• WAVE1 and WAVE2 have distinct and overlapping roles in controlling actin assembly at the leading edge.

      Tang, Qing; Schaks, Matthias; Koundinya, Neha; Yang, Changsong; Pollard, Luther W; Svitkina, Tatyana M; Rottner, Klemens; Goode, Bruce L; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (American Society for Cell Biology, 2020-07-22)
      SCAR/WAVE proteins and Arp2/3 complex assemble branched actin networks at the leading edge. Two isoforms of SCAR/WAVE, WAVE1 and WAVE2, reside at the leading edge, yet it has remained unclear whether they perform similar or distinct roles. Further, there have been conflicting reports about the Arp2/3-independent biochemical activities of WAVE1 on actin filament elongation. To investigate this in vivo, we knocked out WAVE1 and WAVE2 genes, individually and together, in B16-F1 melanoma cells. We demonstrate that WAVE1 and WAVE2 are redundant for lamellipodia formation and motility. However, there is a significant decrease in the rate of leading edge actin extension in WAVE2 KO cells, and an increase in WAVE1 KO cells. The faster rates of actin extension in WAVE1 KO cells are offset by faster retrograde flow, and therefore do not translate into faster lamellipodium protrusion. Thus, WAVE1 restricts the rate of actin extension at the leading edge, and appears to couple actin networks to the membrane to drive protrusion. Overall, these results suggest that WAVE1 and WAVE2 have redundant roles in promoting Arp2/3-dependent actin nucleation and lamellipodia formation, but distinct roles in controlling actin network extension and harnessing network growth to cell protrusion.
    • Diversely Functionalised Cytochalasins through Mutasynthesis and Semi-Synthesis.

      Wang, Chongqing; Lambert, Christopher; Hauser, Maurice; Deuschmann, Adrian; Zeilinger, Carsten; Rottner, Klemens; Stradal, Theresia E B; Stadler, Marc; Skellam, Elizabeth J; Cox, Russell J; et al. (Wiley-VCH, 2020-06-02)
      Mutasynthesis of pyrichalasin H from Magnaporthe grisea NI980 yielded a series of unprecedented 4'-substituted cytochalasin analogues in titres as high as the wild-type system (≈60 mg L-1 ). Halogenated, O-alkyl, O-allyl and O-propargyl examples were formed, as well as a 4'-azido analogue. 4'-O-Propargyl and 4'-azido analogues reacted smoothly in Huisgen cycloaddition reactions, whereas p-Br and p-I compounds reacted in Pd-catalysed cross-coupling reactions. A series of examples of biotin-linked, dye-linked and dimeric cytochalasins was rapidly created. In vitro and in vivo bioassays of these compounds showed that the 4'-halogenated and azido derivatives retained their cytotoxicity and antifungal activities; but a unique 4'-amino analogue was inactive. Attachment of larger substituents attenuated the bioactivities. In vivo actin-binding studies with adherent mammalian cells showed that actin remains the likely intracellular target. Dye-linked compounds revealed visualisation of intracellular actin structures even in the absence of phalloidin, thus constituting a potential new class of actin-visualisation tools with filament-barbed end-binding specificity.
    • The Arp2/3 complex is critical for colonisation of the mouse skin by melanoblasts.

      Papalazarou, Vassilis; Swaminathan, Karthic; Jaber-Hijazi, Farah; Spence, Heather; Lahmann, Ines; Nixon, Colin; Salmeron-Sanchez, Manuel; Arnold, Hans-Henning; Rottner, Klemens; Machesky, Laura M; et al. (Company of Biologists, 2020-10-07)
      The Arp2/3 complex is essential for the assembly of branched filamentous actin but its role in physiology and development is surprisingly little understood. Melanoblasts deriving from the neural crest migrate along the developing embryo and traverse the dermis to reach the epidermis colonising the skin and eventually homing within the hair follicles. We have previously established that Rac1 and Cdc42 direct melanoblast migration in vivo We hypothesised that the Arp2/3 complex might be the main downstream effector of these small GTPases. Arp3 depletion in the melanocyte lineage results in severe pigmentation defects in dorsal and ventral regions of the mouse skin. Arp3 null melanoblasts demonstrate proliferation and migration defects and fail to elongate as their wild-type counterparts. Conditional deletion of Arp3 in primary melanocytes causes improper proliferation, spreading, migration and adhesion to extracellular matrix. Collectively, our results suggest that the Arp2/3 complex is absolutely indispensable in the melanocyte lineage in mouse development, and indicate a significant role in developmental processes that require tight regulation of actin-mediated motility.
    • The Small GTPase Rac1 Increases Cell Surface Stiffness and Enhances 3D Migration Into Extracellular Matrices.

