{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/364540"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/364540","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"High-resolution optical analyses of inositol 1,4,5-trisphosphate receptors and the Ca²⁺ puffs they evoke","abstract":"Ca²⁺ is an essential and near-universal intracellular messenger. Many intracellular Ca²⁺ signals are initiated by inositol 1,4,5-trisphosphate receptors (IP₃Rs) which respond to IP₃ produced when cell-surface receptors stimulate phospholipase C. IP₃Rs are regulated by both IP₃ and Ca²⁺, a property which allows Ca²⁺-induced Ca²⁺ release (CICR) between neighbouring IP₃Rs on the endoplasmic reticulum membrane. The assembly of IP₃Rs into small clusters allows local CICR to generate brief, localised increases in cytosolic Ca²⁺ concentration ([Ca²⁺]<sub>c</sub>), known as Ca²⁺ puffs, which arise from the coordinated opening of a few IP₃Rs within a cluster. IP₃R clusters that are immobilised near to the plasma membrane are preferentially licensed to respond to IP₃ with Ca²⁺ puffs. Ca²⁺ puffs can regulate local Ca²⁺ sensors and, importantly, contribute to the genesis of global cytosolic Ca²⁺ signals that can regulate diverse cellular processes. Since high [Ca²⁺]<sub>c</sub> inhibits IP₃R activity, negative feedback by Ca²⁺ probably contributes to terminating Ca²⁺ puffs. However, the complex mechanisms governing the generation, propagation, and, particularly, the termination of Ca²⁺ puffs are not completely understood. In this project, I aimed to address these issues. By expressing a SNAP-tagged IP₃R3 construct (SNAP-IP₃R3) in HEK cells without endogenous IP₃Rs and using high-resolution total internal reflection fluorescence (TIRF) microscopy, I was able to visualise both IP₃Rs and the Ca²⁺ puffs they evoke following photolysis of a caged analogue of IP₃. I optimised fluorescent labelling of SNAP-IP₃R3, and confirmed that its fluorescence reliably reports IP₃R expression level and subcellular distribution. I confirmed that, when expressed at near-endogenous levels, SNAP-IP₃R3 can evoke Ca²⁺ puffs whose properties resemble those evoked by endogenous IP₃R3. After developing these tools, I aimed to explore the relationship between the spatial organisation of IP₃Rs and the properties of Ca²⁺ puffs. I found that increased IP₃R expression levels caused cells to assemble more clusters, each of which contained more IP₃Rs. Ca²⁺ puffs occurred with higher frequencies and shorter latencies at higher expression levels, however, properties of individual Ca²⁺ puffs, most notably the mean amplitude (indicative of the number of IP₃Rs open during a Ca²⁺ puff), were unaltered. Using correlative imaging of individual Ca²⁺ puff sites and the IP₃R clusters underlying them, I found there was no relationship between IP₃R cluster size and the amplitude, duration, or frequency of Ca²⁺ puffs at that site. I concluded that the number of IP₃Rs recruited during the rising phase of a Ca²⁺ puff varies independently of the number of IP₃Rs in a cluster. I then aimed to introduce mutations in ligand-binding domains of IP₃R to examine effects of manipulating regulation by IP₃ and Ca²⁺ on Ca²⁺ puffs. I found that Ca²⁺ puffs evoked by a mutant IP₃R with a reduced affinity for IP₃ were less frequent, had undiminished amplitudes, and significantly shorter decay times. Exposing normal IP₃R to a lower concentration of IP₃ mimicked the effect of the mutant on Ca²⁺ puff frequency, but not on decay time. This suggests that the former effect is attributable to a decreased occupancy of IP₃Rs by IP₃, but the latter to a faster rate of dissociation of IP₃ from IP₃R. Finally, I found that Ca²⁺ puffs evoked by a mutant IP₃R with a reduced sensitivity to Ca²⁺ activation and inhibition were slightly less frequent but otherwise unchanged. The role of Ca²⁺-binding in controlling Ca²⁺ puff activity remains to be fully explored, but from my findings I concluded that dissociation of IP₃ from IP₃R contributes to the termination of Ca²⁺ puffs, potentially by rendering clustered IP₃Rs susceptible to inhibition by high local [Ca²⁺]<sub>c</sub>.","abstract_html":"Ca²⁺ is an essential and near-universal intracellular messenger. Many intracellular Ca²⁺ signals are initiated by inositol 1,4,5-trisphosphate receptors (IP₃Rs) which respond to IP₃ produced when cell-surface receptors stimulate phospholipase C. IP₃Rs are regulated