Rsu1 contributes to cell adhesion and spreading in MCF10A cells via effects on P38 map kinase signaling

The ILK, PINCH, Parvin (IPP) complex regulates adhesion and migration via binding of ILK to β1 integrin and α−parvin thus linking focal adhesions to actin cytoskeleton. ILK also binds the adaptor protein PINCH which connects signaling proteins including Rsu1 to the complex. A recent study of Rsu1 and PINCH1 in non-transformed MCF10A human mammary epithelial cells revealed that the siRNA-mediated depletion of either Rsu1 or PINCH1 decreased the number of focal adhesions (FAs) and altered the distribution and localization of FA proteins. This correlated with reduced adhesion, failure to spread or migrate in response to EGF and a loss of actin stress fibers and caveolae. The depletion of Rsu1 caused significant reduction in PINCH1 implying that Rsu1 may function in part by regulating levels of PINCH1. However, Rsu1, but not PINCH1, was required for EGF-induced activation of p38 Map kinase and ATF2 phosphorylation, suggesting a Rsu1 function independent from the IPP complex. Reconstitution of Rsu1-depleted cells with a Rsu1 mutant (N92D) that does not bind to PINCH1 failed to restore FAs or migration but did promote IPP-independent spreading and constitutive as well as EGF-induced p38 activation. In this commentary we discuss p38 activity in adhesion and how Rsu1 expression may be linked to Map kinase kinase (MKK) activation and detachment-induced stress kinase signaling.     

Actin-binding protein G (AbpG) participates in modulating the actin cytoskeleton and cell migration in Dictyostelium discoideum

Cell migration is involved in various physiological and pathogenic events, and the complex underlying molecular mechanisms have not been fully elucidated. The simple eukaryote Dictyostelium discoideum displays chemotactic locomotion in stages of its life cycle. By characterizing a Dictyostelium mutant defective in chemotactic responses, we identified a novel actin-binding protein serving to modulate cell migration and named it actin-binding protein G (AbpG); this 971–amino acid (aa) protein contains an N-terminal type 2 calponin homology (CH2) domain followed by two large coiled-coil regions. In chemoattractant gradients, abpG⁻ cells display normal directional persistence but migrate significantly more slowly than wild-type cells; expressing Flag-AbpG in mutant cells eliminates the motility defect. AbpG is enriched in cortical/lamellipodial regions and colocalizes well with F-actin; aa 401–600 and aa 501–550 fragments of AbpG show the same distribution as full-length AbpG. The aa 501–550 region of AbpG, which is essential for AbpG to localize to lamellipodia and to rescue the phenotype of abpG⁻ cells, is sufficient for binding to F-actin and represents a novel actin-binding protein domain. Compared with wild-type cells, abpG⁻ cells have significantly higher F-actin levels. Collectively our results suggest that AbpG may participate in modulating actin dynamics to optimize cell locomotion.     

Traction stress in focal adhesions correlates biphasically with actin retrograde flow speed

How focal adhesions (FAs) convert retrograde filamentous actin (F-actin) flow into traction stress on the extracellular matrix to drive cell migration is unknown. Using combined traction force and fluorescent speckle microscopy, we observed a robust biphasic relationship between F-actin speed and traction force. F-actin speed is inversely related to traction stress near the cell edge where FAs are formed and F-actin motion is rapid. In contrast, larger FAs where the F-actin speed is low are marked by a direct relationship between F-actin speed and traction stress. We found that the biphasic switch is determined by a threshold F-actin speed of 8–10 nm/s, independent of changes in FA protein density, age, stress magnitude, assembly/disassembly status, or subcellular position induced by pleiotropic perturbations to Rho family guanosine triphosphatase signaling and myosin II activity. Thus, F-actin speed is a fundamental regulator of traction force at FAs during cell migration.     

ZF21 Protein,a Regulator of the Disassembly of Focal Adhesions and Cancer Metastasis,Contains a Novel Noncanonical Pleckstrin Homology Domain

Directional migration of adherent cells on an extracellular matrix requires repeated formation and disassembly of focal adhesions (FAs). Directional migration of adherent cellsWe have identified ZF21 as a regulator of disassembly of FAs and cell migration, and increased expression of the gene has been linked to metastatic colon cancer. ZF21 is a member of a protein family characterized by the presence of the FYVE domain, which is conserved among Fab1p, YOPB, Vps27p, and EEA1 proteins, and has been shown to mediate the binding of such proteins to phosphoinositides in the lipid layers of cell membranes. ZF21 binds multiple factors that promote disassembly of FAs such as FAK, β-tubulin, m-calpain, and SHP-2. ZF21 does not contain any other known protein motifs other than the FYVE domain, but a region of the protein C-terminal to the FYVE domain is sufficient to mediate binding to β-tubulin. In this study, we demonstrate that the C-terminal region is important for the ability of ZF21 to induce disassembly of FAs and cell migration, and to promote an early step of experimental metastasis to the lung in mice. In light of the importance of the C-terminal region, we analyzed its ternary structure using NMR spectroscopy. We demonstrate that this region exhibits a structure similar to that of a canonical pleckstrin homology domain, but that it lacks a positively charged interface to bind phosphatidylinositol phosphate. Thus, ZF21 contains a novel noncanonical PH-like domain that is a possible target to develop a therapeutic strategy to treat metastatic cancer.     