      Kunschmann, Tom; Puder, Stefanie; Fischer, Tony; Steffen, Anika; Rottner, Klemens; Mierke, Claudia Tanja; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Nature research, 2019-05-22)
      Membrane ruffling and lamellipodia formation promote the motility of adherent cells in two-dimensional motility assays by mechano-sensing of the microenvironment and initiation of focal adhesions towards their surroundings. Lamellipodium formation is stimulated by small Rho GTPases of the Rac subfamily, since genetic removal of these GTPases abolishes lamellipodium assembly. The relevance of lamellipodial or invadopodial structures for facilitating cellular mechanics and 3D cell motility is still unclear. Here, we hypothesized that Rac1 affects cell mechanics and facilitates 3D invasion. Thus, we explored whether fibroblasts that are genetically deficient for Rac1 (lacking Rac2 and Rac3) harbor altered mechanical properties, such as cellular deformability, intercellular adhesion forces and force exertion, and exhibit alterations in 3D motility. Rac1 knockout and control cells were analyzed for changes in deformability by applying an external force using an optical stretcher. Five Rac1 knockout cell lines were pronouncedly more deformable than Rac1 control cells upon stress application. Using AFM, we found that cell-cell adhesion forces are increased in Rac1 knockout compared to Rac1-expressing fibroblasts. Since mechanical deformability, cell-cell adhesion strength and 3D motility may be functionally connected, we investigated whether increased deformability of Rac1 knockout cells correlates with changes in 3D motility. All five Rac1 knockout clones displayed much lower 3D motility than Rac1-expressing controls. Moreover, force exertion was reduced in Rac1 knockout cells, as assessed by 3D fiber displacement analysis. Interference with cellular stiffness through blocking of actin polymerization by Latrunculin A could not further reduce invasion of Rac1 knockout cells. In contrast, Rac1-expressing controls treated with Latrunculin A were again more deformable and less invasive, suggesting actin polymerization is a major determinant of observed Rac1-dependent effects. Together, we propose that regulation of 3D motility by Rac1 partly involves cellular mechanics such as deformability and exertion of forces.
    • Actin-binding protein cortactin promotes pathogenesis of experimental autoimmune encephalomyelitis by supporting leukocyte infiltration into the central nervous system.

      Samus, Maryna; Li, Yu-Tung; Sorokin, Lydia; Rottner, Klemens; Vestweber, Dietmar; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Society for Neuroscience, 2020-01-06)
      Leukocyte entry into the central nervous system (CNS) is essential for immune surveillance, but is also the basis for the development of pathologic inflammatory conditions within the CNS such as multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). The actin-binding protein, cortactin, in endothelial cells is an important player in regulating the interaction of immune cells with the vascular endothelium. Cortactin has been shown to control the integrity of the endothelial barrier and to support neutrophil transendothelial migration in vitro and in vivo in the skin. Here we employ cortactin gene inactivated (cortactin--/--) male and female mice to study the role of this protein in EAE. Inducing EAE by immunization with a myelin oligodendrocyte glycoprotein peptide (MOG35-55) revealed an ameliorated disease course in cortactin--/-- female mice compared to WT mice. However, proliferation capacity and expression of IL-17A and IFNγ by cortactin-deficient and wildtype splenocytes did not differ, suggesting that the lack of cortactin does not affect induction of the immune response. Rather, cortactin deficiency caused decreased vascular permeability and reduced leukocyte infiltration into the brains and spinal cords of EAE mice. Accordingly, cortactin gene-deficient mice had smaller numbers of proinflammatory cuffs, less extensive demyelination and reduced expression levels of proinflammatory cytokines within the neural tissue compared to wildtype littermates. Thus, cortactin contributes to the development of neural inflammation by supporting leukocyte transmigration through the blood-brain barrier and, therefore, represents a potential candidate for targeting CNS autoimmunity.SIGNIFICANCE STATEMENTMultiple sclerosis (MS) is an autoimmune neuroinflammatory disorder, based on the entry of inflammatory leukocytes into the central nervous system (CNS) where these cells cause demyelination and neurodegeneration. Here, we use a mouse model for MS, experimental autoimmune encephalomyelitis (EAE), and show that gene inactivation of cortactin, an actin binding protein that modulates actin dynamics and branching, protects against neuroinflammation in EAE. Leukocyte infiltration into the CNS was inhibited in cortactin deficient mice and lack of cortactin in cultured primary brain endothelial cells inhibited leukocyte transmigration. Expression levels of proinflammatory cytokines in the CNS and induction of vascular permeability were reduced. We conclude that cortactin represents a novel potential target for the treatment of MS.
    • N-WASP Guides Cancer Cells toward LPA.

