03 CELL BIOLOGY_unlocked.pdf

For Your Eyes Only CELL BIOLOGY TYPES of ORGANISMS A. Prokaryotes - lack a nucleus - includes all bacteria - archaebacteria - eubacteria B. Eukaryotes - contain a nucleus - includes...

For Your Eyes Only CELL BIOLOGY TYPES of ORGANISMS A. Prokaryotes - lack a nucleus - includes all bacteria - archaebacteria - eubacteria B. Eukaryotes - contain a nucleus - includes - plants - animals - single cells organisms - amoeba - algae - fungi - molds C. Viruses - obligatory cellular parasites that require host cells to survive 1. Bacteriophages - bacterial viruses COMPARISON of EUKARYOTES and PROKARYOTES A. General Prokaryotic Cell Anatomy 1. Cell Wall - rigid - surrounds the plasma membrane of prokaryotes 2. Periplasmic Space - where proteins secreted by the cell accumulate - between plasma membrane and cell wall a. Plasma Membrane - provides selective molecular barrier to the environment - made of phospholipids and proteins - controls entrance and exit of molecules - contains the enzymes of oxidative phosphorylation and electron transport 3. Nucleoids - contains the genomic DNA a. Prokaryotic DNA - single, circular - not packaged with histones or other chromosomal proteins b. Bacterial Plasmid DNA - small, extrachromosomal, circular - confer antibiotic resistance 4. Ribosomes - site of protein synthesis B. General Eukaryotic Cell Anatomy 1. Extracellular Matrix - made of many different combinations of carbohydrates and proteins 2. Plasma/Cell Membrane - separates the cytoplasm of the cell from the interstitial fluid, which is in equilibrium with the plasma - also known as limiting membrane  For Your Eyes Only Cell Lysis - breaking of the cell membrane and release of cell contents - occurs when the continuity of the cell membrane is disrupted 3. Organelles - many distinct internal membranous structures or compartments 4. Cytoskeleton - provides strength and structure to the cell - control intracellular movement - provides mechanistic components needed for extracellular movement EUKARYOTIC MEMBRANES A. Functions - compartmentalize and segregate intracellular events - separate cells from one another - segregate organ functions - mediate the regulation of cellular functions by acting as selective barriers - localize specific enzyme systems - provides semisolid-phase in an aqueous environment - act as barrier with selective permeabilities as provided by - channels and pumps for ions and substrates - specific receptors for signals - exchange of materials with outside by exo- or endocytosis - gap junctions for exchange of materials from adjacent cells - function as integral element in excitation-response coupling - site of energy transduction (photosynthesis, oxidative phosphorylation) B. Clinical Significance 1. Gross Alterations  disturbance of water balance and ion flux 2. Specific Component Deficiency or Alteration  disease states C. Structure of the Plasma Membrane - continuous and sealed so that the hydrophobic lipid bilayer selectively restricts the exchange of polar compounds between the external fluid and the intracellular compartment - referred to as a fluid mosaic because it consists of a mosaic of proteins and lipid molecules that can, for the most part, move laterally in the plane of the membrane a. Two Main Functional Groups Within the Lipid Bilayer i. Lipid Molecules ii. Membrane Proteins For Your Eyes Only 1. Plasma Membrane Lipids - exist as a sheet - undergo lateral diffusion (side-to-side or translational movement, and flexing) a. Major Lipids i. Phospholipids - most abundant lipid molecules - do not “flip-flop” (pass from one side of the bilayer to the other) - asymmetrically distributed ( alteration  trigger inflammatory reactions in surrounding cells) For Your Eyes Only ia. Glycerol Lipids iai. Phosphatidylcholine - constitute the outer face of the bilayer - major plasma membrane lipid in most cell types One of the bacterial toxins secreted by Clostridium perfringens, the bacteria that cause gas gangrene, is a lipase that hydrolyzes phosphocholine from phosphatidylcholine and from sphingomyelin. The resulting lysis of the cell membrane releases intracellular contents that provide the bacteria with nutrients for rapid growth. These bacteria are strict anaerobes and grow only in the absence of oxygen. As their toxins lyse membranes in the endothelial cells of blood vessels, the capillaries are destroyed, and the bacteria are protected from oxygen transported by the red blood cells. They are also protected from antibiotics and components of the immune system carried in the blood. For Your Eyes Only iaii. Amino Phospholipids - constitute the inner or cytoplasmic face of the bilayer iaiia. Phosphatidylethanolamine iaiib. Phosphatidylserine - contains a net negative charge that contributes to the membrane potential - might be important for binding positively charged molecules within the cell iaiii. Phosphatidylinositol - found only in the inner membrane - transfer of information from hormones and neurotransmitters across the cell membrane into the cell ib. Sphingolipid - most variable - ex: sphingomyelin For Your Eyes Only b. Lipids are Amphipathic Molecules - have both hydrophobic and hydrophilic ends - arranged with their hydrophilic head groups facing the aqueous medium and typically two fatty acyl tails forming a hydrophobic membrane core (buried)] - amphipathic nature  spontaneous formation of a lipid bilayer when phospholipids are placed in an aqueous environment c. Permeability - impermeable to most water-soluble molecules since they would be insoluble in the hydrophobic core i. Lipid-Soluble Compounds ia. Gases - oxygen - CO2 - nitrogen ib. Lipid-Derived Molecules - ex: hormones ic. Organic Non-Electrolyte Molecules d. Detergents - amphipathic molecules - used to solubilise membrane proteins during purification - hydrophobic ends bind to hydrophobic region of the protein displacing bound lipids - polar is free  bringing proteins into solution For Your Eyes Only 2. Plasma Membrane Proteins - plasma membrane proteins consist of - structural proteins - antigens - transport proteins - receptors - enzymes a. Integral Proteins - amphipathic - contain transmembrane (span the lipid bilayer) domains with hydrophobic amino acid side chains that interact with the hydrophobic portions of the lipids to seal the membrane - hydrophilic regions of the proteins protrude into the aqueous medium on both sides of the membrane - many of these proteins function as either channels or transporters for the movement of compounds across the membrane, as receptors for the binding of hormones and neurotransmitters, or as structural proteins - comprise most of membrane proteins - usually globular - asymmetrically distributed i. Membrane Channel Proteins ii. Transporters iii. Receptors - 3 major receptor categories iiia. Receptors that Mediate Endocytosis iiib. Anchorage Receptors - e.g. integrins iiic. Signalling Receptors For Your Eyes Only For Your Eyes Only Variations in the Way in which Proteins are Inserted into Membranes LDL Receptor - crosses the membrane once - amino terminal on the exterior - type I transmembrane protein Asialoglycoprotein Receptor - crosses the membrane once - carboxyl terminal on the exterior - type II transmembrane protein Cytochrome P450 (not shown) - type III transmembrane protein - its disposition is similar to type I proteins - does not contain a cleavable signal sequence Various Transporters - cross the membrane a number of times - type IV transmembrane proteins - also referred to as polytopic membrane proteins Two of the prominent integral proteins in the red blood cell membrane are glycophorin, which provides an external negative charge that repels other cells, and band 3, which is a channel for bicarbonate and chloride exchange. The transport of bicarbonate into the red blood cell in exchange for chloride helps to carry the bicarbonate to the lungs, where it is expired as CO2 b. Peripheral Proteins - attached to the membrane surface through hydrogen bonding or electrostatic interactions to