University of Cambridge
High-resolution optical analyses of inositol 1,4,5-trisphosphate receptors and the Ca²⁺ puffs they evoke
Abstract
dc:description.abstractCa²⁺ 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>.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2023
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Smith, Holly
- Advisors dc:contributor.advisor
-
- Taylor, Colin
- Ladds, Graham
Subjects
dc:subject × 4Rights
dc:rightsIdentifiers
dc:identifier.*- DOI dc:identifier.doi
- https://doi.org/10.17863/CAM.106160
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/364540