Regulatory and Functional Studies of Store-Operated Calcium Entry

This dissertation, "Regulatory and Functional Studies of Store-operated Calcium Entry" by Baixia, Hao, 郝佰侠, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Ca2+ signaling is essential for a wide variety of cellular activities, ranging from short term activities, such as synaptic and muscle contraction, to long term processes, such as proliferation and differentiation. Store-operated Ca2+ entry (SOCE), an important Ca2+ influx pathway in non-excitable cells, well coordinates Ca2+ release from ER and Ca2+ influx through plasma membrane. STIM1 and Orai1, serving as ER Ca2+ sensor and pore forming subunit, respectively, are the two essential components of SOCE machinery. In addition to activate Orai1 channel, studies have shown that STIM1 regulates other plasma membrane Ca2+ channels and senses a variety of cellular stresses to regulate SOCE. Therefore, it is of great interests to investigate the mechanisms and physiological functions of STIM1 and Orai1 mediated SOCE. Here, we performed tandem affinity purification to identify STIM1 associated proteins in Hela cells stably expressing STIM1-His6-3Flag. Four candidate proteins, including GRP78, HSP70, IQGAP1, and Actin, were identified by mass spectrometry analyses. Surprisingly, IQGAP1 failed to affect the activity of SOCE. Interestingly, GRP78 knockdown only affected the inactivation phase while exerted no effect on the activation phase of SOCE. In addition, GRP78 knockdown markedly induced cell apoptosis and dramatically increased the ER Ca2+ concentration. Moreover, GRP78 was involved in the regulation of SOCE by the ER stress. These data indicate that GRP78 is an important regulator of SOCE to prevent Ca2+ overload in cells. HSP70, however, significantly reduced the activity of SOCE by inhibiting STIM1 translocation to ER-PM junctions. Future studies will explore the mechanism of GRP78 and HSP70 in regulating SOCE by confocal and TIRF imaging. Embryonic stem (ES) cells proliferate unlimitedly and can differentiate into all fetal and adult cell types. This property endows ES cells to be the promising candidates in the therapy of neurodegenerative diseases. Thus, it is important to identify novel signaling molecules or events that could play a role in the neural commitment of ES cells. Accumulated evidences have documented the role of STIM1 and Orai1 mediated SOCE in neuronal activities. Yet, the role of SOCE in early neural development remains to be determined. Here we examined the role of STIM1 and Orai1 during neural differentiation of mouse ES cells. Both of STIM1 and Orai1 were expressed and functionally active in ES cells, and expressions of STIM1 and Orai1 were dynamically regulated during neural differentiation of mouse ES cells. STIM1 knockdown inhibited the differentiation of mouse ES cells into neural progenitors, neurons, and astrocytes. In addition, STIM1 knockdown caused severe cell death and markedly suppressed the proliferation of neural progenitors. Surprisingly, Orai1 knockdown had little effect on neural differentiation of mouse ES cells, but the neurons derived from Orai1 knockdown ES cells, like those from STIM1 knockdown cells, had defective SOCE. Taken together, our data indicate that STIM1 is required for neural differentiation of mouse ES cells independent of Orai1-mediated SOCE. DOI: 10.5353/th_b5177295 Subjects: Intracellular calcium Cellular signal transduction