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

I.F. Smith, D. Swaminathan and I. Parker, Neurobiology and Behavior, University of California, Irvine, CA 92697, USA. (Introduced by Bradley Launikonis)

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.