Python script for Golgi cytoscape analysis
By onCustom Python script used for the cytoscape network analysis reported in doi.org/10.1101/2022.11.22.517583 (Golgi-IP, a novel tool for multimodal analysis of Golgi molecular content).
POLCAM: Instant molecular orientation microscopy for the life sciences
By onPOLCAM is a simplified single-molecule orientation localization microscopy method, using a polarization camera, enabling fast easy implementation on fluorescence microscopes. Here, we apply it to alpha-synuclein fibrils.
BPK1520-LRRK2-G2019S-ng
By onPlasmid: This resource can be used to introduce LRRK2-G2019S mutation using prime editing, in PE3 approach.
A mono- and intralink filter (mi-filter) to reduce false identifications in cross-linking mass spectrometry data
By onCross-linking mass spectrometry (XL-MS) has become an indispensable tool for the emerging field of systems structural biology over the recent years. However, the confidence in individual protein–protein interactions (PPIs) depends on the correct assessment of individual inter-protein cross-links. In this article, we describe a mono- and intralink filter (mi-filter) that is applicable to any kind of cross-linking data and workflow. It stipulates that only proteins for which at least one monolink or intra-protein cross-link has been identified within a given data set are considered for an inter-protein cross-link and therefore participate in a PPI. We show that this simple and intuitive filter has a dramatic effect on different types of cross-linking data ranging from individual protein complexes over medium-complexity affinity enrichments to proteome-wide cell lysates and significantly reduces the number of false-positive identifications for inter-protein links in all these types of XL-MS data.
H9 ES AAVS1-NGN2 FAM134C/A/B-/-;TEX264-/-; CCPG1-/-; PiggyBac-Keima-REEP5
By onES cells were modified to produce iNeurons lacking ER-phagy receptor genes and expressing Keima-REEP5 reporter. CRISPR/Cas9 was used to introduce NEUROG2 construct in AAVS1 locus, and various genes were knocked out.
pCAG-MBP-Foldon-ATG9 (692-839)
By onPlasmid for expression of ATG9 C-terminal tail trimer in mammalian cells.
LC3 Lipidation assay for ATG3 Mutants
By onProtocol describing the procedure to perform LC3 lipidation reaction assay on liposomes (SUVs, Small Unilamellar Vesicles).
Synaptotagmin-1-dependent phasic axonal dopamine release is dispensable for basic motor behaviors in mice
By onIn Parkinson’s disease (PD), motor dysfunctions only become apparent after extensive loss of DA innervation. This resilience has been hypothesized to be due to the ability of many motor behaviors to be sustained through a diffuse basal tone of DA; but experimental evidence for this is limited. Here we show that conditional deletion of the calcium sensor synaptotagmin-1 (Syt1) in DA neurons (Syt1 cKODA mice) abrogates most activity-dependent axonal DA release in the striatum and mesencephalon, leaving somatodendritic (STD) DA release intact. Strikingly, Syt1 cKODA mice showed intact performance in multiple unconditioned DA-dependent motor tasks and even in a task evaluating conditioned motivation for food. Considering that basal extracellular DA levels in the striatum were unchanged, our findings suggest that activity-dependent DA release is dispensable for such tasks and that they can be sustained by a basal tone of extracellular DA. Taken together, our findings reveal the striking resilience of DA-dependent motor functions in the context of a near-abolition of phasic DA release, shedding new light on why extensive loss of DA innervation is required to reveal motor dysfunctions in PD.
Design and preparation of synthetic reference peptides for APP/Aβ TOMAHAQ proteomics, version 2
By onThis protocol describes the design and preparation of synthetic reference peptides for APP/Aβ TOMAHAQ proteomics.
Thawing of feeder-free hPSCs
By onThis protocol describes the procedure of thawing feeder-free human pluripotent stem cells (hPSCs) using mTeSR-plus or StemFlex.
The chaperone Clusterin in neurodegeneration−friend or foe?
By onThe authors review the diverse functions of Clusterin in the pathogenesis of neurodegenerative diseases, focusing on evidence that Clusterin may act either as a suppressor or enhancer of pathology.
H9 ES AAVS1-NGN2 FAM134C/A/B-/-; PiggyBac-Keima-RAMP4
By onES cells were modified to produce iNeurons lacking FAM134C, A, B receptors and expressing Keima-RAMP4 ER-phagy flux reporter. CRISPR/Cas9 was used to introduce NEUROG2 construct and knockout RETREG1, 2, 3 genes.
H9 ES AAVS1-NGN2 TEX264-/-; PiggyBac-Keima-RAMP4
By onES cells were modified using CRISPR/Cas9 to lack TEX264 and express Keima-RAMP4 ER-phagy flux reporter. NEUROG2 construct was introduced in AAVS1 locus. Cells are human embryonic stem cells with a fluorescent protein marker.
Assay for PhosphoRab activation of LRRK2 Kinase
By onThis protocol includes a method to produce phosphoRab8A protein and remove as much contaminating MST3 as possible, to enable use of the phosphoRab to test subsequent activation of LRRK2 kinase.
Interaction of an α-synuclein epitope with HLA-DRB1*15:01 triggers enteric features in mice reminiscent of prodromal Parkinson’s disease
By onInteraction of α-syn32-46 and HLA-DRB1*15:0 is critical for gut inflammation and CD4+ T cell-mediated loss of enteric neurons in humanized mice, suggesting potential mechanisms of prodromal enteric PD.
Organelle isolation from Mouse Embryonic Fibroblasts (MEFs) stably expressing organelle tags for subsequent immunoblotting or proteomic analysis
By onWe describe here a method to perform rapid isolation of intact organelles (including lysosomes and Golgi) from mouse embryonic fibroblasts stably expressing an organelle tag (TMEM192-3xHA, or LysoTag, and TMEM115-3xHA, or GolgiTag). First, cells are broken using a ball-bearing cell breaker, leading to plasma membrane rupture, while lysosomes and Golgi remain intact. Then, the cell homogenate is incubated with anti-HA magnetic beads to allow for immunopurification of HA-tagged lysosomes or Golgi in less than 15 minutes. The organelles purified using this method are highly enriched, intact, contaminant-free and, depending on solubilisation buffer, can be used for various downstream applications, including immunoblotting analysis and mass spectrometry proteomic analysis (as described here), but also metabolomic or lipidomic analysis. This protocol can be adapted to isolate organelles from commonly cultured cells, such as HEK293 and A549 cells, that express an organelle tag.
