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ScienceWeek
CELL BIOLOGY: ON BAR DOMAINS
The following points are made by M.C. Lee and R. Schekman (Science 2004 303:479):
1) One of the basic needs of any growing cell is the ability to remodel the lipid membranes that both surround the cell and make up its intracellular organelles. At any point in time, a multitude of tiny bubbles of membrane known as vesicles are busy acting as couriers, picking up and delivering lipids and proteins with exquisite specificity. Although there is still much to be learned about how the many distinct flavors of vesicle are generated, one of the central themes that has emerged is that vesicle formation requires an interplay between the membrane lipids themselves and the cytosolic proteins that coat and deform the membrane. Of particular interest is how these coat proteins and additional effector molecules cooperate in a temporal and spatial manner to initiate membrane curvature, sculpt a vesicle of defined size, and pinch off the new bud.
2) The BAR domain -- named for the Bin-Amphiphysin-Rvs proteins in which it was first identified -- is found in proteins implicated in vesicle generation and membrane remodeling in mammals, Drosophila, and yeast (2). The founder of the BAR protein family, amphiphysin I, is enriched at synaptic nerve terminals in the brain where it helps to coordinate vesicle budding from the plasma membrane, a process known as endocytosis. It does this through interactions with a variety of other molecules, including the clathrin coat proteins, the AP2 complex whose job is to populate the vesicle with cargo proteins, and the guanosine triphosphatase (GTPase) dynamin, which is recruited to the constricted "neck" of an almost-budded vesicle to drive fission from the donor membrane (3,4). Amphiphysin thus seems well placed to couple the recruitment of cargo and coat proteins to regions of membrane curvature. But how is membrane bending initiated, and how is progress during budding monitored?
3) If you were to design a protein domain for detecting or imposing membrane curvature, you would likely come up with something that closely resembles the structure of the Drosophila BAR domain solved by Peter et al (1). It is, simply, a curve itself, although the crescent-shaped structure is only revealed upon dimerization of two slightly kinked monomers, which suggests that the domain functions only as a dimer. Intuition would probably tell you that the concave surface of the crescent would face the membrane. Peter et al (1) do show that mutation of positively charged amino acid residues in the concave part of the BAR domain abolishes the ability of the isolated domain to bind to negatively charged membranes and to induce them to form tubes. 4) The story becomes even more interesting as the authors identify previously unsuspected BAR domains in a variety of proteins such as nadrins, oligophrenins, centaurins, and sorting nexins. These proteins all participate in some way in membrane-remodeling events, and they present the BAR domain in the context of other lipid-sensing or effector domains. Thus, BAR-containing proteins are able to integrate multiple signals from their protein partners and from specific lipid species (such as phosphatidylinositol 4,5-bisphosphate) with regions of membrane curvature.(5)
References (abridged):
1. B. J. Peter et al., Science 303, 495 (2004); published online 26 November 2003 (10.1126/science.1092586).
2. B. Zhang, A. C. Zelhof, Traffic 3, 452 (2002)
3. M. Marsh, H. T. McMahon, Science 285, 215 (1999).
4. K. Takei, V. I. Slepnev, V. Haucke, P. de Camilli, Nature Cell Biol. 1, 33 (1999)
5. M. G. J. Ford et al., Nature 419, 361 (2002) [Medline]. K. Farsad, P. De Camilli, Curr. Opin. Cell Biol. 15, 372 (2003)
Science http://www.sciencemag.org
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ON SYNAPTIC VESICLE RELEASE KINETICS AND QUANTAL SIZE
The following points are made by M.E. Graham et al (Proc. Nat. Acad. Sci. 2002 99:7124):
1) It has been debated over many years whether regulated exocytosis always occurs through a pathway involving full fusion of the secretory vesicle or whether it can occur through a transient "kiss-and-run" (1) mechanism as first proposed to occur at the neuromuscular junction (2). In the classical pathway (3), full fusion of the vesicle with the plasma membrane allows complete emptying of the vesicle contents, and vesicle recycling then occurs through a dynamin-dependent pathway (4) involving clathrin-coated vesicles (5). Exocytosis involves the formation of a transient fusion pore. Recycling of the vesicle in kiss-and-run exocytosis could occur by rapid reclosure of such a pore. This mechanism would allow for very rapid recycling and reuse of synaptic vesicles that could be important to maintain neurotransmission. Rapid endocytosis on a time scale of seconds has been directly observed in neurons and neuroendocrine cells, and a dynamin-dependent but clathrin-independent form of endocytosis has been described as potentially underlying kiss-and-run events. It is clear, however, that a much faster type of kiss-and-run event can occur on a time scale of a few milliseconds. This third type of exo/endocytic event, which the authors term "fast kiss-and-run," could be important, if sufficiently rapid, as it could limit the amount released per vesicle and thereby modify quantal size.
