Dynamic Movement of the Calcium Sensor STIM1 and the Calcium Channel Orai1 in Activated T-Cells: Puncta and Distal Caps

Abstract

The proteins STIM1 and Orai1 are the long sought components of the store-operated channels required in T-cell activation. However, little is known about the interaction of these proteins in T-cells after engagement of the T-cell receptor. We found that T-cell receptor engagement caused STIM1 and Orai1 to colocalize in puncta near the site of stimulation and accumulate in a dense structure on the opposite side of the T-cell. FRET measurements showed a close interaction between STIM1 and Orai1 both in the puncta and in the dense cap-like structure. The formation of cap-like structures did not entail rearrangement of the entire endoplasmic reticulum. Cap formation depended on TCR engagement and tyrosine phosphorylation, but not on channel activity or Ca 2؉ influx. These caps were very dynamic in T-cells activated by contact with superantigen pulsed B-cells and could move from the distal pole to an existing or a newly forming immunological synapse. One function of this cap may be to provide preassembled Ca 2؉ channel components to existing and newly forming immunological synapses. INTRODUCTIONT-cell activation in response to antigen induces and requires the elevation of intracellular Ca 2ϩ (Hogan et al., 2003;Quintana et al., 2005;Feske, 2007). T-cell receptor (TCR) engagement leads to activation of well-documented signal transduction pathways that cause a rapid release of Ca 2ϩ from the endoplasmic reticulum (ER; Samelson, 2002;Panyi et al., 2004;Gwack et al., 2007a). The depletion of Ca 2ϩ from this internal store then leads to the opening of ion channels in the plasma membrane (PM) allowing an influx of Ca 2ϩ from outside the cell. The store-operated channel responsible for Ca 2ϩ influx in T-cells is known as the Ca 2ϩ release-activated Ca 2ϩ (CRAC) channel, which has been studied by electrophysiological techniques for many years (Lewis, 2001;Prakriya and Lewis, 2003;Cahalan et al., 2007;Hewavitharana et al., 2007;Hogan and Rao, 2007;Putney, 2007a).Sustained elevation of cytosolic Ca 2ϩ is required for complete T-cell activation (Iezzi et al., 1998;Lewis, 2001;Hogan et al., 2003;Feske, 2007). High levels of intracellular Ca 2ϩ are necessary to maintain the interaction between a T-cell and antigen-presenting cell (APC) that leads to formation of the specialized contact surface known as the immunological synapse (IS). Increased Ca 2ϩ levels are also required for the activation of transcription factors. In particular, elevated Ca 2ϩ maintains prolonged nuclear accumulation of nuclear factor of activated T-cells (NFAT) by activating the phosphatase calcineurin that dephosphorylates NFAT, allowing it to translocate to the nucleus and activate genes such as IL2 (Hogan et al., 2003;Macian, 2005). Several hours of Ca 2ϩ influx are required to complete the T-cell activation program, which involves expression of a large number of activation-associated genes (Lewis, 2001;Macian et al., 2002;Hogan et al., 2003).Although sustained Ca 2ϩ influx in T-cells has been studied for decades, the proteins involved have only been ident...

A Cytosolic Homomerization and a Modulatory Domain within STIM1 C Terminus Determine Coupling to ORAI1 Channels

“…ORAI1 has been assumed to act in concert with STIM1 (10,19,20), activating inward Ca 2ϩ currents after store depletion. We and others have recently provided evidence that store depletion leads to a dynamic coupling of STIM1 to ORAI1 (21)(22)(23), probably involving the putative coiled-coil domain in the C terminus of ORAI1 (22). Furthermore, the C terminus of STIM1 has been established as the key fragment for CRAC as well as ORAI1 activation because its expression alone, without the necessity to deplete ER store, is sufficient for constitutive current activation (18,22,24).…”

Store-Operated Calcium Channels: New Perspectives on Mechanism and Function

“…A particularly interesting example occurs during immune synapse formation between T cells and antigen-presenting cells (APCs), in which the T cell receptors engage peptide-MHC complexes on the APC. Shortly after cell -cell contact, STIM1 and Orai1 change their localization, accumulating initially at the synapse and then appearing to move in tandem to the distal end of the cell (Barr et al 2008;Lioudyno et al 2008). Ca 2þ imaging shows a domain of elevated [Ca 2þ ] i at the synapse, where it could potentially affect synapse stability, cytoskeletal remodeling, secretion, and other Ca 2þ -dependent events (Lioudyno et al 2008).…”

The STIM/Orai coupling machinery

“…We and others demonstrated by FRET microscopy a distance closer than 10 nm between CFP/YFP-labels linked to STIM1 and Orai1 in store-depleted cells. 51,59 Moreover a cytosolic STIM1 C-terminal fragment couples to Orai1 in in vitro as well as in vivo studies and accordingly activates constitutive Orai1 currents. 51 Thus, at least in the case of STIM1 C-terminus we propose a rather direct than indirect interaction of these two proteins via their C-termini.…”