Revisjonen fra 3. mar. 2009 kl. 13:54

Kort om faget

Emnet gir en innføring i cellestruktur, organeller, membraner, kommunikasjon, etc. Kvantitativ beskrivelse av prosesser som diffusjon, transport og membranpotensial. Også gjennomgang av teknikker som celledyrking og mikroskopering (se under). Ingen regneøvinger, obligatorisk lab med (begrenset) rapportskriving.

Lab

Seks obligatoriske laboratorieøvinger:

Elektronmikroskopilab: Demonstrasjonslab som viser bruk av TEM for å se på biologiske prøver, samt gjennomgang av prepareringsteknikker for disse.

Lysmikroskopilab: Optikklab som likner mye på første optics lab i nanoverktøy. Bruk av lysmikroskop til å undersøke epithelceller, ved forskjellige mikroskopinnstillinger.

Celledyrkinglab: Først en demonstrasjonslab der celledyrking blir demonstrert, deretter individuell lab der cellene telles ved gitte tidspunkt.

Fluorescencesmikroskopilab: Cellekjernen og aktinfilamentet merkes med spesielle fargestoffer, deretter avbildes disse i et CSLM mikroskop.

Cellemembraner:

Cellekinetikk:

Core curriculum

Membrane properties

Membranes are important structures in the cells. They serve as boundaries and permeability barriers for cells and organelles. They help regulate transport processes through these membranes, detect signals reaching the cells and aid in inter cellular communication.

Membranes consist of about half and half lipids and proteins (wt %), in addition to a certain percentage of carbohydrates. The commonly excepted model for the membrane is known as 'Singer and Nicolson's fluid mosaic model'. Here the membrane is modeled as a fluid bilayer of lipids with a mosaic of membrane-bound proteins dispersed in it. The main classes of lipids in the membrane are 'phospholipids', 'glycolipids' and 'sterols'.

Membrane lipids

Phospholipids are lipids consisting of long-chain fatty acids, glycerol and phosphate, and sometimes associated molecules (e.g. choline), while sphingolipids consist of fatty acids, sphingosine and phosphate, and sometimes associated molecules. Sterols (steroid alcohols) consist of three six-carbon rings and one five-carbon ring, all interconnected, with a hydroxyl group at carbon-3 on the A ring, and various side-chains.

Common phospholipids are phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine, where the last part of the name signifies what group is bound to the phosphate group. Sphingomyelin is an important sphingolipid, where choline is the phosphate-bound group.

The properties of the membrane is decided by many factors. The ratio between headgroup area and chain length and diameter decides what kind of shape the lipids will self-assemble to. Two fatty-acid chains of 12-20 carbon atoms (with 16 or 18 being most common) are by far the most common in cell membranes, as these form stable lipid bilayers. The bilayers will form closed surfaces to avoid contact between the hydrophobic fatty acids and water. Saturated lipids form stiffer membranes, while unsaturated lipids form more fluid membranes due to lower packing density.

The membrane has two phases: fluid and crystalline. The transition temperature is decided by the same factors that regulate fluidity, but can be offset by cholesterol. Cholesterol (a sterol) will prevent the packing of the other lipids, which reduces the transition temperature of the membrane. This is especially important for cold-blooded animals, where maintenance of membrane fluidity during cold weather is important. The reduction in fluidity also accounts for reduced nerve sensitivity and many other effects of being cold. However, when the membrane is in the fluid phase cholesterol actually decreases fluidity, due to attractive interactions between the the rigid cholesterol molecules and the other lipids. Cholesterol also reduces the membrane permeability to small molecules and ions, probably due to it filling channels that would otherwise spontaneously form in the membrane.

Diffusion is a very important part of the membrane, it is by no means static. Both membrane-proteins and lipids have relatively high (<math>~10^{-12} m^2/s</math>) diffusion constants, in both lateral and rotational directions. This is demonstrated for proteins by cells fusion. Membrane proteins in two different cells are tagged in different colors. A virus is then infused which causes these cells to merge, and the distribution of proteins in the membrane is the imaged using fluorescence microscopy. This shows a rapid (45 minutes) distribution of the proteins on the membrane of the new cell. Most proteins still diffuse more slowly than would be expected of freely diffusing proteins. Many mechanisms are thought to be responsible, among these are aggregation of protein into larger complexes, some proteins becoming barriers to the diffusion of other proteins. The most common mechanism for restricting protein mobility is the anchoring of proteins to intra- or extracellular matrix elements of the cell.

On the other hand, diffusion causing the flipping of lipids in the bilayer almost never occurs. The membrane has different types lipids facing inwards toward cytosol and outward towards extracellular matrix, and due to low switching diffusion this distribution stays rather constant. The composition of the membrane is decided as it is formed in the endoplasmic reticulum (ER), where the enzyme flippase causes certain lipids to end up on a given side of the membrane.

TLC (thin-layer chromatography) can be used to assess the various types of lipids in the cellular membranes.

Membrane proteins

To image the surface of the membranes a method called freeze-fracture analysis is used. The sample is frozen very quickly and the split (or fractured) by a sharp knife. The cells tend to fracture through the middle of the bilayer membranes, which allows molds to be made which mimic the shape of the membrane. When imaged in e.g. a TEM, a large amount of proteins can be seen in the membrane as small lumps or indentations.

These membrane-proteins have many important functions: They catalyze reactions that happen on or close to the membrane (e.g. adenylyl cyclase), they anchor the cells cytoskeleton, they connect the plasmamembrane with the extracellular matrix or other cells (e.g. connexins in electrical synapses), they function as ion channels or ion pumps, they can facilitate the diffusion of molecules that otherwise cannot permeate the membrane, and they function as receptors for a variety of signaling molecules.

Integral membrane proteins

There are three main types of membrane proteins: integral, peripheral and lipid anchored. The integral membrane proteins are amphipathic, i.e. they have both hydrophilic and hydrophobic areas. If the proteins only are embedded in one side of the membrane they are called integral monotopic proteins, while transmembrane proteins span the whole membrane one or more times. Transmembrane proteins are by far the more common the the two. The transmembrane proteins are anchored in the membrane by transmembrane segments. These segments usually consist of an <math>\alpha</math>-sheet segment of about the same length as the lipid bilayer thickness (20-30 amino acid residues), but a few (e.g. porins) have closed <math>\beta</math> sheets known as <math>\beta</math> barrels.

