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.
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.
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.
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.
pAC150- FAM134C-GFP
By onThe text describes a PiggyBac vector used to express a GFP-FAM134C reporter gene.
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.
Phosphosite mapping on PI3Kc1 by ULK1 and TBK1 kinases.
By onPhosphosite mapping on PI3Kc1 by ULK1 and TBK1 kinases.
Detecting Full-Length EccDNA with FLED and long-reads sequencing
By onReconstructing the full-length sequence of extrachromosomal circular DNA (eccDNA) from short sequencing reads has proved challenging given the similarity of eccDNAs and their corresponding linear DNAs. Previous sequencing methods were unable to achieve high-throughput detection of full-length eccDNAs. Here we describe a new strategy that combined rolling circle amplification (RCA) and nanopore long-reads sequencing technology to generate full-length eccDNAs. We further developed a novel algorithm, called Full-Length eccDNA Detection (FLED), to reconstruct the sequence of eccDNAs. We used FLED to analyze seven human epithelial and cancer cell line samples and identified over 5,000 full-length eccDNAs per sample. The structures of identified eccDNAs were validated by both PCR and Sanger sequencing. Compared to other published nanopore-based eccDNA detectors, FLED exhibited higher sensitivity. In cancer cell lines, the genes overlapped with eccDNA regions were enriched in cancer-related pathways and cis-regulatory elements can be predicted in the up-stream or downstream of intact genes on eccDNA molecules, and the expressions of these cancer-related genes were dysregulated in tumor cell lines, indicating the regulatory potency of eccDNAs in biological processes. Our method takes advantage of nanopore long reads and enables unbiased reconstruction of full-length eccDNA sequences. FLED is imple-mented using Python3 which is freely available on GitHub (https://github.com/FuyuLi/FLED).
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
From Policy to Practice: Tracking an Open Science Funding Initiative
By onBy normalizing the open science and compliance process across funding bodies, ASAP hopes to simplify and streamline researcher, institutional, and funder workflows, allowing researchers to focus on science.
Immunoblotting analysis of samples from GolgiTAG (TMEM115-3HA) immunoprecipitation
By onAn immunoblotting method to assess efficient enrichment of Golgi proteins in Golgi immunoprecipitation products compared to whole cell lysates, and the purity of the immunoprecipitated Golgi by monitoring the expression of other organelles’ markers.
Cell lysis and gel electrophoresis for protein analysis of HeLa cells
By onThe authors present multiple protocols used for biochemical analysis of protein expression and association. T
Single cell transcriptomics from DAT-Cre mouse gut tissue
By onColonic myenteric cells were dissociated, and live tdTomato-positive cells were FACS-collected. Colons were isolated from 2 male and female wild type adult DAT-Cre mice. Wild type C57Bl6/J was used as control for gating. Cells were processed according to 10X Genomics Chromium single cell 3' Reagent guidelines.
Native-PAGE analysis of VCP hexamer
By onThis protocol is associated with the following preprint, published on February 19th 2022: The AAA+ chaperone VCP disaggregates Tau fibrils and generates aggregate seeds Itika Saha, Patricia Yuste-Checa, Miguel Da Silva Padilha, Qiang Guo, Roman Körner, Hauke Holthusen, Victoria A. Trinkaus, Irina Dudanova, Rubén Fernández-Busnadiego, Wolfgang Baumeister, David W. Sanders, Saurabh Gautam, Marc I. Diamond, F. Ulrich Hartl, Mark S. Hipp bioRxiv 2022.02.18.481043; doi: https://doi.org/10.1101/2022.02.18.481043
Immunological detection of APP and proteins of the endolysosomal system
By onThis protocol describes the immunological detection of APP and proteins of the endolysosomal system.Here we present a general protocol for immunological detection by Western blotting of APP and proteins of the endolysosomal system, including EEA1, RAB5, PSEN1, LAMP1, LAMP2, TMEM192, and BACE1.
