Biology subjects

De Camilli, P.

Publications and source records attributed to De Camilli, P..

At least 19 recordsLinked to original sources

bioRxiv2026

Dystrophic changes of nigrostriatal axons harboring a Synj1 Parkinson mutation suggest catastrophic failure of endocytic mechanisms

Synaptojanin 1 is a brain enriched phosphoinositide phosphatase implicated in endocytosis at the synapse. A mutation (R258Q) that selectively impairs its Sac1 phosphatase domain causes early onset familial Parkinsonism. Neurons of mice with this mutation display synaptic vesicle traffic defects across the brain, but selective dystrophic changes in a subset of dopaminergic axons in the dorsolateral striatum. Using correlative light microscopy-FIB-SEM of mutant mouse striata to visualize in 3D these abnormal structures we show that they represent clusters of focal axonal dilations harboring massive, onion-like DAT enriched plasma membrane infoldings, generally localized next to cell bodies of neighboring cells, often engulfing evaginations of such cells. This dysmorphia was associated with a deficit in dopamine release in the same striatal region. Given the involvement of Synj1 in endocytic mechanisms, these structures may reflect an imbalance between exocytosis and endocytosis. Their occurrence only in a subset of axons suggest a vulnerability threshold of these axons beyond which the expansion of the plasma membrane is not counteracted by compensatory mechanisms.

neuroscience↗

bioRxiv2026

Molecular insights into bulk lipid transport from structural studies of the bridge-like protein VPS13A complexed with the scramblase XKR1

In eukaryotes, bridge-like lipid-transfer proteins (BLTPs) are central in mediating vesicle-independent lipid transfer between organelles. BLTPs span the cytosolic space between organelles at contact sites, featuring hydrophobic channels for lipids to travel between membranes. How BLTPs cooperate with partner proteins to orchestrate lipid delivery remains mysterious. Here we used cryo-electron microscopy to visualize a complex comprising the prototypical BLTP VPS13A and the plasma membrane localized scramblase XKR1 at near-atomic resolution. VPS13A interacts with XKR1 via its PH-domain, priming VPS13As bridge-like lipid-transfer domain to deliver lipids directly to the cytosolic leaflet of the acceptor membrane. In molecular dynamics simulations, such arrangement allows for robust lipid transfer, accelerated by membrane properties. Newly delivered lipids can then be equilibrated between leaflets of the membrane bilayer by the scramblase, allowing for membrane growth. Mechanistic insights regarding lipid delivery by VPS13A are directly applicable to all VPS13 proteins and all BLTP family members more broadly.

cell biology↗

bioRxiv2025

Insights into the regulation of VPS13 family bridge-like lipid transfer proteins from the structure of VPS13C.

Bridge-like lipid transfer proteins (BLTPs) play central roles in redistributing lipids from their primary site of synthesis in the endoplasmic reticulum to other organelles. They comprise bridge-domains spanning between organelles at contact sites that allow lipids to transit the cytosol between adjacent membranes. The assembly of BLTPs into complexes with adaptor proteins enables their lipid transfer ability. To address the mechanisms underlying assembly and regulation of BLTP complexes, we used cryo-EM to resolve the structure of one such BLTP, the Parkinsons protein VPS13C, at near-atomic resolution. The structure identifies a lipid-transfer-nonpermissive conformation, where the built-in C-terminal VAB adaptor module blocks the end of the lipid transfer bridge, interfering with lipid delivery. We also identify calmodulin, central to calcium signaling, as a VPS13 partner, suggesting calcium regulation of VPS13 function. Altogether, this structure of intact VPS13C serves as starting point to understand its regulation and, more broadly, that of other BLTPs.

cell biology↗

bioRxiv2025

Defect in hematopoiesis and embryonic lethality at midgestation of Vps13a/Vps13c double knockout mice