lipids or to the exposed surface of integral proteins - hydrophilic only i. Ankyrin - bound to integral protein “band 3” of RBC membranes ii. Spectrin Family of Proteins - bound to the intracellular membrane surface - provide mechanical support for the membrane ia. Spectrin - bound to actin to form the inner membrane skeleton or the cortical skeleton For Your Eyes Only All cells contain an inner membrane skeleton of spectrin- like proteins. Red blood cell spectrin was the first member of the spectrin family described. The protein dystrophin present in skeletal muscle cells is a member of the spectrin family. Genetic defects in the dystrophin gene are responsible for Duchenne’s and Becker’s muscular dystrophies. c. Lipid-Anchored Proteins - bound to the inner or outer surface of the membrane - many integral proteins also contain attached lipid groups to increase their stability in the membrane i. Glycophosphatidylinositolglycan (GPI) Anchor - covalently attached lipid that anchors proteins to the external surface of the membrane For Your Eyes Only The prion protein, present in neuronal membranes, provides an example of a protein attached to the membrane through a GPI anchor. This is the protein that develops an altered pathogenic conformation in both mad cow disease and Creutzfeldt-Jakob disease. ii. Other Anchors - a number of proteins involved in hormonal regulation are anchored to the internal surface of the membrane through iia. Palmityl (C16) or Myristyl (C14) Fatty Acyl Groups iib. Geranylgeranyl (C20) or Farnesyl (C15) Isoprenyl d. Movement of Proteins in Plasma Membranes i. Fluid-Mosaic Model by Singer and Nicholson - membrane proteins can move laterally in the matrix of the lipid bilayer - rate of movement depends on the fluidity of the membrane For Your Eyes Only ia. Membrane Fluidity - depends on the nature of the packing and interaction of the fatty acyl chains in membrane phospholipids - fatty acids are aligned or ordered  stiff structure - long chain saturated fatty acids pack closely and interact strongly  rigid structure ib. Transition Temperature (Tm) - temperature at which the structure undergo transition from ordered to disordered (melting) - increased temperature  hydrophobic side chain undergo transition from ordered to disordered - temperatures above melting point, Tm  change in fluidity - the longer the chain length and more saturated the fatty acids  higher the Tm - unsaturated fatty acids with cis double bonds do not pack closely  more fluid, Tm is lowered - the greater the double bonds  the lower the Tm  the greater the fluidity ii. Cholesterol - interspersed between the phospholipids - in the phosphoacylglycerols, unsaturated fatty acid chains bent into the cis conformation form a pocket for cholesterol, which binds with its hydroxyl group in the external hydrophilic region of the membrane and its hydrophobic steroid nucleus in the hydrophobic membrane core For Your Eyes Only iia. Function - maintains membrane fluidity - cholesterol reduces membrane fluidity by preventing the movement of fatty acyl chains - decreases the ability of phospholipids to translate and flex iib. Tm - at temperatures Tm, cholesterol limits the disorder because it is more rigid than the fatty acid hydrocarbon tail  decreased fluidity - high cholesterol : phospholipid ratio  Tm is abolished iic. Cholesterol and Cis Unsaturated Fatty Acids in the Membrane - prevent the hydrophobic chains from packing too closely together  lipid and protein molecules that are not bound to external or internal structural proteins can rotate and move laterally in the plane of the leaflet  enables the plasma membrane to: iici. Partition - between daughter cells during cell division iicii. Deform - as cells pass through capillaries iiciii. Form and Fuse with Vesicle Membranes For Your Eyes Only iii. Membrane Fluidity vs. Function - increased fluidity - increased water permeability - increased lateral mobility of integral proteins - if the function of the protein resides in the hydrophilic segment, fluidity change will have little effect - if the function of the protein resides in the hydrophobic segment, fluidity change may have significant effect - protein-protein interactions may restrict integral protein mobility within the membrane A patient is suffering from both short-term and long-term effects of ethanol on his central nervous system. Data support the theory that the short-term effects of ethanol on the brain partially arise from an increase in membrane fluidity caused when ethanol intercalates between the membrane lipids. The changes in membrane fluidity may affect proteins that span the membrane (integral proteins), such as ion channels and receptors for neurotransmitters involved in conducting the nerve impulse. e. Devices used by cells to limit protein diffusion in the lipid bilayer - confinement to limited areas - cell junctions - increases in mass by aggregation - cross-links by extrinsic elements - links to cytoplasmic components of the cytoskeleton 3. The Glycocalyx of the Plasma Membrane - short chains of carbohydrates (oligosaccharides) that extend into the aqueous medium contained by some of the proteins and lipids on the external surface of the membrane - hydrophilic carbohydrate layer - protects the cell against digestion - restricts the uptake of hydrophobic compounds - complex oligosaccharides play a role in cell-to-cell interactions - constitute 2-10% of the weight of plasma membranes  For Your Eyes Only a. Membrane Glycoproteins - generally contain branched oligosaccharide chains of approximately 15 sugar residues that are attached through i. N-Glycosidic Bonds - to the amide nitrogen of an asparagine side chain ii. Glycosidic Bond - to the oxygen of serine (O-Glycoproteins) - carbohydrate moiety of glycoproteins are in contact with the environment limiting movement of glycoproteins b. Membrane Glycolipids - usually galactosides or cerebrosides - specific carbohydrate chains on the glycolipids serve as cell recognition molecules The variable carbohydrate components of the glycolipids on the cell surface function as cell recognition markers. For example, the A, B, or O blood groups are determined by the carbohydrate composition of the glycolipids. Cell surface glycolipids may also serve as binding sites for viruses and bacterial toxins before penetrating the cell. For example, the cholera AB toxin binds to GM1-gangliosides on the surface of the intestinal epithelial cells. The toxin is then endocytosed in caveolae (invaginations or “caves” that can form in specific regions of the membrane). D. Membranes as Asymmetric Structure - irregular protein distribution - external location of carbohydrate attached to membrane proteins - specific enzymes are exclusively located either outside or inside - choline-containing phospholipids and sphingomyelin in the outer layer - aminophospholipids (phosphatidylserine and phosphatidylethanolamine) in the inner layer - cholesterol more outside than inside - in some regions of the membrane - gap junctions - tight junctions - synapses E. Transport of Molecules across the Plasma Membrane - membranes form hydrophobic barriers around cells  control the internal environment by restricting the entry and exit of molecules  cells require transport systems to permit entry of small polar compounds that they need (e.g., glucose) to concentrate compounds inside the cell (e.g. K+) and to expel other compounds (e.g. Ca++, Na+) - cells engage in endocytosis (plasma membrane extends or invaginates to surround a particle, a foreign cell, or extracellular fluid, which then closes into a vesicle that is released into the cytoplasm For Your Eyes Only 1. Transport Proteins - transmembrane proteins that allow small polar molecules (that would otherwise be inhibited by the hydrophobic interior of the plasma membrane) to cross the lipid bilayer a. Carrier Proteins (Transporters) - low capacity - work by binding solute on one side of the membrane which induces a conformational change that exposes the solute binding site on the other