Programme Contents

Imaging the motility of inositol trisphosphate receptors in intact mammalian cells using single particle tracking photoactivated localization microscopy (sptPALM)

Inositol trisphosphate receptors (IP₃Rs) are Ca²⁺-permeable channels in the membrane of the endoplasmic reticulum (ER) that liberate Ca²⁺ sequestered in ER stores to generate cytosolic Ca²⁺ signals that control diverse cellular functions including gene expression, secretion and synaptic plasticity (Berridge, Lipp & Bootman, 2000). These channels are gated by both the second messenger IP₃ and biphasically by Ca²⁺ itself. Activation of IP₃Rs by Ca²⁺ ions diffusing from neighboring channels thus results in a regenerative amplification by Ca²⁺-induced Ca²⁺ release (CICR). The extent of this functional coupling depends strongly upon the spacing between IP₃Rs, so that spatial localization of these channels is a major determinant of cellular Ca²⁺ signals. In particular, Ca²⁺ imaging studies in numerous cell lines and in Xenopus oocytes reveal local IP₃-mediated Ca²⁺ signals ("puffs") that arise through the concerted opening of several IP₃R channels within tight clusters.

The mechanisms underlying the aggregation and maintenance of IP₃Rs within these clusters are controversial. Puffs arise at just a few, fixed locations within a cell, suggesting that the clusters are relatively stable entities; and calcium blips generated by lone IP₃Rs are similarly immotile (Smith & Parker, 2009; Smith et al., 2009). In contrast, imaging studies employing GFP-tagged or immunostained IP₃Rs show a dense distribution throughout the cell. Moreover, the majority IP₃Rs can diffuse freely within the ER membrane, and aggregate into clusters following sustained (minutes) activation of IP₃ signaling and/or cytosolic Ca²⁺ elevation, or even undergo clustering in response to IP₃ within just a few seconds (Taufiq-Ur-Rahman et al., 2009).

These apparently different behaviors may be explained because Ca²⁺ imaging studies detect only functional IP₃Rs (those that mediate Ca²⁺ liberation from the ER), whereas imaging studies utilizing immunostaining or GFP-tagged IP₃Rs report on the behavior of the entire population of IP₃R proteins. We therefore hypothesized that a majority of IP₃Rs are motile, but are either functionally unresponsive or mediate Ca²⁺ liberation only during sustained global elevations of cytosolic [Ca²⁺]. Local Ca²⁺ signals arise, instead, from a small subset of IP₃Rs that are anchored, individually or in clusters, by association with static cytoskeletal structures and which, possibly as a consequence of this anchoring, display high sensitivity to IP₃ to generate Ca²⁺ blips and puffs (Parker & Smith, 2010).

In order to test this hypothesis we have utilized the new generation of photoactivatable genetically encoded proteins to track the motility of thousands of individual IP₃R molecules with nanoscale spatial resolution and millisecond temporal resolution (sptPALM) (Manley et al., 2008). We find that IP₃Rs can be distinguished into two groups with relatively high or low motility and are currently investigating whether there is a spatial correlation to the differences in observed motilities.

Berridge MJ, Lipp P, Bootman MD. (2000) Nature Reviews Molecular Cell Biology 1: 11-21.

Manley S, Gillette JM, Patterson GH, Shroff H, Hess HF, Betzig E, Lippincott-Schwartz J. (2008) Nature Methods 5: 155-157.

Parker I, Smith IF. (2010) Journal of General Physiology 136: 119-127.

Smith IF, Parker I. (2009) Proceedings of the National Academy of Sciences USA 106: 6404-6409.

Smith IF, Wiltgen SM, Shuai J, Parker I. (2009) Science Signaling 2: ra77.

Taufiq-Ur-Rahman, Skupin A, Falcke M, Taylor CW. (2009) Nature 458: 655-659.