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 8

PICK1 resides transiently on insulin ISGs before budding off.

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PICK1 resides transiently on insulin ISGs before budding off. (A) GRINCH...
(A) GRINCH cells were transiently transfected with PICK1-mCherry. Representative GRINCH cell shows a colocalized hPro-CpepsfGFP (magenta) and PICK1-mCherry (cyan) cluster, indicated by the dotted square, with the inset (30% zoom) highlighting the overlap. Scale bar: 10 μm. Right: Profile plot from the inset and time-lapse images of the merged Z-stack (500 nm) during steady-state conditions (11 mM glucose). Time is in seconds. Upper 2 rows present a PICK1-mCherry cluster and hPro-CpepsfGFP cluster, respectively, both shown in gray scale. The third row shows merge images. Time is in seconds. Scale bar: 1 μm. (B and C) INS-1E cells transduced with KD + WT and immunostained for eGFP-PICK1 (cyan) and insulin (magenta). (B) Representative SIM image of INS-1E cells. Bottom: 3D reconstruction. Scale bar: 5 μm. Right: Insets with higher-magnification images of overlapping PICK1 and insulin granules (arrows) in 3D. Scale bar: 500 nm. (C) Representative dSTORM image of INS-1E cells. Scale bar: 5 μm. Bottom: Insets with higher-magnification images of overlapping PICK1 and insulin granules. Scale bar: 250 nm. (D) Insulin CBC shift analysis. PICK1 clusters shifted +500 nm in the x direction, and the CBC distribution of the insulin granules was recalculated (brown) and overlaid on the original CBC distribution (gray). (E) The difference in CBC between the original from B (gray) and the +500 nm shift (brown). We refer to this as rpsCBC distribution. Note that many points are not assigned CBC values (NA) when shifted. n = 5 individual experiments. (F) The 3D images display distinctive colocalized clusters of insulin (colored by CBC scale) and eGFP-PICK1 (black), ordered by CBC values ranging from 0.78 to 0.20 and indicative of PICK1 fission from insulin granules. Scale bar: 200 nm.