Glycoproteins

Many of the integral membrane proteins are glycoproteins, which are proteins that have carbohydrate groups attached to their extracellular segments. The carbohydrates on the glycoproteins serve as a protective sheet (both chemically and mechanically) around the cell, by strongly binding with water. This surface coating is known as the 'glycocalyx' They are also important as receptors and for intercellular contact. The hydrocarbons can be N-linked to the amino-group of aspargine or O-linked to the hydroxyl groups of serine, threonine, hydroxylysine or hydroxyproline. This glycosylation happens in the ER and in the Golgi apparatus, more details will be described in the sections concerning them.

Peripheral membrane proteins

The second type of membrane proteins are the peripheral membrane proteins. These do not have specific hydrophobic segments, and are instead bound to one side of the bilayer by weak electrostatic attractions and hydrogen bonding. This type of proteins form a meshwork on the inside of the plasma membrane, stabilizing the shape and structure of the membrane, and also have important roles in endo- and exocytosis.

Lipid anchored membrane proteins

The final type of membrane proteins are called lipid anchored membrane proteins. These proteins are protrude from one side of the membrane, but are covalently bound to a lipid molecule which is part of the membrane. An important example is glycosylphosphatidylinositol (GPI), which is an regulatory protein causing increased intracellular Ca2+ concentration to rise in responce to protein kinase C activity.

Analysis of membrane proteins

Membrane proteins can often be characterized using gel electrophoresis to separate them. Once isolated, they can be assessed with x-ray diffraction (XRD), which can be used to recreate the 3-dimensional structure. Integral membrane proteins can be studied using hydropathy, where the hydrophobicity/hydrophilicity of each amino acid in the chain is assessed to see how many transmembrane segments there are.

Membrane Transport

There is a constant flux of material into and out of the cell, nucleus and organelles. Nutrition, ions, waste and excretions all have to pass through the membrane, but the membrane itself is only permeable to a small amount of molecules, the rest have to be transported through various processes. If one looks at a cell membrane without any proteins (i.e. a synthetic lipid bilayer) one sees that small hydrophobic molecules are soluble in the membrane, which means they also can diffuse across it. Examples are O2, N2 and CO2, as well as small organic molecules, such as benzene. Small polar, but uncharged molecules such as H2O, urea and glycerol are somewhat soluble, but will not diffuse rapidly. Larger uncharged polar molecules such as glucose and sucrose diffuse across the membrane to a very, almost negligible, extent. Ions do not diffuse in the membrane at all.

Types of transport

There are many ways material can enter and exit the cell through the membrane. The first, which is mentioned above, is called simple diffusion. Then molecules (or solutes) that can diffuse across the membrane will do so, as long as it is down their concentration gradient, or more precisely: until their chemical potential is equalized on both sides of the membrane. The two remaining types of transport involve proteins.

Facilitated diffusion

A second form of transport is called facilitated diffusion or passive transport. Here solutes come across the membrane either by through a channel protein or a carrier protein.

Channel proteins, or transporters or permeases, form hydrophilic channels through the membrane which allows molecules, which could not otherwise pass, to diffuse down their electrochemical gradient. A very important class of channel proteins is the ion channels. Ion channels can be voltage gated (such as the Na+ and K+ channels in action potentials), ligand gated (such as acetylcholine-gated Na+/K+ channels in muscular cells) and mechanically gated (such as in sensory Meissner-corpuscles).

Carrier proteins bind a solute molecule, then undergo a conformal change which ends up transporting the solute across the membrane. This process is also driven by the electrochemical gradient of the solute. Carrier proteins follow typical Michaelse-Menten enzyme kinematics, and are also highly specific. Carrier proteins can carry either one or two solutes. When only one molecule is carried it is called uniport transport, while if two are carried it is called coupled transport. Couple transport can further be divided into symport and antiport transport, where the two solutes move the same or opposite ways, respectively. The carrier proteins that perform these functions are known as symporters and antiporters. These terms are also used for active transport carrier proteins (see below). A typical example of a uniport carrier protein is the glucose transport protein GLUT1, which specifically binds glucose, then shifts conformation, in which it releases glucose and reverts to it's original conformation. It is important to note that this process will work the same both ways, it is only the chemical potential of the glucose over the membrane which determines the net flux.

Active transport

A very important method of transport across the membrane is active transport. In active transport energy is used to move solute molecules up their chemical or electrochemical potential gradient. There are two main types of active transport: direct and indirect. Direct active transport uses the energy from hydrolysis of a high-energy molecule to drive the transport. Usually this molecule is ATP, so these transport proteins are known as ATPases, or ATPase pumps. There any many different ATPases transporting both ions and larger solute molecules. Important examples are the Na+/K+ exchange pump to maintain the resting membrane potential, Ca2+ pump to keep a low intracellular Ca2+ concentration and the H+ ATPases in the mitochondrial inner membranes which drive ATP synthesis. Indirect active transport involves utilizing the electrochemical gradient of one ion type to transport a solute against it's electrochemical gradient. An example of this is the Na+/glucose symporter which exists in the epithelial cells of the intestine.

A very important ATPase is the Na+/K+ exchange pump. A proposed mechanism for this pump is as follows: I the first comformal state (E1) three intracellular Na+ atoms are bound. The pump is then phosphorylated by ATP, which causes a conformal change to state E2. In this state the pump has higher affinity for the extracellular K+, and two K+ ions are bound while the three Na+ ions are released. A subsequent hydrolyzation of the phosphate group causes a new conformal change, in which K+ is expelled into cytosol, and the pump goes back to the starting point, E1.

Chemotropic Energy Metabolism - The Mitochondrion

Anatomy

The mitochondrion is a cellular organelle with a central role in aerobic respiration. The mitochondrion is thought to originate from a bacteria phagocytized by an ancestral eukaryotic cell. The mitochondrion has it's own circular DNA, like many bacterium, but also relies on proteins from the cell to function, probably as a result of the long evolution since the incorporation. The mitochondrion has a double layer membrane. Between the outer membrane and the inner membrane is the intermembrane space. The inner matrix is folded to a large degree into the mitrochondrion, and the space within these folds is called the cristae. Inside the inner membrane is the matrix space or just matrix.

