Redistribution Of STIM1 After Ca²⁺ Store Depletion In Endothelial Cell

Calcium is an essential signaling messenger in every eukaryotic cell, regulating diverse and kinetically distinct cellular phenomena.Store-operated Ca²⁺ entry (SOCE), a major mechanism for Ca²⁺ entry in non-excitable cells. Store-operated Ca²⁺ (SOC) influx is an important process in cellular physiology that controls such diverse functions as refilling of intracellular CaPPP2+ PPPstores activation of enzymatic activity, gene transcription , and release of cytokines. The last 2 years have seen remarkable progress in defining the critical roles of STIM1(stromal interaction molecule 1)in the regulation of SOC entry into cells.STIM1 was originally discovered as tumour suppressor, and it plays an important role in immunologic deficiency diseases and many kinds of tumours. However, recent studies suggest that STIM1 might be involved in the activation of SOCE .STIM1 is a Ca²⁺-binding protein and a transmembrane protein that functions as a Ca²⁺ sensor in the endoplasmic reticulum, including several conservative and important structural domain: EF-hand and sterile a motif(SAM)in the ER lumen ; the cytoplasmic coiled-coil domain and the cytoplasmic proline-rich region and lysine-rich tail.EF-hand is a Ca²⁺ sensor-- responsible for Ca²⁺ sensing in the ER lumen. In the presence of Ca²⁺ the EF hand/SAM domain exists as a monomer that has a compact alpha-helical structure. In contrast, when Ca²⁺ is depleted, the EF hand/SAM domain changes to a less alpha-helical, less compact conformation that promotes aggregation to dimers and oligomers. Each of these three domains (coiled-coil, Ser_Thr-rich, and SAM ) was essential for activating SOC channels.Using energy from ATP hydrolysis, SERCA( Ca-ATPase of the ER) moves Ca into the ER. TG blocks Ca²⁺ ATPases, thereby prevents pumping of Ca²⁺ into the endoplasmic reticulum that leads to store.The ER store is full, STIM1 appears to be localised intracellularly within the ER in the cytoplasmic regions with the Immunofluorescent staining and the Overexpression of STIM1 tagged with YFP. EF-hand is a Ca²⁺ sensor, which CaPPP2+ PPPbinding on. When Ca²⁺ is depleted with TG, STIM1 undergo a conformational change in response to dissociation of Ca²⁺, causes aggregation of STIM1 beneath the PM(plasma membrane) and activative the SOC channel.STIM1 protein was proposed as a"sensor"of Ca²⁺ within stores : 1) this function being mediated via the EF-hand Ca²⁺-binding domain on the N-terminal ER luminal portion of STIM1; 2) Decreased ER Ca²⁺ results in a profound intracellular redistribution of STIM1 from a uniform ER pattern to spatially discrete areas termed puncta; 3) STIM1 puncta in close proximity to the PM causes Orai1 aggregation and activative the SOC channel. Concretely, studies provide evidence for a four-step activation process for STIM1 that can be understood as a"STIM1 ER-to-PM signaling relay"These four steps are as follows. 1)Receptor-induced reduction of ER CaPPP2+ PPPconcentration lowers the Ca²⁺ occupancy of the luminal EF hand of STIM1. 2) STIM1 without bound Ca²⁺ rapidly forms oligomers along the ER network. 3) The oligomerization of STIM1 exposes a C-terminal polybasic PM-targeting motif, which leads to the recruitment of STIM1 oligomers to nearby ER–PM junctions in a diffusion-limited step. 4) At ER–PMjunctions, STIM1 recruits and activates the Ca²⁺ channel Orai1 and possibly other PM signaling proteins.For the first time, we detect that STIM1 is mainly localised in the cytoplasm in Human Umbilical Vein Endothelial cell(HUVEC) with immunofluorescence and Western Blot. Upon Ca²⁺ depletion with TG STIM1 translocate to nucleus and distribute like oscillation from the cytoplasm to nucleus or from nucleus to the cytoplasm with time increasing. In order to confirm that STIM1 translocate to nucleus after Ca²⁺ store depletion , we transfect STIM1 cDNA into HUVEC and monitor the mobility of STIM1 by fluorescence after TG stimulation using confocal microscope .However, other studies indicate that in Jurkat T cell STIM1 translocate to plasma membrane and maintain a long time. So, what mechanisms cause STIM1 protein redistributing difference? What influence has STIM1 on nuclear calcium signal ? These are our research content. There is a debate regarding nuclear calcium signal, so our further investigation will provide new theory foundation for nuclear calcium signal regulation.Objective:Many studies have detected that STIM1 translocating to the plasma membrane result in activation of functional SOC channels and entry of Ca2+ into the cytoplasm. Our experimental studies detect whether there is STIM1 in the nuclear membrane or intranuclear and investigate the mechanisms underlying STIM1 translocate to nucleus in HUVEC using STIM1 cDNA constructs.Methods:1. Human Umbilical Vein Endothelial cells(HUVEC)were cultured and passaged using trypsinzation method .2. Cells were identified by growth pattern and immunofluorescent staining.3. STIM1 redistribution after TG stimulation by immunofluorescent staining.4. Nucelus and plasm protein abstraction and Western Blot detect STIM1 distribution.5. Transfections or infection of STIM1 cDNA into HUVEC were performed using Transfection Reagent.6. monitor the mobility of STIM1 by fluorescence after TG stimulation using confocal microscope.Results:1. Human Umbilical Vein Endothelial cells(HUVEC) were comfirmed by typical"slabstone"growth pattern and Endothelial cells specificâ…§factor immunofluorescent staining, and purity at 100%.2. In Human Umbilical Vein Endothelial cell(HUVEC) STIM1 is mainly localised in the cytoplasm and no expression in intranuclear before TG stimulation. At 1 minute, STIM1 redistribution have no obvious change. At 3 minute, STIM1 aggregate towards nuclear membrane. At 10 minute, STIM1 restore to condition without stimulation, in other words, mainly locate in the cytoplasm. At 30 minute, STIM1 again aggregate towards nuclear membrane. At 60 minute, STIM1 is mainly localised in the cytoplasm. In short, upon Ca2+ depletion with TG STIM1 translocate to nucleus and distribute like oscillation from the cytoplasm to nucleus or from nucleus to the cytoplasm with time increasing.3. Western Blot detect that STIM1 is mainly localised in the cytoplasm and no expression in intranuclear.4. Successfully detection of STIM1 cDNA translocation:without TG stimulation, GFP-STIM1 have no change ; after TG stimulation, GFP-STIM1 change from uniformity to puncta, and translocate to nucleus.Conclusion:1. Human Umbilical Vein Endothelial cells(HUVEC) can be cultured and passaged using trypsinzation method .2.STIM1 is mainly localised in the cytoplasm in HUVEC, and upon Ca²⁺ depletion with TG STIM1 translocate to nucleus and distribute like oscillation from the cytoplasm to nucleus or from nucleus to the cytoplasm with time increasing.3.Translocation of STIM1 after TG stimulation further confirm that STIM1 translocate to nucleus after CaPPP2+ store depletion.