Age-related defects in moesin/ezrin cytoskeletal signals in mouse CD4 T cells

Cytoskeletal proteins of the ezrin-radixin-moesin (ERM) family contribute to T cell activation in response to Ag, and also to T cell polarization in response to connective tissue matrix proteins and chemokine gradients. Previous work has shown that T cells from aged mice are defective in their ability to develop molecular linkages between surface macromolecules and the underlying cytoskeletal framework, both for proteins that move to the synapse and those that are excluded from the site of T cell-APC interaction. T cells from aged mice also show defective cytoskeletal rearrangements and lamellipodia formation when placed in contact with slides coated with Abs to the TCR/CD3 complex. In this study, we show that old CD4 T cells differ from young CD4 T cells in several aspects of ERM biochemistry, including ERM phosphorylation and ERM associations with CD44, CD43, and EBP50. In addition, CD4 T cells from aged mice show defects in the Rho GTPase activities known to control ERM function.     

Apoptosis paves the detour path for CD8 T cell activation against intracellular bacteria

Intracellular bacteria such as Mycobacterium tuberculosis primarily infect macrophages. Within these host cells, the pathogens are confined to phagosomes and their antigens are secluded from the classical MHC I presentation pathway. Moreover, macrophages fail to express certain antigen presenting molecules like CD1 proteins. As a result of this intracellular lifestyle, the pathways for the induction of MHC I- and CD1-restricted CD8 T cells by such microorganisms remain elusive. Based on recent findings in tuberculosis and salmonellosis, we propose a new detour pathway for CD8 T cell activation against intracellular bacteria through apoptotic blebs from infected macrophages. Pathogen-derived antigens including proteins and lipids are delivered from infected cells to non-infected dendritic cells. Subsequently, these professional antigen presenting cells display microbial antigens through MHC I and CD1 to T cells. Thus, cross-priming mediated by apoptotic vesicles is not just a matter of antigen distribution, but an intrinsic immunological function due to the nature of phagosomally located intracellular bacteria. We consider infection-induced apoptosis the conditio sine qua non for antigen-specific CD8 T cell activation by phagosome-enclosed pathogens. This important new function of cell death in antibacterial immunity requires consideration for rational vaccine design.     

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.     

Intracellular Theileria annulata Promote Invasive Cell Motility through Kinase Regulation of the Host Actin Cytoskeleton

The intracellular, protozoan Theileria species parasites are the only eukaryotes known to transform another eukaryotic cell. One consequence of this parasite-dependent transformation is the acquisition of motile and invasive properties of parasitized cells in vitro and their metastatic dissemination in the animal, which causes East Coast Fever (T. parva) or Tropical Theileriosis (T. annulata). These motile and invasive properties of infected host cells are enabled by parasite-dependent, poorly understood F-actin dynamics that control host cell membrane protrusions. Herein, we dissected functional and structural alterations that cause acquired motility and invasiveness of T. annulata-infected cells, to understand the molecular basis driving cell dissemination in Tropical Theileriosis. We found that chronic induction of TNFα by the parasite contributes to motility and invasiveness of parasitized host cells. We show that TNFα does so by specifically targeting expression and function of the host proto-oncogenic ser/thr kinase MAP4K4. Blocking either TNFα secretion or MAP4K4 expression dampens the formation of polar, F-actin-rich invasion structures and impairs cell motility in 3D. We identified the F-actin binding ERM family proteins as MAP4K4 downstream effectors in this process because TNFα-induced ERM activation and cell invasiveness are sensitive to MAP4K4 depletion. MAP4K4 expression in infected cells is induced by TNFα-JNK signalling and maintained by the inhibition of translational repression, whereby both effects are parasite dependent. Thus, parasite-induced TNFα promotes invasive motility of infected cells through the activation of MAP4K4, an evolutionary conserved kinase that controls cytoskeleton dynamics and cell motility. Hence, MAP4K4 couples inflammatory signaling to morphodynamic processes and cell motility, a process exploited by the intracellular Theileria parasite to increase its host cell''s dissemination capabilities.     

