Protein transduction as a means of effective manipulation of Cdc42 activity in primary T cells

Abstract

The Rho family GTPase Cdc42 is a critical regulator of cellular polarization from yeast to man. An analysis of its function in T cell activation is therefore of interest. This analysis poses two substantial challenges, similar to the analysis of many other critical T cell signaling intermediates. First, Cdc42 is required for development and cell survival, necessitating short-term manipulation of its activity. Second, Cdc42 is likely involved in multiple signaling pathways, requiring approaches to distinguish multiple roles. To address these challenges, we first determined and quantified spatio-temporal patterns of Cdc42 activity using live cell video fluorescence microscopy. This generates hypotheses at which times and locations Cdc42 might play possibly distinct roles. Second and as the focus of this manuscript, we employed protein transduction to manipulate Cdc42 activity for the generation of causality. Protein transduction allows such manipulation to be short-term, quantitative, and with multiple reagents. Here, we characterize uptake, retention, and subcellular distribution of protein transduction reagents. We describe how a more quantitative single cell analysis of Cdc42 activity provides superior distinction between experimental conditions. And we show how we have used dose responses of the protein transduction reagents to minimize side effects while retaining efficacy. We suggest that our strategy is an important complement to more established techniques to study protein function in primary T cells, in particular in the investigation of signaling intermediates that are essential for cell survival and regulate multiple aspects of T cell activation.

Itk Controls the Spatiotemporal Organization of T Cell Activation

“…We used the V12 constitutively active mutant of Cdc42 (Cdc42ca), because we were unsure whether proteins that mediate Cdc42 activation would properly localize in Itk-deficient T cells. We used protein transduction with the HIV Tat-derived transduction peptide (tat) (26, 27) to enable dose-dependent delivery of Cdc42ca to cells (Fig. 3).…”

“…The antibody against Vav1 was obtained from Cell Signaling Technology. The protein transduction version of constitutively active Cdc42 (V12), fusions thereof with different recruitment domains, and the Tec-PHTHSH3 domains were generated, purified under native conditions from Escherichia coli by immobilized metal-affinity chromatography, and applied to T cells as described previously (27). …”

Transience in polarization of cytolytic effectors is required for efficient killing and controlled by Cdc42

“…To address roles of Cdc42 in cytolytic effector function, we interfered with the activation of Cdc42, and Rac as a control, with well established dominantnegative mutants (Cdc42dn, Rac1dn), as applied short-term and quantitatively as protein transduction reagents at a concentration of 100 nM (24,25). As expected (22), both Cdc42dn and Rac1dn blocked interface actin accumulation, by more than 70% in NK cells and a bit less in CTLs (Fig.…”

“…Protein transduction versions of Cdc42dn and Rac1dn, their purification, and their functionality in primary lymphocytes are established (24,25). Cytolytic effectors were incubated with the protein transduction reagents for 30 min at 37°C at the indicated concentrations before the functional assays (25).…”

“…Later reductions in Cdc42 activity and actin accumulation at the cytolytic effector/target cell interface likely allow dissociation of nonproductive cell couples. To test this notion, we interfered with Cdc42 activation (24,25) such that initial polarization was preserved while sustained actin accumulation was blocked. This enhanced the frequency of cytolytic effectors involved in killing and secretion of IFN-γ.…”