2) Recent work has established the existence of kiss-and-run exocytosis of dense-core granules in adrenal chromaffin cells, and various experimental data are consistent with regulation of quantal size in these cells by partial release of vesicle contents because of fast kiss-and-run events. In addition, morphological evidence for partial release has come from the demonstration of the presence of extracellular markers after exocytosis in intracellular vesicles similar in size to chromaffin granules that still retained an electron-dense core. The use of lipophilic FM dyes to follow vesicle cycling has also suggested the existence of fast kiss-and-run exocytosis in synaptosomes and in synapses of hippocampal neurons based on discrepancies between dye destaining and neurotransmitter release. The existence of kiss-and-run exocytosis in neurons is, however, controversial and not supported by other evidence. The use of carbon-fiber amperometry to detect release of catecholamines from single granules of chromaffin cells has shown more directly that the kinetics of release events and the extent of release can be modified by manipulation of the expression of proteins involved in the exocytotic machinery and by changes in second messenger pathways. Importantly, the continuous variation in amounts released per exocytotic event after acute changes in stimulation parameters is consistent with control through fast kiss-and-run exocytosis rather than by recruitment of different sized granules.
3) In summary: Accumulating evidence suggests that the kinetics of release from single secretory vesicles can be regulated and that quantal size can be modified during fast kiss-and-run fusion. Multiple pathways for vesicle retrieval have been identified involving clathrin and dynamin. It has been unclear whether dynamin could participate in a fast kiss-and-run process to reclose a transient fusion pore and thereby limit vesicle release. The authors report they have disrupted dynamin function in adrenal chromaffin cells by expression of the amphiphysin Src-homology domain 3 (SH3) or by application of guanosine 5'-[-thio]triphosphate (GTPS), and have monitored single vesicle release events, evoked by digitonin and Ca2+, by using carbon-fiber amperometry. Under both conditions, there was an increase in mean quantal size accompanying an increase in the half-width of amperometric spikes and a slowing of the fall time. The authors suggest these data indicate the existence of a dynamin-dependent process that can terminate vesicle release under basal conditions. Protein kinase C activation changed release kinetics and decreased quantal size by shortening the release period. The effects of phorbol ester treatment were not prevented by expression of the amphiphysin SH3 domain or by GTPS, suggesting the existence of alternative dynamin-independent process underlying fast kiss-and-run exocytosis.
References (abridged):
1. Fesce, R. , Grohovaz, F. , Valtorta, F. & Meldolesi, J. (1994) Trends Cell Biol. 4, 1-4
2. Ceccarelli, B. , Hurlburt, W. P. & Mauro, A. (1973) J. Cell Biol. 57, 499-524
3. Heuser, J. E. & Reese, T. S. (1973) J. Cell Biol. 57, 315-344
4. Koenig, J. H. & Ikeda, K. (1989) J. Neurosci. 11, 3844-3860
5. Cremona, O. & De Camilli, P. (1997) Curr. Opin. Cell Biol. 7, 323-330
Proc. Nat. Acad. Sci. http://www.pnas.org
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CAPACITANCE MEASUREMENTS REVEAL STEPWISE FUSION EVENTS IN DEGRANULATING MAST CELLS.
The following points are made by J.M. Fernandez et al (Nature 1984 312:453):
1) Mast cells undergo an extensive and violent morphological transformation on stimulation. The authors describe the dynamics of fusion of the secretory granules in individual mast cells during exocytosis. The cell membrane capacitance (proportional to the cell surface area) was measured using the whole-cell patch-pipette technique, in which the intracellular space is dialyzed with the solutions used to fill the patch pipette.
2) The results show that degranulation occurs spontaneously and reproducibly if the GTP analogue, GTP-gamma-S, and Mg-ATP are present in the pipette filling solution. Contrary to previous reports, in these conditions Ca2+ (and/or Ca2+ buffers) is not required for degranulation. Although electrogenic Ca2+ entry was not detected before or during degranulation and membrane conductance remained low, the capacitance, and by implication the area of the membrane of degranulating cells, increased sigmoidally and stepwise.
3) The authors conclude that stepwise increases of capacitance are due to the fusion of individual secretory granules with the plasma membrane, and that guanine nucleotide regulatory proteins are involved in the control of this process.
Nature http://www.nature.com/nature
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