In situ structural analysis reveals membrane shape transitions during autophagosome formation
By onThe authors combined cell biology with correlative cryo-electron tomography in yeast cells to show a high resolution stepwise structural progression of autophagosome biogenesis.
Endosome isolation
By onSubcellular fractionation to isolate early and late endosomes (EEs and LEs) by performing a series of centrifugation steps.
Differentiation of human cortical neurons (CNs) from induced pluripotent stem cells (iPSCs) and their coculture with rat astrocytes
By onThis protocol described the production of human cortical neurons from induced pluripotent stem cells in cocultured with commercially available rat astrocytes.
Structure of wild-type PPM1H phosphatase at 3.1 Angstrom resolution
By onStructure of wild-type PPM1H phosphatase at 3.1 Angstrom resolution (as reported in 10.15252/embr.202152675) deposited in the Protein Data Bank with code 7KPR.
Generation of knock-in and knock-out CRISPR/Cas9 editing in mammalian cells
By onThis protocol is to help with generation of knock-in and knock-out CRISPR/Cas9 editing in mammalian cells
Confocal imaging and digital image analysis
By onProtocol for confocal imaging on Nikon A1R microscope & digital image analysis is detailed.
Membrane remodeling properties of the Parkinson’s disease protein LRRK2
By onPreprint: 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.
pCAG-MBP-TSF-ULK1(K46I)
By onPlasmid for the expression and purification of human ULK1 kinase dead complex
Generation of induced pluripotent stem cells and gene correction
By oniPSC generation and gene correction (CRISPR-CAS9) protocol.
Surface protein biotinylation
By onThis 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.
Western blotting for LRRK2 signalling in macrophages
By onThis protocol describes the immunoblotting for components of the LRRK2 signalling pathway (LRRK2, LRRK2 pS935 and phospho-Rabs) using Invitrogen NuPage SDS-PAGE reagents and the BioRad Turbo Blot transfer system.
Deficiency of the frontotemporal dementia gene GRN results in gangliosidosis.
By onHomozygous mutations of granulin precursor (GRN) lead to neuronal ceroid lipofuscinosis1, a severe neurodevelopmental disease, in humans and neuroinflammation in mice2. Haploinsufficiency of GRN almost invariably causes frontotemporal dementia (FTD)3,4. The GRN locus produces progranulin (PGRN), a lysosomal precursor protein that is cleaved to granulin peptides5,6. Despite intensive investigation, the function of granulins and the reason why their absence causes neurodegeneration remain unclear. Here, we investigated PGRN function in lipid degradation, a major function of lysosomes. We show that PGRN-knockout human cells, PGRN-deficient murine brain, and frontal lobes of human brains from patients with GRN mutation-related FTD have increased levels of gangliosides, highly abundant sialic acid–containing glycosphingolipids (GSL) that are degraded in lysosomes. Probing how PGRN deficiency causes these changes, we found normal levels and activities of enzymes that catabolize gangliosides. However, levels of bis(monoacylglycero)phosphate (BMP), a lysosomal lipid required for ganglioside catabolism7, were markedly reduced in PGRN-deficient cells and patient brain tissues. These data indicate that granulins are required to maintain BMP levels, which regulate ganglioside catabolism, and that PGRN deficiency in lysosomes leads to gangliosidosis. This aberrant accumulation of gangliosides may contribute to neuroinflammation and neurodegeneration susceptibility.
RNA isolation and qRT-PCR
By onThis protocol describes the RNA isolation from cultured cells and quantitative RT PCR.
SHIP164 is a chorein motif lipid transfer protein that controls endosome-Golgi membrane traffic
By onCellular membranes differ in protein and lipid composition as well as in the protein-lipid ratio. Thus, progression of membranous organelles along traffic routes requires mechanisms to control bilayer lipid chemistry and their abundance relative to proteins. The recent structural and functional characterization of VPS13-family proteins has suggested a mechanism through which lipids can be transferred in bulk from one membrane to another at membrane contact sites, and thus independently of vesicular traffic. Here, we show that SHIP164 (UHRF1BP1L) shares structural and lipid transfer properties with these proteins and is localized on a subpopulation of vesicle clusters in the early endocytic pathway whose membrane cargo includes the cation-independent mannose-6-phosphate receptor (MPR). Loss of SHIP164 disrupts retrograde traffic of these organelles to the Golgi complex. Our findings raise the possibility that bulk transfer of lipids to endocytic membranes may play a role in their traffic.
Collection of protocols for paper: “Glucocerebrosidase, a Parkinson´s disease-associated protein, is imported into mitochondria and regulates complex I assembly and function”
By onThis is a collection of protocols used in a recent preprint by the Deleidi Lab, Team Schapira. You can access pre-print at https://doi.org/10.21203/rs.3.rs-1521848/v1
