HIV-1 Nef Employs Two Distinct Mechanisms to Modulate Lck Subcellular Localization and TCR Induced Actin Remodeling

The Nef protein acts as critical factor during HIV pathogenesis by increasing HIV replication in vivo via the modulation of host cell vesicle transport and signal transduction processes. Recent studies suggested that Nef alters formation and function of immunological synapses (IS), thereby modulating exogenous T-cell receptor (TCR) stimulation to balance between partial T cell activation required for HIV-1 spread and prevention of activation induced cell death. Alterations of IS function by Nef include interference with cell spreading and actin polymerization upon TCR engagement, a pronounced intracellular accumulation of the Src kinase Lck and its reduced IS recruitment. Here we use a combination of Nef mutagenesis and pharmacological inhibition to analyze the relative contribution of these effects to Nef mediated alterations of IS organization and function on TCR stimulatory surfaces. Inhibition of actin polymerization and IS recruitment of Lck were governed by identical Nef determinants and correlated well with Nef's association with Pak2 kinase activity. In contrast, Nef mediated Lck endosomal accumulation was separable from these effects, occurred independently of Pak2, required integrity of the microtubule rather than the actin filament system and thus represents a distinct Nef activity. Finally, reduction of TCR signal transmission by Nef was linked to altered actin remodeling and Lck IS recruitment but did not require endosomal Lck rerouting. Thus, Nef affects IS function via multiple independent mechanisms to optimize virus replication in the infected host

Unravelling the Actin Cytoskeleton:A New Competitive Edge?

Dynamic rearrangements in the actin cytoskeleton underlie a wide range of cell behaviours, which in turn contribute to many aspects of human health including embryogenesis, cancer metastasis, wound healing, and inflammation. Precise control of the actin cytoskeleton requires the coordinated activity of a diverse set of different actin regulators. However, our current understanding of the actin cytoskeleton has focused on how individual actin regulatory pathways function in isolation from one another. Recently, competition has emerged as a means by which different actin assembly factors can influence each other's activity at the cellular level. Here such findings will be used to explore the possibility that competition within the actin cytoskeleton confers cellular plasticity and the ability to prioritise multiple conflicting stimuli

Essential and unique roles of PIP5K-γ and -α in Fcγ receptor-mediated phagocytosis

The actin cytoskeleton is dynamically remodeled during Fcγ receptor (FcγR)-mediated phagocytosis in a phosphatidylinositol (4,5)-bisphosphate (PIP2)-dependent manner. We investigated the role of type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) γ and α isoforms, which synthesize PIP2, during phagocytosis. PIP5K-γ−/− bone marrow–derived macrophages (BMM) have a highly polymerized actin cytoskeleton and are defective in attachment to IgG-opsonized particles and FcγR clustering. Delivery of exogenous PIP2 rescued these defects. PIP5K-γ knockout BMM also have more RhoA and less Rac1 activation, and pharmacological manipulations establish that they contribute to the abnormal phenotype. Likewise, depletion of PIP5K-γ by RNA interference inhibits particle attachment. In contrast, PIP5K-α knockout or silencing has no effect on attachment but inhibits ingestion by decreasing Wiskott-Aldrich syndrome protein activation, and hence actin polymerization, in the nascent phagocytic cup. In addition, PIP5K-γ but not PIP5K-α is transiently activated by spleen tyrosine kinase–mediated phosphorylation. We propose that PIP5K-γ acts upstream of Rac/Rho and that the differential regulation of PIP5K-γ and -α allows them to work in tandem to modulate the actin cytoskeleton during the attachment and ingestion phases of phagocytosis

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

Cytolytic effectors polarize toward target cells for effective killing and IFN-γ secretion. The spatiotemporal features of this polarization and their importance for cytolysis have not been resolved. In cytotoxic T cells and natural killer (NK) cells, transient polarization was consistently associated with effective killing. Polarization was regulated by Cdc42, a small Rho family GTPase universally critical for cytoskeletal dynamics. Transient accumulation of active Cdc42 at the cytolytic effector/target cell interface and focus of such accumulation on the interface center were closely related to cytolysis. Surprisingly, however, the intensity of Cdc42 activation was not. We interfered with Cdc42 activation in NK cells such that sustained polarization in long lasting nonkilling cell couples was selectively blocked. Thus the proportion of the NK cell population displaying transient polarization was increased. As a consequence, cytolytic responder frequency and IFN-γ production were enhanced upon such interference with Cdc42 activation. These data support the notion that transience in polarization is critical for cytolytic effector function, likely by preventing cytolytic effectors from becoming trapped in nonproductive target cell interactions

T/B-cell interactions are more transient in response to weak stimuli in SLE-prone mice

Changes in immune function during the course of systemic lupus erythematosus (SLE) are well characterized. Class-switched antinuclear antibodies are the hallmark of SLE, and T/B- cell interactions are thus critical. However, changes in immune function contributing to disease susceptibility are unknown. Here, we have analyzed primary T and B cells from a mouse model of SLE prior to the onset of disease. To allow cognate T-cell activation with low affinity, we have developed a lower potency peptide ligand for the OTII T-cell receptor. T and B-cell couples formed less frequently and retained their polarity less efficiently preferentially in response to low affinity stimulation in SLE-prone mice. This matched decreased recruitment of actin and Vav1 and an enhanced PKCΘ recruitment to the cellular interface in T cells. The induction of the germinal center B-cell marker GL7 was increased in T/B cell couples from SLE-prone mice when the T-cell numbers were limited. However, the overall gene expression changes were marginal. Taken together, the enhanced cell couple transience may allow a more efficient sampling of a large number of T/B cell couples, preferentially in response to limiting stimuli, therefore enhancing the immune reactivity in the development of SLE