Smooth muscle is an involuntary non-striated muscle. It is divided into two subgroups. Within single-unit cells, the whole bundle or sheet contracts as a syncytium. Smooth muscle cells are found in the walls of hollow organs, including the stomach, urinary bladder and uterus, in the walls of passageways, such as the arteries and veins of the circulatory system, the tracts of the respiratory and reproductive systems; these cells are present in the eyes and are able to change the size of the iris and alter the shape of the lens. In the skin, smooth muscle cells cause hair to stand erect in response to cold fear. Most smooth muscle is of the single-unit variety, that is, either the whole muscle contracts or the whole muscle relaxes, but there is multiunit smooth muscle in the trachea, the large elastic arteries, the iris of the eye. Single unit smooth muscle, however, is most common and lines blood vessels, the urinary tract, the digestive tract. However, the terms single- and multi-unit smooth muscle represents an oversimplification.
This is due to the fact that smooth muscles for the most part are controlled and influenced by a combination of different neural elements. In addition, it has been observed that most of the time there will be some cell to cell communication and activators/ inhibitors produced locally; this leads to a somewhat coordinated response in multiunit smooth muscle. Smooth muscle is fundamentally different from skeletal muscle and cardiac muscle in terms of structure, regulation of contraction, excitation-contraction coupling. Smooth muscle cells known as myocytes, have a fusiform shape and, like striated muscle, can tense and relax. However, smooth muscle tissue tends to demonstrate greater elasticity and function within a larger length-tension curve than striated muscle; this ability to stretch and still maintain contractility is important in organs like the intestines and urinary bladder. In the relaxed state, each cell is 20 -- 500 micrometers in length. A substantial portion of the volume of the cytoplasm of smooth muscle cells are taken up by the molecules myosin and actin, which together have the capability to contract, through a chain of tensile structures, make the entire smooth muscle tissue contract with them.
Myosin is class II in smooth muscle. Myosin II contains two heavy chains which constitute the tail domains; each of these heavy chains contains the N-terminal head domain, while the C-terminal tails take on a coiled-coil morphology, holding the two heavy chains together. Thus, myosin II has two heads. In smooth muscle, there is a single gene that codes for the heavy chains myosin II, but there are splice variants of this gene that result in four distinct isoforms. Smooth muscle may contain MHC, not involved in contraction, that can arise from multiple genes. Myosin II contains 4 light chains, resulting in 2 per head, weighing 20 and 17 kDa; these bind the heavy chains in the "neck" region between the head and tail. The MLC20 is known as the regulatory light chain and participates in muscle contraction. Two MLC20 isoforms are found in smooth muscle, they are encoded by different genes, but only one isoform participates in contraction; the MLC17 is known as the essential light chain. Its exact function is unclear, but it's believed that it contributes to the structural stability of the myosin head along with MLC20.
Two variants of MLC17 exist as a result of alternative splicing at the MLC17 gene. Different combinations of heavy and light chains allow for up to hundreds of different types of myosin structures, but it is unlikely that more than a few such combinations are used or permitted within a specific smooth muscle bed. In the uterus, a shift in myosin expression has been hypothesized to avail for changes in the directions of uterine contractions that are seen during the menstrual cycle; the thin filaments that form part of the contractile machinery are predominantly composed of α- and γ-actin. Smooth muscle α-actin is the predominant isoform within smooth muscle. There are lots of actin that does not take part in contraction, but that polymerizes just below the plasma membrane in the presence of a contractile stimulant and may thereby assist in mechanical tension. Alpha actin is expressed as distinct genetic isoforms such as smooth muscle, cardiac muscle and skeletal muscle specific isoforms of alpha actin.
