pCAG-OSF-ATG13 (2-197)-E83L
By onPlasmid for expression of human ATG13 HORMA E83L mutant in mammalian cells
Lipid transfer assay
By onThis protocol details how to perform a lipid transfer assay with 3xFLAG-SHIP164Δ901-1099
H9 ES AAVS1-NGN2 CCPG1-/-
By onES cells modified using CRISPR/Cas9 lack the ER-phagy receptor CCPG1. NEUROG2 gene was introduced using a TRE3G promoter in AAVS1 locus. The cells are human embryonic stem cells derived from blastocysts.
HEK293T FBXO7-/-
By onHEK293T cells were genetically modified using CRISPR/Cas9 to delete the FBXO7/PARK15 gene.
Dopamine transporter and synaptic vesicle sorting defects underlie auxilin-associated Parkinson’s disease
By onAuxilin participates in clathrin uncoating to facilitate presynaptic endocytosis. Loss-of-function mutations of auxilin (PARK19) cause Parkinson’s disease. Using auxilin KO mice, Vidyadhara et al. (2023) show that synaptic vesicle sorting deficits, cytoplasmic dopamine accumulation, dopamine transporter mistrafficking, and synaptic autophagic overload may lead to pathogenesis of Parkinson’s disease in PARK19 patients. This file contains the data set used to generate all the main figures.
H9 ES AAVS1-NGN2 FAM134C/A-/-; PiggyBac-Keima-REEP5
By onES cells were modified using CRISPR/Cas9 to express NEUROG2 and Keima-REEP5 for ER-phagy studies. Knockouts RETREG2 and RETREG3 were created, and cells were derived from a blastocyst stage female human embryonic stem cell line.
Custom open-chamber microfluidic fabrication
By onThis protocol described the fabrication of two- and three-open chambered microfluidics suitable for cell culturing using commercially acquired master mould.
Proteomics workflow for whole cell lysate, endosome, and lysosome fractions
By onA protocol for sample preparation for LC-MS analysis of whole cell lysates and for lysosomal and endosomal fractions purified by Lyso-IP and Endo-IP.
Cell-based Tau seeding assay
By onProtocol outlines Tau seeding techniques in cellular model for research purposes.
A Markov random field model-based approach for differentially expressed gene detection from single-cell RNA-seq data
By onSingle-cell RNA-sequencing technology enables the identification of cell-type-specific differential gene expressions. MARBLES, a new statistical model, effectively detects DE genes across conditions.
Membrane interaction and mechanism of LC3 lipidation machinery in autophagy – Raw GUV data
By onRaw GUV data of fluorescent protein imaged on a Nikon A1 confocal microscope with a 63 × Plan Apochromat 1.4 NA objective. Three biological replicates were performed for each experimental condition. Identical laser power and gain settings used.
Preparing plasmids for nucleofection of hPSCs
By onThis protocol describes the standard procedure for the preparation of plasmids to be delivered into human pluripotent stem cells (hPSCs) using nucleofection.
A possible role for VPS13-family proteins in bulk lipid transfer, membrane expansion and organelle biogenesis
By onAt organelle–organelle contact sites, proteins have long been known to facilitate the rapid movement of lipids. Classically, this lipid transport involves the extraction of single lipids into a hydrophobic pocket on a lipid transport protein. Recently, a new class of lipid transporter has been described with physical characteristics that suggest these proteins are likely to function differently. They possess long hydrophobic tracts that can bind many lipids at once and physically span the entire gulf between membranes at contact sites, suggesting that they may act as bridges to facilitate bulk lipid flow. Here, we review what has been learned regarding the structure and function of this class of lipid transporters, whose best characterized members are VPS13 and ATG2 proteins, and their apparent coordination with other lipid-mobilizing proteins on organelle membranes. We also discuss the prevailing hypothesis in the field, that this type of lipid transport may facilitate membrane expansion through the bulk delivery of lipids, as well as other emerging hypotheses and questions surrounding these novel lipid transport proteins.
Optogenetic experiments with iLID system
By onThis protocol details experiments with the iLID optogenetic system as performed to acutely recruit Miro to mitochondria.
Endosomal escape of RNA therapeutics: How do we solve this rate-limiting problem?
By onWith over 15 FDA approved drugs on the market and numerous ongoing clinical trials, RNA therapeutics, such as small interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs), have shown great potential to treat human disease. Their mechanism of action is based entirely on the sequence of validated disease-causing genes without the prerequisite knowledge of protein structure, activity or cellular location. In contrast to small molecule therapeutics that passively diffuse across the cell membrane's lipid bilayer, RNA therapeutics are too large, too charged, and/or too hydrophilic to passively diffuse across the cellular membrane and instead are taken up into cells by endocytosis. However, endosomes are also composed of a lipid bilayer barrier that results in endosomal capture and retention of 99% of RNA therapeutics with 1% or less entering the cytoplasm. Although this very low level of endosomal escape has proven sufficient for liver and some CNS disorders, it is insufficient for the vast majority of extra-hepatic diseases. Unfortunately, there are currently no acceptable solutions to the endosomal escape problem. Consequently, before RNA therapeutics can be used to treat widespread human disease, the rate-limiting delivery problem of endosomal escape must be solved in a nontoxic manner.