Interaction between Mitochondria and the Actin Cytoskeleton in Budding Yeast Requires Two Integral Mitochondrial Outer Membrane Proteins,Mmm1p and Mdm10p

Transfer of mitochondria to daughter cells during yeast cell division is essential for viable progeny. The actin cytoskeleton is required for this process, potentially as a track to direct mitochondrial movement into the bud. Sedimentation assays reveal two different components required for mitochondria–actin interactions: (1) mitochondrial actin binding protein(s) (mABP), a peripheral mitochondrial outer membrane protein(s) with ATP-sensitive actin binding activity, and (2) a salt-inextractable, presumably integral, membrane protein(s) required for docking of mABP on the organelle. mABP activity is abolished by treatment of mitochondria with high salt. Addition of either the salt-extracted mitochondrial peripheral membrane proteins (SE), or a protein fraction with ATP-sensitive actin-binding activity isolated from SE, to salt-washed mitochondria restores this activity. mABP docking activity is saturable, resistant to high salt, and inhibited by pre-treatment of salt-washed mitochondria with papain. Two integral mitochondrial outer membrane proteins, Mmm1p (Burgess, S.M., M. Delannoy, and R.E. Jensen. 1994. J.Cell Biol. 126:1375–1391) and Mdm10p, (Sogo, L.F., and M.P. Yaffe. 1994. J.Cell Biol. 126:1361– 1373) are required for these actin–mitochondria interactions. Mitochondria isolated from an mmm1-1 temperature-sensitive mutant or from an mdm10 deletion mutant show no mABP activity and no mABP docking activity. Consistent with this, mitochondrial motility in vivo in mmm1-1 and mdm10Δ mutants appears to be actin independent. Depolymerization of F-actin using latrunculin-A results in loss of long-distance, linear movement and a fivefold decrease in the velocity of mitochondrial movement. Mitochondrial motility in mmm1-1 and mdm10Δ mutants is indistinguishable from that in latrunculin-A–treated wild-type cells. We propose that Mmm1p and Mdm10p are required for docking of mABP on the surface of yeast mitochondria and coupling the organelle to the actin cytoskeleton.Mitochondria are indispensable organelles for normal eukaryotic cell function. Since mitochondria cannot be synthesized de novo, these organelles are inherited, i.e., transferred from mother to daughter during cell division. In the yeast Saccharomyces cerevisiae, vegetative cell division occurs by budding, a form of proliferation in which growth is directed toward the developing bud. Previous studies indicate that mitochondria undergo a series of cell cycle–linked motility events during normal inheritance in yeast (Simon et al., 1997). These are: (a) polarization of mitochondria towards the site of bud emergence in G₁ phase; (b) linear, polarized movement of mitochondria from mother cells to developing buds in S phase; (c) immobilization of newly inherited mitochondria in the bud tip during S and G₂ phases; and (d) release of immobilized mitochondria from the bud tip during M phase.There is mounting evidence that the actin cytoskeleton controls mitochondrial morphology and inheritance during vegetative yeast cell growth. The two major actin structures of yeast observed by light microscopy are patches and cables. Actin cables are bundles of actin filaments that extend from the mother into the bud. Mitochondria colocalize with these actin cables (Drubin et al., 1993; Lazzarino et al., 1994). Moreover, mutations such as deletion of the tropomyosin I gene, TPM1, or the mitochondrial distribution and morphology gene, MDM20, which selectively destabilize actin cables, result in the loss of polarized mitochondrial movement and reduce transfer of mitochondria into buds (Herman et al., 1997; Simon et al., 1997). Together, these studies indicate that normal mitochondrial inheritance in yeast requires association of mitochondria with actin cables.Cell-free studies reveal a possible mechanism underlying actin control of mitochondrial inheritance. Sedimentation assays document binding of mitochondria to the lateral surface of F-actin. This mitochondrial actin-binding activity is ATP-sensitive, saturable, reversible, and mediated by protein(s) on the mitochondrial surface (Lazzarino et al., 1994). In addition, ATP-driven, actin-dependent motor activity has been identified on the surface of mitochondria (Simon et al., 1995). These observations support a model of mitochondrial inheritance whereby mitochondria use an actin-dependent motor to drive their movement from mother to daughter cells along actin cable tracks.Yeast genetic screens have revealed several genes, collectively referred to as mdm (mitochondrial distribution and morphology) and mmm (maintenance of mitochondrial morphology), which are required for mitochondrial inheritance (McConnell et al., 1990; Burgess et al., 1994; Sogo and Yaffe, 1994). We have focused on two of these genes: MDM10 and MMM1. Deletion of MDM10 leads to the development of giant spherical mitochondria, presumably by the collapse of elongated mitochondria into a spherical mass (Sogo and Yaffe, 1994). Deletion of MMM1 (Burgess et al., 1994) produces a similar phenotype. In both mutants, the fraction of buds without mitochondria is high, indicating defective mitochondrial inheritance. The proteins encoded by these genes, Mdm10p and Mmm1p, appear to be integral membrane proteins in the mitochondrial outer membrane. Here, we report tests of the hypothesis that Mmm1p and Mdm10p are required to link mitochondria to the cytoskeleton.     