VPS13 is the founding member of a family of proteins that mediate lipid transfer at intracellular membrane contact sites by a bridge-like mechanism. Mammalian genomes comprise 4 VPS13 genes encoding proteins with distinct localizations and function. The gene duplication resulting in VPS13A and VPS13C is the most recent in evolution and, accordingly, these two proteins are the most similar to each other. However, they have distinct subcellular localizations and their loss of function mutations in humans are compatible with life but result in two different age-dependent neurodegenerative diseases, chorea-acanthocytosis and Parkinsons disease, respectively. Thus, it remains unclear whether these two proteins have overlapping functions. Here, we show that while Vps13a KO and Vps13c KO mice are viable, embryonic development of Vps13a/Vps13c double knockout (DKO) mice is arrested at midgestation. Prior to death, DKO embryos were smaller than controls, were anemic and had a smaller liver, the key erythropoietic site at this developmental stage. Further analyses of erythroid precursor cells showed that their differentiation was impaired and that this defect was accompanied by activation of innate immunity as revealed by upregulation of interferon stimulated genes (ISGs). Additionally, the RIG-I and MDA5 components of dsRNA triggered innate immunity were found upregulated in the DKO fetal liver. Activation of innate immunity may result from loss of integrity of the membranes of intracellular organelles, such as mitochondria and autophagic lysosomes, due to the absence of these lipid transport proteins. The surprising and striking synthetic effect resulting for the combined loss of VPS13A and VPS13C suggests that despite of the different localization of these two proteins, the lipid fluxes that they mediate are partially redundant.

cell biology↗

bioRxiv2025

ATG2A engages Rab1a and ARFGAP1 positive membranes during autophagosome biogenesis

Autophagosomes form from seed membranes that expand through bulk-lipid transport via the bridge-like lipid transporter ATG2. The origins of the seed membranes and their relationship to the lipid transport machinery are poorly understood. Using proximity labeling and a variety of fluorescence microscopy techniques, we show that ATG2A localizes to extra-Golgi ARFGAP1 puncta during autophagosome biogenesis. ARFGAP1 itself is dispensable during macroautophagy, but among other proteins associating to these membranes, we find that RAB1 is essential. ATG2A co-immunoprecipitates strongly, albeit indirectly, with RAB1A, and siRNA-mediated depletion of RAB1A/B blocks autophagy downstream of LC3B lipidation, similar to ATG2A depletion. Further, when either autophagosome formation or the early secretory pathway is perturbed, ARFGAP1 and RAB1A accumulate at ectopic locations with autophagic machinery. Our results indicate that ATG2A engages a RAB1A complex on select early secretory membranes in support of autophagosome biogenesis. Significance StatementThis study expounds upon the role of early secretory membranes in autophagosome biogenesis. The authors demonstrate that RAB1/ARFGAP1-positive membranes are essential to autophagy and are recruited to the phagophore assembly site at an early step of autophagosome biogenesis. These membranes interact with the bridge-like lipid transport protein ATG2A and are positive for LC3B and WIPI2, suggesting that RAB1 membranes are a direct source for autophagosome formation.

cell biology↗

bioRxiv2025

Multiple interactions mediate the localization of BLTP2 at ER-PM contacts to control plasma membrane dynamics

BLTP2/KIAA0100, a bridge-like lipid transfer protein, was reported to localize at contacts of the endoplasmic reticulum (ER) with either the plasma membrane (PM) or recycling tubular endosomes depending on the cell type. Our findings suggest that mediating bulk lipid transport between the ER and the PM is a key function of this protein as BLTP2 tethers the ER to tubular endosomes only after they become continuous with the PM and that it also tethers the ER to macropinosomes in the process of fusing with the PM. We further identify interactions underlying binding of BLTP2 to the PM, including phosphoinositides, the adaptor proteins FAM102A and FAM102B, and also N-BAR domain proteins at membrane-connected tubules. The absence of BLTP2 results in the accumulation of intracellular vacuoles, many of which are connected to the plasma membrane, pointing to a role of the lipid transport function of BLTP2 in the control of PM dynamics.