Function

After glucolysis in the cytosol the end product is pyruvate. Pyruvate is transported into the mitochondrion where it is oxidized to acetyl coenzyme A and enters the citric acid cycle (aka TCA cycle or Krebb's cycle). The fate of fatty acids is the same. In the citric acid cycle NAD and FAD are reduced to NADH and FADH2, in addition to some ATP production. This takes place in the matrix space of the mitochondrion. These reduced molecules, known as electron carriers, are oxidized in a tightly controlled and stepwise manner with O2 as the final electron acceptor (O2 is reduced to H2O). This process moves protons from the matrix into the intermembrane space. More specifically, Complex I, aka NADH dehydrogenase or NADH-coenzyme Q oxidoreductase, accepts two electrons from NADH. The electrons are transferred to CoQ via two carrier proteins. This process transports 4 H+ across the membrane. In complex III, Cyctochrome c is reduced by CoQH2 via an electron transport from cyt b, and Fe-S protein and cyt c1. This causes 6 new H+ to be pumped or transported across the membrane. Cytochrome c is a peripheral membrane protein that easily diffuses in the membrane to complex IV. Here cyt c finally causes the reduction of O2 via a reduction chain of cyt a, cyt a3 and a Fe-Cu protein, which causes two more H+ to cross the membrane. Simultaneously ATP synthase uses this proton gradient to drive ATP synthesis, where 3 H+ are transported for every ADP that is phosphorylated to ATP. ATP synthase consists of and F0 and F1 complex. The F0 complex is embedded in the inner membrane, while the F1 complex is connected to F0 with a stalk into the matrix space. When a proton passes through the complexes the c-subunit of F0 rotates, which causes the <math>\alpha and \beta</math>-subunits of F1 to rotate. The conditions within the ATP synthesis site of F1 is such that ADP is spontaneously phosphorylated, but energy from the proton gradient is used to drive the continuous synthesis.

Mitochondrial DNA

The mitochondrial DNA codes for many tRNAs and rRNAs needed for protein synthesis, as well as key proteins of the electron transport chain and ATP synthesis. Specifically, the DNA codes for complexes I (NADH dehydrogenase), III (Coenzyme Q - cytochrome c reductase) and IV (cytochrome c oxidase) as well as ATP synthase. All other needed enzymes, e.g. for the citric acid cycle, replicating enzymes for mitochondrial DNA and other supporting enzymes and proteins are synthesized by free ribosomes in the cytosol and transportet in by carrier proteins. Mitochondrial DNA is often used for genetic population tracking, due to it only being inherited from the mother (all of the sperms mitochondrion are discarded when it enters the egg).

Acquisition of cytosolic proteins

Many mitochondrial proteins are synthesized by the free ribosomes in the cytosol. Following a water-soluble protein: A protein of the heat shock protein family (HSP70) binds and stabilizes these proteins, which also have a specific sequence known as a transit sequence. This transit sequence binds to a receptor known as the translocase of the outer mitochondrial membrane (TOM). As the polypeptide passes through the pores of TOM and TIM (transit protein of the inner mitochondrial membrane) in the membrane the HSP70 proteins dissociate (via ATP hydrolyzation). The transit sequence is cleaved off the polypeptide by a membrane bound protein called transit peptidase, while the rest of the polypeptide entering the matrix is bound and stabilized by HSP70 and folds by with the aid of HSP60.

Various signal sequences cause the proteins to be targeted to other compartments of the mitochondrion, similar to the start and stop transfer sequences (see below).

The Endomembrane System, Peroxisomes and Protein Synthesis and Sorting

In addition to the mitochondrion is a range of organelles important for cell function. In the laboratory these organelles can be isolated using centrifugation techniques, either differential or density centrifugation. In differential centrifugation the distribution is given by the sedimentation coefficients of the organelles, while density centrifugation puts up a density gradient, and where the organelles end up depend on their density alone (as opposed to density and other factors in the sedimentation coefficient).

The endoplasmic reticulum has two parts: The smooth and rough ER, different both in appearance and function. Rough ER consists of folded sacks with a bilayer membrane and an inner lumen which is bound directly to the nuclear envelope. The smooth ER is a set of tubules connected to the rough ER. The Golgi apparatus is a nearby network of bilayer sacks responsible for further protein packing and sorting as well as protein modification.

Smooth Endoplasmic Reticulum

Smooth ER is a versatile organelle, responsible for the following functions in a cell:

• Drug detoxification by hydroxylization done by cytochrome P450 enzymes.
• Synthesis of phospholipids in membranes, synthesis of cholesterol-derived steroids, and synthesis of lipoproteins. The phospholipds are synthesized from conjugated fatty acids and glycerol phosphate or sphingocide (in the case of sphingomyelin) in cytosol. The enzymes that carry out the reactions are bound to the outer membrane of the ER. Since the lipids are only synthesized in the outer membrane, special enzymes called phospholipid translocators or simply flippases flip specific lipids (phosphatidylcholine and sphingomyelin) to the other side of the membrane. This keeps the area of the two membranes the same, and also accounts of the heterogenicity of the membranes in the cells.
• Transforming glucose into glycogen or opposite depending on metabolic needs. This is done by dephosphorylating glucose-6-phosphate from glucogen so it can diffuse out of the cell.
• Storage of Ca2+ ions to keep the concentration in the cytosol low.

In muscle cells smooth ER is called sarcoplasmic reticulum, here the calcium storage is of great importance. Smooth ER gets it's name for it's appearance in contrast with rough ER (below).

Rough Endoplasmic Reticulum

Rough ER gets it's name for it rough appearance compared to smooth ER. This is due to the high number of membrane-bound ribosomes in the ER. The main functions of rough ER are:

• Protein synthesis
• Glycolation of proteins
• Quality control of proteins
• Assembly of protein complexes

Protein synthesis in cells happens at two sites: Free ribosomes in the cytosol and membrane bound ribosomes in the rough ER. Proteins from free ribosomes enter the nucleus, mitochondrion, peroxisomes and plastids. Proteins for endosomes, lysosomes, Golgi and ER, as well as proteins for exocytosis and membrane proteins are all synthesized in ER. In short, all proteins that have something to do with membranes or vesicles are manufactured there.

To understand protein synthesis and tagging in the ER we follow a water soluble protein being synthesized: An mRNA binds to a ribosome in the vicinity of the ER. A signal recognition particle (SRP, consists of six polypeptides and an RNA strand) binds to a specific "ER-tag" component of the protein being synthesized. The SRP has one domain that recognizes this sequence, one domain that binds to the ribosome and inhibits further translation, and one part that binds to and SRP receptor protein in the ER membrane. The ER membrane also contains a ribosome receptor, a signal peptidase and a pore protein, where further protein synthesis will happen. The whole complex is known as a translocon. When GTP binds to SRP and the SRP receptor, the pore protein opens and the short polypeptide sequence that has been translated is transferred from SRP to the pore protein. GTP is then hydrolyzed, which causes SRP to dissociate from the ribosome and allows further translation. The signal pepsidase cleaves the signal sequence of the polypeptide as the ribosome keeps translating the protein, and the protein enters the ER through the pore protein. This process is called cotranslational import, because the protein is imported into the ER at the same time as it is translated.