The ratio of actin to myosin is between 10:1 in smooth muscle. Conversely, from a mass ratio standpoint, myosin is the dominant protein in striated skeletal muscle with the actin to myosin ratio falling in the 1:2 to 1:3 range. A typical value for healthy young adults is 1:2.2.. Tropomyosin is present in smooth muscle, spanning seven actin monomers and is laid out end to end over the entire length of the thin filaments. In striated muscle, tropomyosin serves to block actin–myosin interactions until calcium is present, but in smooth muscle, its function is unknown. Calponin molecules may exist in equal number as actin, has been proposed to be a load-bearing protein. Caldesmon has been suggested to be involved in tethering actin and tropomyosin, thereby enhance the ability of smooth muscle to maintain tension. All three of these proteins may have a role in inhibiting the ATPase activity of the m
Low-density lipoprotein is one of the five major groups of lipoprotein which transport all fat molecules around the body in the extracellular water. These groups, from least dense, compared to surrounding water to most dense, are chylomicrons low-density lipoprotein, intermediate-density lipoprotein, low-density lipoprotein and high-density lipoprotein. LDL delivers fat molecules to the cells and can drive the progression of atherosclerosis if they become oxidized within the walls of arteries, it is important to note that LDL is not "bad cholesterol". LDL is not cholesterol at all, not bad, it is an essential transport system for lipids the human body needs to survive, including cholesterol. There is both "large" and "small" particle LDL, while only small is associated with cholesterol-related issues, neither is "bad". "small" LDL is necessary to conduct nutrients to vessels that "large" LDL can't reach. Lipoproteins transfer lipids around the body in the extracellular fluid, making fats available to body cells for receptor-mediated endocytosis.
Lipoproteins are complex particles composed of multiple proteins 80–100 proteins/particle. A single LDL particle is about 220–275 angstroms in diameter transporting 3,000 to 6,000 fat molecules/particle, varying in size according to the number and mix of fat molecules contained within; the lipids carried include all fat molecules with cholesterol and triglycerides dominant. For years, it was believed that LDL particles posed a risk for cardiovascular disease when they invaded the endothelium and became oxidized, since the oxidized forms would be more retained by the proteoglycans, but there is growing evidence that this belief was supported by bad methodology, that there is no actual correlation between LDL and heart disease. A complex set of biochemical reactions regulates the oxidation of LDL particles, chiefly stimulated by presence of necrotic cell debris and free radicals in the endothelium. Increased concentrations of LDL particles is associated with the development of atherosclerosis over time.
Each native LDL particle enables emulsification, i.e. surrounding/packaging all fatty acids being carried, enabling these fats to move around the body within the water outside cells. Each particle contains a single apolipoprotein B-100 molecule, along with 80 to 100 additional ancillary proteins; each LDL has a hydrophobic core consisting of polyunsaturated fatty acid known as linoleate and hundreds to thousands esterified and unesterified cholesterol molecules. This core carries varying numbers of triglycerides and other fats and is surrounded by a shell of phospholipids and unesterified cholesterol, as well as the single copy of Apo B-100. LDL particles are 22 nm to 27.5 nm in diameter and have a mass of about 3 million daltons. Since LDL particles contain a variable and changing number of fatty acid molecules, there is a distribution of LDL particle mass and size. Determining the structure of LDL has been a tough task because of its heterogeneous structure; the structure of LDL at human body temperature in native condition, with a resolution of about 16 Angstroms using cryogenic electron microscopy, has been described.
LDL particles are formed as VLDL lose triglyceride through the action of lipoprotein lipase and they become smaller and denser, containing a higher proportion of cholesterol esters. When a cell requires additional cholesterol, it synthesizes the necessary LDL receptors as well as PCSK9, a proprotein convertase that marks the LDL receptor for degradation. LDL receptors are inserted into the plasma membrane and diffuse until they associate with clathrin-coated pits; when LDL receptors bind LDL particles in the bloodstream, the clathrin-coated pits are endocytosed into the cell. Vesicles containing LDL receptors bound to LDL are delivered to the endosome. In the presence of low pH, such as that found in the endosome, LDL receptors undergo a conformation change, releasing LDL. LDL is shipped to the lysosome, where cholesterol esters in the LDL are hydrolysed. LDL receptors are returned to the plasma membrane, where they repeat this cycle. If LDL receptors bind to PCSK9, transport of LDL receptors is redirected to the lysosome, where they are degraded.
