Protocol for establishing knockout cell clones by deletion of a large gene fragment using CRISPR-Cas9 with multiple guide RNAs

STAR Protocols, Journal Year: 2024, Volume and Issue: 5(3), P. 103179 - 103179

Published: July 6, 2024

Genome editing is a powerful tool for establishing gene knockout or mutant cell lines. Here, we present protocol clones by deletion of large fragments using CRISPR-Cas9 with multiple guide RNAs. We describe steps designing RNAs, cloning them into vectors, seeding, transfection cultured cells, clonal selection, and screening assays. This can delete regions over 100 kbp, including GC-rich domains, applicable to various For complete details on the use execution this protocol, please refer Saito et al.,

Language: Английский

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Rho-ROCK liberates sequestered claudin for rapid de novo tight junction formation

Published: Nov. 22, 2024

The epithelial cell sheet maintains its integrity as a barrier while undergoing turnover of constituent cells. To sustain the continuously, it’s essential to preserve ‘old’ tight junctions (TJs) between cells being excluded from and their neighbors simultaneously forming de novo TJs newly adjacent However, molecular mechanisms involved in formation remain largely unknown. This study investigates two scenarios: during removal apoptotic monolayer sheets differentiation granular layer stratified epidermis. We revealed that rapid claudin assembly is achieved by actively regulating dissociation EpCAM/TROP2-claudin complex both situations. Furthermore, we found Rho-ROCK pathway initiates activation matriptase, which cleaves EpCAM/TROP2, resulting supply polymerizable stockpiled at plasma membrane induce TJ formation.

Language: Английский

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Structure and function of a Notch-like proteolytic switch domain in the extracellular matrix receptor dystroglycan

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2022, Volume and Issue: unknown

Published: April 4, 2022

ABSTRACT The dystrophin glycoprotein complex (DGC), anchored by the transmembrane protein dystroglycan, functions to mechanically link extracellular matrix actin cytoskeleton drive critical aspects of development and adult homeostasis. Breaking this connection via mutation adaptor or impaired glycosylation dystroglycan are strongly associated with diseases such as Muscular Dystrophy, yet cleavage metalloproteinases (MMPs) remains an understudied mechanism disrupt DGC. We solved X-ray structures membrane-adjacent domain understand molecular underpinnings MMP regulation. Dystroglycan proteolysis occurs within versatile SEAL domain, which supports in diverse receptors facilitate mechanotransduction, protection cell membranes, even viral entry. structure reveals a c-terminal extension that buries site packing into hydrophobic pocket, unique further demonstrate structure-guided disease-associated mutations proteolytic regulation using new cell-surface assay. Finally, we find disruption leads altered cellular mechanics migration high-throughput DNA tension probe wound healing assays. These findings highlight disrupted is relevant for “breaking” DGC contribute disease pathogenesis may offer therapeutic avenues dystroglycanopathies.

Language: Английский

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Clipping EpCAM to release Claudin-7 for the greater good of the epithelial barrier

The Journal of Cell Biology, Journal Year: 2022, Volume and Issue: 222(1)

Published: Dec. 14, 2022

Maintenance of epithelial barrier function requires dynamic repair and remodeling tight junctions. In this issue, Higashi et al. (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202204079) demonstrate that the proteolytic cleavage EpCAM by membrane-anchored serine proteinases releases Claudin-7 to join junctions, suggesting a novel mechanism couples sensing with damaged

Language: Английский

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ESCRT-III-dependent adhesive and mechanical changes are triggered by a mechanism sensing paracellular diffusion barrier alteration inDrosophilaepithelial cells

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: May 24, 2023

Summary Barrier functions of proliferative epithelia are constantly challenged by mechanical and chemical constraints. How respond to cope with disturbances the paracellular diffusion barrier allow tissue integrity maintenance has been poorly characterized. Cellular junctions play an important role in this process intracellular traffic contribute their homeostasis. Here, we reveal that, Drosophila pupal notum, alteration bi- or tricellular septate (SJs) triggers a mechanism two prominent outcomes. On one hand, there is increase levels E-cadherin, F- Actin non-muscle myosin II plane adherens junctions. other β-integrin/Vinculin-positive cell contacts reinforced along lateral basal membranes. We report that weakening SJ integrity, caused depletion components, reduces ESCRT-III/Vps32/Shrub-dependent degradation promotes instead Retromer-dependent recycling components. The consequence reduction Shrub-dependent extends transmembrane protein cargoes. Consequently, trigger increased β- integrin, Crumbs effectors β-Heavy Spectrin Karst. propose which epithelial cells, upon sensing alterations barrier, target Shrub adjust degradation/recycling balance thereby compensate for defects while maintaining integrity.

Language: Английский

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