Down Regulation of Genes Involved in T Cell Polarity and Motility during the Induction of Heart Allograft Tolerance by Allochimeric MHC I

Background

The allochimeric MHC class I molecule [α1h1/u]-RT1.Aa that contains donor-type (Wistar Furth, WF; RT1u) epitopes displayed on recipient-type (ACI, RT1a) administered in conjunction with sub-therapeutic dose of cyclosporine (CsA) induces indefinite survival of heterotopic cardiac allografts in rat model. In vascularized transplantation models, the spleen contributes to graft rejection by generating alloantigen reactive T cells. The immune response in allograft rejection involves a cascade of molecular events leading to the formation of immunological synapses between T cells and the antigen-presenting cells.

Methodology/Principal Findings

To elucidate the molecular pathways involved in the immunosuppressive function of allochimeric molecule we performed microarray and quantitative RTPCR analyses of gene expression profile of splenic T cells from untreated, CsA treated, and allochimeric molecule + subtherapeutic dose of CsA treated animals at day 1, 3 and 7 of post transplantation. Allochimeric molecule treatment caused down regulation of genes involved in actin filament polymerization (RhoA and Rac1), cell adhesion (Catna1, Vcam and CD9), vacuolar transport (RhoB, Cln8 and ATP6v1b2), and MAPK pathway (Spred1 and Dusp6) involved in tubulin cytoskeleton reorganization and interaction between actin and microtubule cytoskeleton. All these genes are involved in T cell polarity and motility, i.e., their ability to move, scan and to form functional immunological synapse with antigen presenting cells (APCs).

Conclusions

These results indicate that the immunosuppressive function of allochimeric molecule may depend on the impairment of T cells'' movement and scanning ability, and possibly also the formation of immunological synapse. We believe that these novel findings may have important clinical implications for organ transplantation.     

T Cell Specific Adapter Protein (TSAd) Interacts with Tec Kinase ITK to Promote CXCL12 Induced Migration of Human and Murine T Cells

Background

The chemokine CXCL12/SDF-1α interacts with its G-protein coupled receptor CXCR4 to induce migration of lymphoid and endothelial cells. T cell specific adapter protein (TSAd) has been found to promote migration of Jurkat T cells through interaction with the G protein β subunit. However, the molecular mechanisms for how TSAd influences cellular migration have not been characterized in detail.

Principal Findings

We show that TSAd is required for tyrosine phosphorylation of the Lck substrate IL2-inducible T cell kinase (Itk). Presence of Itk Y511 was necessary to boost TSAd''s effect on CXCL12 induced migration of Jurkat T cells. In addition, TSAd''s ability to promote CXCL12-induced actin polymerization and migration of Jurkat T lymphocytes was dependent on the Itk-interaction site in the proline-rich region of TSAd. Furthermore, TSAd-deficient murine thymocytes failed to respond to CXCL12 with increased Itk phosphorylation, and displayed reduced actin polymerization and cell migration responses.

Conclusion

We propose that TSAd, through its interaction with both Itk and Lck, primes Itk for Lck mediated phosphorylation and thereby regulates CXCL12 induced T cell migration and actin cytoskeleton rearrangements.     

Alteration of the cortical actin cytoskeleton deregulates Ca2+ signaling, monospermic fertilization, and sperm entry

Background

When preparing for fertilization, oocytes undergo meiotic maturation during which structural changes occur in the endoplasmic reticulum (ER) that lead to a more efficient calcium response. During meiotic maturation and subsequent fertilization, the actin cytoskeleton also undergoes dramatic restructuring. We have recently observed that rearrangements of the actin cytoskeleton induced by actin-depolymerizing agents, or by actin-binding proteins, strongly modulate intracellular calcium (Ca²⁺) signals during the maturation process. However, the significance of the dynamic changes in F-actin within the fertilized egg has been largely unclear.

Methodology/Principal Findings

We have measured changes in intracellular Ca²⁺ signals and F-actin structures during fertilization. We also report the unexpected observation that the conventional antagonist of the InsP₃ receptor, heparin, hyperpolymerizes the cortical actin cytoskeleton in postmeiotic eggs. Using heparin and other pharmacological agents that either hypo- or hyperpolymerize the cortical actin, we demonstrate that nearly all aspects of the fertilization process are profoundly affected by the dynamic restructuring of the egg cortical actin cytoskeleton.

Conclusions/Significance

Our findings identify important roles for subplasmalemmal actin fibers in the process of sperm-egg interaction and in the subsequent events related to fertilization: the generation of Ca²⁺ signals, sperm penetration, cortical granule exocytosis, and the block to polyspermy.     