Bax Forms an Oligomer via Separate,Yet Interdependent,Surfaces

Interactions of Bcl-2 family proteins regulate permeability of the mitochondrial outer membrane and apoptosis. In particular, Bax forms an oligomer that permeabilizes the membrane. To map the interface of the Bax oligomer we used Triton X-100 as a membrane surrogate and performed site-specific photocross-linking. Bax-specific adducts were formed through photo-reactive probes at multiple sites that can be grouped into two surfaces. The first surface overlaps with the BH1–3 groove formed by Bcl-2 Homology motif 1, 2, and 3; the second surface is a rear pocket located on the opposite side of the protein from the BH1–3 groove. Further cross-linking experiments using Bax BH3 peptides and mutants demonstrated that the two surfaces interact with their counterparts in neighboring proteins to form two separated interfaces and that interaction at the BH1–3 groove primes the rear pocket for further interaction. Therefore, Bax oligomerization proceeds through a series of interactions that occur at separate, yet allosterically, coupled interfaces.     

Filopodial focal complexes direct adhesion and force generation towards filopodia outgrowth

Filopodia are key structures within many cells that serve as sensors constantly probing the local environment. Although filopodia are involved in a number of different cellular processes, their function in migration is often analyzed with special focus on early processes of filopodia formation and the elucidation of filopodia molecular architecture. An increasing number of publications now describe the entire life cycle of filopodia, with analyses from the initial establishment of stable filopodium-substrate adhesion to their final integration into the approaching lamellipodium. We and others can now show the structural and functional dependence of lamellipodial focal adhesions as well as of force generation and transmission on filopodial focal complexes and filopodial actin bundles. These results were made possible by new high resolution imaging techniques as well as by recently developed elastomeric substrates and theoretical models. The data additionally provide strong evidence that formation of new filopodia depends on previously existing filopodia through a repetitive filopodial elongation of the stably adhered filopodial tips. In this commentary we therefore hypothesize a highly coordinated mechanism that regulates filopodia formation, adhesion, protein composition and force generation in a filopodia dependent step by step process.Key words: filopodia, focal adhesion, cell force, filopodial focal complex, actinCell protrusion depends on collaborative interactions of lamellipodia and filopodia.¹ Although filopodia cannot drive cell migration alone, in contrast to lamellipodia, they are essential for many cell biological functions such as guidance of neuronal growth cones² or during angiogenesis.³ Furthermore, filopodia are vital to cell-cell contact establishment as described for epithelial cells⁴ or during dorsal closure in Drosophila,⁵ and are also implicated in cancer cell metastasis.^6,7 Lamellipodia as well as filopodia can be formed independently from each other,⁸ and recent results implicate independent basic mechanisms of cytoskeletal regulation for their formation. While lamellipodia protrusion is a well accepted Arp2/3-dependent process where actin branches constantly form the protrusive force at the leading edge of the lamella,⁹ the details of filopodia formation are far from being understood.^10–13 Although earlier experiments indicated Arp2/3 was also involved in filopodia formation,¹⁴ recent results point to a machinery that is far less dependent, or even possibly independent, of Arp2/3 with formins being the central regulating molecules instead.⁸As soon as filopodia start to form, they constantly sense their environment upon elongation. Transmembrane proteins such as cadherins or integrins^15,16 connect filopodia to surrounding cells, extracellular matrix, or even pathogens to form stable contacts. When filopodial adhesion fails, retraction takes place.¹⁷ Although integrins and talin have been shown to be initially present at these sites in an un-clustered but active state, many additional adhesion proteins take part in filopodial focal complexes (filopodial FXs).^16,18 Starting from a small VASP-containing adhesion spot at the tip of filopodia, proteins such as vinculin, paxillin, talin, tensin and even zyxin form an elongated filopodial FX behind the VASP spot along the filopodium. While integrin as well as VASP transport along the filopodia shaft via myosin-X has been described,¹⁹ it is still unclear whether additional adhesion proteins are also actively transported or whether diffusion takes place. Diffusion is typically a non-limiting process during cytoplasmic protein complex formation. However for filopodia, diffusion could have an important regulatory function as already hypothesized in theoretical models,²⁰ because they are small in width and densely filled with actin filaments. Therefore, local concentrations of soluble adhesion molecules might drop within filopodia upon FX formation resulting in a pure physical regulation of filopodial length as well as filopodial FX size.The almost complete focal adhesion site specific protein inventory of filopodia FXs^16,18 as indicated above provided further indications for a dependency of lamellipodial focal adhesions (FAs) on filopodial FXs. This hypothesis was confirmed using fluorescent live cell imaging to identify the transition of filopodial FXs into fully assembled FAs upon FX contact with the leading edge of the lamellipodium. While filopodial FXs were responsible for only a sub-fraction of FAs in fish fibroblasts,¹⁸ stable FAs of human keratinocytes were formed almost exclusively by enlargement of existing filopodial FXs¹⁶ (see scheme, Fig. 1).Open in a separate windowFigure 1Filopodia determine the fate of lamellipodial structures. Filopodia are formed by actin polymerization at their tip. Upon stable adhesion, a small but fully assembled filopodial focal complex (FX) is formed. This FX becomes enlarged in size upon lamellipodial contact to form focal adhesions. In parallel, the filopodial actin cross-linker fascin becomes exchanged by palladin and α-actinin as soon as the filopodial actin bundles are incorporated into the lamellipodium. In a next step, α-actinin becomes partially exchanged by myosin II, leading to enhanced force values applied at filopodial-originated FA sites bound to the substrate. The tight interaction between FAs and filopodial actin bundles reduces the actin retrograde flow within the filopodium in front of the FA (lower inlay) compared to filopodia lacking stable FAs in the lamellipodium (not shown). Adhesion sites formed in the lamellipodium lack connections to distinct actin bundles leading to low force application at these sites and short lifetimes (upper inlay).The structural dependency of lamellipodial complexes on filopodial protein aggregates could be also shown for actin bundles. Here, parallel oriented actin filaments become cross-linked by proteins such as fascin or IRSp53-Eps8-complex upon filopodia formation.^21,22 These tightly packed bundles of 15–30 single actin filaments originate from the lamellipodial actin meshwork.²³ Interestingly, filopodial actin bundles in turn also affect lamellipodial actin structures independent of whether the filopodium adheres in a stable manner or looses contact. Nemethova et al.