HIV-1 Nef is a key factor in AIDS pathogenesis. Here, we report that Nef potently inhibits motility of fibroblasts and chemotaxis of HIV-1-infected primary human T lymphocytes toward the chemokines SDF-1alpha, CCL-19, and CCL-21 ex vivo. Furthermore, Nef inhibits guided motility of zebrafish primordial germ cells toward endogenous SDF-1a in vivo. These migration defects result from Nef-mediated inhibition of the actin remodeling normally triggered by migratory stimuli. Nef strongly induces phosphorylation of cofilin, inactivating this evolutionarily conserved actin-depolymerizing factor that promotes cell motility when unphosphorylated. Nef-dependent cofilin deregulation requires association of Nef with the cellular kinase Pak2. Disruption of Nef-Pak2 association restores the cofilin phosphorylation levels and actin remodeling that facilitate cell motility. We conclude that HIV-1 Nef alters Pak2 function, which directly or indirectly inactivates cofilin, thereby restricting migration of infected T lymphocytes as part of a strategy to optimize immune evasion and HIV-1 replication.

When B cells encounter antigens on the surface of an antigen-presenting cell (APC), B cell receptors (BCRs) are gathered into microclusters that recruit signaling enzymes. These microclusters then move centripetally and coalesce into the central supramolecular activation cluster of an immune synapse. The mechanisms controlling BCR organization during immune synapse formation, and how this impacts BCR signaling, are not fully understood. We show that this coalescence of BCR microclusters depends on the actin-related protein 2/3 (Arp2/3) complex, which nucleates branched actin networks. Moreover, in murine B cells, this dynamic spatial reorganization of BCR microclusters amplifies proximal BCR signaling reactions and enhances the ability of membrane-associated antigens to induce transcriptional responses and proliferation. Our finding that Arp2/3 complex activity is important for B cell responses to spatially restricted membrane-bound antigens, but not for soluble antigens, highlights a critical role for Arp2/3 complex-dependent actin remodeling in B cell responses to APC-bound antigens.

IntroductionReplication of HIV-1 in primary human T lymphocytes is tightly coupled to their activation state. Whereas HIV-1 undergoes early replication events in quiescent CD4 ϩ T lymphocytes, subsequent steps in the viral life cycle require cell activation. 1 T lymphocyte activation is primarily governed by signaling through the TCR complex after engagement in a tight contact with APCs; this is referred to as the immunological synapse (IS).TCR engagement by specific MHC-presented peptides launches highly dynamic and coordinated transport events that recruit specific factors to the IS and exclude others from it. This signal initiation triggers a broad cascade of downstream signaling that include dynamic F-actin remodeling at the IS, tyrosine phosphorylation, release of calcium flux, and activation of transcription. These events increase production of the T-cell survival cytokine IL-2 and are coordinated by the TCR proximal tyrosine kinase Lck, a master switch of TCR signaling. Immediately after TCR engagement, active Lck is recruited to the IS. 2-4 Whereas signal diversification and enhancement occur at the plasma membrane (PM), subsequent TCR signaling is compartmentalized and also occurs at intracellular membranes. An important intracellular arm of the TCR response is regulated by the N-Ras GTPase that is activated at Golgi membranes downstream of Lck. [5][6][7][8][9] T-cell activation is thought to be beneficial to HIV-1 because it allows transcriptional activation of latent provirus and progression of the life cycle. However, activation-induced cell death after TCR engagement runs the risk of limiting the lifespan of productively infected cells and thus the amount of viral progeny produced.Consequently, HIV-1 encodes gene products such as Nef to fine-tune the activation states of infected T lymphocytes. 10,11 Nef is a 25-to 34-kDa myristoylated accessory protein encoded by HIV-1, HIV-2, and SIV. Ex vivo, Nef enhances the single-round infectivity of virus particles and moderately accelerates virus spread over multiple rounds. 12 In vivo, Nef strongly boosts virus replication, particularly during primary infection, when the presence of Nef can elevate virus titers by more than 2 logs, and is critical for rapid disease progression. [13][14][15] This role of Nef as a pathogenicity factor is also revealed in transgenic mice, in which Nef expression induces AIDS-like depletion of CD4 ϩ T lymphocytes. 16 Delineating the mechanisms of Nef action has been hampered by the multitude of interactions with host T-cell proteins suggested to modulate various intracellular transport and signaling pathways. 17,18 This includes modulating exposure of cell-surface receptors such as MHC-I and II, CD4, and chemokine receptors to evade immune recognition and to prevent superinfection of infected cells, respectively (reviewed in Laguette et al 12 ). In addition, Nef affects the basal states of T-cell activation and the responsiveness of T lymphocytes to TCR signaling. [19][20][21][22] Initial studies with overexpression strategies ...

Integrating signals from multiple receptors allows cells to interpret the physiological context in which a signal is received. Here we describe a mechanism for receptor crosstalk in which receptor-induced increases in actin dynamics lower the threshold for signalling by another receptor. We show that the Toll-like receptor ligands lipopolysaccharide and CpG DNA, which are conserved microbial molecules, enhance signalling by the B-cell antigen receptor (BCR) by activating the actin-severing protein cofilin. Single-particle tracking reveals that increased severing of actin filaments reduces the spatial confinement of the BCR within the plasma membrane and increases BCR mobility. This allows more frequent collisions between BCRs and greater signalling in response to low densities of membrane-bound antigen. These findings implicate actin dynamics as a means of tuning receptor signalling and as a mechanism by which B cells distinguish inert antigens from those that are accompanied by indicators of microbial infection.