Inhibition of actin polymerization decreases osteogeneic differentiation of mesenchymal stem cells through p38 MAPK pathway

Background

Mesenchymal Stem Cells (MSC) are important candidates for therapeutic applications due to their ex vivo proliferation and differentiation capacity. MSC differentiation is controlled by both intrinsic and extrinsic factors and actin cytoskeleton plays a major role in the event. In the current study, we tried to understand the initial molecular mechanisms and pathways that regulate the differentiation of MSC into osteocytes or adipocytes.

Results

We observed that actin modification was important during differentiation and differentially regulated during adipogenesis and osteogenesis. Initial disruption of actin polymerization reduced further differentiation of MSC into osteocytes and osteogenic differentiation was accompanied by increase in ERK1/2 and p38 MAPK phosphorylation. However, only p38 MAPK phosphorylation was down regulated upon inhibition of actin polymerization which as accompanied by decreased CD49E expression.

Conclusion

Taken together, our results show that actin modification is a pre-requisite for MSC differentiation into osteocytes and adipocytes and osteogenic differentiation is regulated through p38 MAPK phosphorylation. Thus by modifying their cytoskeleton the differentiation potential of MSC could be controlled which might have important implications for tissue repair and regeneration.     

Inside-Out Regulation of ICAM-1 Dynamics in TNF-α-Activated Endothelium

Background

During transendothelial migration, leukocytes use adhesion molecules, such as ICAM-1, to adhere to the endothelium. ICAM-1 is a dynamic molecule that is localized in the apical membrane of the endothelium and clusters upon binding to leukocytes. However, not much is known about the regulation of ICAM-1 clustering and whether membrane dynamics are linked to the ability of ICAM-1 to cluster and bind leukocyte integrins. Therefore, we studied the dynamics of endothelial ICAM-1 under non-clustered and clustered conditions.

Principal Findings

Detailed scanning electron and fluorescent microscopy showed that the apical surface of endothelial cells constitutively forms small filopodia-like protrusions that are positive for ICAM-1 and freely move within the lateral plane of the membrane. Clustering of ICAM-1, using anti-ICAM-1 antibody-coated beads, efficiently and rapidly recruits ICAM-1. Using fluorescence recovery after photo-bleaching (FRAP), we found that clustering increased the immobile fraction of ICAM-1, compared to non-clustered ICAM-1. This shift required the intracellular portion of ICAM-1. Moreover, biochemical assays showed that ICAM-1 clustering recruited beta-actin and filamin. Cytochalasin B, which interferes with actin polymerization, delayed the clustering of ICAM-1. In addition, we could show that cytochalasin B decreased the immobile fraction of clustered ICAM-1-GFP, but had no effect on non-clustered ICAM-1. Also, the motor protein myosin-II is recruited to ICAM-1 adhesion sites and its inhibition increased the immobile fraction of both non-clustered and clustered ICAM-1. Finally, blocking Rac1 activation, the formation of lipid rafts, myosin-II activity or actin polymerization, but not Src, reduced the adhesive function of ICAM-1, tested under physiological flow conditions.

Conclusions

Together, these findings indicate that ICAM-1 clustering is regulated in an inside-out fashion through the actin cytoskeleton. Overall, these data indicate that signaling events within the endothelium are required for efficient ICAM-1-mediated leukocyte adhesion.     

Actin Turnover Is Required for Myosin-Dependent Mitochondrial Movements in Arabidopsis Root Hairs

Background

Previous studies have shown that plant mitochondrial movements are myosin-based along actin filaments, which undergo continuous turnover by the exchange of actin subunits from existing filaments. Although earlier studies revealed that actin filament dynamics are essential for many functions of the actin cytoskeleton, there are little data connecting actin dynamics and mitochondrial movements.

Methodology/Principal Findings

We addressed the role of actin filament dynamics in the control of mitochondrial movements by treating cells with various pharmaceuticals that affect actin filament assembly and disassembly. Confocal microscopy of Arabidopsis thaliana root hairs expressing GFP-FABD2 as an actin filament reporter showed that mitochondrial distribution was in agreement with the arrangement of actin filaments in root hairs at different developmental stages. Analyses of mitochondrial trajectories and instantaneous velocities immediately following pharmacological perturbation of the cytoskeleton using variable-angle evanescent wave microscopy and/or spinning disk confocal microscopy revealed that mitochondrial velocities were regulated by myosin activity and actin filament dynamics. Furthermore, simultaneous visualization of mitochondria and actin filaments suggested that mitochondrial positioning might involve depolymerization of actin filaments on the surface of mitochondria.