¹⁸ described the contribution of non-adhering filopodia to the construction of concave bundles of actin filaments within the lamellipodium of fish fibroblasts. These bundles often extended in length and interconnected with adjacent bundles. Similar observations were found for fibroblasts of chicken embryos and neuronal growth cones.^24,25 Here, filopodial actin bundles were clearly shown to be the origin of nearly 85% of all actin bundles found in the lamella. These actin filaments typically pointed towards the direction of migration. Additionally, myosin II was associated with these filopodial derived actin filaments to form polarized actin bundles. Of equal importance are findings presented by Schäfer et al. in this issue. The authors analyzed the fate of stably adhered filopodia and identified a stepwise exchange of filopodial fascin against the actin cross-linker proteins palladin and especially α-actinin in areas where filopodia were just overgrown by the lamellipodial leading edge (schematically presented in Fig. 1). α-Actinin further induced incorporation of myosin II into filopodial actin bundles in the lamellipodium. The authors additionally found that FAs displayed an enhanced lifetime when adhered to these myosin containing actin filaments. Therefore, these findings could also explain the unusual stability of filopodial actin filaments in neuronal growth cones observed by Mallavarapu and Mitchison.¹⁷ For keratinocytes, filopodia-dependent actin bundles are the only myosin containing actin structures oriented in the direction of movement within the lamellipodium and the lamella. Sensitivity and resolution improvements in cell force analyses further proved that these actin bundles were responsible for almost the entire force transmitted from the lamellipodium of migrating keratinocytes to the substrate. These forces were transferred at FA sites emerging from filopodial FXs, proving the importance of filopodia in lamellipodial structures and functions. Although filopodia-independent adhesion sites are also formed in keratinocytes right behind the leading edge, these sites are neither connected to detectable actin filament bundles nor do they transmit significant forces (see scheme, Fig. 1). Consequently, their sizes and life spans are strongly reduced (Schäfer et al., this issue).Recent results in keratinocytes additionally close the circle from stably adhered filopodia to the generation of new ones. Our original observations indicated that new filopodia were mainly formed in a direct extension of focal adhesions. Since these adhesion sites also depended on previously adhered filopodial FXs, a closer look revealed a consecutive outgrowth of the same filopodia.¹⁶ These cycles were only interrupted when outgrowing filopodia did not adhere in a stable manner between outgrowth cycles. Present results suggest that the same tip complex is present in all subsequently formed filopodia with a VASP tip signal remaining in place during successive filopodial elongations. As a result, well aligned, consecutive elongated focal adhesions can be found in keratinocytes. We can only speculate whether such an Arp2/3-independent mechanism describes a basic principle in filopodia formation at this point, but similar results have been observed for fish fibroblasts with a repetitive and alternating transition between filopodia and microspikes as filopodia-like structures barely extending over the lamellipodial leading edge.¹⁸The strong interdependency between lamellipodial FAs and stably adhered filopodia is also highlighted by actin retrograde flow analyses in keratinocytes (Schäfer et al. this issue). Retrograde actin flow is generated by actin polymerization at the cell front and myosin activity pulling the filaments rearwards. The interaction of actin with FAs is known to dampen flow rates in front of lamellipodial FAs.²⁶ Furthermore, filamentous-actin dynamics measured in lung epithelial cells showed a fast retrograde actin flow at the leading edge compared to rates within the lamellae. The highest flow rates were in the range of 0.3–0.5 µm/min.²⁷ Interestingly, keratocytes exhibited ten times slower flow rates at the leading edge,²⁸ indicating that retrograde flow strongly depends on the cell type analyzed. Actin filaments polymerizing at the tips of filopodia also undergo retrograde flow, but these flow rates are much faster compared to those found in lamellipodia,²⁴ as shown by bleaching experiments in chick embryo fibroblasts with flow rates approximately two-fold faster in filaments derived from filopodia compared to flow rates measured within the lamellipodium. These flow rates of approximately 1.3 µm/min were similar to those found for filopodia in other studies.²² Furthermore, we could show that this retrograde flow rate strongly depends on stable FAs formed behind the filopodium (Schäfer et al. this issue and Fig. 1). In the absence of these FAs, actin retrograde flow is doubled once more to rates of approximately 2.5 µm/min in filopodia. Therefore, although rates of FAs containing filopodia are still much higher than those found in lamellipodia, these rates are still slowed down indicating an effective connection between FAs and filopodial actin. These results further imply that myosin II incorporation into filopodial-originated actin bundles is responsible for enhanced retrograde flow rates in filopodia compared to rates found in the lamellipodium and that myosin II incorporation does not depend on stably adhered FAs directly behind filopodia. These data also strongly support the hypothesis that new filopodia form in front of stable lamellipodial FAs. It will be an intriguing question for future studies to analyze whether the reduced retrograde flow speeds in front of lamellipodial FAs might even be a prerequisite for efficient assembly and stable adhesion of small filopodial FXs, or perhaps even for filopodia formation in general.Taking into account all the currently known functions of filopodia, the presented results finally indicate that filopodia might be characterized best not only by one but actually two main functions. The first function is environmental sensing. Various transmembrane proteins can be involved leading to various roles for filopodia such as formation of cell-cell or cell-matrix interactions.^5,15 Although these functions in environmental sensing seem to be highly diverse, force generation along filopodial-originated actin bundles as the second function for filopodia might be of universal importance independent of the cell type that forms them. Force transmission along cell-pathogen interacting filopodia have been observed,²⁹ and the formation of adherens junctions after filopodia mediated cell-cell interaction is also a cell force dependent process.⁵ Therefore, these observations fit well to the currently presented data by Schäfer et al. (this issue) proving the importance of filopodia-dependent cell matrix interactions in cell force generation in the direction of migration (see scheme, Fig. 1).Present in almost every moving cell type, filopodia are therefore much more than just sensors for environmental conditions. In fact, these needle-like structures are the starting point for essential structures of adhesion and movement. Independent of whether they adhere stably or not, filopodia define the position of cellular adhesion sites, actin bundles, cell force generation and application, and, finally, the new filopodia to be formed.     