cell biology↗

bioRxiv2024

Bridge-like lipid transfer protein 3A (BLTP3A) is associated with membranes of the late endocytic pathway and is an effector of CASM

Recent studies have identified a family of rod-shaped proteins thought to mediate lipid transfer at intracellular membrane contacts by a bridge-like mechanism. We show one such protein, bridge-like lipid transfer protein 3A (BLTP3A)/UHRF1BP1 binds VAMP7 vesicles via its C-terminal region and anchors them to lysosomes via its chorein domain containing N-terminal region to Rab7. Upon lysosome damage, BLTP3A-positive vesicles rapidly (within minutes) dissociate from lysosomes. Lysosome damage is known to activate the CASM (Conjugation of ATG8 to Single Membranes) pathway leading to lipidation and recruitment to lysosomes of mammalian ATG8 (mATG8) proteins. We find that this process drives the reassociation of BLTP3A with damaged lysosomes via an interaction of its LIR motif with mATG8 which coincides with a dissociation from the vesicles. Our findings reveal that BLTP3A is an effector of CASM, potentially as part of a mechanism to help repair or minimize lysosome damage. TeaserBLTP3A is localized on membranes of the late endocytic system and is CASM effector, pointing to a role of this proteins in the response to lysosome damage

cell biology↗

bioRxiv2024

Lysosome damage triggers acute formation of ER to lysosomes membrane tethers mediated by the bridge-like lipid transport protein VPS13C

Based on genetic studies, lysosome dysfunction is thought to play a pathogenetic role in Parkinsons disease (PD). Here we show that VPS13C, a bridge-like lipid transport protein and a PD gene, is a sensor of lysosome stress/damage. Upon lysosome membrane perturbation, VPS13C rapidly relocates from the cytosol to the surface of lysosomes where it tethers their membranes to the ER. This recruitment depends on Rab7 and requires a signal at the damaged lysosome surface that releases an inhibited state of VPS13C which hinders access of its VAB domain to lysosome-bound Rab7. While another PD protein, LRRK2, is also recruited to stressed/damaged lysosomes, its recruitment occurs at much later stages and by different mechanisms. Given the role of VPS13 proteins in bulk lipid transport, these findings suggest that lipid delivery to lysosomes by VPS13C is part of an early protective response to lysosome damage.

cell biology↗

bioRxiv2024

Periodic ER-plasma membrane junctions support long-range Ca2+ signal integration in dendrites

Neuronal dendrites must relay synaptic inputs over long distances, but the mechanisms by which activity-evoked intracellular signals propagate over macroscopic distances remain unclear. Here, we discovered a system of periodically arranged endoplasmic reticulum-plasma membrane (ER-PM) junctions tiling the plasma membrane of dendrites at [~]1 m intervals, interlinked by a meshwork of ER tubules patterned in a ladder-like array. Populated with Junctophilin-linked plasma membrane voltage-gated Ca2+ channels and ER Ca2+-release channels (ryanodine receptors), ER-PM junctions are hubs for ER-PM crosstalk, fine-tuning of Ca2+ homeostasis, and local activation of the Ca2+/calmodulin-dependent protein kinase II. Local spine stimulation activates the Ca2+ modulatory machinery facilitating voltage-independent signal transmission and ryanodine receptor-dependent Ca2+ release at ER-PM junctions over 20 m away. Thus, interconnected ER-PM junctions support signal propagation and Ca2+ release from the spine-adjacent ER. The capacity of this subcellular architecture to modify both local and distant membrane-proximal biochemistry potentially contributes to dendritic computations. HighlightsO_LIPeriodic ER-PM junctions tile neuronal dendritic plasma membrane in rodent and fly. C_LIO_LIER-PM junctions are populated by ER tethering and Ca2+ release and influx machinery. C_LIO_LIER-PM junctions act as sites for local activation of CaMKII. C_LIO_LILocal spine activation drives Ca2+ release from RyRs at ER-PM junctions over 20 m. C_LI

neuroscience↗

bioRxiv2024

Overlapping role of synaptophysin and synaptogyrin family proteins in determining the small size of synaptic vesicles