Conclusions/Significance

Base on these results we propose a mechanism for the regulation of mitochondrial speed of movements, positioning, and direction of movements that combines the coordinated activity of myosin and the rate of actin turnover, together with microtubule dynamics, which directs the positioning of actin polymerization events.     

Characterization of engineered actin binding proteins that control filament assembly and structure

Background

Eukaryotic cells strictly regulate the structure and assembly of their actin filament networks in response to various stimuli. The actin binding proteins that control filament assembly are therefore attractive targets for those who wish to reorganize actin filaments and reengineer the cytoskeleton. Unfortunately, the naturally occurring actin binding proteins include only a limited set of pointed-end cappers, or proteins that will block polymerization from the slow-growing end of actin filaments. Of the few that are known, most are part of large multimeric complexes that are challenging to manipulate.

Methodology/Principal Findings

We describe here the use of phage display mutagenesis to generate of a new class of binding protein that can be targeted to the pointed-end of actin. These proteins, called synthetic antigen binders (sABs), are based on an antibody-like scaffold where sequence diversity is introduced into the binding loops using a novel “reduced genetic code” phage display library. We describe effective strategies to select and screen for sABs that ensure the generated sABs bind to the pointed-end surface of actin exclusively.

Conclusions/Significance

From our set of pointed-end binders, we identify three sABs with particularly useful properties to systematically probe actin dynamics: one protein that caps the pointed end, a second that crosslinks actin filaments, and a third that severs actin filaments and promotes disassembly.     

Actin cytoskeleton regulates calcium dynamics and NFAT nuclear duration

T-cell activation by antigen-presenting cells is accompanied by actin polymerization, T-cell receptor (TCR) capping, and formation of the immunological synapse. However, whether actin-dependent events are required for T-cell function is poorly understood. Herein, we provide evidence for an unexpected negative regulatory role of the actin cytoskeleton on TCR-induced cytokine production. Disruption of actin polymerization resulted in prolonged intracellular calcium elevation in response to anti-CD3, thapsigargin, or phorbol myristate acetate plus ionomycin, leading to persistent NFAT (nuclear factor of activated T cells) nuclear duration. These events were dominant, as the net effect of actin blockade was augmented interleukin 2 promoter activity. Increased surface expression of the plasma membrane Ca(2+) ATPase was observed upon stimulation, which was inhibited by cytochalasin D, suggesting that actin polymerization contributes to calcium export. Our results imply a novel role for the actin cytoskeleton in modulating the duration of Ca(2+)-NFAT signaling and indicate that actin dynamics regulate features of T-cell activation downstream of receptor clustering.     

Microtubule dynamics and signal transduction at the immunological synapse: new partners and new connections

