The cytoplasm of eukaryotic cells is compartmentalized by intracellular membranes that are specialized for distinct functions and that are functionally interconnected by membrane traffic. We study mechanisms underlying their dynamics, with emphasis on membrane traffic reactions involved in neurotransmission. Our long-term goal is to advance our 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 membrane dynamics and transport. A major focus of our research is to elucidate the mechanisms responsible for the biogenesis and traffic of synaptic vesicles—the secretory organelles that store and secrete fast-acting neurotransmitters.
To understand these events and their regulation, we use a variety of complementary approaches, including biochemical and structural studies, cell-free systems, dynamic light microscopy imaging of live cells, super-resolution microscopy methods, and mouse genetics.
Synaptic Vesicle Recycling
The formation of synaptic vesicles represents an event of fundamental importance in neurobiology. The fidelity of synaptic transmission depends on mechanisms that conserve the homogeneous properties and size of synaptic vesicles—key factors ensuring the vesicles' constant neurotransmitter content. Synaptic vesicles are generated and then continually regenerated by endocytosis of membrane components that have been incorporated into the plasma membrane by exocytosis. Vesicle formation implies a well orchestrated series of events leading to membrane shape changes, cargo protein selection by the newly formed membrane bud, scission of the bud neck to generate an endocytic vesicle, and endocytic vesicle maturation to a new, fully functional synaptic vesicle. Our lab has contributed to the elucidation of mechanisms involved in some of these events—clathrin-mediated endocytosis in particular. Current work of the lab continues to focus on (1) the role and mechanisms of action of the GTPase dynamin and its accessory factors in membrane fission, (2) the function of BAR domain–containing proteins as sensors and stabilizers of membrane curvature that help recruit/regulate other factors at endocytic sites, and (3) the role of metabolic changes in bilayer lipid in vesicle membrane dynamics. We expect that these studies will have broad implications in the field of endocytosis. Additional importance of this work comes from genome-wide association studies implicating endocytic proteins in Alzheimer's disease and from genetic studies in mice and humans that have revealed mutations in endocytic proteins in familial forms of Parkinson's disease. The link between some of the proteins studied in our lab and a protein network whose dysfunction may underlie Parkinson's disease is being investigated.
We are also exploring the role of clathrin-independent re-formation of synaptic vesicles. Strong and prolonged stimulation of synapses leads to synaptic vesicle depletion and a transient accumulation of large vacuoles, which form by bulk endocytosis and only slowly convert to new synaptic vesicles. While the occurrence of these endocytic intermediates has been known for decades, the mechanism through which they generate synaptic vesicles and their relation to endosomes that play housekeeping functions in all cells remain elusive.
Phosphoinositides and Membrane Traffic
Phosphoinositides, the phospholipids that derive from the phosphorylation of phosphatidylinositol at the 3, 4, and 5 position on the inositol ring, are signaling lipids heterogeneously localized on different cell membranes. Our studies of synaptic vesicle recycling demonstrated the role of plasma membrane phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] in the control of exo- and endocytosis and showed that PI(4,5)P2 dephosphorylation after endocytosis is a strict requirement for the maturation of newly formed endocytic vesicles into synaptic vesicles. These findings have converged with results of other labs to demonstrate that phosphoinositides are critical determinants of membrane identity, with major regulatory functions in vesicular transport. Building on these studies, we have become more generally interested in the role of these phospholipids in orchestrating membrane traffic in neurons and other cells, with a special emphasis on the interconversion of various phosphoinositide species in the endocytic pathway.
An important focus of our research in this area continues to be the role of enzymes that dephosphorylate PI(4,5)P2, such as synaptojanin, the major inositol 5-phosphatase at synapses, and other inositol 5-phosphatases, such as OCRL. A mutation in synaptojanin 1 was found to be responsible for familial early-onset progressive Parkinsonism. OCRL mutations are responsible for the oculocerebrorenal syndrome of Lowe, a rare but severe human condition, so named because of the cognitive defects, kidney dysfunction, and congenital cataract observed in affected patients. Our studies of synaptojanin 1 and OCRL demonstrated that a main function of these enzymes is to dephosphorylate PI(4,5)P2 at endocytic sites and on endosomes respectively, so that their absence results in major perturbation of endocytosis and endocytic sorting. Ongoing studies address the link between such perturbations and the clinical manifestations resulting from patients' mutations.
Another project focuses on the protein complex comprising PI 4-kinase type IIIα (PI4KA), which is responsible for the synthesis of the bulk of plasma membrane phosphatidylinositol 4-phosphate (PI4P). This pool of PI4P is of fundamental importance in cell physiology, as it is upstream of the major cellular pool of PI(4,5)P2 and the many signaling metabolites derived from PI(4,5)P2, such as PI(3,4,5)P3, DAG, and IP3. Interestingly, a component of this complex, Rolling Black-out/Efr3, which we showed to be a fatty-acylated membrane adaptor protein, has been implicated in synaptic vesicle traffic by genetic studies in Drosophila. Reports by other labs that the PI4P pool controlled by PI4KA plays an important role in hepatitis C infection add medical relevance to these studies.
Biochemical, genetic, and pharmacological methods have been very useful for analyzing the role of specific phospholipids in cell physiology, but none of these techniques permits acute, reversible modification of specific lipids with high temporal and subcellular resolution. To overcome these problems, we are developing and employing optogenetic (genetic perturbations that allow control of cell function by photostimulation) strategies to spatially and temporally control lipid metabolism.
Direct Cross-Talk Between the ER and the Plasma Membrane
Most membrane lipids, including phosphatidylinositol, are synthesized in the endoplasmic reticulum (ER) and then delivered to other organelles by membrane traffic or cytosolic lipid transfer proteins. Recent studies have further demonstrated that direct contacts between the ER and other membranes, including the plasma membrane, play important functions in membrane-lipid dynamics and homeostasis. Such contacts enable ER-anchored proteins to act on adjacent membranes as well as to transfer lipids between the two apposed membranes independently of membrane fusion and fission reactions. We are interested in the impact that ER–plasma membrane contacts may have on the lipid dynamics underlying exo- and endocytosis. So far, ER–plasma membrane contacts have been investigated primarily because of their role in the control of intracellular Ca2+ homeostasis. We expect that these studies will be particularly relevant to the physiology of nerve terminals, where lipids that turn over very rapidly cannot be efficiently replenished by vesicular transport from the cell body.
We have recently demonstrated an important role in the formation and regulation of ER-plasma membrane contacts of the extended-synaptotagmin protein family. These are ER-anchored proteins that tether the ER to the plasma membrane via interactions that are dependent on PI(4,5)P2 in this membrane and that are regulated by Ca2+. The extended-synaptotagmins have a putative role in lipid transfer across membranes, a hypothesis that we are currently testing. Our studies of ER-plasma membrane cross-talk include investigations of the function of VAPB (vesicle-associated membrane protein-associated protein B), a protein that interacts with a variety of enzymes implicated in lipid metabolism and whose mutations are responsible for sporadic cases of amyotrophic lateral sclerosis (ALS).
This work was supported in part by grants from the National Institutes of Health and by funding from the Ellison Foundation and the Simons Foundation.
As of September 12, 2013