Members of the synaptophysin and synaptogyrin family are vesicle proteins with four transmembrane domains. In spite of their abundance in synaptic vesicle (SV) membranes, their role remains elusive and only mild defects at the cellular and organismal level are observed in mice lacking one or more family members. Here, we show that co-expression with synapsin of each of the four brain-enriched members of this family - synaptophysin, synaptoporin, synaptogyrin1 and synaptogyrin3 - in fibroblasts is sufficient to generate clusters of small vesicles in the same size range of SVs. Moreover, mice lacking all these four proteins have larger SVs. We conclude that synaptophysin and synaptogyrin family proteins play an overlapping function in the biogenesis of SVs and in determining their small size.

neuroscience↗

bioRxiv2024

Ectopic Reconstitution of a Spine-Apparatus Like Structure Provides Insight into Mechanisms Underlying Its Formation

The endoplasmic reticulum (ER) is a continuous cellular endomembrane network that displays focal specializations. Most notable examples of such specializations include the spine apparatus of neuronal dendrites, and the cisternal organelle of axonal initial segments. Both organelles exhibit stacks of smooth ER sheets with a narrow lumen and interconnected by a dense protein matrix. The actin-binding protein synaptopodin is required for their formation. Here, we report that expression in non-neuronal cells of a synaptopodin construct targeted to the ER is sufficient to generate stacked ER cisterns resembling the spine apparatus with molecular properties distinct from the surrounding ER. Cisterns within these stacks are connected to each other by an actin-based matrix that contains proteins also found at the spine apparatus of neuronal spines. These findings reveal a critical role of a synaptopodin-dependent actin matrix in generating cis-ternal stacks. These ectopically generated structures provide insight into spine apparatus morphogenesis.

cell biology↗

bioRxiv2023

VPS13B is localized at the cis-trans Golgi complex interface and is a functional partner of FAM177A1

Mutations in VPS13B, a member of a protein family implicated in bulk lipid transport between adjacent membranes, cause Cohen syndrome. VPS13B is known to be concentrated in the Golgi complex, but its precise location within this organelle and thus the site(s) where it achieves lipid transport remains unclear. Here we show that VPS13B is localized at the interface between cis and trans Golgi sub-compartments and that Golgi complex re-formation after Brefeldin A (BFA) induced disruption is delayed in VPS13B KO cells. This delay is phenocopied by loss of FAM177A1, a Golgi complex protein of unknown function reported to be a VPS13B interactor and whose mutations also result in a developmental disorder. In zebrafish, the vps13b orthologue, not previously annotated in this organism, genetically interacts with fam177a1. Collectively, these findings raise the possibility that bulk lipid transport by VPS13B may play a role in expanding Golgi membranes and that VPS13B may be assisted in this function by FAM177A1.

cell biology↗

bioRxiv2023

DDHD2 is necessary for activity-driven fatty acid fueling of nerve terminal function

HSP54, a hereditary spastic paraplegia associated with cognitive impairment, is caused by mutations in the neuron-specific triglyceride (TG) lipase DDHD2. Loss of DDHD2 function results in lipid accumulation in human brains1 and lipid droplets (LDs) in mouse neurons2. In metabolically demanding tissues, TG lipases generate a fatty acid (FA) flux from LDs to fuel mitochondrial ATP production, but neurons are considered unable to use fat as an energy source. Thus, the basis for cognitive impairment driven by DDHD2 loss remains enigmatic. To resolve this paradox, we took advantage of presynaptic sensitivity to metabolic perturbations to determine if FAs derived from LDs could power local {beta}-oxidation to support synaptic functions and whether DDHD2 activity would be required in the process. We demonstrate that nerve terminals are enriched with DDHD2 and blocking its activity leads to presynaptic accumulation of LDs. Moreover, we show that FAs derived from axonal LDs enter mitochondria in an activity-dependent fashion and drive local mitochondrial ATP production allowing nerve terminals to sustain function in the complete absence of glucose. Our data demonstrate that neurons and their nerve terminals can make use of LDs during electrical activity to provide metabolic support when glucose is in short supply.