EMBO J (2012) 31 21, 4140–4152 doi:10.1038/emboj.2012.242; published online August242012Antigen recognition induces T cells to polarize towards antigen presenting cells (APC) generating an organized cell interface named the immunological synapse. T-cell microtubules (MTs) reorient the MT-organizing centre (MTOC) to the immunological synapse central region, while MT irradiate towards the synapse periphery. Martín-Cófreces et al (2012) describe in this issue that the MT plus-end-binding protein 1 (EB1) interacts with TCR cytosolic regions and mediate the organization of an immunological synapse fully functional to transduce activation signals.The pioneer work of Kupfer and Singer (1989) established that T-cell MTs rearrange in response to specific TCR engagement by APCs, resulting in MTOC orientation to the APC contact site in helper and cytotoxic T cells. MTOC reorientation was shown to be the result of a MT polymerization dynamic process involving MT posttranslational modifications (Kuhn and Poenie, 2002; Serrador et al, 2004). MT reorganization during T-cell antigen recognition is functionally linked to T-cell effector functions, like the polarized secretion of helper cytokines to B cells (Kupfer et al, 1991; Huse et al, 2006), or cytotoxic granules to target cells (Stinchcombe et al, 2006). MTs also transport TCR-carrying endosomes during synapse formation (Das et al, 2004) and TCR signalling complexes at the immunological synapse (Lasserre et al, 2010; Hashimoto-Tane et al, 2011). Altogether, these findings show that the dynamic reorganization of MTs and its related molecular transport are critical for the organization and function of the immunological synapse.Martín-Cófreces et al (2012) present here interesting new insights, unveiling a link between EB1 and the TCR complex. EB1 is one of a series of MT plus-end-associated proteins critical for MT polymerization dynamics (Slep, 2010). The first important finding initially issued from a two-hybrid screening was that EB1 could directly interact with TCR complex cytosolic regions. By GST pull-down and co-immunoprecipitation experiments, the authors narrowed down this interaction to two of the TCR complex subunits, ζ and ɛ, in their ITAM (immuno-receptor tyrosine-based activation motif)-containing regions, and within the C-terminal 82 amino-acid region on EB1. In T cells, EB1–TCR interaction could occur without TCR stimulation, suggesting that EB1 plays a role in TCR dynamics previous to TCR engagement. The authors then investigated EB1 localization and its involvement in synapse organization and function. Live cell imaging showed intense EB1 movement in the synapse area, with MTs growing from the MTOC to the synapse periphery, leading to an apparent concentration of EB1 at the T cell–APC interface. To analyse the relationship between MT dynamics and intracellular transport, the authors followed EB1–GFP and TCRζ–Cherry by total internal reflection fluorescence (TIRF) microscopy in synapses formed on anti-CD3-coated cover slips. They observed transient coincident spots between EB1 and TCRζ+ vesicles, suggesting that growing MTs transport TCRζ-carrying vesicles towards the immunological synapse. Consistently, EB1-silenced cells displayed altered TCRζ vesicle dynamics and TCRζ clustering at the synapse. Likewise, vesicle transport to the synapse of the signalling scaffold molecule LAT and its clustering at the synapse were altered. Finally, they observed transient encounters between TCRζ- and LAT-carrying vesicles inhibited by EB1 silencing. These observations point out to a crucial role of EB1 and MT dynamics in the organization of the immunological synapse.Immunological synapse organization has been related with its capacity to regulate TCR signal transduction. Therefore, Martín-Cófreces et al (2012) investigated how EB1 silencing impacted TCR signalling. EB1-silenced cells were indeed impaired in key TCR signalling events, like LAT tyrosine phosphorylation, which allows LAT interaction with activation effectors, like the phospholipase C (PLC)γ, promoting TCR signal propagation. Consistently, PLCγ activation was impaired in EB1-silenced cells. However, upstream activation events, like tyrosine phosphorylation of TCRζ and of its associated protein tyrosine kinase ZAP70, were not altered. This suggests that MT-dependent LAT vesicle traffic is key for LAT phosphorylation and the generation of TCR signalling complexes.Altogether, Martín-Cófreces'' findings reinforce the idea that polarized vesicle transport via organized MT networks is key to set up the immunological synapse as a signal transduction platform. EB1 interaction with two TCR subunits may link the TCR complex with MTs dynamics. It remains unanswered, however, whether EB1 also interacts with LAT, facilitating the merging at the synapse of distinct TCRζ- and LAT-carrying vesicles.Vesicle traffic on MTs generally occurs via molecular motors from the dynein and kinesin families. The former are associated with minus end-oriented transport, whereas the later mostly ensures plus-end-associated transport. The immunological synapse may use both types of transport. Thus, cytotoxic granule delivery to the synapse may mainly involve dynein-mediated vesicle traffic, since the MTOC translocates very close to the immunological synapse (Stinchcombe et al, 2006). Likewise, centripetal movements of signalling microclusters at the synapse involve dynein (Hashimoto-Tane et al, 2011). Martín-Cófreces et al (2012) show that TCRζ- and LAT-carrying vesicles are transported towards MT plus ends in an EB1-dependent manner. It remains uncertain whether EB1 could play a direct transport role at the immunological synapse, helping the attachment of TCRζ vesicles to growing MT plus ends. Alternatively, EB1 could mediate MT interactions with TCR complexes present at the plasma membrane. Initial TCR clustering at the synapse would help capturing EB1-positive MT plus ends, orienting MTs and MT-mediated traffic of TCRζ- and LAT-carrying vesicles to the synapse by a kinesin-based transport (Figure 1), and promoting TCRζ and LAT encountering and clustering at the synapse. EB1 silencing would perturb MT–plasma membrane interactions impairing this MT orientation and transport loop. MT polymerization kinetic studies on immunological synapses formed by EB1-silenced versus control T cells may help to clarify this mechanism. Although further studies will be necessary to elucidate the detailed mechanism, the work by Martín-Cófreces et al (2012) already highlights the importance of MT dynamics and vesicle traffic in the formation of a functional immunological synapse, raising novel and interesting questions on how the MT network helps to set up complex signal transduction machineries.Open in a separate windowFigure 1Model of the role of EB1 in MT dynamics and TCR signal transduction at the immunological synapse. (A) Initial T cell–APC contact. TCR initial clustering would favour the capture of EB1-containing MT plus ends at the T cell–APC contact. (B) Immune synapse formation. The increase capture of MTs plus ends by TCR clusters would promote the arrival of TCRζ- and LAT-carrying vesicles leading to increase TCR and LAT clustering and encountering at the synapse. Alternatively, EB1 interaction with TCR could also be directly involved in TCRζ vesicle transport to the synapse. In turn, increase TCR clustering would promote additional MT and capture, building an amplification loop for MT dynamics and vesicle transport. (C) Established immunological synapse. A structured MT network would facilitate the continuous arrival of TCRζ- and LAT-carrying vesicles through the MT plus ends at the immunological synapse periphery. Then the centripetal movement of TCR signalling complexes towards the MT minus end at the MTOC close to the synapse centre would bring signalling complexes to signal extinction sites (i.e., endosomes). The right panel in C represents a xy section of the immunological synapse, as it is observed on stimulatory cover slips.     

