This protocol details the procedure of Correlative Light Microscopy and Electron Microscopy (CLEM) with 3D Focus Ion Beam Scanning Electron Microscopy (FIBSEM) technique for mouse brain tissue in Zeiss Crossbeam 550 FIBSEM system.

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.

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 a mystery. Here, we used cryo-electron microscopy to visualize a complex comprising the prototypical BLTP VPS13A and the plasma membrane-localized scramblase XK at near-atomic resolution. VPS13A interacts with XK via its pleckstrin homology domain, priming VPS13A's bridge-like lipid-transfer domain to deliver lipids directly to the cytosolic leaflet of the acceptor membrane. In molecular dynamics simulations, this arrangement allows for robust lipid transfer. 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, more broadly, to all BLTP family members.

This protocol describes the post-staining procedure for ultrathin Epon sections on EM grids.

This protocol details the procedure of our mouse brain immunofluorescence for detecting DAergic axonal dystrophies in Synj^^RQ KI mice.

Bridge-like lipid transfer proteins (BLTPs) play fundamental roles in cellular lipid redistribution between organellar membranes. They comprise bridge domains spanning organelles at contact sites that allow lipids to transit through the cytosol between adjacent membranes. The assembly of BLTPs into complexes with adaptor proteins enables lipid transfer. To address the mechanisms underlying the assembly and regulation of BLTP complexes, we used cryo-EM to resolve the structure of one such BLTP, the Parkinson’s disease protein VPS13C, at near-atomic resolution. The structure identifies a lipid-transfer-nonpermissive conformation, in which the built-in C-terminal VAB adaptor module blocks the end of the lipid transfer bridge, interfering with lipid delivery. We also identify calmodulin (CaM), central to calcium signaling, as a constitutive VPS13C interactor. Calcium induces conformational changes in the VPS13C-CaM complex, suggesting calcium regulation of VPS13 function. Altogether, this structure of intact VPS13C serves as a starting point for understanding its regulation and that of other VPS13 proteins.

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 VPS13A's 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.

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 Parkinson's 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, most likely reflecting defective embryonic erythropoiesis which at this developmental stage occurs primarily in this organ. 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, or to impaired autophagy, 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.

Proper fuelling of the brain is critical to sustain cognitive function, but the role of fatty acid (FA) combustion in this process has been elusive. Here we show that acute block of a neuron-specific triglyceride lipase, DDHD2 (a genetic driver of complex hereditary spastic paraplegia), or of the mitochondrial lipid transporter CPT1 leads to rapid onset of torpor in adult male mice. These data indicate that in vivo neurons are probably constantly fluxing FAs derived from lipid droplets (LDs) through β-oxidation to support neuronal bioenergetics. We show that in dissociated neurons, electrical silencing or blocking of DDHD2 leads to accumulation of neuronal LDs, including at nerve terminals, and that FAs derived from axonal LDs enter mitochondria in an activity-dependent fashion to drive local mitochondrial ATP production. These data demonstrate that nerve terminals can make use of LDs during electrical activity to provide metabolic support and probably have a critical role in supporting neuron function in vivo.

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 μm 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 signal transmission and ryanodine-receptor-dependent Ca2+ release at ER-PM junctions over 20 μm 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.

VPS13C is a ER-lysosome contact site protein that transports lipids between the two organellar membranes and important for lysosomal membrane damage repair. We employed structure-function analysis of purified VPS13C and uncovered novel regulatory mechanisms. Here, we describe the protocol to characterize VPS13C's binding to giant unilamellar vesicles (GUVs), where we discovered VPS13C's binding to membrane depends on its ATG2_C region recognizing membrane packing defects.

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 Parkinson's 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.

In eukaryotes, lipid building blocks for cellular membranes are made largely in the endoplasmic reticulum and then redistributed to other organelles. Lipids are transported between organelles by vesicular trafficking or else by proteins located primarily at sites where different organelles are closely apposed. Here we discuss transport at organelle contact sites mediated by shuttle-like proteins that carry single lipids between membranes to fine-tune their composition and by the more recently discovered bridge-like proteins that tether two organelles and provide a path for bulk lipid movement. Protein-mediated lipid transport is assisted by integral membrane proteins that have roles in (a) lowering the energy barrier for lipid transfer between the membrane and the lipid transfer protein, a key parameter determining the transfer rate, and (b) scrambling lipids to counteract the bilayer asymmetry that would result from such transfer. Advances in this field are shedding new light on a variety of physiological mechanisms.

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 Parkinson's 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.

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 that 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 binding 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 lysosomal recruitment 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.

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.