Temporal switching and cell-to-cell variability in Ca2|[plus]| release activity in mammalian cells

doi:doi:10.1038/msb.2009.6

Subject Categories: Simulation and data analysis | Signal Transduction

Molecular Systems Biology 5 Article number: 247 doi:10.1038/msb.2009.6
Published online: 17 March 2009
Citation: Molecular Systems Biology 5:247

Temporal switching and cell-to-cell variability in Ca²⁺ release activity in mammalian cells

Naotoshi Nakamura¹, Toshiko Yamazawa¹, Yohei Okubo¹ & Masamitsu Iino¹

Department of Pharmacology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan

Correspondence to: Masamitsu Iino¹ Department of Pharmacology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel.: +81 3 5841 3414; Fax: +81 3 5841 3390; Email: iino@m.u-tokyo.ac.jp

Received 25 July 2008; Accepted 20 January 2009; Published online 17 March 2009

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Article highlights

Genetically identical cells in a uniform external environment can exhibit different phenotypes. Such non-genetic cell-to-cell variability has rarely been studied in differentiated mammalian cells. Here, we report that only $approx$ 40% of clonal human embryonic kidney 293 cells respond with an intracellular Ca2+ release when ryanodine receptor Ca2+ release channels in the endoplasmic reticulum are maximally activated by caffeine.
We showed that small cell-to-cell differences in either the levels of RyR channels, which cause Ca2+ release from the ER, or the opposing SERCA channels, which cause Ca2+ uptake to the ER, are amplified by the regenerative property of the Ca2+ release mechanism to give all-or-none differences in Ca2+ response among the cells.
We observed using time-lapse microscopy that individual cells switch between the caffeine-sensitive and caffeine-insensitive states with an average transition time of $approx$ 65 h. This is suggestive of temporal fluctuation in endogenous protein expression levels associated with caffeine response.
These results suggest the significance of regenerative mechanisms that amplify protein expression noise and induce cell-to-cell phenotypic variability in mammalian cells.

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Synopsis

Biochemical processes in cells are inherently noisy. This is due in part to the stochastic nature of gene expression systems, which typically involve small numbers of molecules such as DNA, mRNA and proteins (Kaern et al, 2005; Kaufmann and van Oudenaarden, 2007; Pedraza and Paulsson, 2008). In addition to such 'intrinsic noise', the internal states of cells and the structure of the signalling pathway also contribute to the fluctuation in the concentration of molecules, collectively termed 'extrinsic noise' (Hooshangi et al, 2005; Pedraza and van Oudenaarden, 2005; Rosenfeld et al, 2005; Shahrezaei et al, 2008). Intracellular noise can be exploited to play roles such as in the amplification of signals, the divergence of cell fates and the diversification of phenotypes (Arkin et al, 1998). In support of this idea, several recent reports suggested that individual clonal cells in the same external environment can exhibit qualitatively different phenotypes (Rao et al, 2002; Raser and O'Shea, 2005; Acar et al, 2008). Whereas most studies of intracellular noise so far have focused on unicellular organisms such as Escherichia coli or Saccharomyces cerevisiae, the importance of stochastic processes in multicellular organisms is now widely recognised (Laslo et al, 2006; Wernet et al, 2006; Chang et al, 2008). However, there are very few studies of differentiated cells of multicellular organisms (Ravasi et al, 2002; Feinerman et al, 2008), and the possibility and significance of such cells exhibiting different phenotypes in identical environments have rarely been discussed (Sigal et al, 2006; Cohen et al, 2008).

