Reversible translocation of EYFP-tagged STIM1 is coupled to calcium influx in insulin secreting beta-cells

Cell Calcium. 2008 Dec;44(6):533-44. doi: 10.1016/j.ceca.2008.03.007. Epub 2008 May 2.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Bacterial Proteins / metabolism*
  • Boron Compounds / pharmacology
  • Calcium / pharmacology
  • Calcium Channels
  • Calcium Signaling* / drug effects
  • Cell Membrane / drug effects
  • Cell Membrane / metabolism
  • Endoplasmic Reticulum / drug effects
  • Endoplasmic Reticulum / metabolism
  • Insulin-Secreting Cells / cytology
  • Insulin-Secreting Cells / drug effects
  • Insulin-Secreting Cells / metabolism*
  • Luminescent Proteins / metabolism*
  • Membrane Glycoproteins / metabolism*
  • Mice
  • Protein Transport / drug effects
  • Recombinant Fusion Proteins / metabolism*
  • Stromal Interaction Molecule 1

Substances

  • Bacterial Proteins
  • Boron Compounds
  • Calcium Channels
  • Luminescent Proteins
  • Membrane Glycoproteins
  • Recombinant Fusion Proteins
  • Stim1 protein, mouse
  • Stromal Interaction Molecule 1
  • yellow fluorescent protein, Bacteria
  • diphenylborate
  • Calcium