Revisjonen fra 28. feb. 2009 kl. 20:01

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.

Fluorensmikroskopilab:

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, 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.

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