LDL interfere with the quorum sensing system that upregulates genes required for invasive Staphylococcus aureus infection. The mechanism of antagonism entails binding Apolipoprotein B to a S. aureus autoinducer pheromone, preventing signaling through its receptor. Mice deficient in apolipoprotein B are more susceptible to invasive bacterial infection. LDL can be grouped based on its size: large low density LDL particles are described as pattern A, small high density LDL particles are pattern B. Pattern B has been associated by some with a higher risk for coronary heart disease; this is thought to be because the smaller particles are more able to penetrate the endothelium of arterial walls. Pattern I, for intermediate, indicates that most LDL particles are close in size to the normal gaps in the endothelium. According to one study, sizes 19.0–20.5 nm were designated as pattern B and LDL sizes 20.6–22 nm were designated as pattern A. Other studies have shown no such correlation at all; some evidence suggests the correlation between Pat
A biological membrane or biomembrane is an enclosing or separating membrane that acts as a selectively permeable barrier within living things. Biological membranes, in the form of eukaryotic cell membranes, consist of a phospholipid bilayer with embedded and peripheral proteins used in communication and transportation of chemicals and ions; the bulk of lipid in a cell membrane provides a fluid matrix for proteins to rotate and laterally diffuse for physiological functioning. Proteins are adapted to high membrane fluidity environment of lipid bilayer with the presence of an annular lipid shell, consisting of lipid molecules bound to surface of integral membrane proteins; the cell membranes are different from the isolating tissues formed by layers of cells, such as mucous membranes, basement membranes, serous membranes. The lipid bilayer consists of two layers - an inner leaflet; the components of bilayers are distributed unequally between the two surfaces to create asymmetry between the outer and inner surfaces.
This asymmetric organization is important for cell functions such as cell signaling. The asymmetry of the biological membrane reflects the different functions of the two leaflets of the membrane; as seen in the fluid membrane model of the phospholipid bilayer, the outer leaflet and inner leaflet of the membrane are asymmetrical in their composition. Certain proteins and lipids rest only on one surface of not the other. • Both the plasma membrane and internal membranes have cytosolic and exoplasmic faces • This orientation is maintained during membrane trafficking – proteins, glycoconjugates facing the lumen of the ER and Golgi get expressed on the extracellular side of the plasma membrane. In eucaryotic cells, new phospholipids are manufactured by enzymes bound to the part of the endoplasmic reticulum membrane that faces the cytosol; these enzymes, which use free fatty acids as substrates, deposit all newly made phospholipids into the cytosolic half of the bilayer. To enable the membrane as a whole to grow evenly, half of the new phospholipid molecules have to be transferred to the opposite monolayer.
This transfer is catalyzed by enzymes called flippases. In the plasma membrane, flippases transfer specific phospholipids selectively, so that different types become concentrated in each monolayer. Using selective flippases is not the only way to produce asymmetry in lipid bilayers, however. In particular, a different mechanism operates for glycolipids—the lipids that show the most striking and consistent asymmetric distribution in animal cells; the biological membrane is made up of lipids with hydrophilic heads. The hydrophobic tails are hydrocarbon tails whose length and saturation is important in characterizing the cell. Lipid rafts occur when lipid proteins aggregate in domains in the membrane; these help organize membrane components into localized areas that are involved in specific processes, such as signal transduction. Red blood cells, or erythrocytes, have a unique lipid composition; the bilayer of red blood cells is composed of cholesterol and phospholipids in equal proportions by weight.
Erythrocyte membrane plays a crucial role in blood clotting. In the bilayer of red blood cells is phosphatidylserine; this is in the cytoplasmic side of the membrane. However, it is flipped to the outer membrane to be used during blood clotting. Phospholipid bilayers contain different proteins; these membrane proteins have various functions and characteristics and catalyze different chemical reactions. Integral proteins span the membranes with different domains on either side. Integral proteins hold strong association with the lipid bilayer and cannot become detached, they will dissociate only with chemical treatment. Peripheral proteins are unlike integral proteins in that they hold weak interactions with the surface of the bilayer and can become dissociated from the membrane. Peripheral proteins create membrane asymmetry. Oligosaccharides are sugar containing polymers. In the membrane, they can be covalently bound to lipids to form glycolipids or covalently bound to proteins to form glycoproteins.