      Rottner, Klemens; Schaks, Matthias; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Elsevier, 2019-11-18)
      The actin remodeling factor N-WASP is best known as an Arp2/3 complex activator in processes like endocytosis, extracellular matrix degradation, and host-pathogen interaction. In this issue of Developmental Cell, Juin et al. establish a novel trafficking function for N-WASP in driving lysophosphatidic acid-dependent chemotaxis and metastasis of pancreatic cancer cells.
    • EPLIN-α and -β Isoforms Modulate Endothelial Cell Dynamics through a Spatiotemporally Differentiated Interaction with Actin.

      Taha, Muna; Aldirawi, Mohammed; März, Sigrid; Seebach, Jochen; Odenthal-Schnittler, Maria; Bondareva, Olga; Bojovic, Vesna; Schmandra, Thomas; Wirth, Benedikt; Mietkowska, Magdalena; et al. (Elsevier, 2019-10-22)
      Actin-binding proteins are essential for linear and branched actin filament dynamics that control shape change, cell migration, and cell junction remodeling in vascular endothelium (endothelial cells [ECs]). The epithelial protein lost in neoplasm (EPLIN) is an actin-binding protein, expressed as EPLIN-α and EPLIN-β by alternative promoters; however, the isoform-specific functions are not yet understood. Aortic compared to cava vein ECs and shear stress-exposed cultured ECs express increased EPLIN-β levels that stabilize stress fibers. In contrast, EPLIN-α expression is increased in growing and migrating ECs, is targeted to membrane protrusions, and terminates their growth via interaction with the Arp2/3 complex. The data indicate that EPLIN-α controls protrusion dynamics while EPLIN-β has an actin filament stabilizing role, which is consistent with FRAP analyses demonstrating a lower EPLIN-β turnover rate compared to EPLIN-α. Together, EPLIN isoforms differentially control actin dynamics in ECs, essential in shear stress responses, cell migration, and barrier function.
    • Actin dynamics in cell migration

      Schaks, Matthias; Giannone, Grégory; Rottner, Klemens; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Portland Press Ltd., 2019-09-24)
      Cell migration is an essential process, both in unicellular organisms such as amoeba and as individual or collective motility in highly developed multicellular organisms like mammals. It is controlled by a variety of activities combining protrusive and contractile forces, normally generated by actin filaments. Here, we summarize actin filament assembly and turnover processes, and how respective biochemical activities translate into different protrusion types engaged in migration. These actin-based plasma membrane protrusions include actin-related protein 2/3 complex-dependent structures such as lamellipodia and membrane ruffles, filopodia as well as plasma membrane blebs. We also address observed antagonisms between these protrusion types, and propose a model – also inspired by previous literature – in which a complex balance between specific Rho GTPase signaling pathways dictates the protrusion mechanism employed by cells. Furthermore, we revisit published work regarding the fascinating antagonism between Rac and Rho GTPases, and how this intricate signaling network can define cell behavior and modes of migration. Finally, we discuss how the assembly of actin filament networks can feed back onto their regulators, as exemplified for the lamellipodial factor WAVE regulatory complex, tightly controlling accumulation of this complex at specific subcellular locations as well as its turnover.
    • RhoG and Cdc42 can contribute to Rac-dependent lamellipodia formation through WAVE regulatory complex-binding.