by both IP₃ and Ca²⁺, a property which allows Ca²⁺-induced Ca²⁺ release (CICR) between neighbouring IP₃Rs on the endoplasmic reticulum membrane. The assembly of IP₃Rs into small clusters allows local CICR to generate brief, localised increases in cytosolic Ca²⁺ concentration ([Ca²⁺]&lt;sub&gt;c&lt;/sub&gt;), known as Ca²⁺ puffs, which arise from the coordinated opening of a few IP₃Rs within a cluster. IP₃R clusters that are immobilised near to the plasma membrane are preferentially licensed to respond to IP₃ with Ca²⁺ puffs. Ca²⁺ puffs can regulate local Ca²⁺ sensors and, importantly, contribute to the genesis of global cytosolic Ca²⁺ signals that can regulate diverse cellular processes. Since high [Ca²⁺]&lt;sub&gt;c&lt;/sub&gt; inhibits IP₃R activity, negative feedback by Ca²⁺ probably contributes to terminating Ca²⁺ puffs. However, the complex mechanisms governing the generation, propagation, and, particularly, the termination of Ca²⁺ puffs are not completely understood. In this project, I aimed to address these issues. By expressing a SNAP-tagged IP₃R3 construct (SNAP-IP₃R3) in HEK cells without endogenous IP₃Rs and using high-resolution total internal reflection fluorescence (TIRF) microscopy, I was able to visualise both IP₃Rs and the Ca²⁺ puffs they evoke following photolysis of a caged analogue of IP₃. I optimised fluorescent labelling of SNAP-IP₃R3, and confirmed that its fluorescence reliably reports IP₃R expression level and subcellular distribution. I confirmed that, when expressed at near-endogenous levels, SNAP-IP₃R3 can evoke Ca²⁺ puffs whose properties resemble those evoked by endogenous IP₃R3. After developing these tools, I aimed to explore the relationship between the spatial organisation of IP₃Rs and the properties of Ca²⁺ puffs. I found that increased IP₃R expression levels caused cells to assemble more clusters, each of which contained more IP₃Rs. Ca²⁺ puffs occurred with higher frequencies and shorter latencies at higher expression levels, however, properties of individual Ca²⁺ puffs, most notably the mean amplitude (indicative of the number of IP₃Rs open during a Ca²⁺ puff), were unaltered. Using correlative imaging of individual Ca²⁺ puff sites and the IP₃R clusters underlying them, I found there was no relationship between IP₃R cluster size and the amplitude, duration, or frequency of Ca²⁺ puffs at that site. I concluded that the number of IP₃Rs recruited during the rising phase of a Ca²⁺ puff varies independently of the number of IP₃Rs in a cluster. I then aimed to introduce mutations in ligand-binding domains of IP₃R to examine effects of manipulating regulation by IP₃ and Ca²⁺ on Ca²⁺ puffs. I found that Ca²⁺ puffs evoked by a mutant IP₃R with a reduced affinity for IP₃ were less frequent, had undiminished amplitudes, and significantly shorter decay times. Exposing normal IP₃R to a lower concentration of IP₃ mimicked the effect of the mutant on Ca²⁺ puff frequency, but not on decay time. This suggests that the former effect is attributable to a decreased occupancy of IP₃Rs by IP₃, but the latter to a faster rate of dissociation of IP₃ from IP₃R. Finally, I found that Ca²⁺ puffs evoked by a mutant IP₃R with a reduced sensitivity to Ca²⁺ activation and inhibition were slightly less frequent but otherwise unchanged. The role of Ca²⁺-binding in controlling Ca²⁺ puff activity remains to be fully explored, but from my findings I concluded that dissociation of IP₃ from IP₃R contributes to the termination of Ca²⁺ puffs, potentially by rendering clustered IP₃Rs susceptible to inhibition by high local [Ca²⁺]&lt;sub&gt;c&lt;/sub&gt;.","abstract_has_math":false,"creators":["Smith, Holly"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Taylor, Colin","Ladds, Graham"],"committee_chairs":[],"committee_members":[],"year":2023,"date_issued":"2023-08-05","date_published":"2023-08-05","updated_at":"2026-07-22T22:24:14Z","subjects":["Calcium signalling","Cell signalling","Fluorescence microscopy","Pharmacology"],"languages":["eng"],"rights":[],"rights_urls":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/0596ed32-f498-4092-acb7-69efd3d2cccc/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.106160","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Taylor, Colin","Ladds, Graham"]},{"key":"dc:creator","label":"Author","values":["Smith, Holly"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2023-08-05"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/364540"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Calcium