Nef is an accessory protein and pathogenicity factor of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) which elevates virus replication in vivo. We recently described for HIV type 1 SF2 (HIV-1 SF2 ) the potent interference of Nef with T-lymphocyte chemotaxis via its association with the cellular kinase PAK2. Mechanistic analysis revealed that this interaction results in deregulation of the actin-severing factor cofilin and thus blocks the chemokine-mediated actin remodeling required for cell motility. However, the efficiency of PAK2 association is highly variable among Nef proteins from different lentiviruses, prompting us to evaluate the conservation of this actin-remodeling/cofilin-deregulating mechanism. Based on the analysis of a total of 17 HIV-1, HIV-2, and SIV Nef proteins, we report here that inhibition of chemokine-induced actin remodeling as well as inactivation of cofilin are strongly conserved activities of lentiviral Nef proteins. Of note, even for Nef variants that display only marginal PAK2 association in vitro, these activities require the integrity of a PAK2 recruitment motif and the presence of endogenous PAK2. Thus, reduced in vitro affinity to PAK2 does not indicate limited functionality of Nef-PAK2 complexes in intact HIV-1 host cells. These results establish hijacking of PAK2 for deregulation of cofilin and inhibition of triggered actin remodeling as a highly conserved function of lentiviral Nef proteins, supporting the notion that PAK2 association may be critical for Nef's activity in vivo.

Phase separation drives numerous cellular processes, ranging from the formation of membrane-less organelles to the cooperative assembly of signaling proteins. Features such as multivalency and intrinsic disorder that enable condensate formation are found not only in cytosolic and nuclear proteins, but also in membrane-associated proteins. The ABC transporter Rv1747, which is important for Mycobacterium tuberculosis (Mtb) growth in infected hosts, has a cytoplasmic regulatory module consisting of 2 phosphothreonine-binding Forkhead-associated domains joined by an intrinsically disordered linker with multiple phospho-acceptor threonines. Here we demonstrate that the regulatory modules of Rv1747 and its homolog in Mycobacterium smegmatis form liquid-like condensates as a function of concentration and phosphorylation. The serine/threonine kinases and sole phosphatase of Mtb tune phosphorylation-enhanced phase separation and differentially colocalize with the resulting condensates. The Rv1747 regulatory module also phase-separates on supported lipid bilayers and forms dynamic foci when expressed heterologously in live yeast and M. smegmatis cells. Consistent with these observations, single-molecule localization microscopy reveals that the endogenous Mtb transporter forms higher-order clusters within the Mycobacterium membrane. Collectively, these data suggest a key role for phase separation in the function of these mycobacterial ABC transporters and their regulation via intracellular signaling.

Transmembrane protein 30A (TMEM30A) maintains the asymmetric distribution of phosphatidylserine, an integral component of the cell membrane and 'eat-me' signal recognized by macrophages. Integrative genomic and transcriptomic analysis of diffuse large B-cell lymphoma (DLBCL) from the British Columbia population-based registry uncovered recurrent biallelic TMEM30A loss-of-function mutations, which were associated with a favorable outcome and uniquely observed in DLBCL. Using TMEM30A-knockout systems, increased accumulation of chemotherapy drugs was observed in TMEM30A-knockout cell lines and TMEM30A-mutated primary cells, explaining the improved treatment outcome. Furthermore, we found increased tumor-associated macrophages and an enhanced effect of anti-CD47 blockade limiting tumor growth in TMEM30A-knockout models. By contrast, we show that TMEM30A loss-of-function increases B-cell signaling following antigen stimulation-a mechanism conferring selective advantage during B-cell lymphoma development. Our data highlight a multifaceted role for TMEM30A in B-cell lymphomagenesis, and char-acterize intrinsic and extrinsic vulnerabilities of cancer cells that can be therapeutically exploited. DLBCL is the most common lymphoma subtype worldwide, accounting for 40% of all non-Hodgkin lymphomas 1 . DLBCL presents as an aggressive disease requiring immediate Reprints and permissions information is available at www.nature.com/reprints.

Nef, an accessory protein of the Human Immunodeficiency Virus type 1 (HIV-1), is dispensable for viral replication in cell culture, but promotes virus replication and pathogenesis in the infected host. Acting as protein-interaction adaptor, HIV-1 Nef modulates numerous target cell activities including cell surface receptor expression, cytoskeletal remodeling, vesicular transport, and signal transduction. In infected T-lymphocytes, altering T-cell antigen receptor (TCR) signaling has long been recognized as one key function of the viral protein. However, reported effects of Nef range from inhibition to activation of this cascade. Recent advances in the field begin to explain these seemingly contradictory observations and suggest that Nef alters intracellular trafficking of TCR proximal machinery to disrupt plasma membrane bound TCR signaling while at the same time, the viral protein induces localized signal transduction at the trans-Golgi network. This review summarizes these new findings on how HIV-1 Nef reprograms TCR signalling output from a broad response to selective activation of the RAS-Erk pathway. We also discuss the implications of these alterations in the context of HIV-1 infection and in light of current concepts of TCR signal transduction.