The calcium ion (Ca²⁺) is a ubiquitous intracellular messenger that regulates a diverse array of cellular functions, such as muscle contraction, secretion, fertilisation, immune responses, gene expression and synaptic plasticity (Berridge et al, 2003). The endoplasmic reticulum (ER) is the major intracellular Ca²⁺ store, from which Ca²⁺ is released via two families of Ca²⁺ release channels: ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP₃Rs). RyRs are activated by Ca²⁺ released by themselves (Endo, 1977)—a mechanism known as Ca²⁺-induced Ca²⁺ release (CICR). IP₃Rs are also activated by Ca²⁺ in the presence of IP₃ (Iino, 1990; Bezprozvanny et al, 1991; Finch et al, 1991). As Ca²⁺ response via these channels involves such a positive feedback, individual cells of the same type may show different Ca²⁺ responses with the amplification of intracellular noise.

Here, we report that only approximately 40% of clonal human embryonic kidney 293 cells respond with an intracellular Ca²⁺ increase when RyRs are maximally activated by caffeine. The expression levels of RyRs and sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA) showed a unimodal distribution, which could not explain the existence of two populations in terms of the RyR-mediated Ca²⁺ response. Using a mathematical model of Ca²⁺ release via RyRs, we investigated a mechanism that generates two distinct phenotypes but no significant difference in the amount of molecular components. Model simulations predicted that the difference in the RyR-mediated Ca²⁺ response depends on a critical balance between Ca²⁺ release and Ca²⁺ uptake activities, which is amplified by the regenerative nature of the CICR (Figure 3). This prediction was confirmed by the modulation of RyR-mediated Ca²⁺ response by changing the activities of RyRs and SERCAs.

Figure 3

Mathematical model of intracellular Ca²⁺ dynamics. (A, B) Response of model when RyR activity or SERCA activity changes. (A) RyR activity was increased in a stepwise manner (0.5 (magenta), 2.5 (blue), 4.5 (green), 6.5 (orange) and 8.5 (red) in s^-1) at a constant SERCA activity (3 M s^-1). (B) SERCA activity was decreased in a stepwise manner (5 (magenta), 4 (blue), 3 (green), 2 (orange) and 1 (red) in M s^-1) at a constant RyR activity (4.5 s^-1). (C) The balance between the numbers or activities of RyRs and SERCAs determines Ca²⁺ response via RyRs with threshold characteristics.

Full figure and legend (200K)Figures & Tables index

Fluctuation of protein concentrations in individual cells over time (Rosenfeld et al, 2005; Cai et al, 2006; Sigal et al, 2006; Yu et al, 2006) points to the possibility that individual HEK293 cells switch between the caffeine-sensitive and caffeine-insensitive states. Long-term Ca²⁺ imaging with GCaMP2, a genetically encoded fluorescent Ca²⁺ indicator (Tallini et al, 2006) revealed that individual cells switch between the caffeine-sensitive and caffeine-insensitive states in a flip-flop manner with an average transition time of $approx$ 65 h, suggestive of temporal fluctuation in endogenous protein expression levels associated with caffeine response (Figure 6). These results suggest the significance of regenerative mechanisms that amplify protein expression noise and induce cell-to-cell phenotypic variation in mammalian cells.

Figure 6

Switching maintains the intercellular heterogeneity of caffeine response. (A) Description of model. Black and white cells denote caf-positive and caf-negative cells, respectively. (B) After a sufficiently long time, the proportion of caf-positive cells approaches a constant value regardless of the initial proportion (0, 25, 50, 75 or 100%). In (B), p=0.29 and q=0.13 are used. (C) Schematic showing mechanism of phenotype switching.

Full figure and legend (115K)Figures & Tables index

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Acknowledgements

We thank Dr Junichi Nakai for providing a GCaMP2 construct, and Dr Hideto Oyamada for providing a rabbit RyR1 construct and an anti-RyR1 antibody. We also thank Dr Kazunori Kanemaru, Mr Yusuke Kawashima and Ms Yuri Sato for their technical assistance. This work was supported by Grant-in-Aid for Scientific Research (S) and in part by Global COE Program (Integrative Life Science Based on the Study of Biosignaling Mechanisms), MEXT, Japan.

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Synopsis