Comparison of actin and cell surface dynamics in motile fibroblasts

We have investigated the dynamic behavior of actin in fibroblast lamellipodia using photoactivation of fluorescence. Activated regions of caged resorufin (CR)-labeled actin in lamellipodia of IMR 90 and MC7 3T3 fibroblasts were observed to move centripetally over time. Thus in these cells, actin filaments move centripetally relative to the substrate. Rates were characteristic for each cell type; 0.66 +/- 0.27 microns/min in IMR 90 and 0.36 +/- 0.16 microns/min in MC7 3T3 cells. In neither case was there any correlation between the rate of actin movement and the rate of lamellipodial protrusion. The half-life of the activated CR-actin filaments was approximately 1 min in IMR 90 lamellipodia, and approximately 3 min in MC7 3T3 lamellipodia. Thus continuous filament turnover accompanies centripetal movement. In both cell types, the length of time required for a section of the actin meshwork to traverse the lamellipodium was several times longer than the filament half-life. The dynamic behavior of the dorsal surface of the cell was also observed by tracking lectin-coated beads on the surface and phase-dense features within lamellipodia of MC7 3T3 cells. The movement of these dorsal features occurred at rates approximately three times faster than the rate of movement of the underlying bulk actin cytoskeleton, even when measured in the same individual cells. Thus the transport of these dorsal features must occur by some mechanism other than simple attachment to the moving bulk actin cytoskeleton.     

ADP-ribosylation by exoenzyme T of Pseudomonas aeruginosa induces an irreversible effect on the host cell cytoskeleton in vivo

Pseudomonas aeruginosa utilises a type III secretion system (TTSS) to introduce exoenzyme S and exoenzyme T into host cells to subvert host cell signalling and thereby promote infection. In this study, we have employed the heterologous TTSS of Yersinia to deliver different mutants of ExoT into HeLa cells. Wild-type ExoT and ExoT variants expressing either GAP (GTPase activating protein) or ADP-ribosyltransferase activity mediated changes in cell morphology, which correlated to disruption of the actin microfilaments of the infected cells. ExoT expressing ADP-ribosylating activity gave an irreversible effect on HeLa cell morphology, while ExoT expressing only GAP activity displayed a reversible effect where the cells regained normal cell morphology after killing of the infecting bacteria. This shows that ExoT can modify and inactivate host cell proteins involved in maintaining the actin cytoskeleton in vivo by two independent mechanisms.     

Modulation of ubiquitin dynamics and suppression of DALIS formation by the Legionella pneumophila Dot/Icm system

Legionella pneumophila is an intracellular pathogen that uses effector proteins translocated by the Dot/Icm type IV secretion system to modulate host cellular processes. Here we investigate the dynamics of subcellular structures containing ubiquitin during L. pneumophila infection of phagocytic host cells. The Dot/Icm system mediated the formation of K48 and K63 poly-ubiquitin conjugates to proteins associated with L. pneumophila -containing vacuoles in macrophages and dendritic cells, suggesting that regulatory events and degradative events involving ubiquitin are regulated by bacterial effectors during infection. Stimulation of TLR2 on the surface of macrophages and dendritic cells by L. pneumophila- derived molecules resulted in the production of ubiquitin-rich dendritic cell aggresome-like structures (DALIS). Cells infected by L. pneumophila with a functional Dot/Icm system, however, failed to produce DALIS. Suppression of DALIS formation did not affect the accumulation of ubiquitinated proteins on vacuoles containing L. pneumophila. Examining other species of Legionella revealed that Legionella jordanis was unable to suppress DALIS formation after creating a ubiquitin-decorated vacuole. Thus, the L. pneumophila Dot/Icm system has the ability to modulate host processes to promote K48 and K63 ubiquitin conjugates on proteins at the vacuole membrane, and independently suppress cellular events required for the formation of DALIS.     

Growth of Theileria annulata and Theileria parva macroschizont-infected bovine cells in immunodeficient mice: effect of irradiation and tumour load on lymphocyte subsets.

