A fluorescence-based in vitro scrambling assay for yeast MCP1 and human XK
By onVPS13 proteins are proposed to function at contact sites between organelles as bridges for lipids to move directionally and in bulk between organellar membranes. VPS13s are found to interact with integral membrane proteins, like MCP1 in yeast or XK in humans. We showed that MCP1 and XK scramble phospholipids in vitro. Here I describe the detailed procedure of purification, reconstitution, and scrambling assay for both MCP1 and XK.
In vitro GCase activity assay (total cell lysate)
By onGlucocerebrosidase is a lysosomal enzyme that catalyzes the hydrolysis of glucosylceramide (GlcCer), a membrane glyco-sphingolipid, to ceramide and glucose. This assay detects GBA activity by using a fluorogenic substrate that reacts with cell lysates previously treated with or without CBE (GBA1 inhibitor). This protocol is part of a Collection of protocols (dx.doi.org/10.17504/protocols.io.8epv593dng1b/v1) for the paper "Glucocerebrosidase, a Parkinson´s disease-associated protein, is imported into mitochondria and regulates complex I assembly and function" (https://doi.org/10.21203/rs.3.rs-1521848/v1)
ENS Quantification
By onLabelled enteric neurons in living mice with recombinant adeno-associated viruses (rAAVs) expressing fluorescent proteins, and used tissue clearing techniques to enhance visualization of intact GI tissue.
Induction of non-selective bulk autophagy
By onThis protocol describes how to induce bulk (non-selective) autophagy in HeLa cells through nutrient starvation.
Reconstitution of cargo-induced LC3 lipidation in mammalian selective autophagy
By onGUV quantification
Reduced Thalamic Excitation to Motor Cortical Pyramidal Tract Neurons in Parkinsonism
By onThe current data suggests that cell subtype- and synapse-specific adaptations in M1 contribute to altered cortical outputs in parkinsonism and are important aspects of PD pat
Glucocerebrosidase is imported into mitochondria and preserves complex I integrity and energy metabolism
By onMutations in GBA1, the gene encoding the lysosomal enzyme β-glucocerebrosidase (GCase), which cause Gaucher’s disease, are the most frequent genetic risk factor for Parkinson’s disease (PD). Here, we employ global proteomic and single-cell genomic approaches in stable cell lines as well as induced pluripotent stem cell (iPSC)-derived neurons and midbrain organoids to dissect the mechanisms underlying GCase-related neurodegeneration. We demonstrate that GCase can be imported from the cytosol into the mitochondria via recognition of internal mitochondrial targeting sequence-like signals. In mitochondria, GCase promotes the maintenance of mitochondrial complex I (CI) integrity and function. Furthermore, GCase interacts with the mitochondrial quality control proteins HSP60 and LONP1. Disease-associated mutations impair CI stability and function and enhance the interaction with the mitochondrial quality control machinery. These findings reveal a mitochondrial role of GCase and suggest that defective CI activity and energy metabolism may drive the pathogenesis of GCase-linked neurodegeneration.
Structural basis for the specificity of PPM1H phosphatase for Rab GTPases
By onLRRK2 acts by adding a phosphate group to enzymes known as Rab GTPases, which causes new biological events. The authors analyzed the structure of an enzyme, PPM1H, that counteracts LRRK2 by removing the phosphate group it adds to Rab GTPases.
VPS13D DNA plasmid generation
By onThis protocol describes the basic mole cular cloning technique utilized for the generation of VPS13D constructs in https://doi.org/10.1083/jcb.202010004. This protocol and the enzymes included in it are commercialized by Takara Bio.Due to low expression of big DNA constructs (u003e10kb), we decided to generate a codon-optimized cDNA encoding for human VPS13D, including an mScarlet fluorescent protein after aminoacid 1576 flanked by BamHI restriction enzyme sites. The construct was generated by and purchased from Genscript. From this initial construct, we generated several VPS13D constructs. The specific primers and enzymes used for each construct are included in Table 1 of our manuscript: https://doi.org/10.1083/jcb.202010004" . For most of our cloning procedures, Infusion cloning (Takara) was used.
pAC150- FAM134C-GFP
By onThe text describes a PiggyBac vector used to express a GFP-FAM134C reporter gene.
ASAP Blueprint for Collaborative Open Science
By onThis Blueprint presents initial findings on how ASAP’s approach to open science has solidified and evolved over its first three years, data and metrics on progress, and CC-BY versions of assets that can be adopted and adapted by others.
pCAG- WIPI2dI92E-cs- TEV -STREP
By onPlasmid: Mammalian expression of human WIPI2d I92E with C-terminal Strep.
Purifying Vps13-VAB domain proteins
By onIn 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.
Detection of mosaic and population-level structural variants with Sniffles2
By onSniffles2 is a fast and accurate tool for identifying complex genomic alterations using long -read data.
Conserved and cell type-specific transcriptional responses to IFN-γ in the ventral midbrain
By onNeurons present antigens when exposed to IFN-γ, leading to MHC-I expression. IFN-γ induces MHC-I in neurons, glia, and microglia, with glia showing a stronger response. Neuronal response to IFN-γ is dependent on IFNGR signaling.
Generation of human-induced pluripotent-stem-cell-derived cortical neurons for high-throughput imaging of neurite morphology and neuron maturation
By onA STAR protocol that describes how to differentiate cryopreserved human cortical neuronal progenitors into mature cortical neurons for high-throughput imaging analysis
Phosphosite mapping on PI3Kc1 by ULK1 and TBK1 kinases.
By onPhosphosite mapping on PI3Kc1 by ULK1 and TBK1 kinases.
