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.
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.
Surface protein biotinylation
This protocol describes surface protein labeling with biotin using EZ-Link Sulfo-NHSLC-Biotin. This chemical reacts with primary amines such as lysine but does not permeate cell membranes because of the charge. Thus, it only biotinylates surface proteins.
SHIP164 is a Chorein Motif Containing Lipid Transport Protein that Controls Membrane Dynamics and Traffic at the Endosome-Golgi Interface
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.
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.
Membrane remodeling properties of the Parkinson’s disease protein LRRK2
Preprint: The authors examine how purified LRRK2 directly binds acidic lipid bilayers in a cell-free system and can deform them into narrow tubules in a guanylnucleotide-dependent but ATP-independent way.
Cell culture, transfection, and imaging
This protocol details the general preparation of cells for imaging and also for imaging experiments involving cellular hypotonic shock and cytosolic Ca2+ changes as they were performed in https://doi.org/10.1083/jcb.202010004.
Cell culture, transfection, and imaging
This protocol describes general procedures for culturing HeLa cells, transient transfection, and imaging using an Andor Dragonfly spinning disk confocal system.
This protocol describes the procedure of plunge freezing for subsequent cryo-electron tomography.
Purifying Vps13-VAB domain proteins
In this protocol, we have listed the steps to purify the Vps13 adaptor binding domain (or VAB) with or without PxP motif fusion peptide derived from adaptor proteins Mcp1, Ypt35 or Spo71.
Crystallization, data collection and structure solution of PxP-VAB
Protocol lists the steps to obtain PxP(Mcp1ct)-VABct crystals, X-ray diffraction data acquisition and obtaining the structure solution using molecular replacement with a model generated by AlphaFold2.
Endoplasmic Reticulum Membrane Contact Sites, Lipid Transport, and Neurodegeneration
Review: Several mutations of genes that encode proteins localized at the endoplasmic reticulum membrane contact sites result in familial neurodegenerative diseases. Here, the authors provide an overview of such diseases, with a specific focus on proteins that directly or indirectly impact lipid transport.
VPS13D with deleted DHPH domain.
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.
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.