Transmembrane proteins are synthesized in a similar way, but they contain additional sequences known as start and stop transfer sequences, which are hydrophobic sequences that fit into the ER membrane. When the stop sequence reaches the pore protein is incorporated into the membrane, while further synthesis happens on the outside of the membrane. Conversely, a start sequence also binds to the membrane, but causes the following part of the polypeptide chain to be pushed through the pore protein into the ER lumen. In that way transmembrane proteins become part of the ER membrane.

Protein folding is controlled in the ER lumen by chaperones, especially a heat shock protein (HSP) known as Binding Protein (BiP). This small protein binds to hydrophobic regions of the polypeptide being translated, preventing folding and aggregation from hydrophobic interaction. BiP then dissociates from the polypeptide by ATP hydrolysis, and the hydrophobic regions fold into the interior of the protein. If the protein folds incorrectly, so the hydrophobic regions still protude, the BiP binds again and allows the protein to refold. This process also is facilitated by protein disulfide isomerase, which catalyses the formation and breaking of disulfide bonds in the protein, until the most stable conformation (and thus correctly folded protein) is found - this will be the one that forms the most often and breaks the least often.

The Golgi Apparatus

The Golgi apparatus, also called the Golgi complex, consists of several folded bilayer sacks called cisternae in what is called a Golgi stack. The stack has to faces, the cis-Golgi network (CGN), which is closest to the ER, and the trans-Golgi network (TGN), which is furthest from the ER. It is at the faces that transitions vesicles from the ER and transport vesicles to other cell locations come and leave the Golgi apparatus, respectively. There are two models for the transport of proteins through the Golgi apparatus: The static and dynamic cisternea models. In the static model, it is vesicles which transport the proteins from cisternea to cisternea, while the cisternea themselves remains stationary. In the dynamic model, or the cisternea maturation model, the cisternea move as a whole and gradually change from CGN to medial to TGN cisternea. Enzymes that belong in the cisternea are then shuttled back from TGN to CGN, while TGN dissolves into vesicles to be transported out. Some recent fluorescence microscopy supports the cisternea maturation model. The reactions in the Golgi apparatus are localized to specific cisternea, which is a continuous reaction after the initial processing in the ER. The two main reaction types in Golgi are modification of the carbohydrates of the glycoproteins from the ER and phosphorylation of lysosomal proteins, in addition to some other sorting functions.

Protein glycosylation

The process of forming glycoproteins, where carbohydrates are bound to specific amino acid residues, is performed in the ER and Golgi apparatus. As mentioned earlier there are two main types: N- and O- linked glycosylation. To exemplify how this works we look more closely at the N-linked glycosylation, where the carbohydrate is attached to the amine group of an aspargine residue. In the ER there is initially attached a core oligosaccharide, common for all the varieties of N-linked glycoproteins. This oligosaccharide consists of two N-acetyl-<math>\beta</math>-D-glucosamine (GlcNAc), nine mannose and three glucose sugars. This reaction is initiated by the insertion of dolichol phosphate (a long phosphorylated fatty acid) into the ER membrane. Then sugars carried by UDP and GDP (GlcNAc and mannose, respectively) are attached until there are two GlcNAc and five mannose. Then the glycosylated phospholipid is flipped by the flippase enzyme, and further addition of sugars continues in the ER lumen until the core oligosaccharide is bound. The core oligosaccharide is transferred to an aspargine residue of a protein being synthesized in the ER membrane, catalyzed by oligosaccharyl transferase. This cotranslational glycosylation helps in the process of protein folding. Finally, removal of certain glucose and mannose units completes the glycosylation in the ER and the glycoprotein is ready for shipping to the Golgi apparatus. Here there is some trimming of the core oligosaccharide, and then a large variety of further modification can happen, catalyzed by hundreds of different glycosyl transferases. Since all these proteins are in the lumen side of the ER and Golgi, they always end up facing to the exterior of the cell membrane.

As mentioned above, the carbohydrate groups help in protein folding by providing binding sites for chaperones and di-sulfide catalysts until the protein is correctly folded. They also provide recognition sites for protein sorting, and protect the protein from being metabolized by proteases.

Protein sorting and packing

Proteins produced in the ER and at free ribosomes have a wide variety of specific locations. For free ribosomes an example of a mechanism concerning mitochondrial import has been explained. Proteins from the ER have even greater need for specific targeting. This is accomplished by certain tags on the protein. These tags can be specific amino acids sequences, certain types of bound oligosaccharides in the glycoproteins, or a certain type of hydrophobic domain. This tagging can also extend to membrane lipids, to make sure vesicles are targeted to correct destinations. Proteins that are to be retained in the ER can either have specific sequences, e.g. and RXR (Arg-X-Arg) sequence (a retention tag), or other tags such as KDEL (Lys-Asp-Glu-Leu, a retrival tag), which allows the protein to be trafficked to the Golgi and then back to the ER. Also, certain large complexes seem to be excluded from vesicle transport entirely, which ensures their retention. Similarly, proteins to be retained in Golgi can have either retention or retrieval tags, or form large complexes inhibiting vesicle transport. A third, Golgi-distinct retention system is tagging by length of hydrophobic domain. All Golgi-specific proteins are integral membrane proteins, and this domain gets longer the further into the Golgi stack the protein is supposed to sit, which reflects the fact that the thickness of the Golgi cisternea bilayer membranes increases from 5nm to 8nm from CGN to TGN.

An illustrative example of protein sorting for other organelle compounds is the targeting of water-soluble lysosomal proteins to endosomes and lysosomes. In Golgi, these glycoproteins are modified and phosphorylated, producing oligosaccharides containing mannose-6-phosphate. The TGN membrane has specific receptors for mannose-6-phosphate, which the proteins bind to with high affinity at TGN pH. These parts of the membrane form transport vesicles, which later fuses with endosomes. As the endosomes evolve from early to late endosomes the pH drops, which causes the proteins to dissociate from their receptors, activating them and preventing retrograde transport. This is only one of many mechanisms that exists to target proteins to specific locations, but this gives an idea of how it can work.

