Cell Calcium (2009) 45, 89—97

journal homepage: www.elsevier.com/locate/ceca

Rapid downregulation of the Ca2+-signal after exocytosis stimulation in Paramecium cells: Essential role of a centrin-rich filamentous cortical network, the infraciliary lattice Ivonne M. Sehring a, Catherine Klotz b,c,d, Janine Beisson b,c,d, Helmut Plattner a,∗ a

Department of Biology, University of Konstanz, P.O. Box 5560, 78457 Konstanz, Germany Centre de Génétique Moléculaire, CNRS, 91190 Gif-sur-Yvette, France c Université Paris-Sud, Orsay F-91405, France d Université Pierre et Marie Curie, Paris 6, Paris F-75005, France b

Received 29 February 2008; received in revised form 15 April 2008; accepted 17 June 2008 Available online 23 July 2008

KEYWORDS 2+

Ca ; Calcium; Centrin; Centrin-binding proteins; Ciliates; Infraciliary lattice; Exocytosis; Paramecium

∗

Summary We analysed in Paramecium tetraurelia cells the role of the infraciliary lattice, a cytoskeletal network containing numerous centrin isoforms tightly bound to large binding proteins, in the re-establishment of Ca2+ homeostasis following exocytosis stimulation. The wild type strain d4-2 has been compared with the mutant cell line -PtCenBP1 which is devoid of the infraciliary lattice (‘‘-PtCenBP1’’ cells). Exocytosis is known to involve the mobilization of cortical Ca2+ -stores and a superimposed Ca2+ -influx and was analysed using Fura Red ratio imaging. No difference in the initial signal generation was found between wild type and -PtCenBP1 cells. In contrast, decay time was greatly increased in -PtCenBP1 cells particularly when stimulated, e.g., in presence of 1 mM extracellular Ca2+ , [Ca2+ ]o . Apparent halftimes of f/f0 decrease were 8.5 s in wild type and ∼125 s in -PtCenBP1 cells, requiring ∼30 s and ∼180 s, respectively, to re-establish intracellular [Ca2+ ] homeostasis. Lowering [Ca2+ ]o to 0.1 and 0.01 mM caused an acceleration of intracellular [Ca2+ ] decay to t1/2 = 33 s and 28 s, respectively, in -PtCenBP1 cells as compared to 8.1 and 5.6, respectively, for wild type cells. We conclude that, in Paramecium cells, the infraciliary lattice is the most efficient endogenous Ca2+ buffering system allowing the rapid downregulation of Ca2+ signals after exocytosis stimulation. © 2008 Elsevier Ltd. All rights reserved.

Corresponding author. Tel.: +49 7531 88 2228; fax: +49 7531 88 2245. E-mail address: [email protected] (H. Plattner).

0143-4160/$ — see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.ceca.2008.06.004

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Introduction In eukaryotic cells, the universal second messenger, Ca2+ , can be activated from different sources and then activates a variety of processes including secretion [1—4]. Thereby cells operate with a very large excess of Ca2+ to achieve sufficient local concentrations although few molecules suffice for local activation [5—7]. Spatio-temporal precision is a prerequisite of specific functional activation, as is the subsequent downregulation of the Ca2+ signal. Depending on the system, during stimulated exocytosis, rapidly increasing values of intracellular Ca2+ concentrations, [Ca2+ ]i , can be counteracted by widely different mechanisms, such as Ca2+ -pumps (Ca2+ -ATPases in the cell membrane and in the membranes of Ca2+ -stores), cation exchangers (notably Na+ /Ca2+ exchangers) and/or binding to buffer proteins such as calsequestrin and calreticulin in the Sarcoplasmatic and Endoplasmic Reticulum, respectively, as well as calbindin or parvalbumin and some others in the cytosolic compartment [1,8—13]. Within the cytosol, due to Ca2+ -binding proteins, only 90 s after stimulation) corresponds to the decay seen in wt. cells, while the preceding phase is much slower (Fig. 4, Table 1). The most reasonable explanation of the slow decay from 0 to 90 s is the lack of Ca2+ -binding in the absence of the cortical centrins, thus underscoring their key-function in rapid [Ca2+ ]i downregulation. How do our data compare with those obtained with other cells? Disposal of Ca2+ after stimulation greatly varies in different cell types and it may also vary depending on the type of stimulation [1—4,8—13,36]. In Paramecium, Ca2+ regulation has been well analysed in two types of processes, i.e. ciliary beating and stimulated exocytosis performance. For instance, the Ca2+ signal generated during depolarization-mediated ciliary beat reversal is normally restricted to cilia. It results in inactivation of ciliary voltage-dependent Ca2+ -influx channels by the very same Ca2+ [42,43], probably followed by Ca2+ binding within the cilium and by its subsequent slow elimination (only overstimulation causes a spillover into the cell soma [44]). No such negative feedback is known from signals generated by exocytosis stimulation. When Ca2+ signals are generated in the somatic (non-ciliary) part of a Paramecium cell this encompasses first a Ca2+ release from alveolar sacs and second a superimposed Ca2+ -influx [28,30,32,33]. This signal can sweep deep inside cells, as we observe (Fig. 5) for both wt. and wt.-derived -PtCenBP1 cells. Internal Ca2+ concentrations following exocytosis can be appreciated as follows. The local [Ca2+ ]i required for exocytosis is ∼5 ␮M [35]. Since this is only partially achieved by Ca2+ release from alveolar sacs, but considerably complemented by Ca2+ -influx [33], one may estimate the relative contribution of the influx component at different [Ca2+ ]o values. Alveolar sacs contain a total calcium concentration, [Ca], of ∼40 mM of which 40% are mobilized upon AED stimulation [31]. Assuming that the signal is finally distributed over the entire cell volume, a theoretical increase to 0.25 mM would result [33]. Together with a larger influx component, measured at [Ca2+ ]o = 0.04 mM [29], a global increase of [Ca2+ ]i to 0.85 mM can be theoretically calculated (disregarding that published data sets were obtained under slightly varying experimental conditions and that rapid Ca2+ binding, sequestration and extrusion would counteract and, thus, blur these theoretical estimations) [33]. With increasing [Ca2+ ]o , [Ca2+ ]i values resulting from AED stimulation would be accordingly higher. The question arose, therefore, how this signal can be reduced after exocytosis stimulation. Paramecium cells possess a plasma membrane Ca2+ ATPase, PMCA [45], as well as a Sarcoplasmic/Endoplasmic Reticulum-type Ca2+ -ATPase, SERCA [46], that is enriched in subplasmalemmal alveolar sacs [47]. The refilling of these alveolae by SERCA activity after Ca2+ depletion, as determined by widely different methods, is rather slow [48], as is the PMCA mechanism [45]. In fact, we have calculated that neither the PMCA nor the SERCA could ensure the rapid decay of the Ca2+ signal generated during exocytosis stim-

