A liquid phase of synapsin and lipid vesicles Milovanovic, Dragomir; Wu, Yumei; Bian, Xin ...
Science (American Association for the Advancement of Science),
08/2018, Letnik:
361, Številka:
6402
Journal Article
Recenzirano
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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.
Clusters of tightly packed synaptic vesicles (SVs) are a defining feature of nerve terminals. While SVs are mobile within the clusters, the clusters have no boundaries consistent with a liquid phase. ...We previously found that purified synapsin, a peripheral SV protein, can assemble into liquid condensates and trap liposomes into them. How this finding relates to the physiological formation of SV clusters in living cells remains unclear. Here, we report that synapsin alone, when expressed in fibroblasts, has a diffuse cytosolic distribution. However, when expressed together with synaptophysin, an integral SV membrane protein previously shown to be localized on small synaptic-like microvesicles when expressed in non-neuronal cells, is sufficient to organize such vesicles in clusters highly reminiscent of SV clusters and with liquid-like properties. This minimal reconstitution system can be a powerful model to gain mechanistic insight into the assembly of structures which are of fundamental importance in synaptic transmission.
VAP (VAPA and VAPB) is an evolutionarily conserved endoplasmic reticulum (ER)-anchored protein that helps generate tethers between the ER and other membranes through which lipids are exchanged across ...adjacent bilayers. Here, we report that by regulating PI4P levels on endosomes, VAP affects WASH-dependent actin nucleation on these organelles and the function of the retromer, a protein coat responsible for endosome-to-Golgi traffic. VAP is recruited to retromer budding sites on endosomes via an interaction with the retromer SNX2 subunit. Cells lacking VAP accumulate high levels of PI4P, actin comets, and trans-Golgi proteins on endosomes. Such defects are mimicked by downregulation of OSBP, a VAP interactor and PI4P transporter that participates in VAP-dependent ER-endosomes tethers. These results reveal a role of PI4P in retromer-/WASH-dependent budding from endosomes. Collectively, our data show how the ER can control budding dynamics and association with the cytoskeleton of another membrane by direct contacts leading to bilayer lipid modifications.
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•An interaction between VAP and the retromer subunit SNX2 tethers the ER to endosomes•Segregation of TGN proteins from endosomes is impaired in cells that lack VAP•A PI4P pool regulated by OSBP accumulates on endosomes of VAP double KO cells•WASH mediates potent actin nucleation from endosomes in cells lacking VAP or OSBP
Contacts between the membranes of two organelles influences vesicle budding through changes in lipid composition that affect actin dynamics.
Mitochondria, which are excluded from the secretory pathway, depend on lipid transport proteins for their lipid supply from the ER, where most lipids are synthesized. In yeast, the outer ...mitochondrial membrane GTPase Gem1 is an accessory factor of ERMES, an ER-mitochondria tethering complex that contains lipid transport domains and that functions, partially redundantly with Vps13, in lipid transfer between the two organelles. In metazoa, where VPS13, but not ERMES, is present, the Gem1 orthologue Miro was linked to mitochondrial dynamics but not to lipid transport. Here we show that Miro, including its peroxisome-enriched splice variant, recruits the lipid transport protein VPS13D, which in turn binds the ER in a VAP-dependent way and thus could provide a lipid conduit between the ER and mitochondria. These findings reveal a so far missing link between function(s) of Gem1/Miro in yeast and higher eukaryotes, where Miro is a Parkin substrate, with potential implications for Parkinson's disease pathogenesis.
The endoplasmic reticulum (ER) has a broad localization throughout the cell and forms direct physical contacts with all other classes of membranous organelles, including the plasma membrane (PM). A ...number of protein tethers that mediate these contacts have been identified, and study of these protein tethers has revealed a multiplicity of roles in cell physiology, including regulation of intracellular Ca
2+
dynamics and signaling as well as control of lipid traffic and homeostasis. In this review, we discuss the cross talk between the ER and the PM mediated by direct contacts. We review factors that tether the two membranes, their properties, and their dynamics in response to the functional state of the cell. We focus in particular on the role of ER-PM contacts in nonvesicular lipid transport between the two bilayers mediated by lipid transfer proteins.