Structural Basis for Type VI Secretion Effector Recognition by a Cognate Immunity Protein

The type VI secretion system (T6SS) has emerged as an important mediator of interbacterial interactions. A T6SS from Pseudomonas aeruginosa targets at least three effector proteins, type VI secretion exported 1–3 (Tse1–3), to recipient Gram-negative cells. The Tse2 protein is a cytoplasmic effector that acts as a potent inhibitor of target cell proliferation, thus providing a pronounced fitness advantage for P. aeruginosa donor cells. P. aeruginosa utilizes a dedicated immunity protein, type VI secretion immunity 2 (Tsi2), to protect against endogenous and intercellularly-transferred Tse2. Here we show that Tse2 delivered by the T6SS efficiently induces quiescence, not death, within recipient cells. We demonstrate that despite direct interaction of Tsi2 and Tse2 in the cytoplasm, Tsi2 is dispensable for targeting the toxin to the secretory apparatus. To gain insights into the molecular basis of Tse2 immunity, we solved the 1.00 Å X-ray crystal structure of Tsi2. The structure shows that Tsi2 assembles as a dimer that does not resemble previously characterized immunity or antitoxin proteins. A genetic screen for Tsi2 mutants deficient in Tse2 interaction revealed an acidic patch distal to the Tsi2 homodimer interface that mediates toxin interaction and immunity. Consistent with this finding, we observed that destabilization of the Tsi2 dimer does not impact Tse2 interaction. The molecular insights into Tsi2 structure and function garnered from this study shed light on the mechanisms of T6 effector secretion, and indicate that the Tse2–Tsi2 effector–immunity pair has features distinguishing it from previously characterized toxin–immunity and toxin–antitoxin systems.     

Gene Related to Anergy in Lymphocytes (GRAIL) Expression in CD4+ T Cells Impairs Actin Cytoskeletal Organization during T Cell/Antigen-presenting Cell Interactions

GRAIL (gene related to anergy in lymphocytes), is an E3 ubiquitin ligase with increased expression in anergic CD4+ T cells. The expression of GRAIL has been shown to be both necessary and sufficient for the induction of T cell (T) anergy. To date, several subsets of anergic T cells have demonstrated altered interactions with antigen-presenting cells (APC) and perturbed TCR-mediated signaling. The role of GRAIL in mediating these aspects of T cell anergy remains unclear. We used flow cytometry and confocal microscopy to examine T/APC interactions in GRAIL-expressing T cells. Increased GRAIL expression resulted in reduced T/APC conjugation efficiency as assessed by flow cytometry. Examination of single T/APC conjugates by confocal microscopy revealed altered polarization of polymerized actin and LFA-1 to the T/APC interface. When GRAIL expression was knocked down, actin polarization to the T/APC interface was restored, demonstrating that GRAIL is necessary for alteration of actin cytoskeletal rearrangement under anergizing conditions. Interestingly, proximal TCR signaling including calcium flux and phosphorylation of Vav were not disrupted by expression of GRAIL in CD4+ T cells. In contrast, interrogation of distal signaling events demonstrated significantly decreased JNK phosphorylation in GRAIL-expressing T cells. In sum, GRAIL expression in CD4+ T cells mediates alterations in the actin cytoskeleton during T/APC interactions. Moreover, in this model, our data dissociates proximal T cell signaling events from functional unresponsiveness. These data demonstrate a novel role for GRAIL in modulating T/APC interactions and provide further insight into the cell biology of anergic T cells.     