Vesicles

There are two main types of secretion from the cell: Regulated and constitutive secretion. Contitutive secretion is a continuous and unregulated secretion of proteins with specific short amino-acids tags via vesicles from the TGN. Regulated secretion, on the other hand, forms vesicles from the TGN in a similar way, but these vesicles accumulate in the cell until some extracellular signal initiates fusion with the membrane. In regulated secretion concentration of proteins presumably causes them to form aggregates which is part of what tags these proteins as secretory proteins. Receptors for proteins in TGN could also be responsible for targeting these vesicles. Proteins can also mature in these vesicles, which will not fuse with the membrane until the proteins are complete.

Exocytosis

When the vesicles of either constitutive or regulated secretion fuse with the plasma membrane the process is called exocytosis. In general, when the vesicle start fusing with the plasma membrane, the membrane ruptures and the contents of the vesicle exits the cell, as the vesicle itself, with it's membrane proteins, becomes part of the plasma membrane.

Endocytosis

Endocytosis is the process of material entering the cell by making a vesicle formed of the plasma membrane. Endocytosis starts with the formation of a pocket in the membrane. This pocket pinches off and seperates from the plasma membrane, forming an endocytic vesicle. There are three types of endocytosis: Pinocytosis, where a liquid containing a solute is taken up, phagocytosis, where large solid particles are taken up, and receptor mediated endocytosis, where endocytosis occurs after triggering by certain extracellular signals. Endocytosis and exocytosis have to balance each other out, otherwise a net change in cell volume and area will result. This change can be very rapid, with an area equivalent of an entire plasma membrane changed in a hour or less for certain types of cells.

Receptor-mediated endocytosis is the way most macromolecules are imported into the cell. The molecules bind to certain receptors in the membrane. The receptor-ligand complex then diffuses laterally in the membrane until they reach specific areas called coated pits, where they accumulate. This accumulation also causes the accumulation of specific proteins on the cytosolic side of the membrane. These proteins (adaptor protein, clathrin and dynamin) bind in such a way as to promote membrane curvature and vesicle formation. Specifically, adaptin binds to the cytosolic part of the receptor-ligand complex in the coated pits. Clathrin, a structurally rigid protein consisting of three bent arms, forms hexagonal lattices on vesicle surfaces, but only when it can bind to the surface via adaptin. Dynamin binds around a narrow piece of plasma membrane, which helps pinch of the clathrin-coated vesicle.

Vesicle targeting and transport

In addition to the clathrin coating described above, there are other types of coatings for different vesicles. The clathrin-coated vesicles transport specific types of proteins, such as the substances of receptor-mediated endocytosis. Other proteins, such as COPI and II bind to general vesicles that do not contain specific proteins. These are more strongly involved in retrograde transport within Golgi and from Golgi to the ER.

A mechanism for the formation of COPII-coated vesicles is as follows: In the vicinity of a forming vesicle there is a number of GEF (guanine exchanging factors). Proteins called SarI are bound to GDP in an inactive state. When they encounter GEF in the membrane GDP is switched with GTP, which causes the SarI to enter an active state where, where a lipid subcomponent can bind to the membrane. The COPII can the bind to the now membrane-bound SarI, which causes the formation of a vesicle. After the vesicle is formed, hydrolysis of the GTP in SarI by a COPII component causes the dissociation of SarI and thus dissembles the coat, allowing further processing of the vesicle.

Membrane fusion is a very specific process which has many proteins regulating it. Generally, the specific targeting of vesicles follows what is known as the SNARE hypothesis. v-SNAREs (vesicle SNAP receptors) on transport vesicles are complementary to t-SNAREs (target SNAP receptors) on the target membranes. Another conjugate pair, Rab GTPases (which is a GTP activated membrane bound protein family) and Rab receptors on the membrane ensure specificy, as different types of Rab proteins are on the different membranes in a cell. When a vesicles is near it's target membrane a tether protein recognizes the vesicle and binds to it. The Rab protein stimulates the association between the v-SNARE and t-SNARE proteins of the vesicle and membrane, which facilitates membrane fusion. Binding of N-ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment proteins (SNAPs) help release the SNARE complex completing membrane fusion. In neurons, it is though that the SNAREs there have Ca2+ binding sites, which accounts for some of the Ca2+-induced secretion of neurotransmitters.

Lysosomes

Lysosomes are a class of organelles that compartmentalize much of the digestive enzymes in the cells. The pH in lysosomes is low (4.0-5.0), which is the optimal pH for the acid hydrolase enzymes of the lysosomes. The low pH also denatures som of the proteins that are digested there. Late endosomes form when the vesicles containing acid hydrolases fuse with early endosomes. Endosomes can fuse with existing lysosomes, making endolysosomes. These break down the material within them, and form the spherical shapes known as lysosomes, which can then fuse with new endosomes. Lysosomes receive material from endocytosis, phagocytosis and autophagy, which is material from within the cell that should be broken down.

Peroxisomes

Peroxisomes are similar to lysosomes in function, as they also help break down harmfull and excess material in a cell, and like mitochondrion in the way that they probably originated as separate bacteria that entered a symbiotic relationship with the cell. The most important enzyme of peroxisomes is catalase, which breaks down hydrogen perokside which is formed by the activity oxidases. Since oxidases also are contained to the peroxisomes, this protects the cell from this harmful activity. Peroxisomes can be visually identified in electron microscopes by their characteristic crystalline cores of urate oxidase.

As indicated above, peroxisomes are important for hydrogen peroxide metabolism. This includes detoxification reactions, in which certain toxic organic molecules are oxidized to less harmful forms (this includes methanol, ethanol, formaldehyde, etc.), fatty-acid oxidation (they are broken down to Acetyl-CoA to enter the citric acid cycle), metabolism of nitrogen-containing compounds and catabolism of xenobiotics.

How new peroxisomes are formed is debated, but the most common model is that proteins and lipids are imported from the cytosol and vesicles from the ER, and become activated on entering the peroxisome. When enough proteins and lipids have gathered, the peroxisome divides and forms two new peroxisomes.

Signal Transduction - Intracellular Cascades

There are 4 main types of inter cellular signaling:

• Endocrine, specific cells secrete hormones into the blood stream.
• Paracrine, cells secrete signal molecules into the extracellular space to nearby cells.
• Contact-dependent.
• Synaptic.

