Reversible translocation of EYFP-tagged STIM1 is coupled to calcium influx in insulin secreting beta-cells.
Calcium (Ca(2+)) signaling regulates insulin secretion in pancreatic beta-cells. STIM1 has been proposed to function as an endoplasmic reticulum (ER) Ca(2+) sensor regulating store-operated Ca(2+) entry (SOCE). Here we studied the translocation of EYFP-STIM1 in response to ER calcium depletion in mouse insulinoma MIN6 cells by fluorescent microscopy. While in resting cells EYFP-STIM1 is co-localized with an ER marker, in thapsigargin (Tg)-stimulated cells it occupied highly defined areas of the peri-PM space in punctae adjacent to, but not entirely coincident with the ER. Co-staining with fluorescent phalloidin revealed that EYFP-STIM1 punctae was located in actin-poor areas. Use of the SOCE blocker in MIN6 cells, 2-aminoethoxy diphenylborate (2-APB), prevented store depletion-dependent translocation of EYFP-STIM1 to the PM in a concentration-dependent (3.75-100muM) and reversible manner. TIRF microscopy revealed that 2-APB treatment led to the reversible disappearance of peri-PM EYFP-STIM1 punctae, while the ER structure in this compartment remained grossly unaffected. We conclude from this data that in these cells EYFP-STIM1 is delivered to a peri-PM location from the ER upon store depletion and this trafficking is reversibly blocked by 2-APB.
Tamarina
Natalia A
NA
Department of Medicine, University of Chicago, 5841 S. Maryland Avenue, Chicago, IL 60637, USA.
Kuznetsov
Andrey
A
Philipson
Louis H
LH
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D001896:Boron Compounds / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D015220:Calcium Channels; D020013:Calcium Signaling / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D000071737:Stromal Interaction Molecule 1
D001426:Bacterial Proteins; D001896:Boron Compounds; D015220:Calcium Channels; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; C100757:Stim1 protein, mouse; D000071737:Stromal Interaction Molecule 1; C054562:yellow fluorescent protein, Bacteria; C035757:diphenylborate; D002118:Calcium