Membranes contain sugar-containing lipid molecules known as glycolipids. In the bilayer, the sugar groups of glycolipids are exposed at the cell surface, where they can form hydrogen bonds. Glycolipids provide the most extreme example of asymmetry in the lipid bilayer. Glycolipids perform a vast number of functions in the biological membrane that are communicative, including cell recognition and cell-cell adhesion. Glycoproteins are integral proteins, they play an important role in the immune protection. The phospholipid bilayer is formed due to the aggregation of membrane lipids in aqueous solutions. Aggregation is caused by the hydrophobic effect, where hydrophobic ends come into contact with each other and are sequestered away from water; this arrangement maximises hydrogen bonding between hydrophilic heads and water while minimising unfavorable contact between hydrophobic tails and water. The increase in available hydrogen bonding increases the entropy of the system, creating a spontaneous process.
Biological molecules are amphiphilic or amphipathic, i.e. are hydrophobic and hydrophilic. The phospholipid bilayer contains charged hydrophilic headgroups; the lipids contain hydrophobic tails, which meet with the hydrophobic tails of the complementary layer. The hydrophobic tails are fatty acids that differ in lengths; the interactions of lipids the hydrophobic tails, determine the lipid bilayer physical properties such as fluidity. Membranes in cells define enclosed spaces or compartments in which
An electron microscope is a microscope that uses a beam of accelerated electrons as a source of illumination. As the wavelength of an electron can be up to 100,000 times shorter than that of visible light photons, electron microscopes have a higher resolving power than light microscopes and can reveal the structure of smaller objects. A scanning transmission electron microscope has achieved better than 50 pm resolution in annular dark-field imaging mode and magnifications of up to about 10,000,000x whereas most light microscopes are limited by diffraction to about 200 nm resolution and useful magnifications below 2000x. Electron microscopes have electron optical lens systems that are analogous to the glass lenses of an optical light microscope. Electron microscopes are used to investigate the ultrastructure of a wide range of biological and inorganic specimens including microorganisms, large molecules, biopsy samples and crystals. Industrially, electron microscopes are used for quality control and failure analysis.
Modern electron microscopes produce electron micrographs using specialized digital cameras and frame grabbers to capture the images. In 1926 Hans Busch developed the electromagnetic lens. According to Dennis Gabor, the physicist Leó Szilárd tried in 1928 to convince him to build an electron microscope, for which he had filed a patent; the first prototype electron microscope, capable of four-hundred-power magnification, was developed in 1931 by the physicist Ernst Ruska and the electrical engineer Max Knoll. The apparatus was the first practical demonstration of the principles of electron microscopy. In May of the same year, Reinhold Rudenberg, the scientific director of Siemens-Schuckertwerke, obtained a patent for an electron microscope. In 1932, Ernst Lubcke of Siemens & Halske built and obtained images from a prototype electron microscope, applying the concepts described in Rudenberg's patent. In the following year, 1933, Ruska built the first electron microscope that exceeded the resolution attainable with an optical microscope.
Four years in 1937, Siemens financed the work of Ernst Ruska and Bodo von Borries, employed Helmut Ruska, Ernst's brother, to develop applications for the microscope with biological specimens. In 1937, Manfred von Ardenne pioneered the scanning electron microscope. Siemens produced the first commercial electron microscope in 1938; the first North American electron microscope was constructed in 1938, at the University of Toronto, by Eli Franklin Burton and students Cecil Hall, James Hillier, Albert Prebus. Siemens produced a transmission electron microscope in 1939. Although current transmission electron microscopes are capable of two million-power magnification, as scientific instruments, they remain based upon Ruska’s prototype; the original form of the electron microscope, the transmission electron microscope, uses a high voltage electron beam to illuminate the specimen and create an image. The electron beam is produced by an electron gun fitted with a tungsten filament cathode as the electron source.
