Publications of RG Cytoskeleton Dynamics (CYD)http://hdl.handle.net/10033/68802024-03-28T17:30:33Z2024-03-28T17:30:33ZRac function is crucial for cell migration but is not required for spreading and focal adhesion formation.Steffen, AnikaLadwein, MarkusDimchev, Georgi AHein, AnkeSchwenkmezger, LisaArens, StefanLadwein, Kathrin IMargit Holleboom, JSchur, FlorianVictor Small, JSchwarz, JanettGerhard, RalfFaix, JanStradal, Theresia E BBrakebusch, CordRottner, Klemenshttp://hdl.handle.net/10033/3061702019-08-30T11:25:11Z2013-10-15T00:00:00ZRac function is crucial for cell migration but is not required for spreading and focal adhesion formation.
Steffen, Anika; Ladwein, Markus; Dimchev, Georgi A; Hein, Anke; Schwenkmezger, Lisa; Arens, Stefan; Ladwein, Kathrin I; Margit Holleboom, J; Schur, Florian; Victor Small, J; Schwarz, Janett; Gerhard, Ralf; Faix, Jan; Stradal, Theresia E B; Brakebusch, Cord; Rottner, Klemens
Cell migration is commonly accompanied by protrusion of membrane ruffles and lamellipodia. In two-dimensional migration, protrusion of these thin sheets of cytoplasm is considered relevant to both exploration of new space and initiation of nascent adhesion to the substratum. Lamellipodium formation can be potently stimulated by Rho GTPases of the Rac subfamily, but also by RhoG or Cdc42. Here we describe viable fibroblast cell lines genetically deficient for Rac1 that lack detectable levels of Rac2 and Rac3. Rac-deficient cells were devoid of apparent lamellipodia, but these structures were restored by expression of either Rac subfamily member, but not by Cdc42 or RhoG. Cells deficient in Rac showed strong reduction in wound closure and random cell migration and a notable loss of sensitivity to a chemotactic gradient. Despite these defects, Rac-deficient cells were able to spread, formed filopodia and established focal adhesions. Spreading in these cells was achieved by the extension of filopodia followed by the advancement of cytoplasmic veils between them. The number and size of focal adhesions as well as their intensity were largely unaffected by genetic removal of Rac1. However, Rac deficiency increased the mobility of different components in focal adhesions, potentially explaining how Rac - although not essential - can contribute to focal adhesion assembly. Together, our data demonstrate that Rac signaling is essential for lamellipodium protrusion and for efficient cell migration, but not for spreading or filopodium formation. Our findings also suggest that Rac GTPases are crucial to the establishment or maintenance of polarity in chemotactic migration.
2013-10-15T00:00:00ZArp2/3 complex is essential for actin network treadmilling as well as for targeting of capping protein and cofilin.Koestler, Stefan ASteffen, AnikaNemethova, MariaWinterhoff, MoritzLuo, NingningHolleboom, J MargitKrupp, JessicaJacob, SonjaVinzenz, MarleneSchur, FlorianSchlüter, KaiGunning, Peter WWinkler, ChristophSchmeiser, ChristianFaix, JanStradal, Theresia E BSmall, J VictorRottner, Klemenshttp://hdl.handle.net/10033/3046022019-08-30T11:37:00Z2013-09-01T00:00:00ZArp2/3 complex is essential for actin network treadmilling as well as for targeting of capping protein and cofilin.
Koestler, Stefan A; Steffen, Anika; Nemethova, Maria; Winterhoff, Moritz; Luo, Ningning; Holleboom, J Margit; Krupp, Jessica; Jacob, Sonja; Vinzenz, Marlene; Schur, Florian; Schlüter, Kai; Gunning, Peter W; Winkler, Christoph; Schmeiser, Christian; Faix, Jan; Stradal, Theresia E B; Small, J Victor; Rottner, Klemens
Lamellipodia are sheet-like protrusions formed during migration or phagocytosis and comprise a network of actin filaments. Filament formation in this network is initiated by nucleation/branching through the actin-related protein 2/3 (Arp2/3) complex downstream of its activator, suppressor of cAMP receptor/WASP-family verprolin homologous (Scar/WAVE), but the relative relevance of Arp2/3-mediated branching versus actin filament elongation is unknown. Here we use instantaneous interference with Arp2/3 complex function in live fibroblasts with established lamellipodia. This allows direct examination of both the fate of elongating filaments upon instantaneous suppression of Arp2/3 complex activity and the consequences of this treatment on the dynamics of other lamellipodial regulators. We show that Arp2/3 complex is an essential organizer of treadmilling actin filament arrays but has little effect on the net rate of actin filament turnover at the cell periphery. In addition, Arp2/3 complex serves as key upstream factor for the recruitment of modulators of lamellipodia formation such as capping protein or cofilin. Arp2/3 complex is thus decisive for filament organization and geometry within the network not only by generating branches and novel filament ends, but also by directing capping or severing activities to the lamellipodium. Arp2/3 complex is also crucial to lamellipodia-based migration of keratocytes.
