Elsevier

Cell Calcium

Volume 45, Issue 1, January 2009, Pages 65-76
Cell Calcium

Localization of puff sites adjacent to the plasma membrane: Functional and spatial characterization of Ca2+ signaling in SH-SY5Y cells utilizing membrane-permeant caged IP3

https://doi.org/10.1016/j.ceca.2008.06.001Get rights and content

Summary

The Xenopus oocyte has been a favored model system in which to study spatio-temporal mechanisms of intracellular Ca2+ dynamics, in large part because this giant cell facilitates intracellular injections of Ca2+ indicator dyes, buffers and caged compounds. However, the recent commercial availability of membrane-permeant ester forms of caged IP3 (ci-IP3) and EGTA, now allows for facile loading of these compounds into smaller mammalian cells, permitting control of [IP3]i and cytosolic Ca2+ buffering. Here, we establish the human neuroblastoma SH-SY5Y cell line as an advantageous experimental system for imaging Ca2+ signaling, and characterize IP3-mediated Ca2+ signaling mechanisms in these cells. Flash photo-release of increasing amounts of i-IP3 evokes Ca2+ puffs that transition to waves, but intracellular loading of EGTA decouples release sites, allowing discrete puffs to be studied over a wide range of [IP3]. Puff activity persists for minutes following a single photo-release, pointing to a slow rate of i-IP3 turnover in these cells and suggesting that repetitive Ca2+ spikes with periods of 20–30 s are not driven by oscillations in [IP3]. Puff amplitudes are independent of [IP3], whereas their frequencies increase with increasing photo-release. Puff sites in SH-SY5Y cells are not preferentially localized near the nucleus, but instead are concentrated close to the plasma membrane where they can be visualized by total internal reflection microscopy, offering the potential for unprecedented spatio-temporal resolution of Ca2+ puff kinetics.

Introduction

A major mechanism of cellular Ca2+ signaling involves the liberation of Ca2+ ions from the endoplasmic reticulum through inositol 1,4,5-trisphosphate receptor-channels (IP3Rs) [1], [2], [3]. Opening of the IP3R channel requires binding of the second messenger IP3 together with Ca2+ to receptor sites on the cytosolic face. Gating by Ca2+ is biphasic, such that small elevations of cytosolic Ca2+ induce channel opening whereas larger elevations cause inactivation [4], [5]. The positive feedback by Ca2+ underlies the process known as Ca2+-induced Ca2+ release (CICR), whereby Ca2+ is released in a regenerative manner that may remain restricted to a cluster of IP3R producing local Ca2+ signals known as Ca2+ puffs [6], or propagate throughout the cell as a saltatory wave involving the recruitment of multiple puff sites by successive cycles of Ca2+ diffusion and CICR. Thus, IP3-mediated Ca2+ signaling represents a hierarchy of Ca2+ events of differing magnitudes [7], [8].

Xenopus oocytes have been a favored model cell system in which to study the physiology of fundamental and elementary IP3-mediated elementary Ca2+ release events [9], [10], [11], [12], [13], [14] and their roles in supporting global Ca2+ waves [15], [16]. Advantages of the oocyte include its lack of ER Ca2+ release channels other than IP3Rs (e.g. ryanodine receptors (RyRs) and cADP-ribose receptors) [17] and its large size (∼1 mm diameter), which facilitates experimental procedures such as injection of cell-impermeant compounds. In particular, intracellular injection of caged IP3 permits precise control of [IP3]i via flash photolysis [18], [19] and, in conjunction with intracellular loading of the ‘slow’ Ca2+ buffer EGTA to disrupt CICR between puff sites and thereby ‘balkanize’ global Ca2+ events into multiple independent puff loci, has enabled detailed analysis of puff dynamics as a function of [IP3]i [10], [20].

Following the original discovery of Ca2+ puffs in Xenopus oocytes [21], [22], analogous events have been observed in numerous cell types, indicating that puffs are a ubiquitous feature of cellular Ca2+ signaling [15], [23], [24]. They form the building blocks from which global Ca2+ signals are constructed [1], [25] and may also serve local signaling functions in their own right, such as controlling action potential propagation at neuritic branch points by activating Ca2+-dependent K+ channels to reduce neuronal excitability [26]. However, studies of Ca2+ puffs in neurons and other mammalian cells of ‘normal’ size have been greatly hindered by inability to control [IP3]i. Bootman and colleagues [2], [15], [27] made extensive studies in HeLa and other cultured cell lines employing extracellular agonists to evoke IP3 production, but this method provides only an imprecise regulation of [IP3]i within the narrow concentration ‘window’ required to evoke puffs; as does an alternative approach of incubating cells with membrane-permeant IP3 esters to cause a slow increase in [IP3]i [23]. Although caged IP3 can be introduced into small cells by whole-cell patch-clamping [28], [29], [30], this method is technically demanding and severely limits the throughput at which multiple cells can be examined.

In order to circumvent this problem, we describe here the use of a membrane-permeant caged IP3 (ci-IP3) [31], [32], [33], [34] to characterize elementary Ca2+ release events in the human neuroblastoma SH-SY5Y cell line. We show that strong UV flash photolysis of ci-IP3 causes the generation of Ca2+ oscillations, whereas weaker photo-release evokes puffs. Intracellular loading of EGTA via a membrane-permeant ester devolves the waves evoked by even strong photo-release of IP3 into discrete, highly localized and transient puffs, that persist for several minutes. The frequency of Ca2+ puff sites is highly sensitive to increasing [IP3]i and there is also a significant heterogeneity between sites with some evoking a high frequency and others a lower frequency of puffs to similar [IP3]i. Puff sites in the SH-SY5Y cells are not preferentially localized near the nucleus, but instead are concentrated very close to the plasma membrane where they can be visualized with high resolution by total internal reflection (TIRF) microscopy.

Section snippets

Cell culture

Human neuroblastoma SH-SY5Y cells were cultured in a mixture of Ham's F12 medium and Eagle's minimal essential medium (1:1 mixture), supplemented with 10% (v/v) fetal calf serum and 1% nonessential amino acids. Cells were incubated at 37 °C in a humidified incubator gassed with 95% air and 5% CO2, passaged every 7 days and used for up to 20 passages. When required for fluorimetric studies, cells were harvested in phosphate-buffered saline (PBS) without Ca2+ or Mg2+ and sub-cultured on glass

Local and global Ca2+ signals evoked by agonist activation and by photo-released IP3

We first characterized the patterns of Ca2+ signals evoked in SH-SY5Y cells by agonist activation, employing wide-field epi-fluorescence imaging of the Ca2+ indicator dye fluo-4. Bath application of relatively high (100 μM) concentrations of the muscarinic agonist carbachol evoked global cellular Ca2+ elevations that oscillated in 32% of cells (n = 45) with periods of 20–30 s (Fig. 1A). The remaining non-oscillatory cells displayed a single Ca2+ transient that decayed to baseline with a half time

Tools for imaging local Ca2+ signaling in mammalian cells

Several reports describe local IP3/Ca2+ signaling in mammalian cells [2], [15], [19], [23], [24], [27], [34], [40], but quantitative studies have been hindered technical problems in controlling [IP3]i within the narrow range that evokes local puffs without triggering global Ca2+ waves and neither approaches using low concentrations of Ca2+-mobilizing agonists [27], [40] nor membrane-permeant IP3 [23] have proved entirely satisfactory. We had previously circumvented this problem by utilizing

Acknowledgements

This work was supported by grant GM 48071 from the National Institutes of Health, and by an NIH training fellowship (T32 NS07444) to S.M.W.

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