Shootin1–cortactin interaction mediates signal–force transduction for axon outgrowth

Motile cells transduce environmental chemical signals into mechanical forces to achieve properly controlled migration. This signal–force transduction is thought to require regulated mechanical coupling between actin filaments (F-actins), which undergo retrograde flow at the cellular leading edge, and cell adhesions via linker “clutch” molecules. However, the molecular machinery mediating this regulatory coupling remains unclear. Here we show that the F-actin binding molecule cortactin directly interacts with a clutch molecule, shootin1, in axonal growth cones, thereby mediating the linkage between F-actin retrograde flow and cell adhesions through L1-CAM. Shootin1–cortactin interaction was enhanced by shootin1 phosphorylation by Pak1, which is activated by the axonal chemoattractant netrin-1. We provide evidence that shootin1–cortactin interaction participates in netrin-1–induced F-actin adhesion coupling and in the promotion of traction forces for axon outgrowth. Under cell signaling, this regulatory F-actin adhesion coupling in growth cones cooperates with actin polymerization for efficient cellular motility.     

The molecular complexity of mu and pi symbiont DNA of Paramecium aurelia

The molecular size of mu and pi symbionts of Parameciumaurelia has been calculated from renaturation kinetic data. Observed values were 0.78 × 10⁹ daltons for mu particle DNA and 0.81 × 10⁹ daltons for pi particle DNA. Estimates of analytical complexity were 4.45 × 10⁹ and 5.05 × 10⁹ daltons respectively. Based on these data, mu and pi symbionts appear to possess multiple genomes and contain a minimum of 5 or 6 copies of each DNA sequence.     

A Highlights from MBoC Selection: CARMIL2 is a novel molecular connection between vimentin and actin essential for cell migration and invadopodia formation

Cancer cell migration requires the regulation of actin networks at protrusions associated with invadopodia and other leading edges. Carcinomas become invasive after undergoing an epithelial–mesenchymal transition characterized by the appearance of vimentin filaments. While vimentin expression correlates with cell migration, the molecular connections between vimentin- and actin-based membrane protrusions are not understood. We report here that CARMIL2 (capping protein, Arp2/3, myosin-I linker 2) provides such a molecular link. CARMIL2 localizes to vimentin, regulates actin capping protein (CP), and binds to membranes. CARMIL2 is necessary for invadopodia formation, as well as cell polarity, lamellipodial assembly, membrane ruffling, macropinocytosis, and collective cell migration. Using point mutants and chimeras with defined biochemical and cellular properties, we discovered that localization to vimentin and CP binding are both essential for the function of CARMIL2 in cells. On the basis of these results, we propose a model in which dynamic vimentin filaments target CARMIL2 to critical membrane-associated locations, where CARMIL2 regulates CP, and thus actin assembly, to create cell protrusions.     

Structural and biochemical studies of SLIP1–SLBP identify DBP5 and eIF3g as SLIP1-binding proteins

In metazoans, replication-dependent histone mRNAs end in a stem-loop structure instead of the poly(A) tail characteristic of all other mature mRNAs. This specialized 3′ end is bound by stem-loop binding protein (SLBP), a protein that participates in the nuclear export and translation of histone mRNAs. The translational activity of SLBP is mediated by interaction with SLIP1, a middle domain of initiation factor 4G (MIF4G)-like protein that connects to translation initiation. We determined the 2.5 Å resolution crystal structure of zebrafish SLIP1 bound to the translation–activation domain of SLBP and identified the determinants of the recognition. We discovered a SLIP1-binding motif (SBM) in two additional proteins: the translation initiation factor eIF3g and the mRNA-export factor DBP5. We confirmed the binding of SLIP1 to DBP5 and eIF3g by pull-down assays and determined the 3.25 Å resolution structure of SLIP1 bound to the DBP5 SBM. The SBM-binding and homodimerization residues of SLIP1 are conserved in the MIF4G domain of CBP80/20-dependent translation initiation factor (CTIF). The results suggest how the SLIP1 homodimer or a SLIP1–CTIF heterodimer can function as platforms to bridge SLBP with SBM-containing proteins involved in different steps of mRNA metabolism.     

Ubiquitin Regulates GGA3-mediated Degradation of BACE1

BACE1 (β-site amyloid precursor protein-cleaving enzyme 1) is a membrane-tethered member of the aspartyl proteases, essential for the production of β-amyloid, a toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. The BACE1 C-terminal fragment contains a DXXLL motif that has been shown to bind the VHS (VPS27, Hrs, and STAM) domain of GGA1–3 (Golgi-localized γ-ear-containing ARF-binding proteins). GGAs are trafficking molecules involved in the transport of proteins containing the DXXLL signal from the Golgi complex to endosomes. Moreover, GGAs bind ubiquitin and traffic synthetic and endosomal ubiquitinated cargoes to lysosomes. We have previously shown that depletion of GGA3 results in increased BACE1 levels and activity because of impaired lysosomal degradation. Here, we report that the accumulation of BACE1 is rescued by the ectopic expression of GGA3 in H4 neuroglioma cells depleted of GGA3. Accordingly, the overexpression of GGA3 reduces the levels of BACE1 and β-amyloid. We then established that mutations in the GGA3 VPS27, Hrs, and STAM domain (N91A) or in BACE1 di-leucine motif (L499A/L500A), able to abrogate their binding, did not affect the ability of ectopically expressed GGA3 to rescue BACE1 accumulation in cells depleted of GGA3. Instead, we found that BACE1 is ubiquitinated at lysine 501 and is mainly monoubiquitinated and Lys-63-linked polyubiquitinated. Finally, a GGA3 mutant with reduced ability to bind ubiquitin (GGA3L276A) was unable to regulate BACE1 levels both in rescue and overexpression experiments. These findings indicate that levels of GGA3 tightly and inversely regulate BACE1 levels via interaction with ubiquitin sorting machinery.     

