Research

The cytoplasm of eukaryotic cells is compartmentalized by intracellular membranes that are specialized for distinct roles and are interconnected by membrane traffic. We study mechanisms underlying their dynamics, with an emphasis on membrane traffic reactions involved in neuronal physiology. 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 neurons to learn about general principles of membrane dynamics and transport.

Membrane Traffic at the Synapse

Exchange of signals between neurons at synapses critically depends on membrane traffic. Neurotransmitters are stored in synaptic vesicles and are released into the synaptic cleft by fusion (exocytosis) of their membranes with the plasma membrane. Rapid reuptake (endocytosis) and recycling of these membranes ensures adequate supply of neurotransmitter loaded synaptic vesicles even during intense activity. Our lab has focused extensively on mechanisms underlying the generation of synaptic vesicles and their regeneration after every cycle of secretion. This process must occur with great fidelity, as the homogeneous properties and size of synaptic vesicles are key factors in ensuring their consistent neurotransmitter content. Vesicle formation involves 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 some of these events, with an emphasis on clathrin-mediated endocytosis. Importantly, we have demonstrated the role of metabolic changes in lipids of the bilayer, phosphoinositides in particular (see below), in the progression of the vesicle cycle.

These studies have broad implications in the field of endocytosis. Currently, we are investigating how principles of liquid-liquid phase separation contribute to the structural organization of the presynapse. We also study the interplay of endocytic traffic at synapses with autophagy and the involvement of dysfunction of these processes in neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease.

Phosphoinositides and Membrane Traffic

Phosphoinositides, the phosphorylated metabolites of phosphatidylinositol, are signaling lipids heterogeneously localized on intracellular 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 by the PI(4,5)P₂ phosphatase synaptojanin after endocytosis is a strict requirement for the maturation of newly formed endocytic vesicles into synaptic vesicles. Building on these studies, we have become more generally interested in the roles of these phospholipids in the control of signaling and membrane traffic in neurons and other cells. These findings have converged with results of other labs, primarily the Emrlab, in demonstrating that phosphoinositides are critical determinants of membrane identity, with major regulatory functions in vesicular transport.

We continue to study the role of specific phosphoinositides and phosphoinositide metabolizing enzymes in the control of membrane dynamics and membrane traffic.

Current projects focus on PI4P metabolism and more specifically on Sac domain containing PI4P phosphatases and on the PI 4-kinase type IIIα (PI4KA) complex, which is responsible for the synthesis of the bulk of plasma membrane PI4P. This PI4P pool is of fundamental importance in cell physiology, as it is upstream of the major cellular pool of PI(4,5)P₂ and of the many signaling metabolites derived from PI(4,5)P₂, such as PI(3,4,5)P₃, DAG, and IP₃. We are also studying diseases resulting from perturbation of these enzymes.

Membrane contact sites in the control of membrane lipid homeostasis

Most membrane lipids, including phosphatidylinositol, are synthesized in the endoplasmic reticulum (ER) and then delivered to other organelles by membrane traffic or lipid transport proteins (LTPs) that bypass membrane traffic.

Membrane traffic-independent lipid transport is essential to maintain the unique lipid composition of different membranes and to mediate lipid transport between the ER and mitochondria, as mitochondria are not connected to the ER via vesicular transport. Recent studies have demonstrated that many LTPs act at contacts between membranes that do not lead to membrane fusion.

These contacts are expected to be particularly relevant to the physiology of neuronal compartments distant from the cell body, where lipids that turn-over very rapidly cannot be efficiently replenished by vesicular transport from the cell body, but can be provided by synthesis in the local ER.

Prompted by our findings that membrane lipids play key regulatory roles in membrane dynamics, we became interested in this field and helped to advance it.

Our studies in this area have included the characterization and functional analysis of several ER proteins with lipid transport modules of the SMP domain (extended synaptotagmins, TMEM24/C2CD2L, C2CD2) and ORD domain (ORP5 and ORP8) families. These proteins tether the ER membrane to the plasma membrane while their lipid transport module shuttles back and forth between the two adjacent membranes. We have also studied the role of VAP, an ER protein which functions as an anchor at the ER surface for a variety of LTPs and enzymes implicated in lipid metabolism. Currently, a major emphasis of our studies in this area is on VPS13 family proteins, whose mutations in humans results in several neurodegenerative and neurodevelopmental disorders, including a neuroacanthocytosis (a Huntington-like condition) and Parkinson’s disease.

VPS13 isoforms are very large proteins, which we have shown to function as lipid transport protein at membrane contact sites.

Structural data and structural predictions (collaboration with the Reinisch lab) raise the interesting possibility that these proteins act by providing hydrophobic bridges between adjacent membranes, a novel concept in membrane biology.

Their similarity to the autophagy protein ATG2, which is implicated in the growth of the autophagic membrane, add further interest to these studies.