The electron beam is accelerated by an anode at +100 keV with respect to the cathode, focused by electrostatic and electromagnetic lenses, transmitted through the specimen, in part transparent to electrons and in part scatters them out of the beam. When it emerges from the specimen, the electron beam carries information about the structure of the specimen, magnified by the objective lens system of the microscope; the spatial variation in this information may be viewed by projecting the magnified electron image onto a fluorescent viewing screen coated with a phosphor or scintillator material such as zinc sulfide. Alternatively, the image can be photographically recorded by exposing a photographic film or plate directly to the electron beam, or a high-resolution phosphor may be coupled by means of a lens optical system or a fibre optic light-guide to the sensor of a digital camera; the image detected by the digital camera may be displayed on a computer. The resolution of TEMs is limited by spherical aberration, but a new generation of hardware correctors can reduce spherical aberration to increase the resolution in high-resolution transmission electron microscopy to below 0.5 angstrom, enabling magnifications above 50 million times.
The ability of HRTEM to determine the positions of atoms within materials is useful for nano-technologies research and development. Transmission electron microscopes are used in electron diffraction mode; the advantages of electron diffraction over X-ray crystallography are that the specimen need not be a single crystal or a polycrystalline powder, that the Fourier transform reconstruction of the object's magnified structure occurs physically and thus avoids the need for solving the phase problem faced by the X-ray crystallographers after obtaining their X-ray diffraction patterns. One major disadvantage of the transmission electron microscope is the need for thin sections of the specimens about 100 nanometers. Creating these thin sections for biological and materials specimens is technically challenging. Semiconductor thin sections can be made using a focused ion beam. Biological tissue specimens are chemically fixed and embedded in a polymer resin to stabilize them sufficiently to allow ultrathin sectioning.
Sections of biological specimens, organic polymers, similar materials may require staining with heavy atom labels in order to achieve the required image contrast. One application of TEM is serial-section electron microscopy, for example in analyzing the connectivity in volumetric samples of brain tissue by imaging many thin sections in sequence; the SEM produces imag
Michael Stuart Brown
Michael Stuart Brown ForMemRS is an American geneticist and Nobel laureate. He was awarded the Nobel Prize in Physiology or Medicine with Joseph L. Goldstein in 1985 for describing the regulation of cholesterol metabolism. Brown was born in Brooklyn, New York, the son of Evelyn, a homemaker, Harvey Brown, a textile salesman, he graduated from Cheltenham High School. Brown graduated from the University of Pennsylvania in 1962 and received his M. D. from the University of Pennsylvania School of Medicine in 1966. Moving to the University of Texas Health Science Center in Dallas, now the UT Southwestern Medical Center and colleague Joseph L. Goldstein researched cholesterol metabolism and discovered that human cells have low-density lipoprotein receptors that extract cholesterol from the bloodstream; the lack of sufficient LDL receptors is implicated in familial hypercholesterolemia, which predisposes for cholesterol-related diseases. In addition to explaining the underlying pathology of this disease, their work uncovered a fundamental aspect of cell biology - receptor-mediated endocytosis.
Their findings led to the development of statin drugs, the cholesterol-lowering compounds that today are used by 16 million Americans and are the most prescribed medications in the United States. Their discoveries are improving more lives every year, both around the world. New federal cholesterol guidelines will triple the number of Americans taking statin drugs to lower their cholesterol, reducing the risk of heart disease and stroke for countless people. Following these important advances, their team of dedicated researchers elucidated the role of lipid modification of proteins in cancer. In 1984 he was awarded the Louisa Gross Horwitz Prize from Columbia University together with Joseph L. Goldstein. In 1988, Brown received the National Medal of Science for his contributions to medicine. In 1993, their trainees Xiaodong Wang and Michael Briggs purified the sterol regulatory element binding proteins. Since 1993, Drs. Brown and their colleagues have described the unexpectedly complex machinery by which cells maintain the necessary levels of fats and cholesterol in the face of varying environmental circumstances.