The Actin Targeting Compound Chondramide Inhibits Breast Cancer Metastasis via Reduction of Cellular Contractility

Background

A major player in the process of metastasis is the actin cytoskeleton as it forms key structures in both invasion mechanisms, mesenchymal and amoeboid migration. We tested the actin binding compound Chondramide as potential anti-metastatic agent.

Methods

In vivo, the effect of Chondramide on metastasis was tested employing a 4T1-Luc BALB/c mouse model. In vitro, Chondramide was tested using the highly invasive cancer cell line MDA-MB-231 in Boyden-chamber assays, fluorescent stainings, Western blot and Pull down assays. Finally, the contractility of MDA-MB-231 cells was monitored in 3D environment and analyzed via PIV analysis.

Results

In vivo, Chondramide treatment inhibits metastasis to the lung and the migration and invasion of MDA-MB-231 cells is reduced by Chondramide in vitro. On the signaling level, RhoA activity is decreased by Chondramide accompanied by reduced MLC-2 and the stretch induced guanine nucleotide exchange factor Vav2 activation. At same conditions, EGF-receptor autophosphorylation, Akt and Erk as well as Rac1 are not affected. Finally, Chondramide treatment disrupted the actin cytoskeleton and decreased the ability of cells for contraction.

Conclusions

Chondramide inhibits cellular contractility and thus represents a potential inhibitor of tumor cell invasion.     

The Function of Cortactin in the Clustering of Acetylcholine Receptors at the Vertebrate Neuromuscular Junction

Background

Postsynaptic enrichment of acetylcholine receptors (AChRs) at the vertebrate neuromuscular junction (NMJ) depends on the activation of the muscle receptor tyrosine MuSK by neural agrin. Agrin-stimulation of MuSK is known to initiate an intracellular signaling cascade that leads to the clustering of AChRs in an actin polymerization-dependent manner, but the molecular steps which link MuSK activation to AChR aggregation remain incompletely defined.

Methodology/Principal Findings

In this study we used biochemical, cell biological and molecular assays to investigate a possible role in AChR clustering of cortactin, a protein which is a tyrosine kinase substrate and a regulator of F-actin assembly and which has also been previously localized at AChR clustering sites. We report that cortactin was co-enriched at AChR clusters in situ with its target the Arp2/3 complex, which is a key stimulator of actin polymerization in cells. Cortactin was further preferentially tyrosine phosphorylated at AChR clustering sites and treatment of myotubes with agrin significantly enhanced the tyrosine phosphorylation of cortactin. Importantly, forced expression in myotubes of a tyrosine phosphorylation-defective cortactin mutant (but not wild-type cortactin) suppressed agrin-dependent AChR clustering, as did the reduction of endogenous cortactin levels using RNA interference, and introduction of the mutant cortactin into muscle cells potently inhibited synaptic AChR aggregation in response to innervation.

Conclusion

Our results suggest a novel function of phosphorylation-dependent cortactin signaling downstream from agrin/MuSK in facilitating AChR clustering at the developing NMJ.     

The non-catalytic carboxyl-terminal domain of ARFGAP1 regulates actin cytoskeleton reorganization by antagonizing the activation of Rac1

Background

The regulation of the actin cytoskeleton and membrane trafficking is coordinated in mammalian cells. One of the regulators of membrane traffic, the small GTP-binding protein ARF1, also activates phosphatidylinositol kinases that in turn affect actin polymerization. ARFGAP1 is a GTPase activating protein (GAP) for ARF1 that is found on Golgi membranes. We present evidence that ARFGAP1 not only serves as a GAP for ARF1, but also can affect the actin cytoskeleton.