Neurabin: A Novel Neural Tissue–specific Actin Filament–binding Protein Involved in Neurite Formation

We purified from rat brain a novel actin filament (F-actin)–binding protein of ∼180 kD (p180), which was specifically expressed in neural tissue. We named p180 neurabin (neural tissue–specific F-actin– binding protein). We moreover cloned the cDNA of neurabin from a rat brain cDNA library and characterized native and recombinant proteins. Neurabin was a protein of 1,095 amino acids with a calculated molecular mass of 122,729. Neurabin had one F-actin–binding domain at the NH₂-terminal region, one PSD-95, DlgA, ZO-1–like domain at the middle region, a domain known to interact with transmembrane proteins, and domains predicted to form coiled-coil structures at the COOH-terminal region. Neurabin bound along the sides of F-actin and showed F-actin–cross-linking activity. Immunofluorescence microscopic analysis revealed that neurabin was highly concentrated in the synapse of the developed neurons. Neurabin was also concentrated in the lamellipodia of the growth cone during the development of neurons. Moreover, a study on suppression of endogenous neurabin in primary cultured rat hippocampal neurons by treatment with an antisense oligonucleotide showed that neurabin was involved in the neurite formation. Neurabin is a candidate for key molecules in the synapse formation and function.During the development of the nervous system, the distal tip of the elongating axon—the growth cone—actively migrates toward its target cell in response to the combined actions of attractive and repulsive guidance molecules in the extracellular environment (Garrity and Zipursky, 1995; Keynes and Cook, 1995; Chiba and Keshishian, 1996; Culotti and Kolodkin, 1996; Friedman and O''Leary, 1996; Tessier-Lavigne and Goodman, 1996). When the growth cone contacts with the target cell, it is transformed into the functional presynaptic terminal (Garrity and Zipursky, 1995; Chiba and Kishishian, 1996). The actin cytoskeleton has been shown to play crucial roles in these processes of the synapse formation (Mitchison and Kirschner, 1988; Smith, 1988; Bentley and O''Connor, 1994; Lin et al., 1994; Mackay et al., 1995; Tanaka and Sabry, 1995).In the developing nervous system, the actin cytoskeleton is prominent in two structural domains of the growth cone, filopodia and lamellipodia (Mitchison and Kirschner, 1988; Smith, 1988; Bentley and O''Connor, 1994; Lin et al., 1994; Mackay et al., 1995; Tanaka and Sabry, 1995). In these domains, actin filament (F-actin)¹ assembled at the leading edge are transported into the center of the growth cone and disassembled there. It has been suggested that this retrograde flow of F-actin is crucial for the growth cone motility. Drugs that disrupt F-actin have also been shown to cause the lamellipodial and filopodial collapse and block the ability of neurons to extend the growth cone in the correct direction (Marsh and Letourneau, 1984; Forscher and Smith, 1988; Bentley and Toroian-Raymond, 1986; Chien et al., 1993). These results suggest that the actin cytoskeleton regulates not only the growth cone motility but also the growth cone directionality. Recently, a variety of guidance molecules and their receptors have been identified (Garrity and Zipursky, 1995; Keynes and Cook, 1995; Chiba and Keshishian, 1996; Culotti and Kolodkin, 1996; Friedman and O''Leary, 1996; Tessier-Lavigne and Goodman, 1996). However, which molecules of the actin cytoskeleton are essential for the growth cone motility and directionality is not well understood.When the growth cone contacts with the target cell, the target cell regulates the development of the presynaptic nerve terminal and the formation of the functional synapse (Bowe and Fallon, 1995; Chiba and Keshishian, 1996). In the established nervous system, the presynaptic and postsynaptic membranes get aligned in space and constitute the synaptic junction (Burns and Augustine, 1995; Garner and Kindler, 1996). Electron microscopic studies have revealed the ultrastructural features of the synaptic junction (Burns and Augustine, 1995; Garner and Kindler, 1996). The presynaptic cytoplasm is characterized by synaptic vesicles (SVs). SVs are not distributed uniformly; SVs cluster together in the vicinity of the presynaptic plasma membrane, where F-actin forms a network and is associated with the presynaptic plasma membrane (Hirokawa et al., 1989). Most SVs within the cluster are linked through thin strands to each other, to F-actin, or to both (Hirokawa et al., 1989). A subset of SVs within the cluster are attached by fine filamentous threads to neurotransmitter release zone at the presynaptic plasma membrane (Hirokawa et al., 1989). The presynaptic submembranous cytoskeleton is assumed to be involved in recruiting Ca²⁺ channels and the components of the SV fusion complex, delivering SVs to the neurotransmitter release zone, and keeping them in place (Burns and Augustine, 1995; Garner and Kindler, 1996). At the inner surface of the post-synaptic plasma membrane, there is an electron dense thickening, called postsynaptic density. The postsynaptic density is assumed to be involved in the selective targeting and accumulation of ion channels and receptors (Burns and Augustine, 1995; Garner and Kindler, 1996). It is also assumed that the presynaptic and postsynaptic submembranous cytoskeleton elements are linked to cell adhesion molecules to regulate the synaptic stabilization and plasticity (Fields and Itoh, 1996; Garner and Kindler, 1996). The presynaptic and postsynaptic submembranous cytoskeleton elements are thought to be composed of spectrin/fodrin, ankyrin, α-adducin, and protein 4.1 isoforms and to be linked to F-actin through these cytoskeleton proteins (Garner and Kindler, 1996). However, little is known about which molecules of the submembranous cytoskeleton are essential for the synaptic transmission and/or the synaptic stabilization.To understand the regulation of the actin cytoskeleton during and after the development of the nervous system, it is of crucial importance to identify F-actin–binding proteins implicated in the synapse formation and function. Therefore, we attempted here to isolate neural tissue–specific F-actin–binding proteins. We isolated a novel neural tissue–specific F-actin–binding protein from rat brain, which may be involved in neurite formation, and named it neurabin (neural tissue–specific F-actin–binding protein).     