      Schaks, Matthias; Döring, Hermann; Kage, Frieda; Steffen, Anika; Klünemann, Thomas; Blankenfeldt, Wulf; Stradal, Theresia; Rottner, Klemens; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Taylor and Francis, 2019-08-26)
      Cell migration frequently involves the formation of lamellipodial protrusions, the initiation of which requires Rac GTPases signalling to heteropentameric WAVE regulatory complex (WRC). While Rac-related RhoG and Cdc42 can potently stimulate lamellipodium formation, so far presumed to occur by upstream signalling to Rac activation, we show here that the latter can be bypassed by RhoG and Cdc42 given that WRC has been artificially activated. This evidence arises from generation of B16-F1 cells simultaneously lacking both Rac GTPases and WRC, followed by reconstitution of lamellipodia formation with specific Rho-GTPase and differentially active WRC variant combinations. We conclude that formation of canonical lamellipodia requires WRC activation through Rac, but can possibly be tuned, in addition, by WRC interactions with RhoG and Cdc42.
    • Role of Src and Cortactin in Pemphigus Skin Blistering.

      Kugelmann, Daniela; Rötzer, Vera; Walter, Elias; Egu, Desalegn Tadesse; Fuchs, Michael Tobias; Vielmuth, Franziska; Vargas-Robles, Hilda; Schnoor, Michael; Hertl, Michael; Eming, Rüdiger; et al. (Frontiers, 2019-01-01)
      Autoantibodies against desmoglein (Dsg) 1 and Dsg3 primarily cause blister formation in the autoimmune disease pemphigus vulgaris (PV). Src was proposed to contribute to loss of keratinocyte cohesion. However, the role and underlying mechanisms are unclear and were studied here. In keratinocytes, cell cohesion in response to autoantibodies was reduced in Src-dependent manner by two patient-derived PV-IgG fractions as well as by AK23 but not by a third PV-IgG fraction, although Src was activated by all autoantibodies. Loss of cell cohesion was progredient in a timeframe of 24 h and AK23, similar to PV-IgG, interfered with reconstitution of cell cohesion after Ca2+-switch, indicating that the autoantibodies also interfered with desmosome assembly. Dsg3 co-localized along cell contacts and interacted with the Src substrate cortactin. In keratinocytes isolated from cortactin-deficient mice, cell adhesion was impaired and Src-mediated inhibition of AK23-induced loss of cell cohesion for 24 h was significantly reduced compared to wild-type (wt) cells. Similarly, AK23 impaired reconstitution of cell adhesion was Src-dependent only in the presence of cortactin. Likewise, Src inhibition significantly reduced AK23-induced skin blistering in wt but not cortactin-deficient mice. These data suggest that the Src-mediated long-term effects of AK23 on loss of cell cohesion and skin blistering are dependent on cortactin-mediated desmosome assembly. However, in human epidermis PV-IgG-induced skin blistering and ultrastructural alterations of desmosomes were not affected by Src inhibition, indicating that Src may not be critical for skin blistering in intact human skin, at least when high levels of autoantibodies targeting Dsg1 are present.
    • High cortactin expression in B-cell acute lymphoblastic leukemia is associated with increased transendothelial migration and bone marrow relapse.

      Velázquez-Avila, Martha; Balandrán, Juan Carlos; Ramírez-Ramírez, Dalia; Velázquez-Avila, Mirella; Sandoval, Antonio; Felipe-López, Alfonso; Nava, Porfirio; Alvarado-Moreno, José Antonio; Dozal, David; Prieto-Chávez, Jessica L; et al. (Nature publishing group, 2018-12-20)
      Cancer is a major cause of death in children worldwide, with B-lineage cell acute lymphoblastic leukemia (B-ALL) being the most frequent childhood malignancy. Relapse, treatment failure and organ infiltration worsen the prognosis, warranting a better understanding of the implicated mechanisms. Cortactin is an actin-binding protein involved in cell adhesion and migration that is overexpressed in many solid tumors and in adult B-cell chronic lymphocytic leukemia. Here, we investigated cortactin expression and potential impact on infiltration and disease prognosis in childhood B-ALL. B-ALL cell lines and precursor cells from bone marrow (BM) and cerebrospinal fluid (CSF) of B-ALL patients indeed overexpressed cortactin. In CXCL12-induced transendothelial migration assays, transmigrated B-ALL cells had highest cortactin expression. In xenotransplantation models, only cortactin
    • On the relation between filament density, force generation, and protrusion rate in mesenchymal cell motility.

