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1. 0000000 0000000 THE CELL Life is a whim of several billion cells to be you for a while. Groucho Marx

2. INTRODUCTION TO THE CELL • The cell theory (Matthias Schleiden and Theodor Schwann): the cell is the smallest component of life that can exist independently • The fundamental unit of a living organism is the cell • Cells can grow, reproduce, process information, respond to stimuli and carry out a multitude of chemical reactions • Rudolf Virchow: cells can arise only from pre-existing cells • On the basis of cell structure, living organisms can be divided into two groups, prokaryotes and eukaryotes • Prokaryotes have no distinct compartment to house their DNA; instead, the DNA is found as a highly-folded aggregate known as a nucleoid

3. Prokaryotes often have a tough protective coat, called a cell wall, beneath which a plasma membrane encloses a single cytoplasmic compartment containing DNA, RNA, proteins and the many small molecules needed for life • Most prokaryotic cells are small and simple in outward appearance and they live mostly as independent individuals or in loosely organized communities rather than as multicellular organisms • Bacteria account for an estimated 1–1.5 kg of the average human’s weight • Although bacterial cells do not have membrane-bounded compartments, many proteins are precisely localized in their aqueous interior, or cytosol, indicating the presence of some internal organization

4. In addition to the DNA proper , bacteria often contain smaller DNA molecules known as plasmids • Prokaryotic cells live in an enormous variety of ecological niches, and they are astonishingly varied in their biochemical capabilities. They could be: • Organotrophic – utilize organic molecules as food • Phototrophic – can harness light energy • Lithotrophic – feed on inorganic nutrients • Escherichia coli is an ordinarily harmless inhabitant of the intestinal tract • It is easy to grow in the laboratory and has become the best understood organism at the molecular level

5. E. Coli – “the biochemist’s best friend”

6. Eukaryotic cells, unlike prokaryotic cells, contain a defined membrane-bound nucleus (eu –true, karyon –kernel) and extensive internal membranes that enclose other compartments, the organelles • The cytoplasm comprises the cytosol and the organelles • Eukaryotic cells are generally much larger than bacteria • All cells are thought to have evolved from a common progenitor because the molecules and structures in all cells have so many similarities • Because DNA is subject to random changes (mutations) that accumulate over long periods of time, the number of differences between the DNA sequences of two organisms can be a good indicator of the evolutionary distance between them

7. Detailed analysis of the DNA sequences from a variety of prokaryotic organisms has revealed two distinct types: the so-called “true” bacteria, or eubacteria, and archaea (also called archaebacteria or archaeans) • Many archaeans grow in unusual, often extreme, environments that may resemble ancient conditions when life first appeared on earth –halophiles (“salt loving”); thermoacidophiles (“heat and acid loving”);…

8. CELL STRUCTURE • The plasma membrane • Composed of a lipid bilayer with embedded proteins • It is referred to as a fluid mosaic because the mosaic of proteins and lipid molecules can, for the most part, move laterally in the plane of the membrane • The hydrophobic lipid bilayer selectively restricts the exchange of polar compounds between the external fluid and the intracellular compartment • The proteins are classified as integral proteins, which span the cell membrane, or peripheral proteins, which are attached to the membrane surface through electrostatic bonds to lipids or integral proteins

9. Some of the proteins facilitate the transport of molecules and ions through the membrane, while others are receptors for extracellular molecules which provide information about conditions in adjacent cells, blood and elsewhere in the body • Many of the proteins and lipids on the external leaflet of the bilayer contain covalently bound carbohydrate chains and therefore are glycoproteins and glycolipids. • This layer of carbohydrate on the outer surface of the cell is called the glycocalyx • The glycocalyx protects cells from mechanical and chemical damage and serves as a site for attachment of proteins involved in cell recognition • Each layer of the plasma membrane lipid bilayer is formed primarily by phospholipids, which are arranged with their hydrophilic head groups facing the aqueous medium and their fatty acyl tails forming a hydrophobic membrane core

10. Cholesterol, which is interspersed between the phospholipids, maintains membrane fluidity

11. TRANSPORT OF MOLECULES ACROSS THE PLASMA MEMBRANE • Different mechanisms are used for the transport of substances with different chemical properties • Simple diffusion: gases such as O2 and CO2 and lipid-soluble substances can cross membranes by simple diffusion • Movement of substances is down concentration gradient and does not require energy • Water also enters the cells through simple diffusion • In addition, cells have selective water channels, or aquaporins, embedded in their plasma membrane to allow water to move at a faster rate across this membrane • Facilitative diffusion: the transported molecule binds to a specific carrier or transporter protein in the membrane

