres from different organisms has shown that most are repetitive sequences with a high G content in the strand with its 3' end. Origin of Replication  The origin of replication (also called the replication origin) is a particular sequence in a chromosome at which replication is initiated. One chromosome contains multiples origin replication. 14-09-2024 Cell Morphology 19 Chromosome Number Parts and Function of Nucleus  All eukaryotic cells have multiple linear chromosomes.  Every cell maintains a characteristic number of chromosomes.  The number of chromosomes in a species has no specific significance nor does it indicate any relationship between two species which may have the same chromosome number.  The majority of eukaryotic cells are diploid that is they contain two copies of each chromosome. Function of Nucleus 1. Contains genetic information 2. Governs genetic expression and other vital functions of cell 3. Facilitates DNA replication for mitosis 4. Produces ribosomes responsible for protein synthesis 14-09-2024 Cell Morphology 20  Cell Organelles: Part II Ribosomes and Endoplasmic Reticulum Contents 1. Ribosomes (Cell Protein Factories) 2. Eukaryotic Ribosomes 3. Biogenesis of Ribosomes 4. Differences between Prokaryotic and Eukaryotic Ribosomes 5. Functions of Ribosomes 6. Ribosomal Protein Targeting and Translocation 7. Endoplasmic Reticulum: Basics 8. Structure of Endoplasmic Reticulum 9. Structural Conformation and Function of Endoplasmic Reticulum 10. Functions of Endoplasmic Reticulum 10.1. Essential Functions of Endoplasmic Reticulum  Ribosomes (Cells Protein Factory)  The ribosomes are large ribonucleoproteins consisting of RNAs and proteins, ubiquitous in all cells, that translate genetic information stored in the messenger RNA into polypeptides.  The ribosome is approximately globular structure, its average diameter ranging from 2.5 nm (Escherichia coli) to 2.8 nm (mammalian cells).  The functional ribosomes consists of two subunits of unequal size , known as the large and small subunits.  Ribosomes consist of rRNA and r-proteins. The r-proteins are termed as L and S depending on whether the protein is from the large or small subunit. 27-08-2023 Cell Morphology 2  Ribosomes (Cells Protein Factory)  In prokaryotes such as Escherichia coli, there are three ribosomal RNAs (16S, 23S and 5S), which are organized as single transcription unit.  In all eukaryotes studied so far, the organizational of the ribosomal RNA genes is recognizably similar to that of prokaryotes, but with major differences; the size of the small subunit RNA has increased from 16S to 18S , and that of the large subunit from 23S and 28S ; a new small 5.8S rRNA has become interspersed between the 18S and the 28S rRNA , and the 5S rRNA has become separated from the other rRNAs in a different transcription unit.  5S rRNA genes are transcribed by a different RNA polymerase from rRNA genes (RNA polymerase III rather than RNA polymerase I).  The human genome contains about 100 copies of rRNA genes per haploid set. Many other species including most plants, have several thousand copies. 27-08-2023 Cell Morphology 3 Eukaryotic Ribosomes:  Most eukaryotes contain two distinct types of ribosomes: 1). Cytosolic and 2). Organellar.  The cytosolic ribosomes of eukaryotic cells are 80S types.  Organellar ribosomes from mitochondria and chloroplast are smaller than cytosolic ribosomes and bear resemblance to the bacterial 70S ribosomes.  There are two spatially separate populations of ribosomes in the cytosol 1). membrane bound ribosomes, attached to the cytosolic side of the ER membrane and 2). free ribosomes which are unattached to any membrane.  Membrane bound and free ribosomes are structurally and functionally identical. They differ only in the proteins they are making at any given time.  In cytosol, a single mRNA usually has a number of ribosomes translocating in 5' to 3' direction, each making a separate but identical polypeptide chain; the entire structure is known as a polyribosome or polysomes.  In the cytosol, proteins are synthesized on membrane-bound as well as membrane free ribosomes.  Proteins that are imported into organelles such as mitochondria, chloroplasts and peroxisome are synthesized on membrane free ribosomes in the cytosol , whereas proteins that are imported into the ER-Golgi system are synthesized on the membrane -bound ribosomes. Integral plasma membrane proteins are also synthesized on membrane-bound ribosomes. 27-08-2023 Cell Morphology 4  Biogenesis of Ribosomes  Ribosomes are not self-replicating particles. The synthesis of various components of ribosomes such as rRNAs and proteins are under genetic control.  In eukaryotes, the biogenesis of ribosomes is the result of the coordinated assembly of several molecular products that converge upon the nucleolus.  The 18S, 5.8S and 28S RNAs are synthesized as part of a much longer precursor molecule in the nucleolus. 5S RNA are and ribosomal proteins are synthesized in the cytoplasm and migrate to the nucleolus, where they are assembled into ribosomal subunits and transported to the cytoplasm. 27-08-2023 Cell Morphology 5  Difference between Prokaryotic and Eukaryotic Ribosome 27-08-2023 Cell Morphology 6  Functions of Ribosomes  Ribosomes take part in protein synthesis. Two or more ribosomes simultaneously engaged in protein synthesis on the same mRNA-strand forming polyribosomes. Interaction of the tRNA-amino acid complex with mRNA brings about translation of the genetic code, coordinated by the ribosomes.  During protein synthesis, ribosomes play a protective function. The mRNA strand which passes between two subunits of the ribosome is protected from the action of nucleases. Similarly the nascent polypeptide chains passing through the tunnel or channel of the larger subunit of ribosome are protected against the action of protein digesting enzymes. 27-08-2023 Cell Morphology 7  Ribosomal Protein Targeting and Translocation  The fate of proteins synthesized by cytosol