neuroscience↗

bioRxiv2023

A complex of the lipid transport ER proteins TMEM24 and C2CD2 with band 4.1 at cell-cell contacts

Junctions between the ER and the plasma membrane (ER/PM junctions) are implicated in calcium homeostasis, non-vesicular lipid transfer and other cellular functions. Two ER proteins that function both as membrane tethers to the PM via a polybasic motif in their C-terminus and as phospholipid transporters are brain-enriched TMEM24 (C2CD2L) and its paralog C2CD2. Based on an unbiased proximity ligation analysis, we found that both proteins can also form a complex with band 4.1 family members, which in turn can bind a variety of plasma membrane proteins including cell adhesion molecules such as SynCAM 1. This complex results in the enrichment of TMEM24 and C2CD2 containing ER/PM junctions at sites of cell contacts. Dynamic properties of TMEM24-dependent ER/PM contacts are impacted when in complex as TMEM24 present at cell adjacent junctions is not shed by calcium rise, unlike TMEM24 at non-cell adjacent junctions. These findings suggest that cell-contact interactions control ER/PM junctions via TMEM24 complexes involving band 4.1 proteins. SUMMARYThe ER-anchored lipid transfer proteins TMEM24/C2CD2L and its paralog C2CD2 mediate formation of ER-plasma membrane junctions at sites of cell-cell contacts by interacting with band 4.1 family members and indirectly with cell adhesion proteins.

cell biology↗

bioRxiv2023

Parkinsonism Sac domain mutation in Synaptojanin-1 affects ciliary properties in iPSC-derived dopaminergic neurons

Synaptojanin-1 (SJ1) is a major neuronal-enriched PI(4,5)P2 4- and 5-phosphatase implicated in the shedding of endocytic factors during endocytosis. A mutation (R258Q) that impairs selectively its 4-phosphatase activity causes Parkinsonism in humans and neurological defects in mice (SJ1RQKI mice). Studies of these mice showed, besides an abnormal assembly state of endocytic factors at synapses, the presence of dystrophic nerve terminals selectively in a subset of nigro-striatal dopamine (DA)-ergic axons, suggesting a special lability of DA neurons to the impairment of SJ1 function. Here we have further investigated the impact of SJ1 on DA neurons using iPSC-derived SJ1 KO and SJ1RQKI DA neurons and their isogenic controls. In addition to the expected enhanced clustering of endocytic factors in nerve terminals, we observed in both SJ1 mutant neuronal lines increased cilia length. Further analysis of cilia of SJ1RQDA neurons revealed abnormal accumulation of the Ca2+ channel Cav1.3 and of ubiquitin chains, suggesting an impaired clearing of proteins from cilia which may result from an endocytic defect at the ciliary base, where a focal concentration of SJ1 was observed. We suggest that SJ1 may contribute to the control of ciliary protein dynamics in DA neurons, with implications on cilia-mediated signaling.

neuroscience↗

bioRxiv2023

Phosphoglycerate kinase 1 is a central leverage point in Parkinson's disease driven neuronal metabolic deficits

Phosphoglycerate kinase 1 (PGK1), the first ATP producing glycolytic enzyme, has emerged as a therapeutic target for Parkinsons Disease (PD), since a potential enhancer of its activity was reported to significantly lower PD risk. We carried out a suppressor screen of hypometabolic synaptic deficits and demonstrated that PGK1 is a rate limiting enzyme in nerve terminal ATP production. Increasing PGK1 expression in mid-brain dopamine neurons protected against hydroxy-dopamine driven striatal dopamine nerve terminal dysfunction in-vivo and modest changes in PGK1 activity dramatically suppressed hypometabolic synapse dysfunction in vitro. Furthermore, PGK1 is cross-regulated by PARK7 (DJ-1), a PD associated molecular chaperone, and synaptic deficits driven by PARK20 (Synaptojanin-1) can be reversed by increasing local synaptic PGK1 activity. These data indicate that nerve terminal bioenergetic deficits may underly a spectrum of PD susceptibilities and the identification of PGK1 as the limiting enzyme in axonal glycolysis provides a mechanistic underpinning for therapeutic protection. O_LISuppressor screen identifies PGK1 as the rate limiting enzyme in axon metabolism C_LIO_LIPGK1 expression in-vivo protects against striatal dopaminergic axon degeneration C_LIO_LILoss of DJ-1 impairs axonal ATP production C_LIO_LIPGK1 function is regulated by PARK7/DJ-1, and can reverse PARK20 synaptic deficits C_LI