When a signaling molecule binds to a receptor a variety of responses are available. Some of these are

• Metabolic change
• Secretion of a substance
• Change cell growth and division
• Initiate contraction of muscles
• Change membrane properties
• Make changes to the cytoskeleton
• Change the expression of genes

There are two main receptors: Cell-surface and intracellular receptors. The ones on the surface directly bind a signaling molecule. This signaling molecule is typically hydrophilic and so can travel in the blood on it's own. For intracellular receptors the signalling molecule first is transported by a carrier protein. The typically small, hydrophobic signaling molecule (steroid or thyroid hormones) and diffuse through the plasma membrane of the cell when it reaches it. Once through the membrane, it binds to a intracellular receptor protein and activates it. This activated protein can then perform some function, such as causing the transcription of certain genes.

Extracellular (surface) receptors can cause changes either rapidly or slowly, this depends on whether gene transcription is involved or not. There are three types of surface receptors: Ion-channel-linked receptors, which open or close ion channels when a ligand binds, G-protein-linked receptors, which activate a G-protein when the ligand binds, or enzyme linked receptors, which activates an enzyme on binding.

G-protein-linked receptors

The guanine-nucleotide binding protein (G-proteins) act to a large extent like molecular switches, where "on" and "off" corresponds to GTP or GDP being bound, respectively. G-proteins are either large heterotrimeric G proteins or small monomeric G proteins. The large heterotrimeric G proteins contain three subunits, known as the G alpha, G beta and G gamma. These G-proteins mediate signals via G-protein linked receptors. We will come back to the monomeric G proteins later. The G-protein linked receptor consists of seven trans-membrane domains, with a specific messenger binding site, a G-protein interaction site and places where inhibition of the receptor as a negative feedback responce can occur.

When a G-protein linked receptor attaches to a ligand, the G alpha subunit dissociates with GDP and instead binds GTP, which causes the whole G alpha subunit to dissociate. After that either the G beta and G gamma subunits or the free G alpha subunit can initiate further reactions in the cell. When the GTP is hydrolyzed to GDP by the G alpha subunit, the G-protein can associate again, and also bind to the receptor. If the ligand is still bound the same procedure repeats. An example of a G-protein coupled receptor is the lowering of heart beat frequency. Acetyl choline from parasympathetic neurons binds to a G protein receptor. The beta-gamma subunit dissociates and opens K+ ion channels. This hyperpolarizes the heart cells, reducing heart beat frequency.

cAMP

An important secondary messenger in the cell is cyclic adenosin monophosphate (cAMP). It is generated by adenylyl cyclase from ATP. Adenylyl cyclase is activated by the alpha subunit of an activated G-protein. cAMP is responsible for many effects within cells, usually do to it activating protein kinases. Examples are glucogen breakdown in liver, fatty acid production in adipose tissue, heart rate and blood pressure, water reabsorption in the kindey and bone resorption. If the effect of cAMP is increased or decreased activity depends on the effect of the protein kinases it activates. cAMP is continuously degraded by phosphodiesterase, so as long as the G-protein is not bound to the ligand, activating adenylyl cyclase, the signal will rapidly decline.

An example of cAMP activity is the breakdown of glycogen in the liver. Following binding of epinephrin to a G-protein linked receptor, adenylyl cyclase is activated and generates cAMP. cAMP binds to domains in protein kinase A (PKA), causing it to dissociate and activating a subunit of PKA. PKA phosphorylates phosphorylase kinase, which in it's turn phosphorylates phosphorylase a. This enzyme phosphorylates glucose to glucose-6-phosphate, which makes in unavailable for glucogen storage. Each activated enzyme can phosphorylate tens or hundreds of molecules, so the end result is that a single epinephrine can cause the phosphorylation of millions of glucose molecules. Thus the response is greatly amplified through the signaling cascade.

Another example is the regulation of gene transcription by cAMP. Again PKA is activated, but this time PKA phosphorylates CREB, which is a transcription factor enabling DNA transcription. This activates CREB so it can bind with CREB binding protein (CRB), a transcription co-factor. This results in the transcription of DNA. Which gene is transcribed depends on other factors.

IP3

Inositol triphosphate (IP3) is another secondary messenger important for many regulatory processes in the cell, especially calcium regulation. The cascade starts when the alpha subunit of an activated G-protein complex binds to phospholipase C. The phospholipase C cleaves a bond in phosphatidylinositolbiphosphate (PIP2) resulting in the formation of a free inositol triphosphate and the lipid diacylglycerol (DAG). In the case of calcium regulation, IP3 then goes on to bind to an IP3-gated Ca2+ channel in the ER, which causes the release of Ca2+ into the cell. DAG also plays a role, in that it, together with the increase of Ca2+ concentration in the cell, activates protein kinase C, which in it's turn regulates many processes. The increase in Ca2+ in itself also can cause certain effects. Some effects of this cascade are:

• Platelet activation
• Muscle contraction
• Insulin secretion
• Amylase secretion
• Glycogen degradation
• Antibody production

Other secondary messengers

Calcium in itself is an important secondary messenger, many proteins have Ca2+ binding sites. One important one is calmodulin, which when activated by Ca2+ binds to other proteins, often activating them.

An example of an entire cascade involving many secondary messengers is the dilation of blood vessels by relaxation of smooth muscle cells. Acetylcholine in the blood causes IP3 to be produced in the epithelial cells lining the blood vessel, which increases the Ca2+ concentration. Ca2+ activates calmodulin, which activates NO synthase. NO synthase produces nitric oxide from arginine, which diffuses across the membrane and into the smooth muscle cell. Here NO activates guanylyl cyclase (similar to adenylyl cyclase), which produces cGMP from GTP. cGMP activates protein kinase G, which causes the muscle cells to relax.

Tyrosin Kinase Receptors

An alternative type of surface receptor is the tyrosin kinase receptors. These are receptors for ligands such as insulin and various growth factors. The mechanism is different from the G-protein coupled receptor. Tyrosin kinase receptors bind to ligands, and then aggregate in the membrane. Thus the protein kinases are bound together two and two and activated by the ligand. They then phosphorylate each other's tyrosine residues (autophosphorylate). The phosphate groups provide binding sites for proteins with SH2 domains, such as GRB2. GRB2 can bind to Sos, a guanine exchange factor (GEF) for Ras. Ras is the monomeric G-protein mentioned above, which means that it is activated by binding GTP, which happens when it comes into contact with the tyrosin-coupled Sos GEF. The activated Ras causes a chain of phosphorylations. The first is Raf, which goes on to phosphorylate another protein kinase called MEK, which phosphorylate MAPKs. This activates a cascade of MAPKs phosphorylating other MAPKs, but in the end MAPKs activate certain transcription factors, thus regulating gene expression, or change protein activity.

