2024-03-29T11:00:54Zhttp://repository.helmholtz-hzi.de/oai/requestoai:repository.helmholtz-hzi.de:10033/141222019-08-30T11:33:57Zcom_10033_6807com_10033_6799col_10033_6884
Filopodia formation in the absence of functional WAVE- and Arp2/3-complexes.
Steffen, Anika
Faix, Jan
Resch, Guenter P
Linkner, Joern
Wehland, Juergen
Small, J Victor
Rottner, Klemens
Stradal, Theresia E B
Cell migration is initiated by plasma membrane protrusions, in the form of lamellipodia and filopodia. The latter rod-like projections may exert sensory functions and are found in organisms as distant in evolution as mammals and amoeba such as Dictyostelium discoideum. In mammals, lamellipodia protrusion downstream of the small GTPase Rac1 requires a multimeric protein assembly, the WAVE-complex, which activates Arp2/3-mediated actin filament nucleation and actin network assembly. A current model of filopodia formation postulates that these structures arise from a dendritic network of lamellipodial actin filaments by selective elongation and bundling. Here, we have analyzed filopodia formation in mammalian cells abrogated in expression of essential components of the lamellipodial actin polymerization machinery. Cells depleted of the WAVE-complex component Nck-associated protein 1 (Nap1), and, in consequence, of lamellipodia, exhibited normal filopodia protrusion. Likewise, the Arp2/3-complex, which is essential for lamellipodia protrusion, is dispensable for filopodia formation. Moreover, genetic disruption of nap1 or the WAVE-orthologue suppressor of cAMP receptor (scar) in Dictyostelium was also ineffective in preventing filopodia protrusion. These data suggest that the molecular mechanism of filopodia formation is conserved throughout evolution from Dictyostelium to mammals and show that lamellipodia and filopodia formation are functionally separable.
2007-10-17T11:25:31Z
2007-10-17T11:25:31Z
2006-06-01
Article
Mol. Biol. Cell 2006, 17(6):2581-91
1059-1524
16597702
10.1091/mbc.E05-11-1088
http://hdl.handle.net/10033/14122
en
oai:repository.helmholtz-hzi.de:10033/181552019-08-30T11:36:04Zcom_10033_6807com_10033_6799col_10033_6884
c-Met is essential for wound healing in the skin.
Chmielowiec, Jolanta
Borowiak, Malgorzata
Morkel, Markus
Stradal, Theresia
Munz, Barbara
Werner, Sabine
Wehland, Jürgen
Birchmeier, Carmen
Birchmeier, Walter
Department of Cancer Biology, Max-Delbrück-Center for Molecular Medicine, 13125 Berlin, Germany.
Wound healing of the skin is a crucial regenerative process in adult mammals. We examined wound healing in conditional mutant mice, in which the c-Met gene that encodes the receptor of hepatocyte growth factor/scatter factor was mutated in the epidermis by cre recombinase. c-Met-deficient keratinocytes were unable to contribute to the reepithelialization of skin wounds. In conditional c-Met mutant mice, wound closure was slightly attenuated, but occurred exclusively by a few (5%) keratinocytes that had escaped recombination. This demonstrates that the wound process selected and amplified residual cells that express a functional c-Met receptor. We also cultured primary keratinocytes from the skin of conditional c-Met mutant mice and examined them in scratch wound assays. Again, closure of scratch wounds occurred by the few remaining c-Met-positive cells. Our data show that c-Met signaling not only controls cell growth and migration during embryogenesis but is also essential for the generation of the hyperproliferative epithelium in skin wounds, and thus for a fundamental regenerative process in the adult.
2008-02-13T14:22:33Z
2008-02-13T14:22:33Z
2007-04-09
Article
c-Met is essential for wound healing in the skin. 2007, 177 (1):151-62 J. Cell Biol.
0021-9525
17403932
10.1083/jcb.200701086
http://hdl.handle.net/10033/18155
The Journal of cell biology
en
oai:repository.helmholtz-hzi.de:10033/478092019-08-30T11:31:23Zcom_10033_6807com_10033_6799col_10033_6884
Free Brick1 is a trimeric precursor in the assembly of a functional wave complex.
Derivery, Emmanuel
Fink, Jenny
Martin, Davy
Houdusse, Anne
Piel, Matthieu
Stradal, Theresia E
Louvard, Daniel
Gautreau, Alexis
Institut Curie, Centre de Recherche, Morphogenesis and Cell Signaling laboratory, Paris, France.
BACKGROUND: The Wave complex activates the Arp2/3 complex, inducing actin polymerization in lamellipodia and membrane ruffles. The Wave complex is composed of five subunits, the smallest of which, Brick1/Hspc300 (Brk1), is the least characterized. We previously reported that, unlike the other subunits, Brk1 also exists as a free form. PRINCIPAL FINDINGS: Here we report that this free form of Brk1 is composed of homotrimers. Using a novel assay in which purified free Brk1 is electroporated into HeLa cells, we were able to follow its biochemical fate in cells and to show that free Brk1 becomes incorporated into the Wave complex. Importantly, incorporation of free Brk1 into the Wave complex was blocked upon inhibition of protein synthesis and incorporated Brk1 was found to associate preferentially with neosynthesized subunits. Brk1 depleted HeLa cells were found to bleb, as were Nap1, Wave2 or ARPC2 depleted cells, suggesting that this blebbing phenotype of Brk1 depleted cells is due to an impairment of the Wave complex function rather than a specific function of free Brk1. Blebs of Brk1 depleted cells were emitted at sites where lamellipodia and membrane ruffles were normally emitted. In Brk1 depleted cells, the electroporation of free Brk1 was sufficient to restore Wave complex assembly and to rescue the blebbing phenotype. CONCLUSION: Together these results establish that the free form of Brk1 is an essential precursor in the assembly of a functional Wave complex.
