Since our beginnings in 1990, the Lewis Lab has explored many aspects of calcium signaling at the molecular and cellular level. A selection of these is summarized below in roughly chronological order.
A biophysical fingerprint of the CRAC channel
Over a number of years we applied patch-clamp techniques to discover the unusual characteristics of the CRAC channel. Together, these characteristics comprise a biophysical "fingerprint" that was essential for identifying the CRAC channel as a unique channel entity, and for testing candidate genes that might encode it. Importantly, many of the characteristics serve to shape the channel's unique physiological functions. These include calcium-dependent inactivation (which feeds back to temper channel activity), calcium-dependent potentiation (which maintains the channel's conductive state), extremely high calcium selectivity (which enables the channel to admit only Ca2+ and not the more abundant Na+ ions), an extremely low calcium conduction rate (which restricts signaling to a nanodomain centered on the channel pore), and bimodal regulation of channel activity by 2-APB (which may suggest strategies to enhance and inhibit calcium influx with drugs).
Calcium oscillations drive gene expression
Intracellular Ca2+ oscillations are widely used for signaling, and most cells create them through the repetitive release of Ca2+ from the ER. In T cells, engagement of the antigen receptor triggers Ca2+ oscillations through a different mechanism: repetitive activation of influx through CRAC channels, which appears to be driven by slow feedback between changes in Ca2+ store content and CRAC channel activity. Ca2+ oscillations are a means of conferring signaling specificity on an otherwise promiscuous messenger. Using a calcium clamp to generate controlled oscillations we showed (1) that oscillations increase the efficiency of signaling through NFAT, a central transcription factor in T cell activation; and (2) oscillation frequency controls the specificity of transcriptional activation through NFAT vs. NFκB. This highly cited work stands as one of the best examples of how cells can extract information encoded in the amplitude and dynamics of calcium signals.
Mitochondria regulate slow inactivation of CRAC channels
Mitochondria in T lymphocytes are close to the plasma membrane - so close that they can sense calcium gradients extending from open CRAC channels. We found that calcium uptake and release from mitochondria are required to enable T cells to maintain high calcium levels in response to store depletion. Further studies ascribed this effect to the ability of energized mitochondria to inhibit slow calcium-dependent inactivation of CRAC channels. These studies were the first to reveal crosstalk between mitochondria and CRAC channel inactivation gating which in turn promotes the activation and nuclear translocation of the transcription factor NFAT.
Calcium pump modulation shapes calcium signals in T lymphocytes
By extruding calcium across the plasma membrane, the Plasma Membrane Ca2+-ATPase (PMCA) plays a major role in maintaining a low basal intracellular calcium level (~100 nM) in cells. In T cells we found that the PMCA is slowly upregulated ("modulated") by rises in [Ca2+]i. In this way, the PMCA creates a high-pass filter effect that enhances Ca2+ dynamics and also extends the dynamic range over which the PMCA can control [Ca2+]i. Interestingly, pumps are located close enough to CRAC channels to experience the local gradients that occur within nanodomains near open channels, enabling private communication. Together, these studies showed that the PMCA, far from being merely a "housekeeping" element, also plays an active role in shaping and filtering calcium signals in T cells.
Calcium signaling during thymocyte motility in thymic tissue slices
The maturation of thymocytes into T cells reactive to foreign but not self antigens involves contact with various types of stromal cells as they migrate through the thymus. Calcium signaling is an essential signal for selection and maturation. To study calcium signaling in the intact 3D tissue environment we developed the first thymic slice preparation in which thymocytes loaded with calcium indicator dyes could be tracked by two-photon microscopy. We found that positive selection was associated with calcium oscillations that generated a "stop" signal to arrest cells at sites of cognate antigen presentation. This crosstalk between signaling and motility may integrate the search for potentially rare self-antigens with the requirement for sustained signaling in T cell maturation.