96 ulation [33]. Also a Na+ /Ca2+ exchanger, as envisaged for the related ciliate, Euplotes [49], is an unlikely explanation for our experiments with Paramecium as they have been performed in the absence of Na+ from the medium. Is there any other potential mechanism for [Ca2+ ]i downregulation in Paramecium after AED stimulation? In fact, Stock et al. [50] have shown the extrusion of substantial amounts of Ca2+ by the contractile vacuole. Thus, spillover of Ca2+ into central cell regions after AED stimulation (Fig. 5) may make this organelle an important component for Ca2+ downregulation. Due to binding of Ca2+ to Paramecium centrin, based on prognosticated EF-hand domains and additional negatively charged amino acids [20,22], Ca2+ might be easily released from some of the binding sites and diffuse to more distant sites for final disposal particularly by the osmoregulatory system and — in our judgement — to a lesser extent by the PMCA- and SERCA-type Ca2+ -pumps. In total, the ICL stands out as an efficient Ca2+ -binding array capable of rapidly buffering the large excess of Ca2+ since we have observed a significant delay in the downregulation of Ca2+ in the -PtCenBP1 cells lacking the infraciliary lattice. Considering the now well established molecular organization of the ICL as a stable association of large centrin-binding proteins with numerous centrin isoforms [21,22], it is possible to consider the whole ICL cytoskeletal network as a high capacity Ca2+ -buffering cell constituent. A summary of potential Ca2+ -fluxes and the role of cortical centrins during stimulus-secretion coupling is presented in Fig. 6.

Conclusions Downregulation of Ca2+ after stimulation is important, not only to restrict the signal and the effect thus generated in space and time, but also because high [Ca2+ ]i levels can severely compromise cell survival [51,52]. Mechanisms of Ca2+ signal downregulation immediately after stimulation are widely different in different cell types. As we now determined, in Paramecium cells rapid downregulation of Ca2+ can be mainly attributed to the centrins contained in the ICL, rather than to any direct or indirect sequestration or extrusion mechanisms. In contrast to the situation in Paramecium, in most other systems Ca2+ -binding proteins are reported to exert only rather mild modulatory functions [53—58]. Although their role is not even discussed in some recent reviews on Ca2+ dynamics [36], studies on parvalbumin in neuronal cells [59,60] are a good positive example of the regulation of Ca2+ -signals by cytosolic Ca2+ -binding proteins. The role of centrins in governing [Ca2+ ]i homeostasis we describe here is novel as these Ca2+ -buffering molecules are immobilized as a large cytoskeletal network. The cortical localization of the ICL provides a strategic position for a Ca2+ buffering system.

Acknowledgements We thankfully acknowledge financial support by the Deutsche Forschungsgemeinschaft (grants to H.P.). C.K. and J.B. have been supported by the Centre National de la Recherche Scientifique.

I.M. Sehring et al.

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