      Dolati, Setareh; Kage, Frieda; Mueller, Jan; Müsken, Mathias; Kirchner, Marieluise; Dittmar, Gunnar; Sixt, Michael; Rottner, Klemens; Falcke, Martin; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Amrican Society for Cell biology, 2018-11-01)
      Lamellipodia are flat membrane protrusions formed during mesenchymal motion. Polymerization at the leading edge assembles the actin filament network and generates protrusion force. How this force is supported by the network and how the assembly rate is shared between protrusion and network retrograde flow determines the protrusion rate. We use mathematical modeling to understand experiments changing the F-actin density in lamellipodia of B16-F1 melanoma cells by modulation of Arp2/3 complex activity or knockout of the formins FMNL2 and FMNL3. Cells respond to a reduction of density with a decrease of protrusion velocity, an increase in the ratio of force to filament number, but constant network assembly rate. The relation between protrusion force and tension gradient in the F-actin network and the density dependency of friction, elasticity, and viscosity of the network explain the experimental observations. The formins act as filament nucleators and elongators with differential rates. Modulation of their activity suggests an effect on network assembly rate. Contrary to these expectations, the effect of changes in elongator composition is much weaker than the consequences of the density change. We conclude that the force acting on the leading edge membrane is the force required to drive F-actin network retrograde flow.
    • Distinct Interaction Sites of Rac GTPase with WAVE Regulatory Complex Have Non-redundant Functions in Vivo.

      Schaks, Matthias; Singh, Shashi P; Kage, Frieda; Thomason, Peter; Klünemann, Thomas; Steffen, Anika; Blankenfeldt, Wulf; Stradal, Theresia E; Insall, Robert H; Rottner, Klemens; et al. (2018-10-25)
      Cell migration often involves the formation of sheet-like lamellipodia generated by branched actin filaments. The branches are initiated when Arp2/3 complex [1] is activated by WAVE regulatory complex (WRC) downstream of small GTPases of the Rac family [2]. Recent structural studies defined two independent Rac binding sites on WRC within the Sra-1/PIR121 subunit of the pentameric WRC [3, 4], but the functions of these sites in vivo have remained unknown. Here we dissect the mechanism of WRC activation and the in vivo relevance of distinct Rac binding sites on Sra-1, using CRISPR/Cas9-mediated gene disruption of Sra-1 and its paralog PIR121 in murine B16-F1 cells combined with Sra-1 mutant rescue. We show that the A site, positioned adjacent to the binding region of WAVE-WCA mediating actin and Arp2/3 complex binding, is the main site for allosteric activation of WRC. In contrast, the D site toward the C terminus is dispensable for WRC activation but required for optimal lamellipodium morphology and function. These results were confirmed in evolutionarily distant Dictyostelium cells. Moreover, the phenotype seen in D site mutants was recapitulated in Rac1 E31 and F37 mutants; we conclude these residues are important for Rac-D site interaction. Finally, constitutively activated WRC was able to induce lamellipodia even after both Rac interaction sites were lost, showing that Rac interaction is not essential for membrane recruitment. Our data establish that physical interaction with Rac is required for WRC activation, in particular through the A site, but is not mandatory for WRC accumulation in the lamellipodium.
    • RhoA, Rac1, and Cdc42 differentially regulate αSMA and collagen I expression in mesenchymal stem cells.

      Ge, Jianfeng; Burnier, Laurent; Adamopoulou, Maria; Kwa, Mei Qi; Schaks, Matthias; Rottner, Klemens; Brakebusch, Cord (2018-06-15)
      Mesenchymal stem cells (MSC) are suggested to be important progenitors of myofibroblasts in fibrosis. To understand the role of Rho GTPase signaling in TGF -induced myofibroblast differentiation of MSC, we generated a novel MSC line and its descendants lacking functional Rho GTPases and Rho GTPase signaling components. Unexpectedly, our data revealed that Rho GTPase signaling is required for TGF -induced expression of -smooth muscle actin (SMA) but not of collagen I 1 (col1a1). Whereas loss of RhoA and Cdc42 reduced SMA expression, ablation of the Rac1 gene had the opposite effect. Although actin polymerization and MRTFa were crucial for TGF -induced SMA expression, neither Arp2/3-dependent actinpolymerizationnorcofilin-dependent severinganddepolymerization of F-actin were required. Instead, F-actin levels were dependent on cell contraction, and TGF -induced actin polymerization correlated with increased cell contraction mediated by RhoA and Cdc42. Finally, we observed impaired collagen I secretion in MSC lacking RhoA or Cdc42. These data give novel molecular insights into the role of Rho GTPases in TGF signaling and have implications for our understanding of MSC function in fibrosis.
    • Assembling actin filaments for protrusion.