side of the membrane for release i. Passive Transporters - work without energy source - can only transport downhill to equilibrium ii. Active Transporters - use energy - can work uphill to concentrate a solute iia. Primary Active Transporters - use the energy derived from binding and hydrolysis of ATP to drive the translocation cycle - energy is directly applied to the transporter iiai. ATP Binding Cassette (ABC) Transporters - major class of primary active transporters - malfunction  human diseases (spectrum of liver, eye and skin diseases, bleeding disorders and adrenoleukodystrophy iiaii. Na+-K+ ATPase or Na+-K+ Pump - most important carrier protein - found in the basolateral membrane of nearly all cells - utilizes the energy released from ATP hydrolysis - pump three Na+ ions out of the cell and two K+ ions into the cell with each cycle - K+ binding triggers hydrolysis of the bound phosphate group and a return to the original conformation, accompanied by release of K+ ions inside the cell - transport cycle depends on the phosphorylation and dephosphorylation of the Na+-K+ pump  maintain a much lower intracellular Na+ concentration and much higher intracellular K+ ion concentration than present in the external fluid For Your Eyes Only iiaiia. Na+ Gradient - maintained by primary active transport - used to power the transport of glucose, amino acids, and many other compounds into the cell through secondary active transport iiaiii. Ca++-ATPase Pump - high levels of Ca++ are associated with irreversible progression from cell injury to cell death The Ca++-ATPase, a calcium pump, uses a mechanism similar to that of Na+-K+-ATPase to maintain intracellular Ca++ concentration below 10-7 M in spite of the high extracellular concentration of 10-3 M. This transporter is inhibited by binding of the regulatory protein calmodulin. When the intracellular Ca++ concentration increases, Ca++ binds to calmodulin, which dissociates from the transporter, thereby activating it to pump Ca++ out of the cell. High levels of intracellular Ca++ are associated with irreversible progression from cell injury to cell death. iib. Secondary Active Pumps - driven by ion gradients which are themselves made and maintained by primary active pumps - if energy is used to establish an ion gradient (ex: Na+ gradient), and the gradient is used to concentrate another compound For Your Eyes Only 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 across the membrane in the same direction. Band 3 in the red blood cell membrane, which exchanges chloride ion for bicarbonate, provides an example of an antiport. The dehydration of cholera is often treated with an oral rehydration solution containing Na+ , K+ , and glucose or a diet of rice (which contains glucose and amino acids). Glucose is absorbed from the intestinal lumen via the Na+-dependent glucose cotransporters, which cotransport Na+ into the cells together with glucose. Many amino acids are also absorbed by Na+-dependent cotransport. With the return of Na+ to the cytoplasm, water efflux from the cell into the intestinal lumen decreases. For Your Eyes Only iii. Uniporters - transport a single solute from one side of the membrane to the other iv. Symporters - transport two solutes across the membrane in the same direction For Your Eyes Only v. Antiporters - transport two solutes across the membrane in opposite directions b. Channel Proteins (Ion Channels) - form solute channels through the membrane - can only work downhill and only to equilibrium - form a narrow, small, highly selective hydrophilic pore to allow passage of small inorganic ions (primarily Na+, K+, Ca++, or Cl-) down their electrochemical gradients (combined force of the electric potential and the solute concentration gradient across the membrane) - do not need to undergo a conformational change - bulk flow can be very high - opening and closing of the channel can be regulated - can be selective for specific solutes - faster than transport via carrier proteins - selectively opened and closed in response to different stimuli which determines the specific ion channel type bi. Voltage Stimulus - voltage-gated ion channels bii. Mechanical Stress Stimulus - mechanical-gated ion channels biii. Ligand Binding Stimulus - ligand-gated ion channels - activity of the majority of these channels is also regulated by protein phosphorylation and dephosphorylation i. K+ Leak Channels - most common ion channels - found in the plasma membrane of almost all animal cells - are open even when unstimulated or in a resting state  plasma membrane much more permeable to K+ than to other ions + - K -selective permeability plays a critical role in maintaining the membrane potential in nearly all cells For Your Eyes Only 2. Simple Diffusion (Free Diffusion) a. Mechanism - molecules move by engaging in random collisions with other like molecules - net movement from a region of high concentration to a region of low concentration because molecules keep bumping into each other where their concentration is highest - compounds that are uncharged eventually reach the same concentrations on both sides of the membrane b. Energy Requirement - none c. Freely Diffusible i. Gases - O2 - CO2 ii. Lipid-Soluble Substances - ex: steroid hormones d. Water - considered to diffuse through membranes by unspecific movement through ion channels, pores, or around proteins embedded in the lipids For Your Eyes Only e. Aquaporins - large protein pores in certain cells (e.g. renal tubule cells) - permit a high rate of water flow from a region of a high water concentration (low solute concentration) to one of low water concentration (high solute concentration) A patient has become dehydrated because he has lost so much water through vomiting and diarrhea. Cholera toxin increases the efflux of sodium and chloride ions from his intestinal mucosal cells into the intestinal lumen. The increase of water in his stools results from the passive transfer of water from inside the cell and body fluids, where it is in high concentration (i.e. intracellular Na+ and Cl- concentrations are low), to the intestinal lumen and bowel, where water is in lower concentration (relative to high Na+ and Cl-). The watery diarrhea is also high in K+ ions and bicarbonate. All of the signs and symptoms of cholera generally derive from this fluid loss. 3. Facilitative Diffusion Through Binding to Transporter Proteins a. Mechanism - transported molecule  bind to a specific carrier or transport protein in the membrane  transporter protein then undergoes a conformational change that allows the transported molecule to be released on the other side of the membrane b. Energy Requirement - none c. Result - compound is transported down an electrochemical gradient (the balance of concentration and charge across a membrane), usually from a high concentration to a low concentration, to equilibrate between the two sides of the membrane d. Transporter Proteins - ex: enzymes - exhibit saturation kinetics - when all the binding sites on all of the transporter proteins in the membrane are occupied, the system is saturated and the rate of transport reaches a plateau (the maximum velocity) - relatively specific for the compounds they bind - can be inhibited by compounds that block their binding sites or change their conformation e. Km - concentration of a transported compound required to reach ½ the maximum velocity All of the cells in the body have facilitative glucose transporters that transport glucose across the plasma membrane down an electrochemical (concentration) gradient as it is rapidly metabolized in the cell. In muscle and adipose tissue, insulin increases the content of facilitative glucose transporters in the cell membrane, thus increasing the ability of these tissues to take up glucose. Patients with type 1 diabetes mellitus, who do not produce insulin have a decreased ability to transport glucose into these tissues, thereby contributing to hyperglycemia (high blood glucose). For Your Eyes Only 4. Gated Channels in Plasma Membranes a. Mechanism - transmembrane proteins form a pore for ions that is either opened or closed in response to a stimulus: i. Voltage Changes Across the Membrane (Voltage-Gated Channels) - ex: conduction of a nerve impulse along the axon depends on the passive flux of Na+ through a voltage-gated channel that is opened by depolarization of the membrane ii. Binding of a Compound (Ligand-Gated Channels) iia. CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) - Cl- channel - transport through a ligand-gated channel is considered diffusion, although ATP is involved, because only a few ATP molecules are being used iii. Regulatory Change in the Intracellular Domain (Phosphorylation-Gated and Pressure-Gated Channels) iiia. CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) - regulated through phosphorylation (phosphorylation-gated) - member of the ABC (adenine nucleotide binding cassette, or ATP binding cassette) superfamily of transport proteins - has two transmembrane domains that form a closed channel, each connected to an ATP binding site - regulatory domain that sits in front of the channel - when phosphorylated by a kinase, its conformation changes and it moves away from the ATP binding domains - as ATP binds and is hydrolyzed, the transmembrane domains change conformation and open the channel, and chloride ions diffuse through - as the conformation reverts back to its original form, the channel closes - many gated channels show saturation kinetics at very high concentrations of the compounds being transported  For Your Eyes Only The cystic fibrosis transmembrane conductance regulator (CFTR) was named for its role in cystic fibrosis. A mutation in the gene encoding its transmembrane subunits results in dried mucus accumulation in the airways and pancreatic ducts. The CFTR is also involved in the dehydration experienced by cholera patients. In intestinal mucosal cells, cholera A toxin indirectly activates phosphorylation of the regulatory domain of CFTR by protein kinase A. Thus, the channel stays open and Cl- and H2O flow from the cell into the intestinal lumen, resulting in dehydration. F. G-Protein Coupled Receptors 1. G-Protein Coupled Receptors - the most important class of cell membrane receptors - proteins that traverse the plasma membrane seven times (seven-pass receptors) - coupled to trimeric GTP-binding proteins (G proteins which serve as relay molecules), which are composed of three subunits (, , ) a. Inactive Receptor -  subunit (active subunit) of the G protein is bound to GDP b. Stimulated Receptor - change in conformation   subunit to exchange GDP for GTP thereby releasing itself from the  complex  binds and activates target proteins - the target proteins activated by the  subunit vary, depending on which of the three main types of G protein is involved For Your Eyes Only i. Gs - stimulatory G protein -  cAMP levels ii. Gi - inhibitory G protein -  cAMP levels iii. Gq - activates phospholipase C (PLC)  For Your Eyes Only G. Vesicular Transport Across the Plasma Membrane 1. Mechanism - 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 a. Endocytosis - vesicular transport into the cell i. Phagocytosis - if the vesicle forms around particulate matter (such as whole bacterial cells or metals and dyes from a tattoo) ii. Pinocytosis - if the vesicle forms around fluid containing dispersed molecules iii. Receptor- Mediated Endocytosis - 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 For Your Eyes Only  For Your Eyes Only iv. Potocytosis - endocytosis that occurs via caveolae (small invaginations or “caves”), which are regions of the cell membrane with a unique lipid and protein composition (including the protein caveolin-1) The vitamin folate provides an example of a compound transported into cells by caveolae, which form around the occupied folate receptor. In contrast, endocytosis of many compounds such as membrane hormone receptors occurs through clathrin-coated pits. The receptors are targeted for these pits by adaptor proteins that bind to a specific amino acid sequence in the receptor. b. Exocytosis - transport out of the cell H. Homeostasis 1. Body Water Compartments - water makes up 60% of lean body mass a. ICF - 2/3 of total water - provides environment for the cell to - make, store, utilize energy - self-repair - replicate - perform special functions b. ECF - 1/3 of total water - plasma - interstitial compartment - delivery system - nutrients - oxygen - ions and trace minerals - regulatory molecules - removes - CO2 - waste products - toxic and detoxified materials  For Your Eyes Only 2. Ionic Composition of the Fluid Compartments EUKARYOTIC ORGANELLES A. Nucleus - largest of the subcellular organelles of animal cells is the nucleus - most of the genetic material of the cell is located in the chromosomes of the nucleus 1. Functions - defining feature of eukaryotes - site of storage and replication of DNA - directs the production of RNA 2. Structure a. Nuclear Envelop - dynamic structure that can grow and contract rapidly depending on the relative nuclear activity and the stage of cell division i. 2 Membranes ia. Outer Nuclear Membrane - dotted with ribosomes - continuous with the rough endoplasmic reticulum ib. Inner Nuclear Membrane ii. Perinuclear Space - separate the inner and outer membranes For Your Eyes Only b. Nuclear Pores i. Functions - connect outer and inner nuclear membranes - forms opening between cytoplasm and nucleoplasm - conduct mRNA, ribosomes, and proteins required for replication, transcription, and other processes into the cytoplasm ii. Specificity and Direction of Travel Through the Nuclear Pore (Import vs. Export) - dictated by iia. Binding Proteins iiai. Importins - proteins transported into the nucleus have a nuclear localization signal that causes them to bind to one of the subunits of cytosolic proteins (importins) - other subunit of the importin molecule binds to cytoplasmic filaments attached to the outer ring of the nuclear pore - as the importin-protein complex enters the nucleus, the small GTP-binding protein Ran binds to an importin subunit, causing release of the transported protein into the nucleus - Ran-importin complex is returned to the cytosol, where Ran-GAP (GTPase activating protein) activates hydrolysis of bound GTP to GDP and phosphate - energy released by GTP hydrolysis changes the conformation of Ran and the complex dissociates - free importin can then bind another protein For Your Eyes Only iiaii. Exportins - RNAs are transported from the nucleus to the cytoplasm as ribonucleoproteins, which are targeted for export by a specific amino acid sequence (nuclear export signal) - nucleoprotein forms a complex with additional proteins (exportins) and with Ran - complex is transported through the pore to the cytoplasm, where RanGAP activates hydrolysis of the bound GTP - absence of GTP  complex dissociates with the release of RNA into the cytoplasm, and the exportins and Ran are transported back to the nucleus iib. Ran - small GTP protein iic. Location of RanGAP Only on the Cytoplasmic Side - regulatory protein - GTPase activating protein For Your Eyes Only The Ras family of monomeric G proteins. Ras and Ran belong to a superfamily of proteins called small G proteins (also called small GTP-binding proteins, small GTPases, or monomeric G proteins). These proteins function as timing regulators for a variety of cell functions. They are referred to as “small” because they are composed of a single subunit with a weight of 20 to 40 kDa, and they are called GTPases because they slowly hydrolyze bound GTP. When small G proteins contain bound GTP, they bind to and activate their target proteins. As their bound GTP is hydrolyzed to GDP and Pi, their conformation changes dramatically, and they dissociate from the target protein. They thus serve as “automatic clocks” that shut themselves off. Many of the monomeric GTP binding proteins are regulated by GAPs (GTPase activating proteins), GEFs (GTP exchange proteins which stimulate GDP dissociation and GTP binding), or GDIs (GDP-dissociation inhibitory proteins.  