Getting the membrane into shape for endocytosis.

Clathrin-mediated endocytosis serves to regulate membrane composition and area in most cells. A specialized cellular structure where this occurs with some speed, and is of immediate interest to neuroscientists, is the synapse. Neurotransmitter release occurs by exocytosis of synaptic vesicles; subsequent endocytosis is essential to clear out the excess membrane and recover the vesicular components for reuse. Assuming an average release rate of one vesicle per second, a rough calculation indicates that an area equivalent to the entire synaptic bouton will turn over in just about 10 min at the small synapses of hippocampal pyramidal neurons. Although the exact proportion of turnover mediated by clathrin-mediated endocytosis is unknown (other modes such as kiss-and-run are thought to exist), a large body of evidence points to a critical role for clathrin-dependent mechanisms in synaptic vesicle recycling.Detailed information about clathrin coat formation has begun to accumulate as a result of many elegant studies (reviewed recently by Marsh and McMahon 1999xMarsh, M. and McMahon, H.T. Science. 1999; 285: 215–220Crossref | PubMed | Scopus (396)See all ReferencesMarsh and McMahon 1999). After fusion of synaptic vesicles with the plasma membrane, adaptor proteins are thought to assist in recruiting clathrin coat components to the vesicular membrane. Through a series of protein–protein and protein–lipid interactions, the coated bud emerges and is pinched off to become a coated vesicle. Several proteins important for clathrin-mediated endocytosis have been identified, and their precise roles at different stages of vesicle formation are under intense experimental scrutiny.What induces the requisite large changes in curvature of the coated bud as it grows from the plasma membrane? To form small synaptic vesicles (diameter around 35 nm), the relatively flat plasma membrane needs to undergo a radical change in its curvature. As the vesicle forms, different curvatures occur in the interior of the bud and the edge (see figure). The negative curvature at the edge deepens as the bud progresses to become a coated vesicle. It is conceivable that this alteration of curvature is driven by the assembly of the clathrin coat. Two recent papers, including one by Ringstad et al. 1999xRingstad, N., Gad, H., Low, P., Di Paolo, G., Brodin, L., Shupliakov, O., and De Camilli, P. Neuron. 1999; 24: 143–154Abstract | Full Text | Full Text PDF | PubMed | Scopus (222)See all ReferencesRingstad et al. 1999 in this issue of Neuron, make a case for a role of lipid modification in the formation of clathrin-coated vesicles.A search for SH3 (Src homology 3) domain–containing proteins, as well as a search for interacting partners of dynamin and synaptojanin (two proteins important for synaptic vesicle endocytosis), led to the identification of endophilins. NowSchmidt et al. 1999xSchmidt, A., Wolde, M., Thiele, C., Fest, W., Kratzin, H., Podtelejnikov, A.V., Witke, W., Huttner, W.B., and Soling, H.-D. Nature. 1999; 401: 133–141Crossref | PubMed | Scopus (421)See all ReferencesSchmidt et al. 1999 report that endophilin I is an enzyme that modifies phospholipids in the bilayer membrane. Recall that phospholipids, which are important constituents of membranes, consist of a polar head group and hydrophobic fatty acid tails. Endophilin I acts to transfer a fatty acyl group to lysophosphatidic acid (a phospholipid with one fatty acid tail missing) to convert it to phosphatidic acid (normal “two-tailed” phospholipid). Since lysophosphatidic acid is thought to have an inverted-cone shape and phosphatidic acid is cone-shaped, endophilin I might serve to modify membrane curvature during coated-vesicle formation. Using a perforated PC12 system that can generate synaptic-like microvesicles (SLMVs), Schmidt et al. also find that adding recombinant endophilin I to protein-depleted cytosol increases formation of SLMVs; this increase can be countered by presenting competing substrates. Other experiments with exogenous lipid substrates for endophilin also suggested that it is the acyl group–transferring activity of endophilin that is important for generation of SLMVs. Finally, the authors also demonstrate that the association of endophilin I with dynamin is essential for the formation of SLMVs.Despite the great degree of similarity between secretory and recycling pathways of different cells, there is always the worry that the highly specific and rapid recycling at neuronal synapses is different in its details. An independent study by Ringstad et al. 1999xRingstad, N., Gad, H., Low, P., Di Paolo, G., Brodin, L., Shupliakov, O., and De Camilli, P. Neuron. 