Imaging the Motility of Inositol Trisphosphate Receptors in Intact Mammalian Cells using Single Particle Tracking Photoactivated Localization Microscopy (Sptpalm)

Inositol trisphosphate receptors (IP3Rs) are calcium-permeable channels in the membrane of the endoplasmic reticulum (ER) that liberate calcium to generate cytosolic calcium signals that control diverse cellular functions including gene expression, secretion and synaptic plasticity. The spatial distribution of these channels is crucial in determining the patterning of intracellular calcium signals. The mechanisms underlying the aggregation and maintenance of IP3Rs into clusters are controversial. Local calcium puffs reflecting concerted openings of clustered IP3Rs arise at just a few, fixed locations within a cell, suggesting clusters are stable entities; and calcium blips generated by ‘lone’ IP3Rs are also immotile. In contrast, GFP-tagged or immunostained IP3Rs show a dense distribution throughout a cell. Moreover, the majority IP3Rs can diffuse freely within the ER membrane, and aggregate into clusters following activation of IP3 signaling and/or cytosolic calcium elevation. These apparently different behaviors may be explained because calcium imaging detects only functional IP3Rs, whereas immunostained or GFP-tagged IP3Rs report the entire population of IP3R proteins. We therefore hypothesize that most IP3Rs are motile, but functionally unresponsive. Local calcium signals arise, instead, from a small subset of IP3Rs that are anchored, individually or in clusters, by association with static cytoskeletal structures and possibly as a consequence of this anchoring, display high sensitivity to IP3 to produce calcium signals. To test this hypothesis we expressed type 1 IP3R tagged with a photoactivatable genetically encoded protein to track the motility of thousands of individual IP3R molecules with nanoscale spatial and millisecond temporal resolution. We find that IP3Rs can be distinguished into two groups with relatively high or low motility, and that the apparently immotile IP3Rs are preferentially grouped within tight clusters.Support: NIH GM40871 (IP) and GM100201 (IFS).