neuroscience↗

bioRxiv2023

Erythroid differentiation dependent interaction of VPS13A with XK at the plasma membrane of K562 cells

Mutations of the bridge-like lipid transport protein VPS13A and of the lipid scramblase XK result in Chorea Acanthocytosis (ChAc) and McLeod syndrome (MLS) respectively, two similar conditions involving neurodegeneration and deformed erythrocytes (acanthocytes). VPS13A binds XK, suggesting a model in which VPS13A forms a lipid transport bridge between the ER and the plasma membrane (PM) where XK resides. However, studies of VPS13A in HeLa and COS7 cells showed that this protein localizes primarily at contacts of the ER with mitochondria. Overexpression of XK in these cells redistributed VPS13A to the biosynthetic XK pool in the ER but not to PM localized XK. Colocalization of VPS13A with XK at the PM was only observed if overexpressed XK harbored mutations that disengage its VPS13A binding site from an intramolecular interaction. As the acanthocytosis phenotype of ChAc and MLS suggests a role of the two proteins in cells of the erythroid lineage, we explored their localization in K562 cells, which differentiate into erythroblasts upon hemin addition. When tagged VPS13A was overexpressed in hemin treated K562 cells, robust formation of ER-PM contacts positive for VPS13A were observed and their formation was abolished in XK KO cells. ER-PM contacts positive for VPS13A were seldomly observed in undifferentiated K562 cells, in spite of the presence of XK in these cells at concentrations similar to those observed after differentiation. These findings reveal that the interaction of VPS13A with XK at ER-PM contacts requires a permissive state which depends upon cell type and/or functional state of the cell.

cell biology↗

bioRxiv2023

End Binding protein 1 promotes specific motor-cargo association in the cell body prior to axonal delivery of Dense Core Vesicles

Axonal transport is key to neuronal function. Efficient transport requires specific motor-cargo association in the soma, yet the mechanisms regulating this early step remain poorly understood. We found that EBP-1, the C. elegans ortholog of the canonical microtubule end binding protein EB1, promotes the specific association between kinesin-3/KIF1A/UNC-104 and Dense Core Vesicles (DCVs) prior to their axonal delivery. Using single-neuron, in vivo labelling of endogenous cargo and EBs, we observed reduced axonal abundance and reduced secretion of DCV cargo, but not other KIF1A/UNC-104 cargo, in ebp-1 mutants. This reduction could be traced back to fewer exit events from the cell body, where EBP-1 colocalized with the DCV sorting machinery at the trans Golgi, suggesting that this is the site of EBP-1 function. In addition to its microtubule binding CH domain, mammalian EB1 interacted with mammalian KIF1A in an EBH domain dependent manner, and expression of mammalian EB1 or the EBH domain was sufficient to rescue DCV transport in ebp-1 mutants. Our results suggest a model in which kinesin-3 binding and microtubule binding by EBP-1 cooperate to transiently enrich the motor near sites of DCV biogenesis to promote motor-cargo association. In support of this model, tethering either EBP-1 or a kinesin-3 KIF1A/UNC-104 interacting domain from an unrelated protein to the Golgi restored the axonal abundance of DCV proteins in ebp-1 mutants. These results uncover an unexpected role for a microtubule associated protein and provide insight into how specific kinesin-3 cargo are delivered to the axon.

cell biology↗