      Rottner, Klemens; Schaks, Matthias; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (2018-09-29)
      Cell migration entails a plethora of activities combining the productive exertion of protrusive and contractile forces to allow cells to push and squeeze themselves through cell clumps, interstitial tissues or tissue borders. All these activities require the generation and turnover of actin filaments that arrange into specific, subcellular structures. The most prominent structures mediating the protrusion at the leading edges of cells include lamellipodia and filopodia as well as plasma membrane blebs. Moreover, in cells migrating on planar substratum, mechanical support is being provided by an additional, more proximally located structure termed the lamella. Here, we systematically dissect the literature concerning the mechanisms driving actin filament nucleation and elongation in the best-studied protrusive structure, the lamellipodium. Recent work has shed light on open questions in lamellipodium protrusion, including the relative contributions of nucleation versus elongation to the assembly of both individual filaments and the lamellipodial network as a whole. However, much remains to be learned concerning the specificity and relevance of individual factors, their cooperation and their site-specific functions relative to the importance of global actin monomer and filament homeostasis.
    • Early cell death induced by Clostridium difficile TcdB: Uptake and Rac1-glucosylation kinetics are decisive for cell fate.

      Beer, Lara-Antonia; Tatge, Helma; Reich, Nicole; Tenspolde, Michel; Olling, Alexandra; Goy, Sebastian; Rottner, Klemens; Alekov, Alexi Kirilov; Gerhard, Ralf; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-06-14)
      Toxin A and Toxin B (TcdA/TcdB) are large glucosyltransferases produced by Clostridium difficile. TcdB but not TcdA induces reactive oxygen species-mediated early cell death (ECD) when applied at high concentrations. We found that nonglucosylated Rac1 is essential for induction of ECD since inhibition of Rac1 impedes this effect. ECD only occurs when TcdB is rapidly endocytosed. This was shown by generation of chimeras using the trunk of TcdB from a hypervirulent strain. TcdB from hypervirulent strain has been described to translocate from endosomes at higher pH values and thus, meaning faster than reference type TcdB. Accordingly, intracellular delivery of the glucosyltransferase domain of reference TcdB by the trunk of TcdB from hypervirulent strain increased ECD. Furthermore, proton transporters such as sodium/proton exchanger (NHE) or the ClC-5 anion/proton exchanger, both of which contribute to endosomal acidification, also affected cytotoxic potency of TcdB: Specific inhibition of NHE reduced cytotoxicity, whereas transfection of cells with the endosomal anion/proton exchanger ClC-5 increased cytotoxicity of TcdB. Our data suggest that both the uptake rate of TcdB into the cytosol and the status of nonglucosylated Rac1 are key determinants that are decisive for whether ECD or delayed apoptosis is triggered.
    • Diverse functions of myosin VI elucidated by an isoform-specific α-helix domain.

      Wollscheid, Hans-Peter; Biancospino, Matteo; He, Fahu; Magistrati, Elisa; Molteni, Erika; Lupia, Michela; Soffientini, Paolo; Rottner, Klemens; Cavallaro, Ugo; Pozzoli, Uberto; et al. (2016-04)
      Myosin VI functions in endocytosis and cell motility. Alternative splicing of myosin VI mRNA generates two distinct isoform types, myosin VI(short) and myosin VI(long), which differ in the C-terminal region. Their physiological and pathological roles remain unknown. Here we identified an isoform-specific regulatory helix, named the α2-linker, that defines specific conformations and hence determines the target selectivity of human myosin VI. The presence of the α2-linker structurally defines a new clathrin-binding domain that is unique to myosin VI(long) and masks the known RRL interaction motif. This finding is relevant to ovarian cancer, in which alternative myosin VI splicing is aberrantly regulated, and exon skipping dictates cell addiction to myosin VI(short) in tumor-cell migration. The RRL interactor optineurin contributes to this process by selectively binding myosin VI(short). Thus, the α2-linker acts like a molecular switch that assigns myosin VI to distinct endocytic (myosin VI(long)) or migratory (myosin VI(short)) functional roles.
    • Diversified actin protrusions promote environmental exploration but are dispensable for locomotion of leukocytes.