For Your Eyes Only c. Nuclear Lamina - lies on the nucleoplasmic side of the inner nuclear membrane - fibrous network made of 3 major proteins (lamins) d. Mitosis - phosphorylation of lamins  nuclear envelop breakdown e. Chromatin - nucleoprotein complex - composed of - DNA - equal weight of small, positively charged proteins (histones) - variable amount of other proteins f. Nucleolus i. Functions - site of synthesis of most of the ribosomal RNA (rRNA) - where ribosome assembly begins ii. Structure - suborganelle located in the nucleus - arise from nuclear organizer regions (NORs) - size directly reflects the cell’s synthetic activity B. Endoplasmic Reticulum (ER) 1. Functions - synthesis of lipids, proteins, carbohydrates and steroids - facilitates the separation of newly synthesized proteins that go to the cytosol from those that are secreted or go to other organelles - facilitates the assembly of proteins synthesized on the ER into their correct tertiary and quarternary structure - precursor N-linked and O-linked oligosaccharides may be added to proteins in the ER 2. Structure - highly convoluted, single membrane sheet forming a closed sac  ER lumen - continuous with the outer nuclear membrane 3. Two Types a. Rough ER i. Functions - carries ribosomes on its cytosolic surface which synthesize proteins destined for secretion or membrane insertion (proteins produced on these ribosomes enter the lumen of the RER, travel to the Golgi complex in vesicles, and are subsequently either secreted from the cell, sequestered within membrane-enclosed organelles such as lysosomes, or embedded in the plasma membrane) - predominates in cells that secrete proteins - posttranslational modifications of these proteins, such as the initiation of N- linked glycosylation and the addition of GPI anchors, occur in the RER Proteins encoded by the nucleus and found in the cytosol, peroxisomes, or mitochondria are synthesized on free ribosomes in the cytosol and are seldom modified by the attachment of oligosaccharides. ii. Structure - appears rough because of attached ribosomes b. Smooth ER - also called transitional ER - where vesicles carrying new proteins and lipids bud-off for intracellular transport For Your Eyes Only i. Functions - predominates in cells specializing in lipid and sterol metabolism (triacylglycerols, phospholipids) - steroid hormones (hydrophobic molecules) are synthesized in expanded smooth ER - smooth ER of liver hepatocytes contain enzymes (cytochrome P450 oxidative enzymes) that catalyze the detoxification of drugs (alcohol), steroid and harmful metabolic products - with sodium pumps and channels in its membrane it is also a calcium store which is released for signalling Chronic ingestion of ethanol has increased the content of MEOS, the microsomal ethanol oxidizing system, in a male patient. MEOS is a cytochrome P450 enzyme that catalyzes the conversion of ethanol, NADPH and O2 to acetaldehyde, NADP+ , and 2H2O. The adjective microsomal is a term derived from experimental cell biology that is sometimes used for processes occurring in the ER. When cells are lysed in the laboratory, the ER is fragmented into vesicles called microsomes, which can be isolated by centrifugation. Microsomes, as such, are not actually present in cells. ii. Structure - network of fine tubules that are continuous with rough ER  For Your Eyes Only c. Sarcoplasmic Reticulum - form of ER found in muscle - release of calcium on excitation is necessary for muscle contraction C. Golgi Complex 1. Functions - principal director of intracellular movement of macromolecules - Golgi vesicles transport proteins to and from the Golgi complex - O-linked oligosaccharides may be added to proteins - tags (addition of phosphate to the mannose sugars of N-linked oligosaccharides) lysosomal- targeted proteins to direct them to the lysosome - modifying proteins produced in the RER - sorting and distributing proteins to the lysosomes, secretory vesicles, or the plasma membrane 2. Structure - stacks of single-membrane bound, fattened, disk-shaped structures (Golgi stack or dictyosome) a. Three Compartments i. Cis-Golgi Network - often convex and faces the nucleus ii. Medial Golgi Stacks iii. Trans Golgi Network - often faces the plasma membrane b. Three Vesicles - transport proteins to and from the Golgi i. Coatomer-Coated COP I Vesicles - COP I vesicles recycle material from the Golgi back to the ER and possibly transfer material from the Golgi to other sites - contain the monomeric G protein Arf (ADP-ribosylating factor) ii. Coatomer-Coated COP II Vesicles - proteins produced on the RER travel in COP II vesicles  endoplasmic reticulum-Golgi intermediate compartment (ERGIC)  cis-Golgi network, where they enter the lumen  N-linked oligosaccharide chains that were added to proteins in the RER are modified, and O-linked oligosaccharides are added - contain the monomeric G protein Sar (another member of the Arf family) - hydrolysis of GTP causes dissociation of the G-protein and disassembly of the vesicle coat - vesicle components are then recycled - COP vesicles are coated with a complex composed of coatomer proteins (COP, Arf family monomeric G protein that mediates vesicle assembly, and other proteins) The monomeric G protein Arf was named for its contribution to the pathogenesis of cholera and not for its normal function in the assembly of COP I vesicles. However, it is also required for the transport of V. cholerae A-toxin. The cholera toxin is endocytosed in caveolae vesicles that subsequently merge with lysosomes (or are transformed into lysosomes), where the acidic pH contributes to activation of the toxin. As the toxin is transported through the Golgi and ER, it is further processed and activated. Arf forms a complex with the A-toxin that promotes its travel between compartments. The A-toxin is actually an ADP-ribosylase (an enzyme that cleaves NAD and attaches the ADP portion to a protein), and hence, Arf became known as the ADP-ribosylating factor. The ADP-ribosylation of For Your Eyes Only proteins regulating the CFTR chloride channel leads to the patient’s dehydration and diarrhea. - vesicles that have lost their coats are ready to fuse with the target membrane iii. Clathrin-Coated Vesicles - vesicles released from the trans face of the Golgi complex travel to endosomes as clathrin-coated vesicles c. v-SNARES (Vesicle-SNARES) - proteins contained by vesicle membranes - each type of v-SNARE is able to recognize and bind to its complementary t-SNARE (target SNARE) on the target membrane ensuring that the vesicle contents are delivered to the right location For Your Eyes Only d. Additional Proteins are Required for Fusion of the Vesicle with the Target Membrane i. Rab - monomeric G protein ii. SNAP (Soluble NSF Attachment Proteins) iii. NSF (N-Ethylmaleimide Sensitive Factor) e. Exocytotic Vesicles - release proteins into the extracellular space after fusion of the vesicular and plasma cell membranes - exocytotic vesicles containing hormones also may contain i. Proteases - cleave the prohormone at a specific site ii. v-ATPases - acidify the vesicle and activate the protease  For Your Eyes Only Secretory vesicles. The hormone insulin is synthesized as a prohormone, proinsulin, which is incorporated into secretory vesicles. These vesicles contain a protease that is activated by the acidic pH of the secretory vesicle. It cleaves proinsulin into the A, B, and C chains. D. Lysosomes 1. Functions - control the intracellular digestion of macromolecules - elimination of unwanted material and recycling their components - destruction of infectious bacteria and yeast - recovery from injury - involution of tissues during development - tissue remodelling - normal turnover of cells and organelles 2. Structure - intracellular organelles of digestion enclosed by a single membrane that prevents the release of its digestive enzymes into the cytosol 3. Lysosomal Acid Hydrolases - enzymes that cleave amide, ester, and other bonds through the addition of water - most lysosomal hydrolases have their highest activity near a pH of approximately 5.5 (pH optimum) - intralysosomal pH is maintained near 5.5 principally by v-ATPases (vesicular ATPases), which actively pump protons into the lysosome - inactive at neutral pH of the cytosol a. Lysosomal Digestive Enzymes - nucleases - phosphatases - glycosidases - esterases - proteases called cathepsins For Your Eyes Only Lysosomal Storage Diseases - genetic defects in lysosomal enzymes, or in proteins such as the mannose 6-phosphate receptors required for targeting the enzymes to the lysosome, lead to an abnormal accumulation of undigested material in lysosomes that may be converted to residual bodies. The accumulation may be so extensive that normal cellular function is compromised, particularly in neuronal cells. 