signalling","Cell signalling","Fluorescence microscopy","Pharmacology"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/0596ed32-f498-4092-acb7-69efd3d2cccc/download","https://www.rioxx.net/licenses/all-rights-reserved/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.17863/CAM.106160"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/b7fa81dd-4442-419e-b23e-3c07ab5ff86b/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Ca²⁺ is an essential and near-universal intracellular messenger. Many intracellular Ca²⁺ signals are initiated by inositol 1,4,5-trisphosphate receptors (IP₃Rs) which respond to IP₃ produced when cell-surface receptors stimulate phospholipase C. IP₃Rs are regulated by both IP₃ and Ca²⁺, a property which allows Ca²⁺-induced Ca²⁺ release (CICR) between neighbouring IP₃Rs on the endoplasmic reticulum membrane. The assembly of IP₃Rs into small clusters allows local CICR to generate brief, localised increases in cytosolic Ca²⁺ concentration ([Ca²⁺]<sub>c</sub>), known as Ca²⁺ puffs, which arise from the coordinated opening of a few IP₃Rs within a cluster. IP₃R clusters that are immobilised near to the plasma membrane are preferentially licensed to respond to IP₃ with Ca²⁺ puffs. Ca²⁺ puffs can regulate local Ca²⁺ sensors and, importantly, contribute to the genesis of global cytosolic Ca²⁺ signals that can regulate diverse cellular processes. Since high [Ca²⁺]<sub>c</sub> inhibits IP₃R activity, negative feedback by Ca²⁺ probably contributes to terminating Ca²⁺ puffs. However, the complex mechanisms governing the generation, propagation, and, particularly, the termination of Ca²⁺ puffs are not completely understood. In this project, I aimed to address these issues. By expressing a SNAP-tagged IP₃R3 construct (SNAP-IP₃R3) in HEK cells without endogenous IP₃Rs and using high-resolution total internal reflection fluorescence (TIRF) microscopy, I was able to visualise both IP₃Rs and the Ca²⁺ puffs they evoke following photolysis of a caged analogue of IP₃. I optimised fluorescent labelling of SNAP-IP₃R3, and confirmed that its fluorescence reliably reports IP₃R expression level and subcellular distribution. I confirmed that, when expressed at near-endogenous levels, SNAP-IP₃R3 can evoke Ca²⁺ puffs whose properties resemble those evoked by endogenous IP₃R3. After developing these tools, I aimed to explore the relationship between the spatial organisation of IP₃Rs and the properties of Ca²⁺ puffs. I found that increased IP₃R expression levels caused cells to assemble more clusters, each of which contained more IP₃Rs. Ca²⁺ puffs occurred with higher frequencies and shorter latencies at higher expression levels, however, properties of individual Ca²⁺ puffs, most notably the mean amplitude (indicative of the number of IP₃Rs open during a Ca²⁺ puff), were unaltered. Using correlative imaging of individual Ca²⁺ puff sites and the IP₃R clusters underlying them, I found there was no relationship between IP₃R cluster size and the amplitude, duration, or frequency of Ca²⁺ puffs at that site. I concluded that the number of IP₃Rs recruited during the rising phase of a Ca²⁺ puff varies independently of the number of IP₃Rs in a cluster. I then aimed to introduce mutations in ligand-binding domains of IP₃R to examine effects of manipulating regulation by IP₃ and Ca²⁺ on Ca²⁺ puffs. I found that Ca²⁺ puffs evoked by a mutant IP₃R with a reduced affinity for IP₃ were less frequent, had undiminished amplitudes, and significantly shorter decay times. Exposing normal IP₃R to a lower concentration of IP₃ mimicked the effect of the mutant on Ca²⁺ puff frequency, but not on decay time. This suggests that the former effect is attributable to a decreased occupancy of IP₃Rs by IP₃, but the latter to a faster rate of dissociation of IP₃ from IP₃R. Finally, I found that Ca²⁺ puffs evoked by a mutant IP₃R with a reduced sensitivity to Ca²⁺ activation and inhibition were slightly less frequent but otherwise unchanged. The role of Ca²⁺-binding in controlling Ca²⁺ puff activity remains to be fully explored, but from my findings I concluded that dissociation of IP₃ from IP₃R contributes to the termination of Ca²⁺ puffs, potentially by rendering clustered IP₃Rs susceptible to inhibition by high local [Ca²⁺]<sub>c</sub>."]