Principal Findings

As cells attach to a culture dish foci of actin appear prior to the cells flattening and spreading. We have observed that overexpression of a truncated ARFGAP1 that lacks catalytic activity for ARF, called GAP273, caused these foci to persist for much longer periods than non-transfected cells. This phenomenon was dependent on the level of GAP273 expression. Furthermore, cell spreading after re-plating or cell migration into a previously scraped area was inhibited in cells transfected with GAP273. Live cell imaging of such cells revealed that actin-rich membrane blebs formed that seldom made protrusions of actin spikes or membrane ruffles, suggesting that GAP273 interfered with the regulation of actin dynamics during cell spreading. The over-expression of constitutively active alleles of ARF6 and Rac1 suppressed the effect of GAP273 on actin. In addition, the activation of Rac1 by serum, but not that of RhoA or ARF6, was inhibited in cells over-expressing GAP273, suggesting that Rac1 is a likely downstream effector of ARFGAP1. The carboxyl terminal 65 residues of ARFGAP1 were sufficient to produce the effects on actin and cell spreading in transfected cells and co-localized with cortical actin foci.

Conclusions

ARFGAP1 functions as an inhibitor upstream of Rac1 in regulating actin cytoskeleton. In addition to its GAP catalytic domain and Golgi binding domain, it also has an actin regulation domain in the carboxyl-terminal portion of the protein.     

Rac Activation by the T-Cell Receptor Inhibits T Cell Migration

Background

T cell migration is essential for immune responses and inflammation. Activation of the T-cell receptor (TCR) triggers a migration stop signal to facilitate interaction with antigen-presenting cells and cell retention at inflammatory sites, but the mechanisms responsible for this effect are not known.

Methodology/Principal Findings

Migrating T cells are polarized with a lamellipodium at the front and uropod at the rear. Here we show that transient TCR activation induces prolonged inhibition of T-cell migration. TCR pre-activation leads to cells with multiple lamellipodia and lacking a uropod even after removal of the TCR signal. A similar phenotype is induced by expression of constitutively active Rac1, and TCR signaling activates Rac1. TCR signaling acts via Rac to reduce phosphorylation of ezrin/radixin/moesin proteins, which are required for uropod formation, and to increase stathmin phosphorylation, which regulates microtubule stability. T cell polarity and migration is partially restored by inhibiting Rac or by expressing constitutively active moesin.

Conclusions/Significance

We propose that transient TCR signaling induces sustained inhibition of T cell migration via Rac1, increased stathmin phosphorylation and reduced ERM phosphorylation which act together to inhibit T-cell migratory polarity.     

Flotillins Are Involved in the Polarization of Primitive and Mature Hematopoietic Cells

Background

Migration of mature and immature leukocytes in response to chemokines is not only essential during inflammation and host defense, but also during development of the hematopoietic system. Many molecules implicated in migratory polarity show uniform cellular distribution under non-activated conditions, but acquire a polarized localization upon exposure to migratory cues.

Methodology/Principal Findings

Here, we present evidence that raft-associated endocytic proteins (flotillins) are pre-assembled in lymphoid, myeloid and primitive hematopoietic cells and accumulate in the uropod during migration. Furthermore, flotillins display a polarized distribution during immunological synapse formation. Employing the membrane lipid-order sensitive probe Laurdan, we show that flotillin accumulation in the immunological synapse is concomittant with membrane ordering in these regions.

Conclusions

Together with the observation that flotillin polarization does not occur in other polarized cell types such as polarized epithelial cells, our results suggest a specific role for flotillins in hematopoietic cell polarization. Based on our results, we propose that in hematopoietic cells, flotillins provide intrinsic cues that govern segregation of certain microdomain-associated molecules during immune cell polarization.     

The scaffolding protein Dlg1 is a negative regulator of cell-free virus infectivity but not of cell-to-cell HIV-1 transmission in T cells

Background

Cell-to-cell virus transmission of Human immunodeficiency virus type-1 (HIV-1) is predominantly mediated by cellular structures such as the virological synapse (VS). The VS formed between an HIV-1-infected T cell and a target T cell shares features with the immunological synapse (IS). We have previously identified the human homologue of the Drosophila Discs Large (Dlg1) protein as a new cellular partner for the HIV-1 Gag protein and a negative regulator of HIV-1 infectivity. Dlg1, a scaffolding protein plays a key role in clustering protein complexes in the plasma membrane at cellular contacts. It is implicated in IS formation and T cell signaling, but its role in HIV-1 cell-to-cell transmission was not studied before.