      Leithner, Alexander; Eichner, Alexander; Müller, Jan; Reversat, Anne; Brown, Markus; Schwarz, Jan; Merrin, Jack; de Gorter, David J J; Schur, Florian; Bayerl, Jonathan; et al. (2016-11)
      Most migrating cells extrude their front by the force of actin polymerization. Polymerization requires an initial nucleation step, which is mediated by factors establishing either parallel filaments in the case of filopodia or branched filaments that form the branched lamellipodial network. Branches are considered essential for regular cell motility and are initiated by the Arp2/3 complex, which in turn is activated by nucleation-promoting factors of the WASP and WAVE families. Here we employed rapid amoeboid crawling leukocytes and found that deletion of the WAVE complex eliminated actin branching and thus lamellipodia formation. The cells were left with parallel filaments at the leading edge, which translated, depending on the differentiation status of the cell, into a unipolar pointed cell shape or cells with multiple filopodia. Remarkably, unipolar cells migrated with increased speed and enormous directional persistence, while they were unable to turn towards chemotactic gradients. Cells with multiple filopodia retained chemotactic activity but their migration was progressively impaired with increasing geometrical complexity of the extracellular environment. These findings establish that diversified leading edge protrusions serve as explorative structures while they slow down actual locomotion.
    • A Genome-Wide siRNA Screen Implicates Spire1/2 in SipA-Driven Salmonella Typhimurium Host Cell Invasion.

      Andritschke, Daniel; Dilling, Sabrina; Emmenlauer, Mario; Welz, Tobias; Schmich, Fabian; Misselwitz, Benjamin; Rämö, Pauli; Rottner, Klemens; Kerkhoff, Eugen; Wada, Teiji; et al. (2016)
      Salmonella Typhimurium (S. Tm) is a leading cause of diarrhea. The disease is triggered by pathogen invasion into the gut epithelium. Invasion is attributed to the SPI-1 type 3 secretion system (T1). T1 injects effector proteins into epithelial cells and thereby elicits rearrangements of the host cellular actin cytoskeleton and pathogen invasion. The T1 effector proteins SopE, SopB, SopE2 and SipA are contributing to this. However, the host cell factors contributing to invasion are still not completely understood. To address this question comprehensively, we used Hela tissue culture cells, a genome-wide siRNA library, a modified gentamicin protection assay and S. TmSipA, a sopBsopE2sopE mutant which strongly relies on the T1 effector protein SipA to invade host cells. We found that S. TmSipA invasion does not elicit membrane ruffles, nor promote the entry of non-invasive bacteria "in trans". However, SipA-mediated infection involved the SPIRE family of actin nucleators, besides well-established host cell factors (WRC, ARP2/3, RhoGTPases, COPI). Stage-specific follow-up assays and knockout fibroblasts indicated that SPIRE1 and SPIRE2 are involved in different steps of the S. Tm infection process. Whereas SPIRE1 interferes with bacterial binding, SPIRE2 influences intracellular replication of S. Tm. Hence, these two proteins might fulfill non-redundant functions in the pathogen-host interaction. The lack of co-localization hints to a short, direct interaction between S. Tm and SPIRE proteins or to an indirect effect.
    • Kindlin-2 recruits paxillin and Arp2/3 to promote membrane protrusions during initial cell spreading.

      Böttcher, Ralph T; Veelders, Maik; Rombaut, Pascaline; Faix, Jan; Theodosiou, Marina; Stradal, Theresia E B; Rottner, Klemens; Zent, Roy; Herzog, Franz; Fässler, Reinhard; et al. (2017-09-14)
      Cell spreading requires the coupling of actin-driven membrane protrusion and integrin-mediated adhesion to the extracellular matrix. The integrin-activating adaptor protein kindlin-2 plays a central role for cell adhesion and membrane protrusion by directly binding and recruiting paxillin to nascent adhesions. Here, we report that kindlin-2 has a dual role during initial cell spreading: it binds paxillin via the pleckstrin homology and F0 domains to activate Rac1, and it directly associates with the Arp2/3 complex to induce Rac1-mediated membrane protrusions. Consistently, abrogation of kindlin-2 binding to Arp2/3 impairs lamellipodia formation and cell spreading. Our findings identify kindlin-2 as a key protein that couples cell adhesion by activating integrins and the induction of membrane protrusions by activating Rac1 and supplying Rac1 with the Arp2/3 complex.