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CRISPR knockout screen implicates three genes in lysosome function

ABSTRACT: Defective biosynthesis of the phospholipid PI(3,5)P2 underlies neurological disorders characterized by cytoplasmic accumulation of large acidic vacuoles. To identify novel genetic causes of lysosomal vacuolization, we developed an assay for enlargement of the lysosome compartment that is amenable to cell sorting and pooled screens. After calibration on cells lacking FIG4, a known PI(3,5)P2 biosynthetic factor, we carried out a genome-wide knockout screen that captured fifteen genes, including VAC14 which is known to cause endosomal vacuolization. We selected three genes not previously associated with lysosome dysfunction for validation: C10orf35, LRRC8A, and MARCH7. We isolated two clonal knockout cell lines for each gene and confirmed loss of protein expression. The knockout lines contained enlarged acidic vesicles with surface labeling of the endolysosomal marker LAMP2. This assay will be useful for characterizing variants of unknown significance in patients with neuromuscular or lysosomal storage disorders. This genome-wide strategy for identification of genes with lysosomal function can also be adapted for drug screens to identify small molecules that correct vacuolization.

ORGANISM(S): synthetic construct Homo sapiens

PROVIDER: GSE129282 | GEO | 2019/05/01

REPOSITORIES: GEO

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Project description:Phosphatase activities on PI(3,4,5)P3 and PI(3,4)P2 This model describes the action of various phosphatases on PI(3,4,5)P3 and PI(3,4)P2, in response to a stimulation by EGF. It contains boolean switches to simulate knock-down and knock-out of phosphatases as well as inhibition of PI3 kinase. This model is described in the article: PTEN Regulates PI(3,4)P2 Signaling Downstream of Class I PI3K Mouhannad Malek, Anna Kielkowska, Tamara Chessa, Karen E. Anderson, David Barneda, P?nar Pir, Hiroki Nakanishi, Satoshi Eguchi, Atsushi Koizumi, Junko Sasaki, Véronique Juvin, Vladimir Y. Kiselev, Izabella Niewczas, Alexander Gray, Alexandre Valayer, Dominik Spensberger, Marine Imbert, Sergio Felisbino, Tomonori Habuchi, Soren Beinke, Sabina Cosulich, Nicolas Le Novère, Takehiko Sasaki, Jonathan Clark, Phillip T. Hawkins and Len R. Stephens Molecular Cell Abstract: The PI3K signaling pathway regulates cell growth and movement and is heavily mutated in cancer. Class I PI3Ks synthesize the lipid messenger PI(3,4,5)P3. PI(3,4,5)P3 can be dephosphorylated by 3- or 5-phosphatases, the latter producing PI(3,4)P2. The PTEN tumor suppressor is thought to function primarily as a PI(3,4,5)P3 3-phosphatase, limiting activation of this pathway. Here we show that PTEN also functions as a PI(3,4)P2 3-phosphatase, both in vitro and in vivo. PTEN is a major PI(3,4)P2 phosphatase in Mcf10a cytosol, and loss of PTEN and INPP4B, a known PI(3,4)P2 4-phosphatase, leads to synergistic accumulation of PI(3,4)P2, which correlated with increased invadopodia in epidermal growth factor (EGF)-stimulated cells. PTEN deletion increased PI(3,4)P2 levels in a mouse model of prostate cancer, and it inversely correlated with PI(3,4)P2 levels across several EGF-stimulated prostate and breast cancer lines. These results point to a role for PI(3,4)P2 in the phenotype caused by loss-of-function mutations or deletions in PTEN. This model is hosted on BioModels Database and identified by: MODEL1704190000. To cite BioModels Database, please use: Chelliah V et al. BioModels: ten-year anniversary. Nucl. Acids Res. 2015, 43(Database issue):D542-8. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

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CRISPR knockout screen implicates three genes in lysosome function

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