Coding variants identified in patients with diabetes alter PICK1 BAR domain function in insulin granule biogenesis
Rita C. Andersen, … , Ulrik Gether, Kenneth L. Madsen
Rita C. Andersen, … , Ulrik Gether, Kenneth L. Madsen
Published January 25, 2022
Citation Information: J Clin Invest. 2022;132(5):e144904. https://doi.org/10.1172/JCI144904.
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Research Article Cell biology Metabolism

Coding variants identified in patients with diabetes alter PICK1 BAR domain function in insulin granule biogenesis

Abstract

Bin/amphiphysin/Rvs (BAR) domains are positively charged crescent-shaped modules that mediate curvature of negatively charged lipid membranes during remodeling processes. The BAR domain proteins PICK1, ICA69, and the arfaptins have recently been demonstrated to coordinate the budding and formation of immature secretory granules (ISGs) at the trans-Golgi network. Here, we identify 4 coding variants in the PICK1 gene from a whole-exome screening of Danish patients with diabetes that each involve a change in positively charged residues in the PICK1 BAR domain. All 4 coding variants failed to rescue insulin content in INS-1E cells upon knock down of endogenous PICK1. Moreover, 2 variants showed dominant-negative properties. In vitro assays addressing BAR domain function suggested that the coding variants compromised BAR domain function but increased the capacity to cause fission of liposomes. Live confocal microscopy and super-resolution microscopy further revealed that PICK1 resides transiently on ISGs before egress via vesicular budding events. Interestingly, this egress of PICK1 was accelerated in the coding variants. We propose that PICK1 assists in or complements the removal of excess membrane and generic membrane trafficking proteins, and possibly also insulin, from ISGs during the maturation process; and that the coding variants may cause premature budding, possibly explaining their dominant-negative function.

Authors

Rita C. Andersen, Jan H. Schmidt, Joscha Rombach, Matthew D. Lycas, Nikolaj R. Christensen, Viktor K. Lund, Donnie S. Stapleton, Signe S. Pedersen, Mathias A. Olsen, Mikkel Stoklund, Gith Noes-Holt, Tommas T.E. Nielsen, Mark P. Keller, Anna M. Jansen, Rasmus Herlo, Massimo Pietropaolo, Jens B. Simonsen, Ole Kjærulff, Birgitte Holst, Alan D. Attie, Ulrik Gether, Kenneth L. Madsen

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Figure 10

The PICK1 coding variants alter fission from insulin granules.

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The PICK1 coding variants alter fission from insulin granules. INS-1E ce...
INS-1E cells were transduced with the coding variants and immunostained for eGFP-PICK (cyan) and insulin (magenta). (A) Representative dSTORM images of eGFP-PICK1 in tubular structures colocalized with insulin. Scale bar: 250 nm. Bottom row: The same data illustrated with 3D; axis values indicate nm. (B) rpsCBC distribution of PICK1 clusters to insulin granules of the 4 coding variants (shades of blue) compared with PICK1 WT (gray). Kolmogorov-Smirnov test was used to test the cumulative distribution, **P < 0.01, ***P < 0.001; n = 4–5 individual experiments. (C) Size distribution of colocalized insulin granules and PICK1 clusters, defined as rpsCBC >0.2, shown with representative dSTORM images of small insulin clusters colocalized with eGFP-PICK1. Scale bar: 250 nm. (D) Proposed model for the PICK1 coding variants in insulin granule biogenesis. We propose that the PICK1 coding variants, with increased abscission efficacy, may cause tubulation and premature budding from the SGs during and/or after the maturation process, generating small clusters that contain excess membrane cargo and insulin.