2013-09-01T00:00:00ZCytotoxic necrotizing factor-y boosts yersinia effector translocation by activating rac protein.Wolters, ManuelBoyle, Erin CLardong, KerstinTrülzsch, KonradSteffen, AnikaRottner, KlemensRuckdeschel, KlausAepfelbacher, Martinhttp://hdl.handle.net/10033/3025012019-08-30T11:36:59Z2013-08-09T00:00:00ZCytotoxic necrotizing factor-y boosts yersinia effector translocation by activating rac protein.
Wolters, Manuel; Boyle, Erin C; Lardong, Kerstin; Trülzsch, Konrad; Steffen, Anika; Rottner, Klemens; Ruckdeschel, Klaus; Aepfelbacher, Martin
Pathogenic Yersinia spp. translocate the effectors YopT, YopE, and YopO/YpkA into target cells to inactivate Rho family GTP-binding proteins and block immune responses. Some Yersinia spp. also secrete the Rho protein activator cytotoxic necrotizing factor-Y (CNF-Y), but it has been unclear how the bacteria may benefit from Rho protein activation. We show here that CNF-Y increases Yop translocation in Yersinia enterocolitica-infected cells up to 5-fold. CNF-Y strongly activated RhoA and also delayed in time Rac1 and Cdc42, but when individually expressed, constitutively active mutants of Rac1, but not of RhoA, increased Yop translocation. Consistently, knock-out or knockdown of Rac1 but not of RhoA, -B, or -C inhibited Yersinia effector translocation in CNF-Y-treated and control cells. Activation or knockdown of Cdc42 also affected Yop translocation but much less efficiently than Rac. The increase in Yop translocation induced by CNF-Y was essentially independent of the presence of YopE, YopT, or YopO in the infecting Yersinia strain, indicating that none of the Yops reported to inhibit translocation could reverse the CNF-Y effect. In summary, the CNF-Y activity of Yersinia strongly enhances Yop translocation through activation of Rac.
2013-08-09T00:00:00ZRhoA is dispensable for skin development, but crucial for contraction and directed migration of keratinocytes.Jackson, BenPeyrollier, KarinePedersen, EsbenBasse, AstridKarlsson, RichardWang, ZhipengLefever, TineOchsenbein, Alexandra MSchmidt, GudulaAktories, KlausStanley, AlannaQuondamatteo, FabioLadwein, MarkusRottner, Klemensvan Hengel, JolandaBrakebusch, Cordhttp://hdl.handle.net/10033/2138112019-08-30T11:37:23Z2011-03-01T00:00:00ZRhoA is dispensable for skin development, but crucial for contraction and directed migration of keratinocytes.
Jackson, Ben; Peyrollier, Karine; Pedersen, Esben; Basse, Astrid; Karlsson, Richard; Wang, Zhipeng; Lefever, Tine; Ochsenbein, Alexandra M; Schmidt, Gudula; Aktories, Klaus; Stanley, Alanna; Quondamatteo, Fabio; Ladwein, Markus; Rottner, Klemens; van Hengel, Jolanda; Brakebusch, Cord
RhoA is a small guanosine-5'-triphosphatase (GTPase) suggested to be essential for cytokinesis, stress fiber formation, and epithelial cell-cell contacts. In skin, loss of RhoA was suggested to underlie pemphigus skin blistering. To analyze RhoA function in vivo, we generated mice with a keratinocyte-restricted deletion of the RhoA gene. Despite a severe reduction of cofilin and myosin light chain (MLC) phosphorylation, these mice showed normal skin development. Primary RhoA-null keratinocytes, however, displayed an increased percentage of multinucleated cells, defective maturation of cell-cell contacts. Furthermore we observed increased cell spreading due to impaired RhoA-ROCK (Rho-associated protein kinase)-MLC phosphatase-MLC-mediated cell contraction, independent of Rac1. Rho-inhibiting toxins further increased multinucleation of RhoA-null cells but had no significant effect on spreading, suggesting that RhoB and RhoC have partially overlapping functions with RhoA. Loss of RhoA decreased directed cell migration in vitro caused by reduced migration speed and directional persistence. These defects were not related to the decreased cell contraction and were independent of ROCK, as ROCK inhibition by Y27632 increased directed migration of both control and RhoA-null keratinocytes. Our data indicate a crucial role for RhoA and contraction in regulating cell spreading and a contraction-independent function of RhoA in keratinocyte migration. In addition, our data show that RhoA is dispensable for skin development.
2011-03-01T00:00:00Z