Gorman, Jacquelyn A.

Abstract IQGAP1 is a scaffolding protein that integrates the actin and microtubule cytoskeletons. Although IQGAP1 has been shown to participate in numerous cellular activities in fibroblasts and epithelial cells including E-cadherin-mediated cell-cell adhesion, cell polarity, motility, proliferation, and MAP kinase signaling, its function in thymocyte development or peripheral T cell effector functions has not yet been investigated. IQGAP1 localizes to the immune synapse in CD4+ and CD8+ T cells suggesting a possible role in T cell activation. Using wild type and IQGAP1-deficient mice, we demonstrate that neither thymocyte development nor peripheral T cells numbers are affected in the absence of IQGAP1. Surprisingly, IQGAP1-deficient CD4+ T cells make significantly more IL-2 compared to wild type cells. Despite this, IQGAP1-deficient T cells show diminished T cell proliferation and increased cell death following TCR and CD28 ligation, which is most likely due to increased activation-induced cell death. Interestingly, IQGAP1-deficient T cells spread abnormally in response to anti-CD3, fail to accumulate F-actin or polarize their microtubule-organizing center (MTOC) toward anti-CD3 coated beads. Consistent with the latter observation, CD8+ T cells do show defective cell-mediated killing. Furthermore, we find that IQGAP1-deficient T cells fail to polarize following integrin stimulation. Taken together, our data identify IQGAP1 as an important mediator of proper cytoskeletal polarization during T cell activation. This work was supported by NIAID grant AI065474 to D.D.B. and NIH -T32-AI07425 pre-doctoral Immunology Training Grant to J.A. Gorman.

The Arp2/3-activator Wiskott–Aldrich syndrome protein and Scar homologue (WASH) is suggested to regulate actin-dependent membrane scission during endosomal sorting, but its cellular roles have not been fully elucidated. To investigate WASH function, we generated tamoxifen-inducible WASH-knockout mouse embryonic fibroblasts (WASHout MEFs). Of interest, although EEA1+endosomes were enlarged, collapsed, and devoid of filamentous-actin and Arp2/3 in WASHout MEFs, we did not observe elongated membrane tubules emanating from these disorganized endomembranes. However, collapsed WASHout endosomes harbored segregated subdomains, containing either retromer cargo recognition complex–associated proteins or EEA1. In addition, we observed global collapse of LAMP1+lysosomes, with some lysosomal membrane domains associated with endosomes. Both epidermal growth factor receptor (EGFR) and transferrin receptor (TfnR) exhibited changes in steady-state cellular localization. EGFR was directed to the lysosomal compartment and exhibited reduced basal levels in WASHout MEFs. However, although TfnR was accumulated with collapsed endosomes, it recycled normally. Moreover, EGF stimulation led to efficient EGFR degradation within enlarged lysosomal structures. These results are consistent with the idea that discrete receptors differentially traffic via WASH-dependent and WASH-independent mechanisms and demonstrate that WASH-mediated F-actin is requisite for the integrity of both endosomal and lysosomal networks in mammalian cells.

Abstract The Ras GTPase-activating-like protein IQGAP1 is a multimodular scaffold that controls signaling and cytoskeletal regulation in fibroblasts and epithelial cells. However, the functional role of IQGAP1 in T cell development, activation, and cytoskeletal regulation has not been investigated. In this study, we show that IQGAP1 is dispensable for thymocyte development as well as microtubule organizing center polarization and cytolytic function in CD8+ T cells. However, IQGAP1-deficient CD8+ T cells as well as Jurkat T cells suppressed for IQGAP1 were hyperresponsive, displaying increased IL-2 and IFN-γ production, heightened LCK activation, and augmented global phosphorylation kinetics after TCR ligation. In addition, IQGAP1-deficient T cells exhibited increased TCR-mediated F-actin assembly and amplified F-actin velocities during spreading. Moreover, we found that discrete regions of IQGAP1 regulated cellular activation and F-actin accumulation. Taken together, our data suggest that IQGAP1 acts as a dual negative regulator in T cells, limiting both TCR-mediated activation kinetics and F-actin dynamics via distinct mechanisms.