Tyrosin Kinase receptors can also activate phospholipase C. It is a different subclass than the G-protein coupled receptors activate, but the effect is the same. Due to such crossovers, the activity of different receptors can influence each other. Some proteins can also require two different phosphorylations, each activated by a different receptor, to that the protein is only activated if two ligands are bound.

Other examples of kinase-type receptors that work by autophosphorylation are the transforming growth factor beta (TGF<math>\beta</math>), which is similar to tyrosin kinase, but instead the threonine and serine residues are phosphorylated. Other proteins called Smads are phosphorylated by the receptor complex, which results in gene expression regulation.

Cytoskeleton of the cell

Microtubulus

Microtubulus have three different modes in a cell. During normal (non-dividing) cell life, they form a network in the cytosol with a centrosome in the middle. During cell division they attach to the chromosomes and help pull them apart. They are also present in cilia. Microtubulus have a positive (alpha) and negative (beta) end due to all the monomers being oriented identically. A model of formation of microtubules in vitro is: first alpha and beta tubulin subunits dimerize and oligomerize. These protofilaments start growing on both ends of the microtubule structure forming as it elongates. When it reaches a certain size, the protofilaments polymerize and depolymerize equally quickly, and it reaches as stable phase. There are 13 protofilaments side by side in a microtubule. In vivo, there is also a dymic polymerizing and depolymerizing, but here it is polarized. The depolarizing happens at the minus end, while the polymerizing happens at the same rate at the positive end. The dynamic instability model shows how microtubuli can both grow and shrink. Alpha and beta tubulin both have GTP binding sites, and when they are bound to GTP they bind more strongly to one another. As GTP is hydrolyzed to GDP, this affinity weakens. Thus a growing microtubule has a large GTP cap at the plus end, while a microtubule lacking such a cap depolymerizes rapidly.

In a regular, non-dividing cell (interphase) the microtubuli are bound to centrosomes near the nucleus. The centrosomes consist of two perpendicular centrioles, which are nine sets of triplet microtubules (very short ones). They also have pericentriolar matter. The microtubules grow out from this pericentrolar matter. The role of the centrioles is not clear, but might be involved in assembling matter into the pericentriolar area for the microtubules. It is always the minus end of the microtubules that is bound to the centrosome, while the plus end extends into the periphery of the cell.

There are two main types of proteins associated with microtubules: the motor proteins and the non-motor proteins. The motor proteins, which include dynesins and kinesin, move along the microtubules towards a certain end (plus or minus). These can also bind vesicles, and thus are a very important way of transporting material quickly in a cell, especially in nerve cells where distances can be very long. The movement of these protein requires the hydrolysis of ATP. Kinesins have two feet, one with an ATP binding site, then a double-helix chain, a stalk, and a light chain area (vesicle binding site). The "feet" bind to the microtubule, and move by directed brownian motion towards the favorable direction. As mentioned above, releasing one of the feet requires ATP hydrolysis. Dynein moves in a similar fashion. Dynein binds to cargo vesicles via other proteins such as spectrin and ankyrin in the light chain ends.

A model for vesicle transport within the cell is as follows. The MTOC (centrosome) is located near the TGN, with microtubules extending in all directions. Kinesins, which move toward the plus end (away from MTOC) transport vesicles for exocytosis, as well as retrograde transport from Golgi to rough ER. Dynein moves the opposite way, transporting vesicles from rough ER to Golgi and endocytized material inward in the cell. The microtubules play an important role in arranging and maintaining cellular structure. ER and and Golgi themselves are attached to the microtubules by motor proteins. If nocodazole, a depolymerizing agent of microtubules, is added, fragmenting of the Golgi apparatus is seen together with the disappearance of microtubules.

Microtubules are also important for the movement of cilia and flagella. Cilia are cellular protusions that wave or beat in a cyclic (0.1-0.2 s/cycle) manner. Cilia consist of a pair of central microtubules connected to nine dimeric microtubules by radial spokes. The doublets are connected to each other by nexin connections and bound to dynein arms. The bending is due to the ATPase activity of dynein as well as the structure of the spokes and connections. A model for how dynein causes this bending is as follows: The dynein arm is bound to an adjacent microtubule from the one it is anchored to. ATP binding causes this to loosen. ATP hydrolysis causes the dynein arm to change conformation, bending 45 degrees axially (parallel to the microtubule). This shape allows the dynein to bind to a new position on the adjacent microtubule, and the dynein goes back to it's regular orthagonal position. This causes the adjacent microtubule to be shifted. Due to the crosslinks and spokes between the microtubule doublets, this sliding motion is transformed into a bending motion of the entire celia.

Microfilament

The microfilament, also called the actin filament, consists of two chains of intertwined filamentous actin (F-actin), which are formed by monomers of globular actin (G-actin) monomers. Like microtubules, actin filaments have a positive and negative end, and play somewhat similar roles in the cell. G-actin has a binding site for ATP, and, similar to the tubulin monomers of microtubules, when ATP is bound the positive end of the microfilament grows, while when ATP hydrolyzes to ADP the binding between the monomers weakens, causing depolymerization.

Proteins can regulate the properties of the microfilament assembly. Profilin increases growth speed by catalyzing the polymerization of G-actin at the plus end, while thymosin binds to G-actin so it can not polymerize, thus inhibiting growth. Capping proteins such as CapZ hinder the further growth, disassembly or linking of actin filaments, leaving individual actin strands. Crosslinking proteins such as filamin make networks, while bundling proteins such as <math>\alpha</math>-actin and fimbrin bundle the actin filaments (contractile or parallel), while proteins such as gelsolin can sever the filaments. GTAases such as Rac, Rho and Cdc42 greatly affect what the type and form of microfilaments that are produced in a cell. These are secondary messengers from growth factors (see above).

There are three main forms of microfilaments: The loose network in the cellular cortex, providing strength and stability to the plasma membrane, parallel bundles in in microvilli and filopodium, and contractile bundles such as stress fibers and in muscle cells. The loose network has filamin as a crossbinder, a protein with two orthagonal arms that crosslinks the actin filaments. The contractile bundles are relatively loosely packed, which allows myosin-II crosslinkers to enter the bundle. These are the main components of muscle contraction (see below). The parallel bundles are tightly packed with the fimbrin crosslinkers, excluding myosin from the bundles.

An example of microfilaments in a cell is in microvilli. Here are parallel bundles of microfilaments bound together by fimbrin and villin. The bundles are attached to the plasmamembrane by myosin I and calmodulin, with the positive end bound in cytosol and the negative end anchored at the tip of the microvilli.

