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Molecular Mechanisms in Membrane Traffic at the Neuronal Synapse
Summary: Pietro De Camilli is interested in understanding molecular mechanisms in presynaptic function and the role of phosphoinositide metabolism in the regulation of membrane traffic.
We study the mechanisms underlying the development and function of neuronal synapses. Synapses are specialized contact sites between neurons, or between neurons and muscles, where electrical signals are propagated from cell to cell via chemical intermediates called neurotransmitters. Our long-term goal is to advance the understanding of nervous system function in health and disease. We also exploit the unique structural and functional features of synapses to learn about general principles of cell biology. A main focus of our research is the elucidation of the mechanisms responsible for the biogenesis and traffic of synaptic vesicles, the secretory organelles that store and secrete fast-acting neurotransmitters. Synaptic vesicles deliver their content into the synaptic space by fusing with the plasma membrane (exocytosis). They are rapidly re-formed by the recycling of their membranes via endocytosis. Studies of these organelles are thus relevant for the understanding of mechanisms involved in the secretory and endocytic pathways in all cells.
Over the past several years, our studies on synaptic vesicles have addressed primarily their recycling by clathrin-dependent endocytosis, although the potential involvement of alternative pathways is also being explored. Generation of a clathrin-coated vesicle implies a precise and ordered sequence of events, such as clustering of protein cargo, acquisition of curvature and invagination, fission, uncoating, and translocation away from endocytic sites. Although most of the players in these reactions have been identified, their mechanisms of action, in many cases, remain unclear. We are using a variety of complementary approaches—including biochemical and structural biology studies, studies of cell-free systems, imaging studies of live cells, and mouse genetics—to understand these events and their regulation by phosphorylation and ubiquitination.
Dynamin and the Fission Reaction
Fission—the physical separation of an endocytic bud from the plasma membrane—is the “defining” event of endocytosis. Dynamin, an unconventional GTPase that oligomerizes into rings and spirals at the neck of endocytic buds, has long been known to be a critical player in this reaction, both at synapses, and in other systems. Its precise action continues to be elusive, however. In recent studies, carried out with liposomes and purified dynamin, we provided strong evidence for a GTP hydrolysis–dependent twisting action of the dynamin spiral, leading to fission. Our data, however, also suggest that additional factors are needed to promote the transition from constriction to fission. We are investigating the role of some of these factors. They include proteins with membrane-deforming properties and the actin-based cytoskeleton.
We are also gaining insight into dynamin's function from gene-knockout studies. So far, much of what was known about the role of dynamin in the physiology of cells and of intact organisms was based on dominant-negative interference studies. Mammals have three dynamin genes, all of which are expressed in the brain. Our work has already yielded surprises and provided new insight into synaptic vesicle–recycling mechanisms. Dynamin 1, a neuron-specific protein and by far the major dynamin isoform in the brain, was expected to be the dynamin specifically dedicated to, and essential for, synaptic vesicle recycling. Unexpectedly, mice lacking dynamin 1 appear normal at birth and have a functioning nervous system, although they subsequently fail to thrive. Studies of their synapses revealed the occurrence of an endocytic defect, such as a prominent, activity-dependent accumulation of clathrin-coated pits. Dynamin 1–knockout mice, however, were also found to have the surprising ability to generate synaptic vesicles by a dynamin 1–independent mechanism. Dynamin 2, the ubiquitous dynamin isoform, is essential for embryonic development but not for neuronal function. No obvious phenotypic defects are observed in dynamin 3–knockout mice. Work is in progress to generate and analyze mice lacking expression of two or all three isoforms in selected tissues.
Endocytic Proteins and Bilayer Deformation
Research from our lab pioneered the concept that protein modules that bind and deform the bilayer play a role in the generation of membrane curvature at endocytic sites. Two such modules are BAR and F-BAR domains. Both BAR and F-BAR domains are present in many proteins that bind dynamin and/or regulate actin function and are therefore thought to cooperate with dynamin in fission. One of our goals is to understand the mechanisms through which these proteins deform membranes. In this field, we are collaborating with Vinzenz Unger (Yale University) to determine, via cryoelectron microscopy, the structure of these domains in their membrane-bound state. Another goal is to use cell biology and mouse genetics to elucidate the function of selected members of these protein families in membrane fission and actin regulation.
In another line of study, we are investigating the function and mechanism of action of epsin, a clathrin adapter identified in our lab. Epsin has also been proposed to play a critical role in the generation of curvature at endocytic clathrin-coated pits via its ENTH domain.
Phosphoinositides and Membrane Traffic
Phosphorylation of phosphatidylinositol at the 3, 4, and 5 position of the inositol ring results in seven phosphoinositides that play a major role in cell signaling. Because of their heterogeneous subcellular distributions and their ability to bind proteins at the membrane-cytosol interface, phosphoinositides are critical determinants of organelle identity and major regulators of vesicular transport. Following our identification of the polyphosphoinositide phosphatase synaptojanin 1 as a protein neighbor of dynamin and clathrin, and then of a cycle of PI(4,5)P2 synthesis and hydrolysis nested within the synaptic vesicle cycle exo-endocytic cycle, we have become more generally interested in the role of these phospholipids in orchestrating membrane traffic in neurons and other cells. We have expanded our work to investigate the role in exo-endocytic traffic of other phosphoinositide-metabolizing enzymes, and of other phosphoinositides besides PI(4,5)P2. Surprisingly, many phosphoinositide-metabolizing enzymes are still poorly characterized, despite the importance of phosphoinositides in cell physiology and growing evidence for the role of abnormal function of some of these enzymes in human diseases.
An important current focus of our lab is the inositol 5-phosphatase OCRL, whose mutations are responsible for the oculocerebrorenal syndrome of Lowe, a rare but severe human condition. We have demonstrated a major site of action of this phosphatase at early stations of the endocytic pathway, including endocytic clathrin-coated pits, where its function may overlap with the function of synaptojanin in coupling endocytosis to PI(4,5)P2 and PI(3,4,5)P3 dephosphorylation. We have also identified and characterized an interaction of OCRL with the endocytic adapter APPL1, which is abolished by patient mutations. We are particularly interested in a protein network that may link OCRL and APPL1 to the scavenger receptor megalin and to the nerve growth factor receptor TrkA, because perturbation of the traffic of these receptors may account for some of the phenotypic manifestations of Lowe syndrome patients. We have also determined the crystal structure of the carboxyl-terminal region of OCRL and shown how this region, which contains a RhoGAP-like domain and a newly defined ASH domain, may help to target and orient the catalytic site of the protein at the membrane interface.
Last updated: March 19, 2008
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