Mitochondria control calcium entry at the immunological synapse

Best known for their role as “cellular powerhouses,” mitochondria do much more than simply provide energy to eukaryotic cells. As integral components of multiple signaling cascades, mitochondria regulate metabolism, the cell cycle, and apoptosis (1). Moreover, these tiny organelles play a major role in controlling Ca²⁺ signals throughout the cell (2). The concentration of free intracellular calcium ([Ca²⁺]_i), the most commonly utilized second messenger in nucleated cells, is maintained at a low level in the cytosol of resting cells. Sharp, transient elevations in [Ca²⁺]_i are used by signaling pathways to control such diverse events as oocyte fertilization, cell cycle entry, apoptosis, and lymphocyte activation. Using a sophisticated series of ion transporters and pores, mitochondria are capable of buffering cytosolic calcium and shaping and tuning calcium signals for diverse purposes (3). Through continuous reshaping and transport, mitochondria are distributed to specific cellular domains where they can carry out defined functions. The work of Quintana et al. (4) in a recent issue of PNAS provides compelling evidence that mitochondria are recruited to the immunological synapse, where they reduce local accumulation of Ca²⁺ so as to maintain the robust influx of Ca²⁺ across plasma membrane channels needed to activate downstream signaling components. The findings of this study argue that mitochondria are an essential component of the signaling complex at the immunological synapse.

Robust elevation of [Ca²⁺]_i is a crucial early step for T cell activation after antigen presentation and other stimuli that cross-link the T cell receptor (TCR) (5). The amplitude and duration of this rise in intracellular Ca²⁺ determine the strength and form of the immune response. However, the molecular events that control Ca²⁺ entry during lymphocyte activation are anything but simple. Rather, the process is reminiscent of some of the imaginary machines once designed by the American cartoonist Rube Goldberg that carry out a simple task through a ridiculously complex and convoluted series of events. That said, remarkable progress has been made in the past few years toward understanding the molecular basis for Ca²⁺ signaling in lymphocytes (6). The process begins when peptide antigens bound to major histocompatibility complex (MHC) proteins on an antigen-presenting cell (APC) engage the TCR. Stimulation of the TCR recruits a series of tyrosine kinases and substrates to the TCR/CD3 complex that eventually results in the phosphorylation and activation of phospholipase C-γ (PLCγ). Once activated, PLCγ hydrolyzes phosphatidylinositol 4,5-bisphosphate in the plasma membrane to generate diacylglycerol and inositol 1,4,5-trisphosphate (IP₃). IP₃ is a diffusible second messenger that induces the release of Ca²⁺ into the cytoplasm by opening IP₃ receptor (IP₃R) channels in the endoplasmic reticulum (ER). Although release of the limited amount of Ca²⁺ stored in the ER generates only a small, transient increase in [Ca²⁺]_i, it is a critical step in opening up the “floodgates” in the plasma membrane that then allow sustained Ca²⁺ influx from extracellular sources.

This second, prolonged, phase of Ca²⁺ elevation is essential for the control of several key activation events that lead to interleukin-2 (IL-2) expression (7, 8), a commitment step beyond which further T cell activation becomes antigen independent (9). Entry of extracellular Ca²⁺ into the lymphocyte occurs when reduction of ER Ca²⁺ stores leads to opening of CRAC (calcium release-activated calcium) channels in the plasma membrane (10). The depletion of free lumen ER Ca²⁺ is sensed by STIM1 (stromal interaction molecule 1), an ER-resident Ca²⁺ binding protein (11, 12). Upon reduction of ER Ca²⁺, STIM1 is believed to undergo a conformational change that stabilizes transient connections between the ER and plasma membrane and leads to activation of CRAC channels. The sustained elevation in [Ca²⁺]_i is required for calcineurin A-dependent dephosphorylation of nuclear factor of activated T cells (NFAT), a key transcriptional regulator of the IL-2 gene and other cytokine genes (13). Dephosphorylation is a necessary step for NFAT to translocate into the nucleus and remain in a transcriptionally active state (14). The importance of this Ca²⁺-dependent pathway in lymphocyte activation is highlighted by the profound immunosuppressive effects one sees with cyclosporin A and FK506, two chemically distinct natural products that are highly specific calcineurin inhibitors (15). Moreover, mutations in the gene encoding Orai1 (an ion-conducting pore subunit of the CRAC channel) result in defective T cell activation and a lethal form of severe combined immunodeficiency (SCID) syndrome in humans (16, 17).

