Genome-wide CRISPR screen identified a role for commander complex mediated ITGB1 recycling in basal insulin secretion

Abstract

Objectives

Pancreatic beta cells secrete insulin postprandially and during fasting to maintain glucose homeostasis. Although glucose-stimulated insulin secretion (GSIS) has been extensively studied, much less is known about basal insulin secretion. Here, we performed a genome-wide CRISPR/Cas9 knockout screen to identify novel regulators of insulin secretion.

Methods

To identify genes that cell autonomously regulate insulin secretion, we engineered a Cas9-expressing MIN6 subclone that permits irreversible fluorescence labeling of exocytic insulin granules. Using a fluorescence-activated cell sorting assay of exocytosis in low glucose and high glucose conditions in individual cells, we performed a genome-wide CRISPR/Cas9 knockout screen.

Results

We identified several members of the COMMD family, a conserved family of proteins with central roles in intracellular membrane trafficking, as positive regulators of basal insulin secretion, but not GSIS. Mechanistically, we show that the Commander complex promotes insulin granules docking in basal state. This is mediated, at least in part, by its function in ITGB1 recycling. Defective ITGB1 recycling reduces its membrane distribution, the number of focal adhesions and cortical ELKS-containing complexes.

Conclusions

We demonstrated a previously unknown function of the Commander complex in basal insulin secretion. We showed that by ITGB1 recycling, Commander complex increases cortical adhesions, which enhances the assembly of the ELKS-containing complexes. The resulting increase in the number of insulin granules near the plasma membrane strengthens basal insulin secretion.

Highlights

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  A MIN6 clone was developed for high throughput assay of insulin secretion in bulk and in single cells.

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  Genome-wide CRISPR screen in the clone identified Commander complex among the top hits.

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  Commd3 ^-/- cells exhibited reduced number of docked Insulin granules and exocytic events in low glucose conditions.

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  ITGB1 recycling was impaired in Commd3 ^-/- cells in low glucose, causing diminished cortical focal adhesions and ELKS complexes.