Bovine cells infected with macroschizonts of the protozoan parasites Theileria annulata and Theileria parva formed solid tumours when injected into irradiated Balb/c and irradiated Balb/c nude mice. T. annulata tumours grew more vigorously than T. parva tumours, when initiated with similar doses of infected cells in mice exposed to the same doses of gamma-irradiation. In irradiated Balb/c mice, tumours of both species of parasites began to regress 2-3 weeks after injection of cells but grew without regression in irradiated Balb/c nude mice. Haemorrhage and necrosis of tumours, induced by macrophages and neutrophils, were seen in both mouse strains but were insufficient to cause regression in Balb/c nude mice. Theileria-infected bovine cells failed to establish in C57 beige mice, which lack functional natural killer (NK) cells. Flow cytometry, using monoclonal antibodies to murine leukocyte/lymphocyte antigens, showed that the radiation dose required to allow establishment of T. annulata tumours in Balb/c mice caused a severe depletion of splenic lymphocytes. B cells, helper T and cytotoxic T cells showed differing levels of susceptibility to irradiation. The presence of a tumour promoted the recovery of lymphocyte populations: this recovery was accompanied by destruction of the tumour.     

ADF/cofilin controls cell polarity during fibroblast migration

To migrate, normally a cell must establish morphological polarity and continuously protrude a single lamellipodium, polarized in the direction of migration. We have previously shown that actin filament disassembly is necessary for protrusion of the lamellipodium during fibroblast migration. As ADF/cofilin (AC) proteins are essential for the catalysis of filament disassembly in cells, we assessed their role in polarized lamellipodium protrusion in migrating fibroblasts. We compared the spatial distribution of AC and the inactive, phosphorylated AC (pAC) in migrating cells. AC, but not pAC, localized to the lamellipodium. To investigate a role for AC in cell polarity, we increased the proportion of pAC in migrating fibroblasts by overexpressing constitutively active (CA) LIM kinase 1. In 87% of cells expressing CA LIM kinase, cell polarity was abolished. In such cells, the single polarized lamellipodium was replaced by multiple nonpolarized lamellipodia, which, in contrast to nonexpressing migrating cells, stained for pAC. Cell polarity was rescued by coexpressing an active, nonphosphorylatable Xenopus AC (CA XAC) with the CA LIMK. Furthermore, overexpressing a pseudophosphorylated (less active) XAC by itself also abolished cell polarity. We conclude that locally maintaining ADF/cofilin in the active, nonphosphorylated state within the lamellipodium is necessary to maintain polarized protrusion during cell migration.     

Infection of bovine cells by the protozoan parasite Theileria annulata modulates expression of the ISGylation system

The apicomplexan parasite, Theileria annulata, dedifferentiates and induces continuous division of infected bovine myeloid cells. Re-expression of differentiation markers and a loss of proliferation occur upon treatment with buparvaquone, implying that parasite factors actively maintain the altered status of the infected cell. The factors that induce this unique transformation event have not been identified. However, parasite polypeptides (TashAT family) that are located in the infected leucocyte nucleus have been postulated to function as modulators of host cell phenotype. In this study differential RNA display and proteomic analysis were used to identify altered mRNA and polypeptide expression profiles in a bovine macrophage cell line (BoMac) transfected with TashAT2. One of the genes identified by differential display was found to encode an ubiquitin-like protease (bUBP43) belonging to the UBP43 family. The bUBP43 gene and the gene encoding its ubiquitin-like substrate, bISG15, were expressed at a low level in T. annulata-infected cells. However, infected cells were refractory to induction of elevated bISG15 expression by lipopolysaccharide or type 1 interferons while TashAT2-transfected cells showed no induction when treated with camptothecin. Modulation of the ISGylation system may be of relevance to the establishment of the transformed infected host cell, as ISGylation is associated with resistance to intracellular infection by pathogens, stimulation of the immune response and terminal differentiation of leukaemic cells.     

Cell migration without a lamellipodium: translation of actin dynamics into cell movement mediated by tropomyosin

The actin cytoskeleton is locally regulated for functional specializations for cell motility. Using quantitative fluorescent speckle microscopy (qFSM) of migrating epithelial cells, we previously defined two distinct F-actin networks based on their F-actin-binding proteins and distinct patterns of F-actin turnover and movement. The lamellipodium consists of a treadmilling F-actin array with rapid polymerization-dependent retrograde flow and contains high concentrations of Arp2/3 and ADF/cofilin, whereas the lamella exhibits spatially random punctae of F-actin assembly and disassembly with slow myosin-mediated retrograde flow and contains myosin II and tropomyosin (TM). In this paper, we microinjected skeletal muscle alphaTM into epithelial cells, and using qFSM, electron microscopy, and immunolocalization show that this inhibits functional lamellipodium formation. Cells with inhibited lamellipodia exhibit persistent leading edge protrusion and rapid cell migration. Inhibition of endogenous long TM isoforms alters protrusion persistence. Thus, cells can migrate with inhibited lamellipodia, and we suggest that TM is a major regulator of F-actin functional specialization in migrating cells.     