1. Tay-Sach’s Disease (GM2 Gangliosidosis) - deficiency in lysosomal -N-acetylhexosaminidase, which breaks down ganglioside GM2 to ganglioside GM3 - very high concentration of ganglioside GM2  severe neurologic problems and early death in children - autosomal recessive 2. I-Cell Disease or Mucolipidosis II - deficiency in large number of lysosomal enzymes  lysosomes contain large amount of undegraded glycolipids and glycosaminoglycans  severe neurologic damage and bone deformities - patients are also deficient in the Golgi complex enzyme activity that is responsible for phosphorylating the mannose residue of glycoproteins to mannose 6-phosphate, which is necessary to direct lysosomal-targeted enzymes to lysosomes instead of extracellular secretion 3. Pompe’s Disease - accumulation of glycogen particles in lysosomes 4. Endocytosis, Phagocytosis, and Autophagy i. Endosomes - digestive vesicles that form lysosomes - involved in receptor-mediated endocytosis - participate in digestion of - foreign cells acquired through phagocytosis - internal cell contents in the process of autophagocytosis a. Receptor-Mediated Endocytosis - lysosomes are involved in the digestion of compounds brought into the cells in endocytotic clathrin-coated vesicles formed by the plasma membrane For Your Eyes Only i. Mechanism - vesicles fuse to form multivesicular bodies (early endosomes) - early endosomes mature into late endosomes as they recycle clathrin, lipids, and other membrane components back to the plasma membrane in vesicles (recycling endosomes) - late endosomes mature into lysosomes as they progressively accumulate newly synthesized acid hydrolases and vesicular proton pumps brought to them in clathrin-coated vesicles from the Golgi - within the Golgi, enzymes are targeted for endosomes (and eventually lysosomes) by addition of mannose 6-phosphate residues that bind to mannose 6- phosphate receptor proteins in the Golgi membrane - mannose 6-phosphate receptors together with their bound acid hydrolases are incorporated into the clathrin-coated Golgi transport vesicles and released - transport vesicles lose their clathrin coat and then fuse with the late endosomal membrane - acidity of the endosome releases the acid hydrolases from the receptors into the vesicle lumen - receptors are eventually recycled back to the Golgi For Your Eyes Only The elevated level of uric acid in a female patient’s blood led to the deposition of monosodium urate crystals in the joint space (synovial fluid) of her right great toe, resulting in podagra (painful great toe). Neutrophils, the mediators of the acute inflammation that followed, attempted to phagocytose the urate crystals. The engulfed urate crystals were deposited in the late endosomes and lysosomes of the neutrophil. Because urate crystals are particles that cannot be degraded by any of the lysosomal acid hydolases, their accumulation caused lysis of the lysosomal membranes, followed by cell lysis and release of lysosomal enzymes into the joint space. The urate crystals also resulted in release of chemical mediators of inflammation that recruited other cells into the area. This further amplified the acute inflammatory reaction in the tissues of the joint capsule (synovitis), leading to the extremely painful swelling of acute gouty arthritis. b. Phagocytosis and Autophagy i. Phagocytosis ia. Major Phagocytic Cells - neutrophils - macrophages - devour pathogenic microorganisms - clean up wound debris and dead cells aiding in repair  For Your Eyes Only ib. Mechanism - as bacteria or other particles are enclosed into clathrin-coated pits in the plasma membrane, these vesicles bud off to form intracellular phagosomes - phagosomes fuse with lysosomes, where the acidity and digestive enzymes destroy the contents - pinocytotic vesicles also may fuse with lysosomes ii. Autophagy (Self-Eating) iia. Mechanism - intracellular components such as organelles or glycogen particles are surrounded by a membrane derived from ER vesicles, forming an autophagosome - autophagosome fuses with a lysosome, and the contents of the phagolysosome are digested by lysosomal enzymes - cells that are damaged but still viable recover, in part, by using autophagy to eliminate damaged components iib. Residual Body - lysosome with remaining undigestible material after the digestion process is completed - may be expelled (exocytosis) or remain indefinitely in the cell as lipofuscin granules that accumulate with age Phagocytosis and autophagy are part of the normal turnover of body components, such as degradation of cells that have a shorter lifespan than the whole organism and remodelling of tissues during pregnancy. For example, phagocytes, located mainly in the spleen and liver, remove approximately 3 x 1011 red blood cells from the circulation each day. During pregnancy, breast tissue is remodelled to develop the capacity for lactation; after weaning of an infant, the lactating breast returns to its original state (involution). 5. Action of Chloroquine - antimalarial and amebicidal - inactivate lysosomal enzymes by neutralizing the lumen of the lysosome - antiparasitic activity results from inhibition of the parasites’ lysosomal hydrolases E. Peroxisomes 1. Functions - involved in oxidative reactions using molecular oxygen  produce the toxic chemical hydrogen peroxide (subsequently used or degraded within the peroxisome by catalase and other enzymes) - oxidation of very long chain fatty acids (containing 20 or more carbons) to shorter chain fatty acids - conversion of cholesterol to bile acids - synthesis of ether lipids (plasmalogens) - contain enzymes that degrade amino acids and fatty acids  formation of H2O2 - contain large amount of catalase  For Your Eyes Only 2. Structure - structurally similar to lysosomes - similar in size to lysosomes - bounded by a single membrane - can replicate by division - dependent on the import of proteins to function - contain no DNA Peroxisomal Diseases. Peroxisomal diseases are caused by mutations affecting either the synthesis of functional peroxisomal enzymes or their incorporation into peroxisomes. For example, adrenoleukodystrophy probably involves a mutation that decreases the content of a transporter in the peroxisomal membrane. Zellweger’s syndrome is caused by the failure to complete the synthesis of peroxisomes. F. Ribosomes 1. Function - site of protein synthesis 2. Structure - generated in the nucleolus - eukaryotic ribosomes are larger and more complex than prokaryotic ribosomes - eukaryotic ribosomes exist in the cytosol singly (monosomes) or as multiple ribosomes attached to a single mRNA (polyribosomes or polysomes) - monosomes can also be found attached to endoplasmic reticulum  synthesize proteins to be secreted or transported to other organelles G. Mitochondria - replicate independently of nuclear replication and cell division 1. Functions - contain enzymes for the pathways of fuel oxidation and electron transport chain and oxidative phosphorylation  generate most of the ATP required by mammalian cells  “power plants” of the cell For Your Eyes Only 2. Structure a. Membrane - double membrane organelle i. Inner Membrane - forms invaginations or folds (cristae) - contain - electron transport chain - ATP synthase - highly impermeable - proton gradient that is built up across this membrane during oxidative phosphorylation is essential for ATP generation from ADP and phosphate - transport of ions occurs principally through facilitative transporters in a type of secondary active transport powered by the proton gradient established by the electron transport chain ii. Outer Membrane - contains pores made from proteins (porins) - permeable to molecules with a molecular weight up to about 1000 g/mole b. Mitochondrial Matrix - contain - most of the enzymes for the TCA cycle - other pathways for oxidation - mtDNA and ribosomes 3. Replication - by division - most of the enzymes and proteins in mitochondria are encoded by nuclear DNA and synthesized on cytoplasmic ribosomes a. mtDNA - encodes for only 13 different subunits of proteins involved in oxidative phosphorylation - genetic code differs from the that of chromosomal DNA - maternally inherited b. mtRNA - most of comes from mtDNA  For Your Eyes Only Mitochondial diseases. Mitochondria contain DNA and can reproduce by replicating their DNA and then dividing in half. Although nuclear DNA encodes most of the enzymes found in mitochondria, mitochondrial DNA encodes some of the subunits of the electron transport chain proteins and ATP synthase. Mutations in mitochondrial DNA result in a number of genetic diseases that affect skeletal muscle, neuronal, and renal tissues. They are implicated in aging. EUKARYOTIC CYTOSKELETON A. Microfilaments 1. Structure - made of actin  actin filaments - 3-6 nm - polar  used to transport secretory vesicles, endosomes and mitochondria, powered by motor proteins, including myosin I and V a. Actin Filaments - form a network controlling the shape of the cell and movement of the cell surface, thereby allowing cells to move, divide, engulf particles, and contract - present in all living cells - in most eukaryotic cells, actin is the most abundant intracellular protein For Your Eyes Only i. F-Actin - polymerized state  filamentous form - composed of a helical arrangement of globular G-actin subunits - dynamic - as F-actin elongates, bound ATP is hydrolyzed to ADP, so that most of the polymer contains G-actin-ADP subunits - conformation of ADP-actin favours dissociation from the minus end of the polymer  polymer is capable of lengthening from the plus end  directional growth can account for certain types of cell movement and shape changes: the formation of pseudopodia that surround other cells during phagocytosis, the migration of cells in the developing embryo, or the movement of white blood cells through tissues - form the thin filaments (microfilaments) in the cell that are organized into compact ordered bundles or loose network arrays by cross-linking proteins ii. G-Actin - in unpolymerized state  actin is a globular protein - each G-actin subunit contains a bound ATP or ADP that holds the actin fold into a closed conformation - new subunits of G-actin containing ATP continuously combine with the assembled F-actin polymer at the plus end - assembly of G-actin subunits into polymers, bundling of fibers, and attachments of actin to spectrin and to the plasma membrane proteins and organelles, are mediated by a number of actin-binding proteins and G-proteins from the Rho family iii. Short Actin Filaments - bind to the cross-linking protein spectrin to form the cortical actin skeleton network iv. Long Actin Filaments - combine with thick filaments, composed of the protein myosin, to produce muscle contraction 2. Function - associate with > 50 proteins (ex: myosin)  perform function actin + myosin + ATP  contractile force - controls cell shape - prevents cellular deformation - involved in - cell-cell and cell-matrix adhesion - cell movements such as crawling and cytokinesis (cell division) - intracellular vesicle transport - form the structural core of cellular protrusions - microvilli - lamellipodia - filopodia - actin microfilament bundles within the cell can associate with myosin II to form contractile stress fibres, similar to muscle sarcomeres  For Your Eyes Only a. Stress Fibres - often found as circumferential belts around the apical surfaces of epithelial cells where cells associate with adjacent cells via adherens junctions, permitting reaction to external stresses as a cellular sheet - also form where actin interacts via accessory proteins with the extracellular matrix at sites of focal adhesion b. Actin-Myosin II Bundles - during cytokinesis - form the contractile ring separating dividing cells 3. Drugs That Affect Microfilaments a. Cytochalasin B - inhibits microfilament assembly  alteration of cell shape and inhibition of cell movement b. Phalloidin - toxin from mushroom Amanita phalloides - inhibit depolymerization of actin filaments c. -Amanitin B. Intermediate Filaments 1. Structure - ~10 nm - composed of fibrous protein polymers that provide structural support to membranes of the cells and scaffolding for attachment of other cellular components - long, rod-like -helical molecules - -helical segments of two subunits coil around each other to form a coiled coil, and then combine with another dimer coil to form a tetramer For Your Eyes Only - filament assembly is partially controlled through phosphorylation - exhibit self-assembly into stable form 2. Five Major Classes - based on the subunit protein a. Vimentin - in fibroblasts and many epithelial cells such as those in blood vessels b. Desmin - in muscle cells c. Neurofilaments - in axons d. Glial Fibrillary Acidic Protein - in glial cells e. Cytokeratins - different types of epithelial cells express different, characteristic combinations of cytokeratins - antibodies to different cytokeratins is useful in determining the epithelial cell type from which a tumor arose 3. Functions - primary role is structural integrity - role in cell-to-cell attachment and help to stabilize the epithelium via desmosomes, and with basement matrix via hemidesmosomes - major structural role in skin and hair cells - maintain the correct register of contractile units in muscle cells - provide strength and rigidity to axons - form a network around the nucleus extending to the periphery of the cell For Your Eyes Only In epidermolysis bullosa simplex, the skin blisters in response to a very slight mechanical stress. The familial form of the disease is generally caused by mutations in either of the two forms of keratin that constitute the keratin heterodimers of the basal layer of the epidermis. The weakened keratin cytoskeleton results in cytolysis when stress is applied. The disease also can be caused by mutations in plektin, the protein that attaches keratin to the membrane protein integrin in hemidesmosomes (a structure involved in attaching cells to the extracellular matrix).  For Your Eyes Only C. Microtubules - largest of the fibrous elements - 20-25 nm diameter - present in all nucleated cells and the platelets in blood 1. Structure - cylindrical tubes made of protein tubulin with 2 types: ,  - made of 13 parallel protofilaments of  dimers  fold  hollow-centered microtubule (tubular structure)  resist bending and stretching a. Microtubule-Associated Proteins (MAPs) - associate with microtubules - component of the regulation of microtubule assembly and disassembly - attach microtubules to other cellular components - can determine cell shape and polarity b. Three Tubulin Polypeptides - - - - of similar amino acid composition - encoded by related genes i. ,  Dimers - polymerize to form most microtubules - bind GTP  conformational change in the dimer that favours addition of dimers to the tubulin polymer - can add to and dissociate from both ends of the tubulin, but the end to which they add more rapidly (the plus end) has a net rate of growth, and the end to which they add more slowly (the minus end) has a net rate of loss - as GTP is hydrolyzed to GDP, the binding of tubulin subunits is weakened, resulting in their dissociation (dynamic instability) For Your Eyes Only ii. -Tubulin - found only in the centrosome 2. Functions - intracellular and extracellular movement - in conjunction with other proteins especially dynein, generate contractile force  beating of cilia and flagella A variety of human cells have cilia and flagella, hairlike projections from the surface that have a stroke-like motion. These projections contain a flexible organized array of microtubules. Fluid or mucus is propelled over the surface of ciliated epithelial cells by the coordinated beating of cilia. A sperm cell swims by means of a flagellum. - form tracts on which intracellular vesicles (including phagocytic vesicles, exocytotic vesicles, and the transport vesicles between the ER, Golgi, and endosomes) and organelles move - driven by proteins called molecular motors a. Motor Proteins - require energy in the form of ATP i. Kinesin - moves molecules, vesicles, and organelles toward the plus end of microtubules, usually toward the plasma membrane - microtubule network (minus end) begins in the nucleus at the centriole and extends outward to the plasma membrane (usually the plus end) ii. Cytoplasmic Dyneins - huge proteins that move vesicles and organelles to the minus end, generally toward the nucleus - involved in the positioning of the Golgi complex - movement of chromosomes (form mitotic spindles) during mitosis b. MAP1c - moves vesicles to opposite direction to kinesin 3. Effects of Antimitotic Drugs on Microtubules - block microtubule assembly (polymerization) or disassembly (depolymerization) - many drugs are antineoplastic - preferentially kill dividing cells by preventing mitosis a. Drugs That Inhibit Microtubule Assembly i. Vinblastine ii. Vincristine iii. Podophyllotoxin iv. Colchicine - treatment of gout - blocks microtubule polymerization  inhibition of phagocytosis of uric acid crystals by WBCs (phagocytosis  cell lysis  increased inflammation) A female patient was given colchicine, a drug that is frequently used to treat gout. One of its actions is to prevent phagocytic activity by binding to dimers of the  and  subunits of tubulin. When the tubulin dimer-colchicine complexes bind to microtubules, further polymerization of the microtubules is inhibited, depolymerization predominates, and the microtubules disassemble. Microtubules are necessary for vesicular movement of urate crystals during phagocytosis and release of mediators that activate the inflammatory response. Thus, colchicine diminishes the inflammatory response, swelling and pain caused by formation of urate crystals.  For Your Eyes Only Colchicine has a narrow therapeutic index (i.e., the amount of drug that produces the desirable therapeutic effect is not much lower than the amount that produces an adverse effect). Its therapeutic effect depends on inhibiting tubulin synthesis in neutrophils, but it can also prevent tubulin synthesis (and, thus, cell division and other cellular processes) in other cells. Fortunately, neutrophils concentrate colchicine, so they are affected at lower intakes than other cell types. Neutrophils lack the transport protein P-glycoprotein, a member of the ABC cassette family (which includes the CFTR channel). In most other cell types, P-glycoprotein exports chemicals such as colchicine, thus preventing their accumulation. b. Taxanes - inhibits microtubule disassembly (stabilize microtubules) CELL SHAPE and MOTILITY A. Cytoskeleton - determines cell shape and surface structures B. Microvilli - ~1 μm long - cover the apical surface of some epithelial cells  brush border of thousands of small finger-like projections of the plasma membrane  increase the surface area for uptake or efflux - 20-30 cross-linked actin microfilaments  core C. Motile Cilia - fine, finger-like protrusions - longer (~10-20 μm long) 1. Axoneme - at the core of motile cilia - bundle of nine cross-linked tubulin microtubule doublets surrounding a central pair 2. Dynein - motor domain - serves to bend the cilium  For Your Eyes Only D. Non-Motile or Primary Cilia - have a variant axoneme with no central pair of microtubules - have dynein but non-motile - for signalling during development and in the adult E. Flagella - single flagellum found on sperm is structurally related to cilia but is longer (~40 μm) - whip-like motion F. Cell Motility - essential in the following processes - during development - macrophages migrate to sites of infection - keratinocytes migrate to close wounds - osteoclasts and osteoblasts tunnel into and remodel bone - fibroblasts migrate to sites of injury to repair the extracellular matrix - most cell motility in the adult human takes the form of cell crawling (dependent on remodelling of the actin cytoskeleton) 1. How the Actin Cytoskeleton is Remodelled Determines the Mode of Migration a. Filopodia - if remodelled essentially in one dimension into a long actin filament, the leading edge of the plasma membrane is pushed forward as spikes, similar to long thin villi b. Lamellipodia - if remodelled in two dimensions  form a network of cross-linked actin microfilaments  broad flat skirt or lamellipodium is formed c. Pseudopodia - more three-dimensional projections as the actin cytoskeleton is remodelled into a gel- like lattice  For Your Eyes Only A male patient has been drinking for 5 years and has begun to exhibit mental and systemic effects of chronic alcohol consumption. In his brain, ethanol has altered the fluidity of neuronal lipids, causing changes in their response to neurotransmitters released from exocytotic vesicles. In his liver, increased levels of MEOS (CYP2E1) located in the smooth ER increased his rate of ethanol oxidation to acetaldehyde, a compound that is toxic to the cell. His liver also continues to oxidize ethanol to acetaldehyde through a cytosolic enzyme, liver alcohol dehydrogenase. One of the toxic effects of acetaldehyde is inhibition of tubulin polymerization. Tubulin is used in the liver for secretion of very low-density lipoprotein (VLDL) particles containing newly synthesized triacylglycerols. As a result, these triacylglycerols accumulate in the liver, and he has begun to develop a fatty liver. Acetaldehyde may also damage protein components of the inner mitochondrial membrane and affect its ability to pump protons to the cytosol. A female patient had a rapid and gratifying clinical response to the hourly administration of colchicine. This drug diminishes phagocytosis and the subsequent release of the lysosomal enzymes that initiate the inflammatory response in synovial tissue. The inflammatory response that causes the symptoms of an acute gout attack begins when neutrophils and macrophages ingest urate crystals. In neutrophils, urate activates the conversion of the polyunsaturated fatty acid arachidonic acid (present in membrane phospholipids) to leukotriene B4. The release of this prostaglandin contributes to the pain. Colchicine, through its effect on tubulin, inhibits phagocytosis, leukotriene B4 release, and recruitment and cell division of additional cells involved in inflammation. Colchicine also inhibits the tubulin-dependent release of histamine from mast cells. As a result, there was a rapid improvement in the pain and swelling in her great toe. After the gout attack subsided, she was placed on allopurinol, a drug that inhibits urate production. During the next 6 months of allopurinol therapy, her blood urate levels decreased. She did not have another gout attack during this time. A male patient was diagnosed with cholera. He was placed on intravenous rehydration therapy, followed by oral rehydration therapy with high glucose and Na+-containing fluids. Vibrio cholerae secrete an A toxin that is processed and transported in the cell in conjunction with the monomeric G protein Arf (ADP-ribosylation factor). The A toxin ADP-ribosylates the G subunit of a heterotrimeric G protein. The net result is activation of protein kinase A, which then phosphorylates the CFTR (cystic fibrosis transmembrane conductance regulator) chloride channel so that it remains permanently open. The subsequent efflux of chloride, sodium, and water into the bowel lumen is responsible for his diarrhea and subsequent dehydration.