Role for Heat Shock Protein 90α in the Proliferation and Migration of HaCaT Cells and in the Deep Second-Degree Burn Wound Healing in Mice

Inflammation, proliferation, and tissue remodeling are essential steps for wound healing. The hypoxic wound microenvironment promotes cell migration through a hypoxia—heat shock protein 90 alpha (Hsp90α)—low density lipoprotein receptor-related protein-1 (LRP-1) autocrine loop. To elucidate the role of this autocrine loop on burn wound healing, we investigated the expression profile of Hsp90α at the edge of burn wounds and found a transient increase in both mRNA and protein levels. Experiments performed with a human keratinocyte cell line—HaCaT also confirmed above results. 17-dimethylaminoethylamino-17demethoxygeldanamycin hydrochloride (17-DMAG), an Hsp90α inhibitor, was used to further evaluate the function of Hsp90α in wound healing. Consistently, topical application of Hsp90α in the early stage of deep second-degree burn wounds led to reduced inflammation and increased tissue granulation, with a concomitant reduction in the size of the wound at each time point tested (p<0.05). Consequently, epidermal cells at the wound margin progressed more rapidly causing an expedited healing process. In conclusion, these results provided a rationale for the therapeutic effect of Hsp90α on the burn wound management.     

The Role of Biological Agents in the Management of Large Vessel Vasculitis (LVV): A Systematic Review and Meta-Analysis

Background

Giant cell arteritis (GCA) and Takayasu''s arteritis (TAA) are large vessel vasculitides (LVV) for which corticosteroids (CS) are the mainstay for treatment. In patients with LVV unable to tolerate CS, biological agents have been used with variable effectiveness.

Objective

To systematically review the effectiveness and safety of biological agents in patients with LVV.

Methods

We searched 5 electronic databases (inception to October 2012) and conference abstracts with no language restrictions. Two reviewers independently selected studies, extracted data and assessed methodological quality. Our protocol was registered in PROSPERO.

Results

We included 25 studies (3 RCTs and 22 case series with ≥2 cases). 95 GCA and 98 TAA patients received biological agents. The RCTs using anti-TNF agents (infliximab, etanercept and adalimumab) did not suggest a benefit in GCA. GCA patients receiving tocilizumab, in case series, achieved remission (19 patients) and reduction of corticosteroid dose (mean difference, –16.55 mg/day (95% CI: –26.24, –6.86)). In case series, 75 patients with refractory TAA treated with infliximab discontinued CS 32% of the time. Remission was variably defined and the studies were clinically heterogeneous which precluded further analysis.

Conclusion

This systematic review demonstrated a weak evidence base on which to assess the effectiveness of biological treatment in LVV. Evidence from RCTs suggests that anti-TNF agents are not effective for remission or reduction of CS use. Tocilizumab and infliximab may be effective in the management of LVV and refractory TAA, respectively, although the evidence comes from case series. Future analytical studies are needed to confirm these findings.     

Vinculin–actin interaction couples actin retrograde flow to focal adhesions,but is dispensable for focal adhesion growth

In migrating cells, integrin-based focal adhesions (FAs) assemble in protruding lamellipodia in association with rapid filamentous actin (F-actin) assembly and retrograde flow. How dynamic F-actin is coupled to FA is not known. We analyzed the role of vinculin in integrating F-actin and FA dynamics by vinculin gene disruption in primary fibroblasts. Vinculin slowed F-actin flow in maturing FA to establish a lamellipodium–lamellum border and generate high extracellular matrix (ECM) traction forces. In addition, vinculin promoted nascent FA formation and turnover in lamellipodia and inhibited the frequency and rate of FA maturation. Characterization of a vinculin point mutant that specifically disrupts F-actin binding showed that vinculin–F-actin interaction is critical for these functions. However, FA growth rate correlated with F-actin flow speed independently of vinculin. Thus, vinculin functions as a molecular clutch, organizing leading edge F-actin, generating ECM traction, and promoting FA formation and turnover, but vinculin is dispensible for FA growth.