13. The transporter then undergoes a conformational change that allows the transported molecule to be released on the other side of the membrane • Although the transported molecules are bound to proteins, the transport process is still classified as diffusion because energy is not required, and the compound equilibrates (achieves a balance of concentration and charge) on both sides of the membrane • Transporter proteins are specific, can be saturated and may be inhibited

14. Gated channels: transmembrane proteins forming a pore for ions that is either opened or closed in response to a stimulus • The channels may be voltage-gated, ligand-gated,… • Even though some types of transporters consume a small amount of ATP, the process is generally taken to be passive • If a transported substance carries a net charge, its movement is influenced by both its concentration gradient and the membrane potential, the electric potential (voltage) across the membrane • The combination of these two forces, called the electrochemical gradient, determines the energetically favorable direction of transport of a charged molecule across a membrane

15. A transporter alternates between two conformations so that the solute-binding site is sequentially accessible on one side of the bilayer and then on the other • In contrast, a channel protein forms a water-filled pore across the bilayer through which specific solutes can diffuse

16. CFTR (cystic fibrosis transmembrane conductance regulator): a ligand-gated channel regulated by phosphorylation

17. Active transport: requires transporters but instead of equilibrating a substance on both sides of the membrane, it concentrates a substance on one side of the membrane using energy • If energy is directly applied to the transporter, the transport is called primary active transport; if energy is used to establish an ion gradient, and the gradient is used to concentrate another compound, the transport is called secondary active transport • Protein-mediated transport systems, whether facilitative or active, are classified as antiports if they specifically exchange compounds of similar charge across a membrane; they are called symports or cotransporters if they simultaneously transport two molecules in the same direction • Some transport systems which simply mediate the transport of a solute across the membrane are called uniports

18. Active transport by Na+, K+ATPase

19. Secondary active transport of glucose by the Na+-glucose cotransporter

20. Vesicular Transport across the Plasma Membrane • Vesicular transport occurs when a membrane completely surrounds a compound, particle or cell and encloses it into a vesicle • When the vesicle fuses with another membrane system, the entrapped compounds are released • Endocytosis refers to vesicular transport into the cell, and exocytosis to transport out of the cell • Endocytosis is further classified as phagocytosis if the vesicle forms around particulate matter (such as whole bacterial cells), and pinocytosisif the vesicle forms around fluid containing dispersed molecules • Receptor-mediated endocytosis is the formation of clathrin-coated vesicles that mediate the internalization of membrane-bound receptors in vesicles coated on the intracellular side with subunits of the protein clathrin

21. The assembly and disassembly of a clathrin coat

22. CELLULAR ORGANELLES • Lysosomes: are the intracellular organelles of digestion enclosed by a single membrane that prevents the release of digestive enzymes into the cytosol • They are central to a wide variety of body functions that involve elimination of unwanted material and recycling their components • This includes the destruction of infectious agents, recovery from injury, tissue remodeling, involution of tissues during development and normal turnover of cells and organelles • The lysosomal digestive enzymes include nucleases, phosphatases, glycosidases, esterases and proteases • These enzymes are all hydrolases, enzymes that cleave bonds through the addition of water • Most of these lysosomalhydrolases have their highest activity near a pH of approximately 5.5 → acid hydrolases

24. Lysosomes are formed from digestive vesicles called endosomes, which are involved in receptor-mediated endocytosis • Many compounds are brought into the cells in endocytotic clathrin-coated vesicles formed from the plasma membrane • These vesicles fuse to form multivesicular bodies called early endosomes • The early endosomes mature into late endosomesas they recycle receptors, clathrin, lipids and other membrane components • The late endosomes fuse with primary lysosomes, vesicles secreted from the Golgi apparatus carryingnewly synthesized acid hydrolases and vesicular proton pumps (for maintaining acidity); mature lysosomes are formed

26. One of the major roles of lysosomes is phagocytosis • Neutrophils and macrophages, the major phagocytic cells, devour pathogenic microorganisms and clean up wound debris and dead cells, thus aiding in repair • As bacteria or other particles are enclosed into pits in the plasma membrane, these vesicles bud off to form intracellular phagosomes • The phagosomes fuse with primary lysosomes, giving phagolysosomes, where the acidity and digestive enzymes destroy the contents • In autophagy (self-eating), intracellular components such as organelles or other particles are surrounded by a membrane derived from endoplasmic reticulum vesicles, forming an autophagosome • The autophagosome fuses with a 10 lysosome, and the contents of the phagolysosome are digested by lysosomal enzymes