Endophilin is a membrane-binding protein with curvature-generating and -sensing properties that participates in clathrin-dependent endocytosis of synaptic vesicle membranes. Endophilin also binds the ...GTPase dynamin and the phosphoinositide phosphatase synaptojanin and is thought to coordinate constriction of coated pits with membrane fission (via dynamin) and subsequent uncoating (via synaptojanin). We show that although synaptojanin is recruited by endophilin at bud necks before fission, the knockout of all three mouse endophilins results in the accumulation of clathrin-coated vesicles, but not of clathrin-coated pits, at synapses. The absence of endophilin impairs but does not abolish synaptic transmission and results in perinatal lethality, whereas partial endophilin absence causes severe neurological defects, including epilepsy and neurodegeneration. Our data support a model in which endophilin recruitment to coated pit necks, because of its curvature-sensing properties, primes vesicle buds for subsequent uncoating after membrane fission, without being critically required for the fission reaction itself.
► Perinatal lethality, but not abolished neurotransmission, in endophilin triple KO mice ► Accumulation of coated vesicles, but not coated pits, in synapses without endophilin ► Mouse KO phenotype implies a key role for endophilin in synaptojanin recruitment ► Partial loss of endophilin results in epilepsy and neurodegeneration
Phosphoinositides (PIs) are minor components of cell membranes, but play key roles in cell function. Recent refinements in techniques for their detection, together with imaging methods to study their ...distribution and changes, have greatly facilitated the study of these lipids. Such methods have been complemented by the parallel development of techniques for the acute manipulation of their levels, which in turn allow bypassing the long-term adaptive changes implicit in genetic perturbations. Collectively, these advancements have helped elucidate the role of PIs in physiology and the impact of the dysfunction of their metabolism in disease. Combining methods for detection and manipulation enables the identification of specific roles played by each of the PIs and may eventually lead to the complete deconstruction of the PI signaling network. Here, we review current techniques used for the study and manipulation of cellular PIs and also discuss advantages and disadvantages associated with the various methods. This article is part of a Special Issue entitled Phosphoinositides.
•Heterogeneous distribution of phosphoinositides (PIs) helps define membrane identity.•Analysis of PI function requires detection of both levels and subcellular localization.•Genetically encoded fluorescent PI binding modules reveal PI dynamics in live cells.•PI levels can be manipulated through chemical, genetic and pharmacological approaches.•Optogenetics enables control of specific PIs with high temporal and spatial precision.
Non-vesicular lipid transport between bilayers at membrane contact sites plays important physiological roles. Mechanistic insight into the action of lipid-transport proteins localized at these sites ...requires determination of the distance between bilayers at which this transport can occur. Here we developed DNA-origami nanostructures to organize size-defined liposomes at precise distances and used them to study lipid transfer by the synaptotagmin-like mitochondrial lipid-binding protein (SMP) domain of extended synaptotagmin 1 (E-Syt1). Pairs of DNA-ring-templated donor and acceptor liposomes were docked through DNA pillars, which determined their distance. The SMP domain was anchored to donor liposomes via an unstructured linker, and lipid transfer was assessed via a Förster resonance energy transfer (FRET)-based assay. We show that lipid transfer can occur over distances that exceed the length of an SMP dimer, which is compatible with the shuttle model of lipid transport. The DNA nanostructures developed here can also be adapted to study other processes occurring where two membranes are closely apposed to each other.
Paul Greengard (1925-2019) De Camilli, Pietro
Science (American Association for the Advancement of Science),
2019-May-24, 2019-05-24, 20190524, Letnik:
364, Številka:
6442
Journal Article
Recenzirano
Pioneer in nerve cell signaling and brain diseases
Paul Greengard, esteemed neuroscientist and beloved mentor, died on 13 April. He was 93. Greengard pioneered the elucidation of intracellular ...signaling in nervous system function and provided key insights into neurological and psychiatric diseases. His work helped shape modern neuroscience and had a broad impact in a variety of other fields.
Dynamin, which is encoded by three genes in mammals, is a GTPase implicated in endocytic membrane fission. Dynamin 1 and 3 are predominantly expressed in brain, whereas dynamin 2 is ubiquitously ...expressed. With the goal of assessing the impact of the lack of dynamin on cell physiology, we previously generated and characterized dynamin 1 and 2 double knockout (DKO) fibroblasts. These DKO cells were unexpectedly viable in spite of a severe impairment of clathrin-mediated endocytosis. As low-level expression of the dynamin 3 gene in these cells could not be excluded, we have now engineered dynamin 1, 2 and 3 triple KO (TKO) fibroblasts. These cells did not reveal any additional defects beyond what was previously observed in DKO fibroblasts. Surprisingly, although fluid-phase endocytosis and peripheral membrane ruffling were not impaired by the lack of all three dynamins, two structurally similar, widely used dynamin inhibitors, dynasore and Dyngo-4a, robustly inhibited these two processes both in wild-type and TKO cells. Dynamin TKO cells will be useful tools for the further exploration of dynamin-dependent processes and the development of more specific dynamin inhibitors.
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