Methodology/Principal Findings

Kinetics of HIV-1 infection in Dlg1-depleted Jurkat T cells show that Dlg1 modulates the replication of HIV-1. Single-cycle infectivity tests show that this modulation does not take place during early steps of the HIV-1 life cycle. Immunofluorescence studies of Dlg1-depleted Jurkat T cells show that while Dlg1 depletion affects IS formation, it does not affect HIV-1-induced VS formation. Co-culture assays and quantitative cell-to-cell HIV-1 transfer analyses show that Dlg1 depletion does not modify transfer of HIV-1 material from infected to target T cells, or HIV-1 transmission leading to productive infection via cell contact. Dlg1 depletion results in increased virus yield and infectivity of the viral particles produced. Particles with increased infectivity present an increase in their cholesterol content and during the first hours of T cell infection these particles induce higher accumulation of total HIV-1 DNA.

Conclusion

Despite its role in the IS formation, Dlg1 does not affect the VS and cell-to-cell spread of HIV-1, but plays a role in HIV-1 cell-free virus transmission. We propose that the effect of Dlg1 on HIV-1 infectivity is at the stage of virus entry.     

A Recombinant Protein Based on Trypanosoma cruzi P21 Enhances Phagocytosis

Background

P21 is a secreted protein expressed in all developmental stages of Trypanosoma cruzi. The aim of this study was to determine the effect of the recombinant protein based on P21 (P21-His₆) on inflammatory macrophages during phagocytosis.

Findings

Our results showed that P21-His₆ acts as a phagocytosis inducer by binding to CXCR4 chemokine receptor and activating actin polymerization in a way dependent onthe PI3-kinase signaling pathway.

Conclusions

Thus, our results shed light on the notion that native P21 is a component related to T. cruzi evasion from the immune response and that CXCR4 may be involved in phagocytosis. P21-His₆ represents an important experimental control tool to study phagocytosis signaling pathways of different intracellular parasites and particles.     

An SK3 channel/nWASP/Abi-1 complex is involved in early neurogenesis

Background

The stabilization or regulated reorganization of the actin cytoskeleton is essential for cellular structure and function. Recently, we could show that the activation of the SK3-channel that represents the predominant SK-channel in neural stem cells, leads to a rapid local outgrowth of long filopodial processes. This observation indicates that the rearrangement of the actin based cytoskeleton via membrane bound SK3-channels might selectively be controlled in defined micro compartments of the cell.

Principal Findings

We found two important proteins for cytoskeletal rearrangement, the Abelson interacting protein 1, Abi-1 and the neural Wiskott Aldrich Syndrome Protein, nWASP, to be in complex with SK3- channels in neural stem cells (NSCs). Moreover, this interaction is also found in spines and postsynaptic compartments of developing primary hippocampal neurons and regulates neurite outgrowth during early phases of differentiation. Overexpression of the proteins or pharmacological activation of SK3 channels induces obvious structural changes in NSCs and hippocampal neurons. In both neuronal cell systems SK3 channels and nWASP act synergistic by strongly inducing filopodial outgrowth while Abi-1 behaves antagonistic to its interaction partners.

Conclusions

Our results give good evidence for a functional interplay of a trimeric complex that transforms incoming signals via SK3-channel activation into the local rearrangement of the cytoskeleton in early steps of neuronal differentiation involving nWASP and Abi-1 actin binding proteins.     

LFA-1 Regulates CD8+ T Cell Activation via T Cell Receptor-mediated and LFA-1-mediated Erk1/2 Signal Pathways

