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 9

Super-resolution imaging implicates PICK1 in an egress route from ISGs.

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Super-resolution imaging implicates PICK1 in an egress route from ISGs. ...
(A and C) Representative dSTORM images of INS-1E cells transduced with KD + WT and immunostained for eGFP-PICK1 (cyan), insulin (magenta), or syntaxin 6 (red). (A) dSTORM image demonstrates eGFP-PICK1 in small clusters around insulin granules; the 3D illustration shows amounts of insulin (magenta) in the small surrounding structure of PICK1 (black). Axis units indicate nm. Scale bar: 250 nm. (B) The size distribution of PICK1 and insulin clusters confirms a high prevalence of small clusters less than 100 nm in the colocalized structures defined as CBC >0.2 insulin in the rpsCBC distribution. (C) Representative dSTORM image of INS-1E cells. Scale bar: 5 μm. Right: Inset with higher-magnification image of overlapping PICK1- and syntaxin 6–positive structures. Scale bar: 250 nm. (D) Representative STED image of INS-1E cells immunostained for PICK1 (cyan), syntaxin 6 (red), and insulin (magenta). Scale bar: 5 μm. Right panels: Insets at higher magnification. Squares represent colocalized PICK1/syntaxin 6 clusters; dashed circles, colocalized PICK1/insulin clusters; whole circles, colocalized PICK1/syntaxin 6/insulin clusters. Scale bar: 1 μm. Top right: Higher-magnification examples of the different colocalized proteins. Scale bar: 250 nm. (E) INS-1E cells were transiently transfected with a Myc-BioID2–PICK1 construct or Myc-BioID2 as control. Biotinylated proteins were pulled down from cell lysates with streptavidin beads and analyzed by immunoblotting for syntaxin 6. n = 3 individual experiments. (F) Putative model for the role of the IPA N-BAR domain proteins in insulin granule biogenesis. Arfaptin 1 controls the neck of growing ISGs at the TGN in a complex with effector and kinases, while PICK1 — in either a homo- or heterodimeric complex with ICA69 — localizes around the growing ISGs, promoting membrane fission. After the ISGs are budded off, they undergo a maturation process. We propose that PICK1, during multiple budding events, removes excess membrane and cargo from the insulin granules in a process complementary to clathrin-dependent egress.