1999; 24: 143–154Abstract | Full Text | Full Text PDF | PubMed | Scopus (222)See all ReferencesRingstad et al. 1999 in this issue of Neuron dispels this worry. They use the lamprey reticulospinal giant axon to show that microinjection of antibodies to endophilin leads to accumulation of coated pits at stimulated synapses. A previous collaborative study by this group of authors had shown that disrupting the SH3 domain binding of amphiphysin leads to the accumulation of coated pits at the synapse—these pits were deeply invaginated and the defect was thought to be in the final fission reaction. What is remarkable in the present study is that these coated pits are arrested at a stage before deep invagination, as if they are unable to become more curved. Quantitative analysis suggested that greater concentrations of antibody at the terminal lead to shallower coated pits with less negative membrane curvature at the edge. Interestingly, Ringstad et al. find that endophilin does not affect coat formation in a cell-free membrane system. They suggest that this might be because free membranes are floppy and perhaps have a higher degree of flexibility. Immunogold electron microscopy indicated that endophilin does not directly associate with clathrin coats—this argues against a direct role for endophilin in altering the curvature of the clathrin coat itself.Ringstad et al. 1999xRingstad, N., Gad, H., Low, P., Di Paolo, G., Brodin, L., Shupliakov, O., and De Camilli, P. Neuron. 1999; 24: 143–154Abstract | Full Text | Full Text PDF | PubMed | Scopus (222)See all ReferencesRingstad et al. 1999 discuss two possible models of endophilin function that are consistent with their results. First, endophilin might alter the tension of the membrane by acting on cytoskeletal elements such as actin and thereby promote invagination of vesicular membrane. Second, endophilin might have a direct effect on membrane lipids. The results of Schmidt et al. 1999xSchmidt, A., Wolde, M., Thiele, C., Fest, W., Kratzin, H., Podtelejnikov, A.V., Witke, W., Huttner, W.B., and Soling, H.-D. Nature. 1999; 401: 133–141Crossref | PubMed | Scopus (421)See all ReferencesSchmidt et al. 1999 support the second model (without necessarily excluding the first). That is, the arrest of coated pits at a shallow stage observed when endophilin is blocked by antibodies may be due to the absence of lipid modification that permits sharp negative curving of the cytoplasmic side of the membrane. Another interesting issue raised by the two studies is the precise role of dynamin. In recent discussions, dynamin has been assigned a role in the late stages of clathrin-coated vesicle formation—particularly in the fission of the budding vesicle. The two studies discussed here, together with the observation that dynamin interacts with endophilin and colocalizes with it, suggest that dynamin is also involved in early stages in addition to its proposed role in fission.Interesting papers tend to raise more questions than they answer. Is endophilin I restricted to the edge of the growing coat? If so, how and why? Some evidence presented by Ringstad and colleagues supports this peripheral localization of endophilin I in the clathrin-coated bud. Perhaps steric hindrance prevents endophilin from being included inside the coat, and clathrin assembly occurs only after the enzyme is displaced. It may also be efficient to have the reaction leading to lipid shape alteration occur at the edge of the growing bud, where the curvature needs to be changed the most. As the bud grows, what was once the edge (with negative curvature) gradually becomes part of the interior membrane (with positive curvature)—so having cone-shaped lipids inside no longer makes sense. Do cone-shaped lipids stay at the edge by continually moving laterally toward the edge of the growing coat? Does endophilin-mediated lipid modification actually provide the driving force to form appropriately curved membrane and clathrin coats? Since the lipid composition of the vesicle membrane is modified during its formation, a reverse modification must occur elsewhere if equilibrium is to be maintained. Does this reverse modification occur after the vesicle fusion event? The pace of investigation in this field guarantees speedy answers to these and other questions.Figure 101Progression of the Bilayer Membrane during EndocytosisGray lines indicate the membrane, black triangles indicate cone-shaped lipids, and white triangles indicated inverted-cone-shaped lipids in the cytoplasmic leaflet of the membrane. The black dots demonstrate how some membrane locations in the inner leaflet have negative curvature in the early stages and positive curvature in the later stages.View Large Image | View Hi-Res Image | Download PowerPoint Slide