Pietro De Camilli MD
Eugene Higgins Professor of Cell Biology and Professor of Neurobiology; Director, Yale Program in Cellular Neuroscience and Neurodegeneration and Repair
Synapses; Exocytosis; Endocytosis; Membranes; Clathrin; Dynamin; Phosphoinositides; Lowe syndrome; BAR proteins; Neurodegeneration
Current ProjectsWe are interested in the cell biology of neuronal synapses, with emphasis on the biogenesis and traffic of synaptic vesicles, the special secretory organelles that store and release neurotransmitters. Our goal is to learn about synaptic transmission but also to advance general knowledge in the field of membrane traffic. Towards this objective we use a variety of neuronal and non-neuronal model systems. We are particularly interested in the role of phospholipid metabolism in the regulation of endocytic membrane traffic. Some of our projects focus on human diseases resulting from abnormal membrane dynamics.
Available rotation projects:
Characterization of synaptic function in mice harboring mutations of genes implicated in synaptic transmission (neuronal cultures, light and electron microscopy, biochemical studies).
Phosphoinositide metabolism in membrane traffic (lipid studies, characterization of phosphoinositide metabolizing enzymes, effect of their disruption on membrane traffic). Mechanisms of human diseases resulting from abnormal phosphoinositide metabolism
Mechanisms in clathrin mediated endocytosis (protein-protein and protein-lipid interaction studies; mechanisms in membrane deformation; cell free assays of vesicle budding).
Our lab capitalizes extensively on advanced imaging methods (light and electron microscopy)
We study mechanisms underlying the development and function of neuronal synapses. Synapses are specialized contact sites between neurons, or between neurons and muscle, 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. In addition, we exploit the unique structural and functional features of synapses to learn about general principles in cell biology and more specifically about mechanisms in intracellular membrane traffic.
Extensive Research Description
CELL BIOLOGY OF SYNAPTIC TRANSMISSION. MECHANISMS IN ENDOCYTIC 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 major 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) and 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.
A major pathway of synaptic vesicle recycling is clathrin-dependent endocytosis. 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 of the deeply invaginated bud, uncoating of the newly formed vesicles, 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 use a variety of complementary approaches - including biochemistry, structural biology methods, cell-free systems, dynamic light microscopy imaging of live cells, super-resolution microscopy methods and mouse genetics - to understand these events and their regulation. We are also investigating the potential occurrence of clathrin independent pathways of synaptic vesicle reformation.
The Fission Reaction of Endocytosis
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. As we continue to investigate the precise mechanism of action of dynamin in fission, we investigate the function of other factors that cooperate with dynamin in this process. They include proteins with membrane-deforming properties (see below) and the actin-based cytoskeleton.
We are also gaining insight into dynamin’s function from the knockout of the three mouse dynamin genes, all of which are expressed in the brain. This work has already yielded surprises and provided new insight into synaptic vesicle–recycling mechanisms. We found that dynamin 1, a neuron-specific protein and by far the major dynamin isoform in the brain, is not essential for synaptic vesicle recycling, but is needed to allow efficient synaptic vesicle reformation under condition of intense activity. Dynamin 2, the ubiquitous dynamin isoform, is essential for embryonic development but not for neuronal function, thus indicating that it does not have an essential house keeping role independent of the other dynamins. No obvious phenotypic defects are observed in dynamin 3 knockout mice. Work is in progress to address the functional consequence of ablating expression of all 3 dynamin genes in neurons or in selected non-neuronal cells.
Properties and function of endosomes in nerve terminals
The role of “endosomes” in synaptic vesicle recycling remains unclear. Strong and prolonged stimulation of synapses leads to synaptic vesicle depletion and to a transient accumulation of cisternae that only slowly convert to new synaptic vesicles. At least some of these structures appear to form by bulk endocytosis, a clathrin independent form of endocytosis. While the occurrence of these endocytic intermediates has been known for decades, their properties and the mechanism through which they generate synaptic vesicles are not known. Furthermore, their relation to endosomes that play housekeeping functions in all cells remains elusive. It is expected that the elucidation of the biochemical, structural and functional characterization of endosome-like structures of the presynapse will shed new light on vesicle recycling mechanisms. Ongoing work investigates the properties of these organelles at synapses of wild type and mutant mice. Such studies are complemented by investigations of endosomal traffic and its regulation in non-neuronal cells. Live cell imaging and electron tomography feature prominently in our tool-box for these projects.
Endocytic Proteins, Bilayer Deformation, Curvature Sensing
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. In collaboration with Vinzenz Unger (Yale University), we continue our structural studies of these proteins with emphasis on their structure in the membrane-bound state. We are also investigating the precise cellular function of selected members of these protein families, the endophilins in particular, in membrane fission and actin regulation.
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 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 the function of 3-phosphorylated phosphoinositides in progression of endocytic traffic. Such studies are closely interconnected with our studies of endosomes at synapses. Surprisingly, 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, including neurological and psychiatric diseases, most phosphoinositide-metabolizing enzymes are still poorly characterized. The involvement of some of these enzymes in diseases of the nervous system is being investigated.
An important current focus of our lab is the inositol 5-phosphatase OCRL, whose mutations are responsible for the oculocerebrorenal syndrome of Lowe and for Dent disease, two rare but severe human conditions involving kidney reabsorption defects and, in the case of Lowe syndrome, also mental retardation and congenital cataract. 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 new interactors of OCRL, including the endocytic adaptor APPL1, and we are currently characterizing their physiological functions and their potential role in disease. We hope to elucidate the precise relationship between OCRL mutations and the clinical manifestations of the Lowe syndrome and Dent disease toward the goal of developing therapeutic strategies for these conditions.