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Catalog
ASAP is committed to accelerating the pace of discovery and informing a path to a cure for Parkinson’s disease through collaboration, research-enabling resources, and data sharing. We’ve created this catalog to showcase the research outputs and tools developed by ASAP-funded programs.
Protocol
Native gel lipid binding assay
This protocol details how to perform a native gel lipid binding assay to evaluate the number of lipids bound per molecule of 3xFLAG-SHIP164Δ901-1099.
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Article
SHIP164 is a chorein motif lipid transfer protein that controls endosome–Golgi membrane traffic
Published: The authors show that SHIP164 shares strong structural similarities with VPS13 and ATG2 and, like these two proteins, functions as a lipid transport protein. SHIP164 localizes to clusters of endocytic vesicles and loss of SHIP164 disrupts retrograde traffic of certain organelles to the Golgi complex. View original preprint.
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Protocol
VPS13D DNA plasmid generation
This protocol describes the basic molecular cloning technique utilized for the generation of VPS13D constructs in VPS13D bridges the ER to mitochondria and peroxisomes via Miro. This protocol and the enzymes included in it are commercialized by Takara Bio.
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Cell culture on EM grids and fluorescence microscopy imaging
This protocol describes the general procedure of seeding mammalian cells on EM grids and confocal fluorescence microscopy imaging of grids for subsequent cryo-tomography experiments, performed.
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A fluorescence-based in vitro scrambling assay for yeast MCP1 and human XK
Detailed procedure of purification, reconstitution, and scrambling assay for both MCP1 and XK.
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Article
Mutations in Parkinsonism-linked endocytic proteins synaptojanin1 and auxilin have synergistic effects on dopaminergic axonal pathology
Published: Mutations in synaptojanin 1 (SJ1) and auxilin have been implicated in PD. The authors show that auxilin knockout mice have similar nigrostriatal changes to SJI mutant mice. Furthermore, auxilin/SJ1 double mutant mice have more severe defects and shower lifespans, showing the mutations have a synergistic interaction. View original preprint.
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Optogenetic experiments with iLID system
This protocol details experiments with the iLID optogenetic system as performed to acutely recruit Miro to mitochondria in https://doi.org/10.1083/jcb.202010004.
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Article
ER-lysosome lipid transfer protein VPS13C/PARK23 prevents aberrant mtDNA-dependent STING signaling
Published: Mutations in VPS13C cause early onset, autosomal recessive PD. VPS13C encodes a lipid transfer protein that is localized to ER-endosome/lysosome contact sites. The authors demonstrate that depletion of VPS13C causes an accumulation of lysosomes and activated STING, suggesting a link between ER-lysosome lipid transfer and innate immune activation. View original preprint.
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Protocol
Protocol for hippocampal neuronal cultures
This protocol details the procedure for preparation of neuronal cultures from mice hippocampi as it was performed in https://doi.org/10.1083/jcb.202010004 but can also be used to prepare cultures of cortical neurons.
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Article
In situ architecture of the lipid transport protein VPS13C at ER-lysosomes membrane contacts
Publication: Loss-of-function mutations in VPS13C are responsible for rare cases of familial early onset Parkinson’s disease. Using cryo-ET, the authors provide in-situ evidence for a bridge-model of VPS13 in lipid transport. View the original preprint.
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Protocol
Protocol for Image processing and analysis of VPS13D recruitment to mitochondria
This protocol details the image processing and analysis of VPS13D recruitment to mitochondria as it was performed in https://doi.org/10.1083/jcb.202010004. The first part details the analysis of acute optogenetic recruitment, and the second part of basal recruitment under different conditions.
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Hippocampal Neuronal Culture
This protocol describes the procedure for hippocampal neuronal cultures from new – born mouse pups.
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Subtomogram averaging and classification
This protocol describes the procedure of subtomogram averaging and 3D classification of VPS13C rod-shaped densities inside cryotomograms of mammalian cells. Sub-tomogram averaging package i3 was used for 3D alignment and classification.
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Plunge freezing
This protocol describes the procedure of plunge freezing for subsequent cryo-electron tomography.
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