Pietro De Camilli studies mechanisms of the dynamics and traffic of cell membranes in physiology and disease, with an emphasis on the role of bilayer lipids, phosphoinositides in particular. Major focuses of his work are the membrane traffic reactions underlying neurotransmission and the impact of the dysfunction of these reactions on neurological and psychiatric conditions.
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 function. 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. As we continue work in this area, we have also begun to explore the role of the clathrin-independent endocytosis of synaptic vesicle membranes. Strong and prolonged stimulation of synapses leads to synaptic vesicle depletion and to a transient accumulation of large vacuoles that form by bulk endocytosis. These vacuoles subsequently convert slowly to new synaptic vesicle membranes. 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 remains elusive.
De Camilli Research Abstract Slideshow 8.30.13
Figure 1: Pathways of synaptic vesicle recycling in nerve terminals.
Schematic diagram of membrane traffic in axon terminals illustrating established and putative pathways of endocytosis of synaptic vesicle (SV) membranes: clathrin-coated pits (CCP) from the plasma membrane and its deep infoldings (1 and 1a),"kiss and run" (2), ultrafast endocytosis (3), and bulk endocytosis (4) followed by vesicle formation via yet unclear mechanisms (?) from endocytic intermediates (EI). This recycling traffic is interconnected with housekeeping membrane recycling (5) involving clathrin-mediated endocytosis and canonical early endosomes (EE) as well as with traffic to the cell body (6) via late endosomes (LE) and multivesicular bodies (MVBs).
Adapted from Saheki, Y., and De Camilli, P. 2012 Cold Spring Harbor Perspectives in Biology. doi:10.1101/cshperspect.a005645. © 2012 Cold Spring Harbor Laboratory Press.
Figure 2: The heterogeneous localization of phosphoinositides on intracellular membranes helps define a code of membrane identity.
Pietro De Camilli
Figure 3: Disruption of synaptic vesicle recycling in dynamin 1 and 3 double-knockout (KO) synapses.
Three-dimensional model of the plasma membrane and clathrin coated endocytic pits derived from a tomographic series from a dynamin 1 and 3 double KO synapse. Lack of these two dynamins results in a dramatic accumulation of clathrin coated endocytic pits on deep plasma membrane invaginations. Virtually all pits shown in the reconstruction originate from, and remain connected to, deep plasma membrane invaginations. At bottom right a conventional EM section is shown, illustrating the presence of clathrin coated vesicular profiles. As shown by electron tomography, all these profiles are connected to the plasma membrane.
From Raimondi, A. et al. 2011 Neuron 70:1100–1114. © 2011, with permission from Elsevier.
Figure 4: Clathrin-mediated endocytosis at the synapse.
Schematic cartoon illustrating putative sites of action of the BAR domain containing protein endophilin and of the PI(4,5)P2 phosphatase synaptojanin 1 in clathrin-coated vesicle fission and uncoating at synapses. Assembly and early maturation of endocytic clathrin coated pits are independent of endophilin. Endophilin is recruited only to the neck of late-stage pits where it binds dynamin, but it is dispensable for dynamin recruitment and for fission. In contrast, the synaptojanin-endophilin interaction is critically important for clathrin uncoating.
From Milosevic, I. et al. 2011 Neuron 72:587–601. © 2011, with permission from Elsevier.
Figure 5: Optogenetic control of phosphoinositide metabolism.
Schematic drawing depicting blue light-mediated recruitment of a phosphoinositide-metabolizing enzyme to a membrane in order to modify a phosphoinositide present in that membrane. See also movie 1. The system is based on the blue-light-dependent heterodimerization between two plant proteins, cryptochrome 2 and the transcription factor CIB1. When the interacting domains of these two proteins (CRY2 and CIBN), fused to an appropriate phosphoinositide metabolizing enzyme and to an appropriate membrane anchor respectively, are expressed in cells, blue-light illumination will recruit the enzyme to the membrane. The example shows the recruitment of an inositol 5-phosphatase to the plasma membrane to dephosphorylate PI(4,5)P2. Tagging of the CRY2 and CIBN fusion proteins to fluorescent proteins, for example GFP and RFP as shown in the cartoon, allows monitoring of their subcellular localization. Expression of a fluorescent PI(4,5)P2 reporter allows PI(4,5)P2 consumption to be monitored.
From Idevall-Hagren, O. et al. 2012 Proceedings of the National Academy of Sciences, USA 109:2316–2323.
Figure 6: Tethering between the endoplasmic reticulum and the plasma membrane mediated by the extended-synaptotagmins.
The extended-synaptotagmins derive their name from their similarity to the synaptotagmins, proteins of secretory vesicle membranes that mediate their interaction and Ca2+-dependent fusion with the plasma membrane. However, they tether the endoplasmic reticulum to the plasma membrane.
From Giordano, F. et al. 2013 Cell 153:1494–1509. © 2013, with permission from Elsevier.
These studies have broad implications in the field of endocytosis. Additional importance of this field comes from genetic studies implicating endocytic proteins in Alzheimer’s disease and familial forms of Parkinson’s disease. We are currently investigating the link between some of the proteins studied in our lab and protein networks whose dysfunction may underlie these conditions.
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. 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 roles of these phospholipids in orchestrating membrane traffic in neurons and other cells.
An important focus of our research in this area continues to be the impact of PI(4,5)P2 dephosphorylating enzymes in phosphoinositide homeostasis and the perturbation of membrane traffic resulting from human disease-causing mutations in these enzymes. A mutation in synaptojanin 1 is responsible for early-onset progressive Parkinsonism, and loss-of-function mutations in the inositol 5-phosphatase OCRL cause a multisystemic disorder termed oculocerebrorenal syndrome of Lowe. We are currently examining the link between perturbations of membrane traffic produced by these mutations and the clinical manifestations of the affected patients.
Other projects focus on the protein complex comprising PI 4-kinase type IIIα (PI4KA), 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. Interestingly, a component of this complex, Rolling Blackout/EFR3, which we showed to be a fatty-acylated membrane adaptor protein, has been implicated in synaptic vesicle traffic by genetic studies in Dropsophila and is a candidate gene in autism. Other complex components have been implicated in human genetic diseases, and PI4KA has been shown to play an important role in hepatitis C infection, thus providing 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 strategies to spatially and temporally control lipid metabolism.
Direct Cross Talk Between the Endoplasmic Reticulum 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 such proteins act primarily at direct contacts between the ER and other membranes that do not lead to membrane fusion. We expect that these contacts 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. Thus, we are investigating how these contacts contribute to neuronal and synaptic function.
We have identified the extended-synaptotagmins (E-syts) as PI(4,5)P2 and Ca2+ regulated components of ER-plasma membrane tethers. We have further shown that the E-Syts harbor glycerophospholipids in their SMP domains pointing to a role in lipid transfer between the two membranes. Ongoing studies address the mechanisms and physiological role of such transfer.
Our studies of ER-plasma membrane cross talk also include investigations of the function of VAPB (vesicle-associated membrane protein-associated protein B), an ER-resident protein that interacts with a variety of enzymes implicated in lipid metabolism and whose mutations are responsible for some cases of familial amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy.
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 April 27, 2016