STIM1 and Orai1 constitute the core machinery of store-operated calcium entry. We used transmission and freeze-fracture electron microscopy to visualize these proteins at endoplasmic reticulum (ER)-plasma membrane (PM) junctions. In thin sections, ER formed frequent extensive cortical junctions in HEK-293 cells overexpressing STIM1 and Orai1 and exposed to thapsigargin (TG), but small and rare junctions in control cells. Extended molecules, most likely STIM1, decorated the ER and bridged the ER-PM gap. Freeze-fracture replicas of cells treated in the same manner showed elevated PM domains (“puncta”) packed with particles at a higher density than on the surface of control cells (2564±443/µm2 versus 1863±331/µm2). Puncta varied in size (∼0.01 to 0.45 µm2) and frequency. Clusters of particles in puncta typically exhibited a clear sharp border, but particle density sometimes declined more gradually to background. Since the puncta most likely correspond to sites of STIM1-Orai1 co-clustering at ER-PM junctions, targeting to puncta was used to guide the classification of particles potentially representing Orai1 channels based upon structural parameters. A high percentage of particles in puncta exhibited a common signature; “large and tall” particles were present at a significantly higher density within puncta than in untransfected cells (average 1300±309 versus 225±84/µm2). Three characteristics of these particles are similar to the tetrameric CaV1.1 channels of skeletal muscle: 1) diameter, only slightly smaller than that of CaV1.1; 2) unusual height indicated by the platinum free “shadow”; 3) square, slightly scalloped outline of the same shadow in some images. It is reasonable to postulate that the “large and tall” particles represent tetrameric Orai1 channels clustered in correspondence with cortical Stim-bearing ER junctions. These observations introduce a new approach to visualize individual unlabeled Stim and Orai molecules in situ. Funded by NIH RO1 HL-48093 (CFA) and NS-14609 (MDC).

Perni, S., Yeromin, A.V., Dynes, J.L., Cahalan, M.D., Franzini-Armstrong, C. (2013). Visualization of STIM1 and Orai1 by Electron Microscopy. BIOPHYSICAL JOURNAL, 104(2), 101A-102A [10.1016/j.bpj.2012.11.597].

Visualization of STIM1 and Orai1 by Electron Microscopy

Perni, S.;
2013

Abstract

STIM1 and Orai1 constitute the core machinery of store-operated calcium entry. We used transmission and freeze-fracture electron microscopy to visualize these proteins at endoplasmic reticulum (ER)-plasma membrane (PM) junctions. In thin sections, ER formed frequent extensive cortical junctions in HEK-293 cells overexpressing STIM1 and Orai1 and exposed to thapsigargin (TG), but small and rare junctions in control cells. Extended molecules, most likely STIM1, decorated the ER and bridged the ER-PM gap. Freeze-fracture replicas of cells treated in the same manner showed elevated PM domains (“puncta”) packed with particles at a higher density than on the surface of control cells (2564±443/µm2 versus 1863±331/µm2). Puncta varied in size (∼0.01 to 0.45 µm2) and frequency. Clusters of particles in puncta typically exhibited a clear sharp border, but particle density sometimes declined more gradually to background. Since the puncta most likely correspond to sites of STIM1-Orai1 co-clustering at ER-PM junctions, targeting to puncta was used to guide the classification of particles potentially representing Orai1 channels based upon structural parameters. A high percentage of particles in puncta exhibited a common signature; “large and tall” particles were present at a significantly higher density within puncta than in untransfected cells (average 1300±309 versus 225±84/µm2). Three characteristics of these particles are similar to the tetrameric CaV1.1 channels of skeletal muscle: 1) diameter, only slightly smaller than that of CaV1.1; 2) unusual height indicated by the platinum free “shadow”; 3) square, slightly scalloped outline of the same shadow in some images. It is reasonable to postulate that the “large and tall” particles represent tetrameric Orai1 channels clustered in correspondence with cortical Stim-bearing ER junctions. These observations introduce a new approach to visualize individual unlabeled Stim and Orai molecules in situ. Funded by NIH RO1 HL-48093 (CFA) and NS-14609 (MDC).
Perni, S., Yeromin, A.V., Dynes, J.L., Cahalan, M.D., Franzini-Armstrong, C. (2013). Visualization of STIM1 and Orai1 by Electron Microscopy. BIOPHYSICAL JOURNAL, 104(2), 101A-102A [10.1016/j.bpj.2012.11.597].
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11365/1216071