SCAR/WAVE-mediated processing of engulfed apoptotic corpses is essential for effective macrophage migration in Drosophila

In vitro studies have shown that SCAR/WAVE activates the Arp2/3 complex to generate actin filaments, which in many cell types are organised into lamellipodia that are thought to have an important role in cell migration. Here we demonstrate that SCAR is utilised by Drosophila macrophages to drive their developmental and inflammatory migrations and that it is regulated via the Hem/Kette/Nap1-containing SCAR/WAVE complex. SCAR is also important in protecting against bacterial pathogens and in wound repair as SCAR mutant embryos succumb more readily to both sterile and infected wounds. However, in addition to driving the formation of lamellipodia in macrophages, SCAR is required cell autonomously for the correct processing of phagocytosed apoptotic corpses by these professional phagocytes. Removal of this phagocytic burden by preventing apoptosis rescues macrophage lamellipodia formation and partially restores motility. Our results show that efficient processing of phagosomes is critical for effective macrophage migration in vivo. These findings have important implications for the resolution of macrophages from chronic wounds and the behaviour of those associated with tumours, because phagocytosis of debris may serve to prolong the presence of these cells at these sites of pathology.     

Lymphocyte egress signal sphingosine-1-phosphate promotes ERM-guided,bleb-based migration

Ezrin, radixin, and moesin (ERM) family proteins regulate cytoskeletal responses by tethering the plasma membrane to the underlying actin cortex. Mutations in ERM proteins lead to severe combined immunodeficiency, but the function of these proteins in T cells remains poorly defined. Using mice in which T cells lack all ERM proteins, we demonstrate a selective role for these proteins in facilitating S1P-dependent egress from lymphoid organs. ERM-deficient T cells display defective S1P-induced migration in vitro, despite normal responses to standard protein chemokines. Analysis of these defects revealed that S1P promotes a fundamentally different mode of migration than chemokines, characterized by intracellular pressurization and bleb-based motility. ERM proteins facilitate this process, controlling directional migration by limiting blebbing to the leading edge. We propose that the distinct modes of motility induced by S1P and chemokines are specialized to allow T cell migration across lymphatic barriers and through tissue stroma, respectively.     

Localized cyclic AMP-dependent protein kinase activity is required for myogenic cell fusion

Multinucleated myotubes are formed by fusion of mononucleated myogenic progenitor cells (myoblasts) during terminal skeletal muscle differentiation. In addition, myoblasts fuse with myotubes, but terminally differentiated myotubes have not been shown to fuse with each other. We show here that an adenylate cyclase activator, forskolin, and other reagents that elevate intracellular cyclic AMP (cAMP) levels induced cell fusion between small bipolar myotubes in vitro. Then an extra-large myotube, designated a "myosheet," was produced by both primary and established mouse myogenic cells. Myotube-to-myotube fusion always occurred between the leading edge of lamellipodia at the polar end of one myotube and the lateral plasma membrane of the other. Forskolin enhanced the formation of lamellipodia where cAMP-dependent protein kinase (PKA) was accumulated. Blocking enzymatic activity or anchoring of PKA suppressed forskolin-enhanced lamellipodium formation and prevented fusion of multinucleated myotubes. Localized PKA activity was also required for fusion of mononucleated myoblasts. The present results suggest that localized PKA plays a pivotal role in the early steps of myogenic cell fusion, such as cell-to-cell contact/recognition through lamellipodium formation. Furthermore, the localized cAMP-PKA pathway might be involved in the specification of the fusion-competent areas of the plasma membrane in lamellipodia of myogenic cells.     

