Cell Calcium 45 (2009) 109–122 Contents lists available at ScienceDirect Cell Calcium journal homepage: www.elsevier.com/locate/ceca Morphological changes of T cells following formation of the immunological synapse modulate intracellular calcium signals Ariel Quintana a,∗ , Carsten Kummerow a , Christian Junker a , Ute Becherer b , Markus Hoth a a Department of Biophysics, University of Saarland, Homburg, Germany b Department of Physiology, University of Saarland, Homburg, Germany article info Article history: Received 25 April 2008 Received in revised form 8 July 2008 Accepted 9 July 2008 Available online 13 September 2008 Keywords: Mitochondria CRAC channels Immunological synapse T cell activation abstract Sustained Ca 2+ influx through plasma membrane Ca 2+ released-activated Ca 2+ (CRAC) channels is essential for T cell activation. Since inflowing Ca 2+ inactivates CRAC channels, T cell activation is only possible if Ca 2+ - dependent inactivation is prevented. We have previously reported that sustained Ca 2+ influx through CRAC channels requires both mitochondrial Ca 2+ uptake and mitochondrial translocation towards the plasma membrane in order to prevent Ca 2+ -dependent channel inactivation. Here, we show that morphologi- cal changes following formation of the immunological synapse (IS) modulate Ca 2+ influx through CRAC channels. Cell shape changes were dependent on the actin cytoskeleton, and they sustained Ca 2+ entry by bringing mitochondria and the plasma membrane in closer proximity. The increased percentage of mitochondria beneath the plasma membrane following shape changes occurred in all 3 dimensions and correlated with an increase in the amplitude of Ca 2+ signals. The shape change-dependent mitochon- drial localization close to the plasma membrane prevented CRAC channel inactivation even in T cells in which dynein motor protein-dependent mitochondria movements towards the plasma membrane were completely abolished, highlighting the importance of the shape change-dependent control of Ca 2+ influx. Our results suggest that morphological changes do not only facilitate an efficient contact with antigen presenting cells but also strongly modulate Ca 2+ dependent T cell activation. © 2008 Elsevier Ltd. All rights reserved. 1. Introduction T lymphocytes engage antigen presenting cells (APCs) in a long- lasting interaction that results in the activation and subsequent proliferation of T cells. A critical event in the activation of T lym- phocytes is the sustained engagement of T cell receptors (TCR) [1]. TCRs are activated by complex molecular mechanisms within the immunological synapse (IS), which consists of a central clus- ter of TCRs (central supramolecular activation clusters [c-SMAC]) that is surrounded by a ring of adhesion molecules (peripheral SMAC [p-SMAC]) [2,3]. In parallel, large molecules like CD43 and CD45 are excluded from the IS and instead are located in the dis- tal supramolecular activation cluster (d-SMAC) [4]. The molecular re-organization of plasma membrane proteins during the forma- tion of a matured synapse concertedly occurs with a complex and dramatic cell shape change, which is driven primarily by active cytoskeletal processes [5–7]. The shape change facilitates a tight and long-lasting conjugation with APC by bringing the mem- ∗ Corresponding author at: Medical Faculty, Saarland University, Building 58, D-66421 Homburg, Germany. Tel.: +49 6841 1626465; fax: +49 6841 1626060. E-mail address: ptaqgo@uks.eu (A. Quintana). brane into sufficiently close apposition at the c-SMAC to allow TCR binding to MHC–peptide [8]. A family of cytoskeleton-associated proteins called the ezrin–radixin–moesin proteins have been pro- posed to reduce the membrane rigidity during formation of the IS through their inactivation by dephosphorylation, allowing the accommodation of T cell surface membrane shape to that of the APC [7,9,10]. The mobility of the plasma membrane is considered essential to determine the quality and quantity of ligands necessary for T cell activation by regulating the formation of the IS [3,6,7]. A necessary step for the activation of T cells following TCR engagement is the stimulation of Ca 2+ entry across the plasma membrane [11,12]. TCR activation increases phospholipase C- activity, generates inositol 1,4,5-trisphosphate, releases Ca 2+ from the endoplasmic reticulum, and promotes activation of store- operated, Ca 2+ release-activated Ca 2+ (CRAC) channels in the plasma membrane [13–16]. Several lines of evidence stress the importance of CRAC channels for T cell function: its absence is par- alleled by a severe immunodeficiency [17,18], its activity is required for transcription of early genes [19,20], for T cell development in the thymus [21], and for the control of antigenic responsiveness [22] and tolerance [23]. In T cells, mitochondria are not only involved in the produc- tion of ATP but also in the control of CRAC channel activity and 0143-4160/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.ceca.2008.07.003