Dr. Brown holds the W. A. Moncrief Distinguished Chair in Arteriosclerosis Research. Mentioned as a candidate for nationally-prominent positions in scientific administration, Dr. Brown, like his colleague Joseph L. Goldstein, elects to continue hands-on involvement with research, leading a research team that includes a dozen doctoral and postdoctoral trainees, he and his colleague are among the most cited scientists in the world. Brown is on the prestigious Prix Galien USA Committee that "recognizes the technical and clinical research skills necessary to develop innovative medicines"; the inauguration of the Prix Galien in the US, the equivalent of the Nobel Prize in this field, was in September 2007, the winners were selected by a preeminent scientific and learned committee that included seven Nobel laureates, of which Brown was one. Brown has won numerous awards and honors, including
Dynamin is a GTPase responsible for endocytosis in the eukaryotic cell. Dynamin is part of the "dynamin superfamily", which includes classical dynamins, dynamin-like proteins, Mx proteins, OPA, GBPs. Members of the dynamin family are principally involved in the scission of newly formed vesicles from the membrane of one cellular compartment and their targeting to, fusion with, another compartment, both at the cell surface as well as at the Golgi apparatus. Dynamin family members play a role in many processes including division of organelles and microbial pathogen resistance. Dynamin itself is a 96 kDa enzyme, was first isolated when researchers were attempting to isolate new microtubule-based motors from the bovine brain. Dynamin has been extensively studied in the context of clathrin-coated vesicle budding from the cell membrane. Beginning from the N-terminus, Dynamin consists of a GTPase domain connected to a helical stalk domain via a flexible neck region containing a Bundle Signalling Element and GTPase Effector Domain.
At the opposite end of the stalk domain is a loop that links to a membrane-binding Pleckstrin homology domain. The protein strand loops back towards the GTPase domain and terminates with a Proline Rich Domain that binds to the Src Homology domains of many proteins. During clathrin-mediated endocytosis, the cell membrane invaginates to form a budding vesicle. Dynamin binds to and assembles around the neck of the endocytic vesicle, forming a helical polymer arranged such that the GTPase domains dimerize in an asymmetric manner across helical rungs; the polymer constricts the underlying membrane upon GTP binding and hydrolysis via conformational changes emanating from the flexible neck region that alters the overall helical symmetry. Constriction around the vesicle neck leads to the formation of a hemi-fusion membrane state that results in membrane fission, pinching off the vesicle from the parent membrane. Constriction may be in part the result of the twisting activity of dynamin, which makes dynamin the only molecular motor known to have a twisting activity.
In mammals, three different dynamin genes have been identified with key sequence differences in their Pleckstrin homology domains leading to differences in the recognition of lipid membranes: Dynamin I is expressed in neurons and neuroendocrine cells Dynamin II is expressed in most cell types Dynamin III is expressed in the testis, but is present in heart and lung tissue. Mutations in Dynamin II have been found to cause dominant intermediate Charcot-Marie-Tooth disease. Epileptic encephalopathy–causing de novo mutations in dynamin have been suggested to cause dysfunction of vesicle scission during synaptic vesicle endocytosis. Dynamins at the US National Library of Medicine Medical Subject Headings
In a neuron, synaptic vesicles store various neurotransmitters that are released at the synapse. The release is regulated by a voltage-dependent calcium channel. Vesicles are essential for propagating nerve impulses between neurons and are recreated by the cell; the area in the axon that holds groups of vesicles is an axon terminal or "terminal bouton". Up to 130 vesicles can be released per bouton over a ten-minute period of stimulation at 0.2 Hz. In the visual cortex of the human brain, synaptic vesicles have an average diameter of 39.5 nanometers with a standard deviation of 5.1 nm. Synaptic vesicles are simple because only a limited number of proteins fit into a sphere of 40 nm diameter. Purified vesicles have a protein:phospholipid ratio of 1:3 with a lipid composition of 40% phosphatidylcholine, 32% phosphatidylethanolamine, 12% phosphatidylserine, 5% phosphatidylinositol, 10% cholesterol. Synaptic vesicles contain two classes of obligatory components: transport proteins involved in neurotransmitter uptake, trafficking proteins that participate in synaptic vesicle exocytosis and recycling.