Cultivation of Three Species of Theileria in Lymphoid Cells in vitro

SYNOPSIS. The intralymphocytic stages of Theileria parva, T. lawrencei and T. annulata have been cultivated for several months in tissue cultures of bovine lymphocytes associated with baby hamster kidney cells. In established cultures the theilerial particles multiplied at about the same rate as the host cells, the percentage of infected cells and the mean number of parasite particles per cell remaining nearly constant.
During mitotic division of the host cell the theilerial body becomes closely associated with the spindle fibres and is pulled apart and distributed to both daughter cells in late anaphase. The single theilerial particles (chromatin) within the theilerial body divide by binary fission; their division is not synchronous with that of the host cell.     

Actin-dependent lamellipodia formation and microtubule-dependent tail retraction control-directed cell migration

Migrating cells are polarized with a protrusive lamella at the cell front followed by the main cell body and a retractable tail at the rear of the cell. The lamella terminates in ruffling lamellipodia that face the direction of migration. Although the role of actin in the formation of lamellipodia is well established, it remains unclear to what degree microtubules contribute to this process. Herein, we have studied the contribution of microtubules to cell motility by time-lapse video microscopy on green flourescence protein-actin- and tubulin-green fluorescence protein-transfected melanoma cells. Treatment of cells with either the microtubule-disrupting agent nocodazole or with the stabilizing agent taxol showed decreased ruffling and lamellipodium formation. However, this was not due to an intrinsic inability to form ruffles and lamellipodia because both were restored by stimulation of cells with phorbol 12-myristate 13-acetate in a Rac-dependent manner, and by stem cell factor in melanoblasts expressing the receptor tyrosine kinase c-kit. Although ruffling and lamellipodia were formed without microtubules, the microtubular network was needed for advancement of the cell body and the subsequent retraction of the tail. In conclusion, we demonstrate that the formation of lamellipodia can occur via actin polymerization independently of microtubules, but that microtubules are required for cell migration, tail retraction, and modulation of cell adhesion.     

LIM kinase has a dual role in regulating lamellipodium extension by decelerating the rate of actin retrograde flow and the rate of actin polymerization

Lamellipodium extension is crucial for cell migration and spreading. The rate of lamellipodium extension is determined by the balance between the rate of actin polymerization and the rate of actin retrograde flow. LIM kinase 1 (LIMK1) regulates actin dynamics by phosphorylating and inactivating cofilin, an actin-depolymerizing protein. We examined the role of LIMK1 in lamellipodium extension by measuring the rates of actin polymerization, actin retrograde flow, and lamellipodium extension using time-lapse imaging of fluorescence recovery after photobleaching. In the non-extending lamellipodia of active Rac-expressing N1E-115 cells, LIMK1 expression decelerated and LIMK1 knockdown accelerated actin retrograde flow. In the extending lamellipodia of neuregulin-stimulated MCF-7 cells, LIMK1 knockdown accelerated both the rate of actin polymerization and the rate of actin retrograde flow, but the accelerating effect on retrograde flow was greater than the effect on polymerization, thus resulting in a decreased rate of lamellipodium extension. These results indicate that LIMK1 has a dual role in regulating lamellipodium extension by decelerating actin retrograde flow and polymerization, and in MCF-7 cells endogenous LIMK1 contributes to lamellipodium extension by decelerating actin retrograde flow more effectively than decelerating actin polymerization.     

Cellular distributions of the ERM proteins in MDCK epithelial cells: regulation by growth and cytoskeletal integrity

The highly homologous ERM (ezrin/radixin/moesin) proteins, molecular cross-linkers which connect the cell membrane with the underlying cytoskeleton, have molecular weights of 81, 80 and 78 kDa respectively. We present data which shows significant variation in the molecular weight and presence of multiple forms of ERM proteins in different cell lines, such that specific antibodies to each protein are essential for unambiguous detection. Biochemical fractionation of MDCK cells demonstrates that although the individual ERM fractionation patterns are unaltered by cell density, the multiple forms of moesin each associate with different subcellular fractions. Since ERM proteins can exist in dormant or active conformations corresponding to their phosphorylation state, we propose that the partitioning of ERM proteins between subcellular compartments may depend on their activation status. In addition, we show that when the co-localization between ezrin and F-actin is disrupted by cytochalasin D, MDCK cells undergo a dramatic morphology change during which long, branching, ezrin-rich protrusions are formed. Consistent with other workers, our data suggest that maintenance of ezrin:F-actin interactions are required for the maintenance of normal cellular morphology.