Showing papers by "Pietro De Camilli published in 2018"

VPS13A and VPS13C are lipid transport proteins differentially localized at ER contact sites.

Abstract: Mutations in the human VPS13 genes are responsible for neurodevelopmental and neurodegenerative disorders including chorea acanthocytosis (VPS13A) and Parkinson's disease (VPS13C). The mechanisms of these diseases are unknown. Genetic studies in yeast hinted that Vps13 may have a role in lipid exchange between organelles. In this study, we show that the N-terminal portion of VPS13 is tubular, with a hydrophobic cavity that can solubilize and transport glycerolipids between membranes. We also show that human VPS13A and VPS13C bind to the ER, tethering it to mitochondria (VPS13A), to late endosome/lysosomes (VPS13C), and to lipid droplets (both VPS13A and VPS13C). These findings identify VPS13 as a lipid transporter between the ER and other organelles, implicating defects in membrane lipid homeostasis in neurological disorders resulting from their mutations. Sequence and secondary structure similarity between the N-terminal portions of Vps13 and other proteins such as the autophagy protein ATG2 suggest lipid transport roles for these proteins as well.

A liquid phase of synapsin and lipid vesicles.

Abstract: Neurotransmitter-containing synaptic vesicles (SVs) form tight clusters at synapses. These clusters act as a reservoir from which SVs are drawn for exocytosis during sustained activity. Several components associated with SVs that are likely to help form such clusters have been reported, including synapsin. Here we found that synapsin can form a distinct liquid phase in an aqueous environment. Other scaffolding proteins could coassemble into this condensate but were not necessary for its formation. Importantly, the synapsin phase could capture small lipid vesicles. The synapsin phase rapidly disassembled upon phosphorylation by calcium/calmodulin-dependent protein kinase II, mimicking the dispersion of synapsin 1 that occurs at presynaptic sites upon stimulation. Thus, principles of liquid-liquid phase separation may apply to the clustering of SVs at synapses.

Ca2+ releases E-Syt1 autoinhibition to couple ER-plasma membrane tethering with lipid transport

Abstract: The extended synaptotagmins (E-Syts) are endoplasmic reticulum (ER) proteins that bind the plasma membrane (PM) via C2 domains and transport lipids between them via SMP domains. E-Syt1 tethers and transports lipids in a Ca2+-dependent manner, but the role of Ca2+ in this regulation is unclear. Of the five C2 domains of E-Syt1, only C2A and C2C contain Ca2+-binding sites. Using liposome-based assays, we show that Ca2+ binding to C2C promotes E-Syt1-mediated membrane tethering by releasing an inhibition that prevents C2E from interacting with PI(4,5)P2-rich membranes, as previously suggested by studies in semi-permeabilized cells. Importantly, Ca2+ binding to C2A enables lipid transport by releasing a charge-based autoinhibitory interaction between this domain and the SMP domain. Supporting these results, E-Syt1 constructs defective in Ca2+ binding in either C2A or C2C failed to rescue two defects in PM lipid homeostasis observed in E-Syts KO cells, delayed diacylglycerol clearance from the PM and impaired Ca2+-triggered phosphatidylserine scrambling. Thus, a main effect of Ca2+ on E-Syt1 is to reverse an autoinhibited state and to couple membrane tethering with lipid transport.

Light-activated protein interaction with high spatial subcellular confinement.

Abstract: Methods to acutely manipulate protein interactions at the subcellular level are powerful tools in cell biology. Several blue-light-dependent optical dimerization tools have been developed. In these systems one protein component of the dimer (the bait) is directed to a specific subcellular location, while the other component (the prey) is fused to the protein of interest. Upon illumination, binding of the prey to the bait results in its subcellular redistribution. Here, we compared and quantified the extent of light-dependent dimer occurrence in small, subcellular volumes controlled by three such tools: Cry2/CIB1, iLID, and Magnets. We show that both the location of the photoreceptor protein(s) in the dimer pair and its (their) switch-off kinetics determine the subcellular volume where dimer formation occurs and the amount of protein recruited in the illuminated volume. Efficient spatial confinement of dimer to the area of illumination is achieved when the photosensitive component of the dimerization pair is tethered to the membrane of intracellular compartments and when on and off kinetics are extremely fast, as achieved with iLID or Magnets. Magnets and the iLID variants with the fastest switch-off kinetics induce and maintain protein dimerization in the smallest volume, although this comes at the expense of the total amount of dimer. These findings highlight the distinct features of different optical dimerization systems and will be useful guides in the choice of tools for specific applications.

The inositol 5-phosphatase INPP5K participates in the fine control of ER organization

Abstract: INPP5K (SKIP) is an inositol 5-phosphatase that localizes in part to the endoplasmic reticulum (ER). We show that recruitment of INPP5K to the ER is mediated by ARL6IP1, which shares features of ER-shaping proteins. Like ARL6IP1, INPP5K is preferentially localized in ER tubules and enriched, relative to other ER resident proteins (Sec61β, VAPB, and Sac1), in newly formed tubules that grow along microtubule tracks. Depletion of either INPP5K or ARL6IP1 results in the increase of ER sheets. In a convergent but independent study, a screen for mutations affecting the distribution of the ER network in dendrites of the PVD neurons of Caenorhabditis elegans led to the isolation of mutants in CIL-1, which encodes the INPP5K worm orthologue. The mutant phenotype was rescued by expression of wild type, but not of catalytically inactive CIL-1. Our results reveal an unexpected role of an ER localized polyphosphoinositide phosphatase in the fine control of ER network organization.

Kv2 potassium channels meet VAP.

Abstract: A defining characteristic of eukaryotic cells is the presence of distinct intracellular membrane-bound compartments Much research has focused on the functional interconnection of these organelles via membrane traffic A flurry of recent studies, however, has brought to center stage the important role of interorganelle communication independent of vesicular transport and mediated by direct contacts (1⇓–3) At these sites, membranes are tethered to each other by protein–protein or protein–lipid interactions not leading to fusion These contacts play a variety of functions, including regulation of ion fluxes across membranes and transport of lipids between participating organelles In PNAS, Johnson et al (4) provide yet another unexpected example of direct communication between two membranes: the binding of the plasma membrane (PM)-localized major delayed-rectifier voltage-gated K+ channels, Kv21 and Kv22, to VAMP-associated protein (VAP), an integral membrane protein of the endoplasmic reticulum (ER) Kv21 and Kv22 ( KCNB1 and KCNB2 ) channels are very abundant in the brain, where they play a major role in neuronal excitability They form large clusters in the PM of neuronal cell bodies, proximal dendrites, and axon initial segments, and such clusters are at sites where the PM is tightly apposed to the ER (5) In the clusters, the cytosolic tails of the channels are in a phosphorylated state and the clusters disperse within minutes due to calcium-dependent dephosphorylation upon excessive excitatory stimulation or exposure to noxious conditions, such as hypoxia (6) While an amino acid stretch, referred to as the PRC (proximal restriction and clustering), was known to be required for clustering (7), how the PRC mediates clustering remained unclear The ER is the most abundant intracellular membranous organelle It comprises a system of tubules and cisterns that are continuous with each other and populate every cell compartment, including axon endings and dendritic spines in neurons … [↵][1]1To whom correspondence should be addressed Email: Pietrodecamilli{at}yaleedu [1]: #xref-corresp-1-1