Muscle contraction

Myosin is a family of motor proteins that bind to actin filaments. Myosin-I has a single head that binds to actin, and associated light chain, and a single tail. Myosin-II has two actin binding heads, two light chains and a double helix heavy-chain tail. Mysosin-IV is similar to myosin-II, but the tail is not fully entwined. Skeletal muscle consists of bundles of muscle cell, which are long and narrow. Within each muscle cell there are tight-packed bundles of myofibrils, which consists of actin and myosin contractile bundles. The repeating unit of the myofibril is called a sarcomer, and is about 2-3 <math>\mu</math>m long. The sarcomer has an isotropic (I-band) and anisotropic (A-band) area. The I-band consists of only actin filaments, or thin-filaments, while the A-band has both myosin and actin, called thick-filament. In the middle of the A band is the H-zone, where the myosin filaments are bound together, while in the middle of the I-zone is the Z-band, where the actin filaments are bound together. Many proteins play a role in maintaining this structure. <math>\alpha</math>-actinine maintains the shape of the actine bundles, while CapZ attaches the bundles to the Z-line (the positive end). Titin attaches the myosin filaments to the Z-line, while nebulin maintains the rigidity of the actin filaments. Tropomodulin binds to the negative end of the actin filaments, stabilizing them and regulating length. Myomesin is in the H-zone and bundles the myosin filaments to each other. The thick filament in the sarcomeres consist of hundreds of myosin-II proteins, all with their heads pointing away from the bundle (towards the actin filaments).

Skeletal muscles

The muscle contraction is explained best by the sliding-filament model. The protein called tropomyosin binds to seven actin monomers (axially), and prevents the binding of the myosin-II heads to the actin filament. A regulatory complex known as the troponin complex is bound between the tropomyesin and the actin filament. This complex consists of three subunits: the tropomyosin binding protein (TnT), the calcium binding troponin (TnC) and the inhibitory troponin (TnI). When the Ca2+ concentration of the cell increases, such as when a muscle contraction is to proceed, Ca2+ binds to the TnC part of troponin. This changes the conformation of troponin, so the binding site for the myosin-II heads on actin is opened. Now a low energy form of myosin (ADP-bound) binds to the actin filament. This binding releases energy, changing the conformation of myosin and releases the ADP. The conformation change causes the myosin bundle to move toward the positive end of the actin filament, i.e. towards the Z-line. This conformation is a low energy conformation, but binding of ATP causes the myosin head to release the actin filament, and ATP hydrolosis completes the conformation change to revert to the starting position. This will keep cycling until Ca2+ is not bound anymore, i.e. the signal for muscle contraction relinquishes. The actin moves 5 nm each of these cycles.

The signal for muscle contraction starts when acetyl choline is released in the synaptic cleft resulting from the action potential of a motor neuron. ACh binds to ligand gated Na+ channels, opening them and locally depolarizing the muscle cell. This causes other voltage gated Na+ channels to open up, and a new action potential along the T-tubules of the muscle cell is initiated. The T-tubules are located near the sarcoplasmic reticulum in the muscle cells, and the action potential causes voltage gated Ca2+ channels to open, releasing Ca2+ from the SR into the cell, which can the bind to the troponin complex.

Smooth muscles

In smooth muscles the actin filaments are organized in a more haphazard fashion, so contraction causes the contraction of the cell in all directions, not only one as in skeletal muscles. Contraction of smooth muscles is initiated by an increase in Ca2+ concentration from extracellular stores. The Ca2+ in the cell binds to calmodulin, which in turn binds to myosin-light-chain-kinase (MLCK). This activates MLK and causes the phosphorylation of the light chain of myosin-II. This allows myosin to bind to actin, and the contraction process explained above proceeds.

Cell migration

Another actin based form of movement i cell migration, or cell crawling. Although the mechanisms are note well understood, the following model seems to hold up:

• Actin at the leading edge polymerizes, causing extention into a lamellipodium.
• Adhesions anchered by actin to the substrate (filopodium) form under the lamellipodium.
• The trailing edge, or tail, of the cell detaches from the surface as the cell body contracts, moving forward.

In a stationary state, the retrograde movement of actin filaments (propelled by stationary myosin moving toward the positive end) is balanced by continued polymerization of the actin at the positive end. When cells move, the actin filament gets anchored to the substrate via integrin, but the polymerization continues. Thus, the myosin move the cell forward compared to the anchored actin. In the filopodium the actin form paralell bundles, but elsewhere in the migrating cells actin form fibrous networks, mediated by WASP and Arp2/3 proteins.

Neutrophils, a type of white blood cell, employ cellular migration to move towards harmful material with a receptor coupled mechanism. When anti-bodies from e.g. a bacteria bind to receptors on one side of the cell (but not the other), the cell activates a G-coupled protein cascade that activates the two kinases Rac and Rho, which travel to enhance or inhibit polymerization in the leading or trailing edge, respectively.

Another method of cell migration is seen in amoebas. These have a gel-like cortex and a liquid cytosol. The gel is much thicker in the trailing end, and it contracts. This causes the liquid part to extend forward, forming pseudopodium.

Intermediate filaments

Intermediate filaments consists of eight protofilaments that are joined end to end, with staggered overlaps. They can be formed by different types of proteins, depending on the function. They help support the cell and the nucleas, e.g. in the nuclear lamina, strengthen axons and bind adjacent cells together.

Intermediate filaments of various types are found at two mains sites: in the cytoplasm or in the nucleus. In the nucleus they form the nuclear lamina, which exists in all animal cells. In the cytoplasm the roles are more diversified: in epithelia they consist of keratins. In connective tissue, muscle cells and neuralglia vimentin and related proteins make up the intermediate filaments. In nerve cells there is a seperate type called neurofilaments.

The different proteins are similar in build up. They have a central domain of about 300 amino acids divided into 4 alpha-helix segments, with small linking areas in between. A model for the assembly of intermediate filaments (in vitro) is as follows. Two identical polypeptides bind together to form a dimer. These dimers align two and two next to each other and form tetramers. The tetramers align axially (along the filament), forming protofilaments. Eight of these protofilaments assemble into a tube 8-12 nm across, the intermediate filament.

Stress vs strain curves show that it is the intermediate filaments that contribute the most to cellular strength, microfilaments and microtubules have more specialized tasks. The intermediate filaments are bound to the other elements of the cytoskeleton by plectin.

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