LFA-1 regulates T cell activation and signal transduction through the immunological synapse. T cell receptor (TCR) stimulation rapidly activates LFA-1, which provides unique LFA-1-dependent signals to promote T cell activation. However, the detailed molecular pathways that regulate these processes and the precise mechanism by which LFA-1 contributes to TCR activation remain unclear. We found LFA-1 directly participates in Erk1/2 signaling upon TCR stimulation in CD8⁺ T cells. The presence of LFA-1, not ligand binding, is required for the TCR-mediated Erk1/2 signal pathway. LFA-1-deficient T cells have defects in sustained Erk1/2 signaling and TCR/CD3 clustering, which subsequently prevents MTOC reorientation, cell cycle progression, and mitosis. LFA-1 regulates the TCR-mediated Erk1/2 signal pathway in the context of immunological synapse for recruitment and amplification of the Erk1/2 signal. In addition, LFA-1 ligation with ICAM-1 generates an additional Erk1/2 signal, which synergizes with the existing TCR-mediated Erk1/2 signal to enhance T cell activation. Thus, LFA-1 contributes to CD8⁺ T cell activation through two distinct signal pathways. We demonstrated that the function of LFA-1 is to enhance TCR signaling through the immunological synapse and deliver distinct signals in CD8⁺ T cell activation.Leukocyte function-associated antigen-1 (LFA-1)² plays an important role in regulating leukocyte adhesion and T cell activation (1, 2). LFA-1 consists of the αL (CD11a) and β2 (CD18) subunits. The ligands for LFA-1 include intercellular adhesion molecular-1 (ICAM-1), ICAM-2, and ICAM-3 (3). LFA-1 participates in the formation of the immunological synapse, which regulates T cell activation synergistically with TCR engagement. The immunological synapse is a specialized structure that forms between the T cell and the APC or target cell (1, 2, 4). The function of the immunological synapse is to facilitate T cell activation and signal transduction. Mice deficient in LFA-1 (CD11a KO) have defects in leukocyte adhesion, lymphocyte proliferation, and tumor rejection (5–7).Upon TCR stimulation, the nascent immunological synapse is initiated with surface receptor clustering and cytoskeleton rearrangement, then followed by mature synapse formation after prolonged stimulation (8, 9). In the mature immunological synapse, LFA-1 forms a ring-like pattern at the peripheral supramolecular activation cluster (pSMAC), which surrounds the central supramolecular activation cluster (cSMAC) containing TCR/CD3/lipid rafts (10, 11). The structure of the mature synapse is stable for hours and thought to be important for sustained TCR signaling (12–14). LFA-1 functions via pSMAC to stabilize the cSMAC and is associated with the induction of T cell proliferation, cytokine production, and lytic granule migration toward cSMAC (1, 15). Although LFA-1-containing pSMAC is self-evident in lipid bilayer systems and cell lines, whether it is required for T cell activation under physiological conditions remains controversial (15).TCR stimulation rapidly induces the functional activity of LFA-1, which then provides unique LFA-1-dependent signals to promote T cell activation (16). The process can be divided into two steps. First, the intracellular signaling from TCR regulating LFA-1 activation is known as “inside-out” signaling; second, activated LFA-1, as a signaling receptor, can feedback to transduce the intracellular signal, the “outside-in” signaling (1, 17). It is widely accepted that TCR stimulation activates LFA-1 through affinity and/or avidity regulation, as supported by increased adhesion to ICAM-1 and pSMAC formation (16, 17). The “inside-out” signal process has been investigated extensively (18–21). The TCR proximal signal molecules, Lck, ZAP-70, and PI3K, are known to be important for TCR signaling to LFA-1 activation (22–26). The molecular mechanisms of LFA-1 “outside-in” signaling have been explored only recently. Perez et al. (27) have demonstrated that LFA-1 and ICAM-1 ligation activates the downstream Erk1/2 MAPK signaling pathway upon TCR stimulation, which ultimately leads to the qualitative modulation of CD4⁺ T cell activation through distinct LFA-1-dependent signals. Another recent study provided compelling evidence that LFA-1 reshapes the Ras MAPK pathway downstream of TCR (28). However, the detailed molecular pathways that regulate these processes are poorly defined. Especially, the evidence in support of a distinctive role for LFA-1 in the T cell signaling pathway has lagged behind; whether the function of LFA-1 is to enhance TCR signaling through the immunological synapse and/or deliver distinct signal in T cell activation and whether LFA-1 is indispensable for or merely assists the existing TCR signal pathway. Furthermore, whether and how TCR proximal signal molecules regulate LFA-1 function remains unknown. Further studies are required to understand the LFA-1 and TCR signaling network.In this study, we found that LFA-1 directly participates in CD8⁺ T cell activation. Upon TCR stimulation, LFA-1 regulates both TCR-mediated and LFA-1-mediated Erk1/2 signal pathways. First, the presence of LFA-1, not ligand binding, is required for the sustained Erk1/2 signaling and TCR/CD3 clustering on the surface of CD8⁺ T cells, subsequently leading to MTOC reorientation, cell cycle progression, and mitosis. Second, LFA-1 ligation with ICAM-1 enhances Erk1/2 signaling, which promotes T cell activation with increased IL-2 production and cell proliferation. This LFA-1-mediated Erk1/2 signal pathway integrates with the existing TCR-mediated Erk1/2 signal pathway to enhance T cell activation.