The endoplasmic reticulum (ER) functions as a storehouse for intracellular Calcium. STIM1, a Calcium sensor localizes mostly to the ER membrane in interphase under resting conditions. Following Ca2+ store depletion, STIM1 forms puncta that localize to the cortical ER and binds Orai1, a Ca2+ channel to allow Ca2+ influx. This mechanism of Ca2+ influx is termed Store-Operated Calcium Entry (SOCE). Orai1 is internalized during meiosis and STIM1 fails to aggregate even when intracellular Ca2+ stores are depleted in both mitosis and meiosis. This causes inhibition of SOCE during mitosis and meiosis. This is an important mechanism to prevent sporadic Ca2+ influx that could disrupt its specialized functions during the cell cycle. We are interested in elucidating the mechanisms that allow STIM1 to cluster during interphase but not during M-phase.

We have taken a proteomics approach to identify proteins that function in hSTIM1 clustering following store depletion, and to determine what makes mitotic cells different in this aspect. We have immunoprecipitated proteins that bind hSTIM1 in HEK 293 cells with the Ca2+ store full or depleted in both mitotic and non-mitotic cells. We used iTRAQ labeling to identify proteins that bind hSTIM1 and to obtain relative quantification among the different treatment groups. This will help in defining function of these proteins under the different experimental conditions. We have identified over 400 proteins that co-immunoprecipitate with hSTIM1. The data suggest that hSTIM1 is a component of distinct complexes within the cell and such may have additional functions apart from SOCE in HEK 293 cells. Gene ontology enrichment is being done on this dataset to assign these proteins to different complexes. Deviating ratios of proteins within complexes under different conditions will provide us with an insight into their functions with hSTIM1 during interphase and mitosis.

We are taking a similar approach to identify proteins that function with hSTIM1 during interphase and meiosis using Xenopus oocytes expressing mCherry-tagged hSTIM1.


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