Reversible translocation of EYFP-tagged STIM1 is coupled to calcium influx in insulin secreting beta-cells |
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Authors: | Tamarina Natalia A Kuznetsov Andrey Philipson Louis H |
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Affiliation: | aDepartment of Medicine, University of Chicago, 5841 S. Maryland Avenue, Chicago, IL 60637, USA |
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Abstract: | Calcium (Ca2+) signaling regulates insulin secretion in pancreatic β-cells. STIM1 has been proposed to function as an endoplasmic reticulum (ER) Ca2+ sensor regulating store-operated Ca2+ 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 (3.75–100 μM) 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. |
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Keywords: | STIM1 Calcium Endoplasmic reticulum Store-operated calcium entry Mouse insulinoma cells 2-Aminoethoxy diphenylborate (2-APB) Thapsigargin Confocal microscopy TIRF |
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