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dc.contributor.authorShen, Yihan
dc.date.accessioned2022-01-25T03:51:24Z
dc.date.available2022-01-25T03:51:24Z
dc.date.submitted2021-09-21
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/332909
dc.description.abstractCa2+ is a ubiquitous intracellular messenger. It regulates a variety of cellular activities, ranging from muscle contraction, neuronal transmission, secretion and cell growth to apoptosis. Store-operated Ca2+ entry (SOCE) is a major pathway of Ca2+ signalling and exists in almost all metazoans. SOCE is activated by loss Ca2+ from the endoplasmic reticulum (ER), which causes stromal interaction molecule 1 (STIM1) to accumulate at junctions between the ER and plasma membrane (PM). Within these membrane contact sites (MCS), STIM1 forms puncta that trap and activate Orai1 Ca2+ channels in the PM, allowing Ca2+ to flow into the cytoplasm and slowly replenish Ca2+ in the ER. Elucidating the binding stoichiometry of STIM1 and Orai1 is essential for understanding Orai1 gating and mechanisms of SOCE. Most previous studies of SOCE used cells overexpressing STIM1 and/or Orai1, which might perturb their behaviours. This study used a HeLa cell line in which one copy of the endogenous STIM1 gene was tagged with EGFP using CRISPR/Cas9 gene-editing to understand the stoichiometry and dynamics of STIM1 and Orai1 in SOCE. I confirmed that SOCE was normal in STIM1-EGFP HeLa cells and that the tagged and untagged versions of STIM1 mixed freely and interacted with Orai1. Total internal reflection fluorescence (TIRF) imaging analyses indicated that there was only a modest increase in the average size of STIM1 puncta after store depletion. Stepwise photobleaching analyses revealed that there was an average of 14.5 STIM1 molecules within each punctum in cells with empty Ca2+ stores. Orai1 was immunostained and the fluorescence intensity distributions of the Orai1 puncta were minimally affected by store depletion. Furthermore, the fluorescence intensities of Orai1 that colocalized with STIM1 puncta were similar to those remote from them. Only a small proportion (26%) of STIM1 colocalized with Orai1 at MCS identified by MAPPER, a fluorescent marker constitutively present in the ER-PM junctions. I conclude that each SOCE complex comprises a small cluster of STIM1 and is likely to include no more than one active Orai1 channel. The presence of a single Orai1 channel within each SOCE junction is estimated to be enough to account for observed SOCE-mediated Ca2+ signals, but it contradicts suggestions that STIM1 promotes clustering of Orai1 within MCS.
dc.rightsAll Rights Reserved
dc.rights.urihttps://www.rioxx.net/licenses/all-rights-reserved/
dc.subjectSOCE
dc.subjectCRISPR/Cas9
dc.subjectCa2+ signalling
dc.subjectSTIM1
dc.subjectStoichiometry
dc.titleStoichiometry of the interactions between endogenous Orai1 and STIM1 during store-operated Ca2+ entry
dc.typeThesis
dc.type.qualificationlevelDoctoral
dc.type.qualificationnameDoctor of Philosophy (PhD)
dc.publisher.institutionUniversity of Cambridge
dc.date.updated2022-01-22T06:00:44Z
dc.identifier.doi10.17863/CAM.80339
rioxxterms.licenseref.urihttps://www.rioxx.net/licenses/all-rights-reserved/
rioxxterms.typeThesis
dc.publisher.collegeSt Johns
cam.supervisorTaylor, Colin
cam.depositDate2022-01-22
pubs.licence-identifierapollo-deposit-licence-2-1
pubs.licence-display-nameApollo Repository Deposit Licence Agreement


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