27. Any indigestible material within the lysosome is normally expelled through the plasma membrane • But as cells grow older, this process functions less effectively so that cells become loaded with unwanted lipid and protein, which is oxidized to produce a complex known as lipofuscin (an age pigment) • The accumulation of lipofuscin over many years may impair cellular function especially in cells like the neurons • Mitochondria : contain most of the enzymes for the pathways of fuel oxidation and oxidative phosphorylation and thus generate most of the ATP required by mammalian cells • All mitochondria have an outer membrane, which is permeable to small molecules, and an inner membrane, which is much less permeable and extensively folded to form cristae that extend into the mitochondrial matrix

29. Some cells (e.g. hepatocytes) contain several thousand mitochondria, whereas others, such as erythrocytes, lack them entirely • Mitochondria can replicate by division; however, most of their proteins must be imported from the cytosol • Mitochondria contain a small amount of DNA, which encodes for only 13 different subunits of proteins involved in oxidative phosphorylation • Most of the enzymes and proteins in mitochondria are encoded by nuclear DNA and synthesized on cytoplasmic ribosomes • Mitochondria are similar in size to small bacteria, and, like bacteria, they have their own genome in the form of a circular DNA molecule, their own ribosomes that differ from those elsewhere in the eukaryotic cell, and their own tRNAs

30. It is now generally accepted that mitochondria originated from free-living aerobic bacteria that were engulfed by an ancestral anaerobic eukaryotic cell • Escaping digestion, these bacteria evolved in symbiosis with the engulfing cell and its progeny, receiving shelter and nourishment in return for the power generation they performed for their hosts

31. Peroxisomes: are spherical vesicles bounded by a single membrane • They contain enzymes that catalyze oxidations that produce hydrogen peroxide • Catalase uses the H2O2 generated by other enzymes in the organelle to oxidize a variety of other substrates • In addition, when excess H2O2 accumulates in the cell, catalase converts it to H2O and O2 • Reactions taking place in the peroxisomes include oxidation of very-long-chain fatty acids, synthesis of certain membrane lipids and bile acid synthesis

32. Nucleus: isthe largest of the subcellular organelles • All eukaryotic cells begin their existence with a nucleus and its loss or removal normally leads to death of the cell • The exception to this is the reticulocyte which, while within the bone marrow, extrudes its nucleus to form an erythrocyte • The nucleus contains the chromosomes , which are composed of DNA, an equal weight of small, positively charged proteins called histones, and a variable amount of other proteins • The nucleolus is the most obvious structure seen in the nucleus of a eukaryotic cell when viewed in the light microscope • The nucleolus is the site for the processing of ribosomal RNAs and their assembly into ribosome subunits; other types of RNAs may also be processed in the nucleolus

33. Material within the nucleus (nucleoplasm) is separated from the cytoplasm by the nuclear membrane (also known as an envelope), a double membrane that is continuous with the endoplasmic reticulum • Immediately inside this membrane is a network of protein filaments (lamina) that defines the shape of the nucleus • The membrane is punctuated by a large number of nuclear pores, which are composed of proteins that permit diffusion of small molecules and limited diffusion of larger molecules • Messenger RNA and ribosomal subunits can leave, and proteins and enzymes that are involved in DNA replication and in mRNA processing can enter the nucleus through an active and specific process

35. Endoplasmic reticulum (ER): typically constitutes more than half of the total membrane of an average animal cell • The ER is organized into a system of branching tubules and flattened sacs that extends throughout the cytosol • Part of the ER is studded with ribosomes and is called the rough endoplasmic reticulum because of its appearance in the electron microscope • Regions of ER that lack bound ribosomes are called smooth endoplasmic reticulum • Smooth endoplasmic reticulum is abundant in hepatocytes • The ER and nuclear membranes form a continuous sheet enclosing a single internal space, called the ER lumen or the ER cisternalspace, which often occupies more than 10% of the total cell volume • The endoplasmic reticulum has four main functions:

37. Synthesis of those proteins that are destined for incorporation into cellular membranes or for export from the cell • Synthesis of phospholipids and steroids (cholesterol derivatives) • Hydroxylation of compounds that are toxic or waste products, which renders them more water soluble, hence they are more rapidly excreted ; these are known as detoxification/ biotransformation reactions • Storage of Ca2+ ions at a concentration 10 000 times greater than in the cytosol (i.e. similar to the extracellular fluid) • The release of Ca2+ into the cytosol from the ER, and its subsequent reuptake, occurs in many rapid responses to extracellular signals • A Ca2+ pump transports Ca2+ from the cytosol into the ER lumen; a high concentration of Ca2+-binding proteins in the ER facilitates Ca2+ storage