Transport proteins are composed of proton pumps that generate electrochemical gradients, which allow for neurotransmitter uptake, neurotransmitter transporters that regulate the actual uptake of neurotransmitters. The necessary proton gradient is created by V-ATPase. Vesicular transporters move neurotransmitters from the cells' cytoplasm into the synaptic vesicles. Vesicular glutamate transporters, for example, sequester glutamate into vesicles by this process. Trafficking proteins are more complex, they include intrinsic membrane proteins, peripherally bound proteins, proteins such as SNAREs. These proteins do not share a characteristic that would make them identifiable as synaptic vesicle proteins, little is known about how these proteins are deposited into synaptic vesicles. Many but not all of the known synaptic vesicle proteins interact with non-vesicular proteins and are linked to specific functions; the stoichiometry for the movement of different neurotransmitters into a vesicle is given in the following table.
It has been discovered that synaptic vesicles contain small RNA molecules, including transfer RNA fragments, Y RNA fragments and mirRNAs. This discovery is believed to have broad impact on studying chemical synapses; some neurotoxins, such as batrachotoxin, are known to destroy synaptic vesicles. The tetanus toxin damages vesicle-associated membrane proteins, a type of v-SNARE, while botulinum toxins damage t-SNARES and v-SNARES and thus inhibit synaptic transmission. A spider toxin called alpha-Latrotoxin binds to neurexins, damaging vesicles and causing massive release of neurotransmitters. Vesicles in the nerve terminal are grouped into three pools: the releasable pool, the recycling pool, the reserve pool; these pools are distinguished by their position in the nerve terminal. The releasable pool are docked to the cell membrane, making these the first group of vesicles to be released on stimulation; the releasable pool is small and is exhausted. The recycling pool is proximate to the cell membrane, tend to be cycled at moderate stimulation, so that the rate of vesicle release is the same as, or lower than, the rate of vesicle formation.
This pool is larger than the releasable pool, but it takes longer to become mobilised. The reserve pool contains vesicles; this reserve pool can be quite large in neurons grown on a glass substrate, but is small or absent at mature synapses in intact brain tissue. The events of the synaptic vesicle cycle can be divided into a few key steps: 1. Trafficking to the synapseSynaptic vesicle components are trafficked to the synapse using members of the kinesin motor family. In C. elegans the major motor for synaptic vesicles is UNC-104. There is evidence that other proteins such as UNC-16/Sunday Driver regulate the use of motors for transport of synaptic vesicles. 2. Transmitter loadingOnce at the synapse, synaptic vesicles are loaded with a neurotransmitter. Loading of transmitter is an active process requiring a neurotransmitter transporter and a proton pump ATPase that provides an electrochemical gradient; these transporters are selective for different classes of transmitters. Characterization of unc-17 and unc-47, which encode the vesicular acetylcholine transporter and vesicular GABA transporter have been described to date.
3. DockingThe loaded synaptic vesicles must dock near release sites, however docking is a step of the cycle that we know little about. Many proteins on synaptic vesicles and at release sites have been identified, however none of the identified protein interactions between the vesicle proteins and release site proteins can account for the docking phase of the cycle. Mutants in rab-3 and unc-18 alter vesicle docking or vesicle organization at release sites, but they do not disrupt docking. SNARE proteins, do not appear to be involved in the docking step of the cycle. 4. PrimingAfter the synaptic vesicles dock, they must be primed before they can begin fusion. Priming prepares the synaptic vesicle so that they are able to fuse in response to a calcium influx; this priming step is thought to involve the formation of assembled SNARE complexes. The proteins Munc13, RIM, RIM-BP participate in this event. Munc13 is thought to stimulate the change of the t-SNARE syntaxin from a closed conformation to an open conformation, which stimulates the assembly of v-SNARE /t-SNARE complexes.
RIM appears to regulate priming, but is not essential for the step. 5. FusionPrimed vesicles fuse quickly in respons