Kinetic Modeling of ABCG2 Transporter Heterogeneity: A Quantitative, Single-Cell Analysis of the Side Population Assay.

The side population (SP) assay, a technique used in cancer and stem cell research, assesses the activity of ABC transporters on Hoechst staining in the presence and absence of transporter inhibition, identifying SP and non-SP cell (NSP) subpopulations by differential staining intensity. The interpretation of the assay is complicated because the transporter-mediated mechanisms fail to account for cell-to-cell variability within a population or adequately control the direct role of transporter activity on staining intensity. We hypothesized that differences in dye kinetics at the single-cell level, such as ABCG2 transporter-mediated efflux and DNA binding, are responsible for the differential cell staining that demarcates SP/NSP identity. We report changes in A549 phenotype during time in culture and with TGFß treatment that correlate with SP size. Clonal expansion of individually sorted cells re-established both SP and NSPs, indicating that SP membership is dynamic. To assess the validity of a purely kinetics-based interpretation of SP/NSP identity, we developed a computational approach that simulated cell staining within a heterogeneous cell population; this exercise allowed for the direct inference of the role of transporter activity and inhibition on cell staining. Our simulated SP assay yielded appropriate SP responses for kinetic scenarios in which high transporter activity existed in a portion of the cells and little differential staining occurred in the majority of the population. With our approach for single-cell analysis, we observed SP and NSP cells at both ends of a transporter activity continuum, demonstrating that features of transporter activity as well as DNA content are determinants of SP/NSP identity.

Calcium Dynamics of Ex Vivo Long-Term Cultured CD8+ T Cells Are Regulated by Changes in Redox Metabolism.

T cells reach a state of replicative senescence characterized by a decreased ability to proliferate and respond to foreign antigens. Calcium release associated with TCR engagement is widely used as a surrogate measure of T cell response. Using an ex vivo culture model that partially replicates features of organismal aging, we observe that while the amplitude of Ca2+ signaling does not change with time in culture, older T cells exhibit faster Ca2+ rise and a faster decay. Gene expression analysis of Ca2+ channels and pumps expressed in T cells by RT-qPCR identified overexpression of the plasma membrane CRAC channel subunit ORAI1 and PMCA in older T cells. To test whether overexpression of the plasma membrane Ca2+ channel is sufficient to explain the kinetic information, we adapted a previously published computational model by Maurya and Subramaniam to include additional details on the store-operated calcium entry (SOCE) process to recapitulate Ca2+ dynamics after T cell receptor stimulation. Simulations demonstrated that upregulation of ORAI1 and PMCA channels is not sufficient to explain the observed alterations in Ca2+ signaling. Instead, modeling analysis identified kinetic parameters associated with the IP3R and STIM1 channels as potential causes for alterations in Ca2+ dynamics associated with the long term ex vivo culturing protocol. Due to these proteins having known cysteine residues susceptible to oxidation, we subsequently investigated and observed transcriptional remodeling of metabolic enzymes, a shift to more oxidized redox couples, and post-translational thiol oxidation of STIM1. The model-directed findings from this study highlight changes in the cellular redox environment that may ultimately lead to altered T cell calcium dynamics during immunosenescence or organismal aging.

Dynamic Redox Regulation of IL-4 Signaling.

Quantifying the magnitude and dynamics of protein oxidation during cell signaling is technically challenging. Computational modeling provides tractable, quantitative methods to test hypotheses of redox mechanisms that may be simultaneously operative during signal transduction. The interleukin-4 (IL-4) pathway, which has previously been reported to induce reactive oxygen species and oxidation of PTP1B, may be controlled by several other putative mechanisms of redox regulation; widespread proteomic thiol oxidation observed via 2D redox differential gel electrophoresis upon IL-4 treatment suggests more than one redox-sensitive protein implicated in this pathway. Through computational modeling and a model selection strategy that relied on characteristic STAT6 phosphorylation dynamics of IL-4 signaling, we identified reversible protein tyrosine phosphatase (PTP) oxidation as the primary redox regulatory mechanism in the pathway. A systems-level model of IL-4 signaling was developed that integrates synchronous pan-PTP oxidation with ROS-independent mechanisms. The model quantitatively predicts the dynamics of IL-4 signaling over a broad range of new redox conditions, offers novel hypotheses about regulation of JAK/STAT signaling, and provides a framework for interrogating putative mechanisms involving receptor-initiated oxidation.