The length of time these CRAC channels spend in an open (e.g., conducting) state determines the amplitude and duration of the rise in [Ca²⁺]_i. Local accumulation of Ca²⁺ quickly leads to inactivation of CRAC channels and limits further Ca²⁺ influx across the plasma membrane. Clearance of cytosolic Ca²⁺ by strategically located mitochondria has been previously shown to modulate the opening of CRAC channels in the plasma membrane in lymphocytes and hence regulate the entry of Ca²⁺ from the extracellular space (18). These prior studies demonstrated that inhibition of mitochondrial Ca²⁺ uptake by compounds that dissipate the intramitochondrial potential results in more rapid inactivation of CRAC channels after TCR cross-linking, which blocks NFAT nuclear translocation and efficient T cell activation. These results established an important role for mitochondria in controlling CRAC channel activity and the transmission of Ca²⁺ signals from the plasma membrane to the nucleus.

The current work by Quintana et al. (4) provides some new and exciting details regarding where mitochondria control CRAC channel activity in T lymphocytes. Using both the Jurkat T helper (T_h) cell line and isolated primary human T_h cells, the authors found that activation conditions that cross-linked TCRs in one area of the cell and generated an immunological synapse (IS) resulted in more sustained increases in [Ca²⁺]_i than conditions that diffusely activated TCRs on the cell surface without IS formation. Through the use of high-powered microscopy, the authors found that mitochondrial localization dramatically changed in T cells after IS formation. Within 15 min after focal TCR cross-linking, a subset of mito-

Robust elevation of [Ca²⁺]_i is a crucial early step for T cell activation.

chondria was directed to an area of the plasma membrane <200 nm from the IS. These mitochondria in the vicinity of the IS accumulated much higher levels of Ca²⁺ than those dispersed throughout the cell. The authors then used a series of inhibitors to show that this form of mitochondrial translocation depends upon the actin cytoskeleton. Moreover, blocking mitochondrial transport or Ca²⁺ uptake adjacent to the IS dramatically reduced CRAC channel conductivity and T cell activation.

These new findings suggest that mitochondria play an important role at the IS by reducing local Ca²⁺ accumulation before it inactivates CRAC/Orai1 channels in the plasma membrane. By keeping CRAC channels open within the region of the IS, mitochondria allow for the steep elevations in intracellular calcium that lead to activation of key downstream transcription factors such as NFAT (Fig. 1). Similar to other instances where mitochondria regulate Ca²⁺ in the cell, this work suggests that only those mitochondria closely juxtaposed to high-Ca²⁺ microdomains are responsible for the uptake (19, 20). This is likely because mitochondria outside these high-Ca²⁺ microdomains never encounter the 10 μM Ca²⁺ necessary for activation of their low-affinity Ca²⁺ uniporter. Hence, rapid transport of mitochondria to the area of the IS is a key role for the actin cytoskeleton in T cell signaling.

Fig. 1.

Efficient T cell activation requires mitochondrial Ca²⁺ uptake at the immunological synapse. (A) Upon formation of the immunological synapse (IS) between an antigen-presenting cell (APC) and T helper (T_h) cell, some mitochondria are normally redistributed within the T_h cell to the immediate vicinity of the IS. Once recruited to the IS, these mitochondria reduce local Ca²⁺ accumulation before it inactivates CRAC/Orai1 channels in the plasma membrane. This local reduction leads to sustained Ca²⁺ influx, high intracellular calcium [Ca²⁺]_i, and activation of key downstream transcription factors such as NFAT. (B) When mitochondria are pharmacologically inhibited from migrating to the IS and/or taking up Ca²⁺, local accumulation of Ca²⁺ near the IS inactivates CRAC/Orai1 channels quickly and blunts Ca²⁺ entry into the cell. In the absence of a sustained Ca²⁺ signal, efficient T_h cell activation does not occur. This illustration was provided by Christine Lin (Department of Pathology, University of California, San Francisco).

Although the work of Quintana et al. (4) adds new insight into the complex series of events that control Ca²⁺ signaling in lymphocytes, many questions remain. For example, it is still unclear exactly how STIM1 activates the CRAC channel in response to the depletion of ER Ca²⁺ stores. Does this occur through direct physical coupling of STIM1 and Orai1 at the junctional zone between the ER and plasma membrane? Are other proteins involved? Moreover, what ultimately leads to the inactivation of CRAC channels? Does this occur only after nearby mitochondria can no longer take up Ca²⁺ such that local [Ca²⁺]_i rises to a level that inactivates the CRAC channels? Alternatively, perhaps Ca²⁺-independent mechanisms are responsible for closing these channels after sufficient Ca²⁺ entry has occurred. It is clear that much more work remains to be done before we will completely understand Ca²⁺ signaling in lymphocytes and which steps are the best targets for treating various immune disorders.