38. Ribosomes: have no membrane but are aggregates of ribonucleic acid (RNA) and protein; they are supramolecular structures • Each ribosome consists of two subunits: large and small • Protein synthesis in a cell takes place on the ribosome • Proteins encoded for by the nucleus and found in the cytosol, peroxisomes or mitochondria are synthesized on free ribosomes in the cytosol and are seldom modified • Proteins synthesized by the ribosomes on the RER, on the other hand, are secreted to the membranes or the exterior of the cell after modification and packaging in the Golgi The 80S ribosome of eukaryotes

39. The Golgi complex/apparatus: comprises a stack of smooth membranes that form flattened sacs • It directs proteins that have been synthesized on the ribosomes and have then entered the endoplasmic reticulum to various parts of the cell • The cis-face of the Golgi complex faces towards the centre of the cell and proteins reach this face inside vesicles that bud off from the endoplasmic reticulum • Within the Golgi these proteins can be modified, e.g. by removal of some amino acids and addition of other compounds (such as sequences of sugars) • Short sequences of amino acids or attached sugar chains (oligosaccharides) serve as addresses for directing them to their correct destination • Vesicles, containing these modified proteins, bud from the trans-face of the Golgi and are then transported to other parts of the cell

40. COP I and II are proteins similar in function with clathrin

41. The cytoskeleton: serves various functions • Pulling chromosomes apart during cell division and splitting the dividing cell into two • It guides the intracellular movement of organelles and materials • It supports the plasma membrane thereby helping maintain the structure of the cell • It helps in the movement of cells (e.g. swimming sperm and crawling white blood cells) • It provides the machinery for muscular contraction and nerve conduction …. • The cytosol of a eukaryotic cell contains three types of filaments each of which is a polymer of proteins:

42. Microtubules composed of tubulin, which move and position vesicles and organelles • Actin filaments or microfilaments , which determine the shape of the cell’s surface and whole-cell locomotion • Intermediate filaments, which provide mechanical strength • There are also several accessory proteins (including motor proteins) that link the filaments with other cell components and each other • Actin and tubulin, which are involved in cell movement, are dynamic structures composed of continuously associating and dissociating globular proteins • Intermediate filaments, which play a structural role, are composed of stable fibrous proteins that turn over more slowly

47. CELL FRACTIONATION • To investigate the individual compartments of the cell, various procedures of to enriching isolating organelles have been developed • These are mainly based on the size, shape and density of the various organelles • The initial step in purifying sub cellular structures is to rupture the plasma membrane and the cell wall, if present, in a suitable buffer; the process is called homogenization: • The cells are first suspended in a solution of appropriate pH and salt content, usually isotonic sucrose (0.25 M) or a combination of salts similar in composition to those in the cell’s interior • Many cells can then be broken by stirring the cell suspension in a high-speed blender or by exposing it to ultrahigh-frequency sound (sonication)

48. Homogenization is followed by filtration through gauze to remove intact cells and connective-tissue fragments • Generally, the cell solution is kept at ooC to best preserve enzymes and other constituents after their release from the stabilizing forces of the cell • The actual fractionation of cellular components is then carried out by centrifugation steps, in which the gravitational force (given as multiples of the earth’s gravity, g) is gradually increased → differential centrifugation • Nuclei sediment at low accelerations; decanting the supernatant and carefully suspending the sediment (or “pellet”) in an isotonic medium yields a fraction that is enriched with nuclei • Particles that are smaller and less dense than the nuclei can be obtained by step-by-step acceleration of the gravity on the supernatant left over from the first centrifugation. This requires very powerful centrifuges

49. The sequence in which the fractions are obtained is: mitochondria, lysosomes and peroxisomes; membrane vesicles and ribosomes • When cells are disrupted by homogenization, the ER breaks into fragments, which reseal to form small closed vesicles called microsomes • Microsomes represent small authentic versions of the ER, still capable of protein translocation, Ca2+ uptake and release and lipid synthesis • Finally, the supernatant from the last centrifugation contains the cytosol with the cell’s soluble components • The relative rate at which each component sediments depends primarily on its size and shape-normally being described in terms of its sedimentation coefficient, or S value • 1 Svedberg = 10-13 sec