},{"key":"dc:format.checksum.md5","label":"Dc Format Checksum Md5","values":["e358475df70db0c93a4ecce84d2538e7","87eda9de84448d1f82354d60eee3eb5f"]},{"key":"dc:title","label":"Title","values":["High-resolution optical analyses of inositol 1,4,5-trisphosphate receptors and the Ca²⁺ puffs they evoke"]}]}],"canonical_facts":{"dc:contributor.advisor":["Taylor, Colin","Ladds, Graham"],"dc:creator":["Smith, Holly"],"dc:date.issued":["2023-08-05"],"dc:description.abstract":["Ca²⁺ is an essential and near-universal intracellular messenger. 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Since high [Ca²⁺]<sub>c</sub> inhibits IP₃R activity, negative feedback by Ca²⁺ probably contributes to terminating Ca²⁺ puffs. However, the complex mechanisms governing the generation, propagation, and, particularly, the termination of Ca²⁺ puffs are not completely understood. In this project, I aimed to address these issues. By expressing a SNAP-tagged IP₃R3 construct (SNAP-IP₃R3) in HEK cells without endogenous IP₃Rs and using high-resolution total internal reflection fluorescence (TIRF) microscopy, I was able to visualise both IP₃Rs and the Ca²⁺ puffs they evoke following photolysis of a caged analogue of IP₃. I optimised fluorescent labelling of SNAP-IP₃R3, and confirmed that its fluorescence reliably reports IP₃R expression level and subcellular distribution. I confirmed that, when expressed at near-endogenous levels, SNAP-IP₃R3 can evoke Ca²⁺ puffs whose properties resemble those evoked by endogenous IP₃R3. After developing these tools, I aimed to explore the relationship between the spatial organisation of IP₃Rs and the properties of Ca²⁺ puffs. I found that increased IP₃R expression levels caused cells to assemble more clusters, each of which contained more IP₃Rs. Ca²⁺ puffs occurred with higher frequencies and shorter latencies at higher expression levels, however, properties of individual Ca²⁺ puffs, most notably the mean amplitude (indicative of the number of IP₃Rs open during a Ca²⁺ puff), were unaltered. Using correlative imaging of individual Ca²⁺ puff sites and the IP₃R clusters underlying them, I found there was no relationship between IP₃R cluster size and the amplitude, duration, or frequency of Ca²⁺ puffs at that site. I concluded that the number of IP₃Rs recruited during the rising phase of a Ca²⁺ puff varies independently of the number of IP₃Rs in a cluster. I then aimed to introduce mutations in ligand-binding domains of IP₃R to examine effects of manipulating regulation by IP₃ and Ca²⁺ on Ca²⁺ puffs. I found that Ca²⁺ puffs evoked by a mutant IP₃R with a reduced affinity for IP₃ were less frequent, had undiminished amplitudes, and significantly shorter decay times. Exposing normal IP₃R to a lower concentration of IP₃ mimicked the effect of the mutant on Ca²⁺ puff frequency, but not on decay time. This suggests that the former effect is attributable to a decreased occupancy of IP₃Rs by IP₃, but the latter to a faster rate of dissociation of IP₃ from IP₃R. Finally, I found that Ca²⁺ puffs evoked by a mutant IP₃R with a reduced sensitivity to Ca²⁺ activation and inhibition were slightly less frequent but otherwise unchanged. The role of Ca²⁺-binding in controlling Ca²⁺ puff activity remains to be fully explored, but from my findings I concluded that dissociation of IP₃ from IP₃R contributes to the termination of Ca²⁺ puffs, potentially by rendering clustered IP₃Rs susceptible to inhibition by high local [Ca²⁺]<sub>c</sub>."],"dc:format.checksum.md5":["e358475df70db0c93a4ecce84d2538e7","87eda9de84448d1f82354d60eee3eb5f"],"dc:identifier.doi":["https://doi.org/10.17863/CAM.106160"],"dc:identifier.uri":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/b7fa81dd-4442-419e-b23e-3c07ab5ff86b/download"],"dc:language":["eng"],"dc:publisher.institution":["University of Cambridge"],"dc:relation.isreferencedby.uri":["https://www.repository.cam.ac.uk/handle/1810/364540"],"dc:rights":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/0596ed32-f498-4092-acb7-69efd3d2cccc/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"dc:subject":["Calcium signalling","Cell signalling","Fluorescence microscopy","Pharmacology"],"dc:title":["High-resolution optical analyses of inositol 1,4,5-trisphosphate receptors and the Ca²⁺ puffs they evoke"],"dc:type":["Thesis"],"dc:type.qualificationlevel":["Doctoral"],"dc:type.qualificationname":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-22T22:24:14Z"}