2009-01-21T15:28:19Z
2009-01-21T15:28:19Z
2008
Article
Free Brick1 is a trimeric precursor in the assembly of a functional wave complex. 2008, 3 (6):e2462 PLoS ONE
1932-6203
18560548
10.1371/journal.pone.0002462
http://hdl.handle.net/10033/47809
PLoS ONE
en
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002462
oai:repository.helmholtz-hzi.de:10033/1068892019-08-30T11:35:39Zcom_10033_6807com_10033_6799col_10033_6884
Rac1 regulates neuronal polarization through the WAVE complex.
Tahirovic, Sabina
Hellal, Farida
Neukirchen, Dorothee
Hindges, Robert
Garvalov, Boyan K
Flynn, Kevin C
Stradal, Theresia E
Chrostek-Grashoff, Anna
Brakebusch, Cord
Bradke, Frank
Axonal Growth and Regeneration Group, Max Planck Institute of Neurobiology, 82152 Martinsried, Germany.
Neuronal migration and axon growth, key events during neuronal development, require distinct changes in the cytoskeleton. Although many molecular regulators of polarity have been identified and characterized, relatively little is known about their physiological role in this process. To study the physiological function of Rac1 in neuronal development, we have generated a conditional knock-out mouse, in which Rac1 is ablated in the whole brain. Rac1-deficient cerebellar granule neurons, which do not express other Rac isoforms, showed impaired neuronal migration and axon formation both in vivo and in vitro. In addition, Rac1 ablation disrupts lamellipodia formation in growth cones. The analysis of Rac1 effectors revealed the absence of the Wiskott-Aldrich syndrome protein (WASP) family verprolin-homologous protein (WAVE) complex from the plasma membrane of knock-out growth cones. Loss of WAVE function inhibited axon growth, whereas overexpression of a membrane-tethered WAVE mutant partially rescued axon growth in Rac1-knock-out neurons. In addition, pharmacological inhibition of the WAVE complex effector Arp2/3 also reduced axon growth. We propose that Rac1 recruits the WAVE complex to the plasma membrane to enable actin remodeling necessary for axon growth.
2010-06-28T08:50:24Z
2010-06-28T08:50:24Z
2010-05-19
Article
Rac1 regulates neuronal polarization through the WAVE complex. 2010, 30 (20):6930-43 J. Neurosci.
1529-2401
20484635
10.1523/JNEUROSCI.5395-09.2010
http://hdl.handle.net/10033/106889
The Journal of neuroscience : the official journal of the Society for Neuroscience
en
oai:repository.helmholtz-hzi.de:10033/1347622019-08-30T11:37:24Zcom_10033_6807com_10033_6799col_10033_6884
Snail regulates cell survival and inhibits cellular senescence in human metastatic prostate cancer cell lines.
Emadi Baygi, Modjtaba
Soheili, Zahra Soheila
Schmitz, Ingo
Sameie, Shahram
Schulz, Wolfgang A
Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
The epithelial-mesenchymal transition (EMT) is regarded as an important step in cancer metastasis. Snail, a master regulator of EMT, has been recently proposed to act additionally as a cell survival factor and inducer of motility. We have investigated the function of Snail (SNAI1) in prostate cancer cells by downregulating its expression via short (21-mer) interfering RNA (siRNA) and measuring the consequences on EMT markers, cell viability, death, cell cycle, senescence, attachment, and invasivity. Of eight carcinoma cell lines, the prostate carcinoma cell lines LNCaP and PC-3 showed the highest and moderate expression of SNAI1 mRNA, respectively, as measured by quantitative RT-PCR. Long-term knockdown of Snail induced a severe decline in cell numbers in LNCaP and PC-3 and caspase activity was accordingly enhanced in both cell lines. In addition, suppression of Snail expression induced senescence in LNCaP cells. SNAI1-siRNA-treated cells did not tolerate detachment from the extracellular matrix, probably due to downregulation of integrin α6. Expression of E-cadherin, vimentin, and fibronectin was also affected. Invasiveness of PC-3 cells was not significantly diminished by Snail knockdown. Our data suggest that Snail acts primarily as a survival factor and inhibitor of cellular senescence in prostate cancer cell lines. We therefore propose that Snail can act as early driver of prostate cancer progression.
2011-06-28T13:47:38Z
2011-06-28T13:47:38Z
2010-12
Article
Snail regulates cell survival and inhibits cellular senescence in human metastatic prostate cancer cell lines. 2010, 26 (6):553-67 Cell Biol. Toxicol.
1573-6822
20397042
10.1007/s10565-010-9163-5
http://hdl.handle.net/10033/134762
Cell biology and toxicology
en
oai:repository.helmholtz-hzi.de:10033/1460102019-08-30T11:25:11Zcom_10033_6807com_10033_6799col_10033_6884
Microtubules as platforms for assaying actin polymerization in vivo.
Oelkers, J Margit
Vinzenz, Marlene
Nemethova, Maria
Jacob, Sonja
Lai, Frank P L
Block, Jennifer
Szczodrak, Malgorzata
Kerkhoff, Eugen
Backert, Steffen
Schlüter, Kai
Stradal, Theresia E B
Small, J Victor
Koestler, Stefan A
Rottner, Klemens
Helmholtz Centre for Infection Research, Braunschweig, Germany.
The actin cytoskeleton is continuously remodeled through cycles of actin filament assembly and disassembly. Filaments are born through nucleation and shaped into supramolecular structures with various essential functions. These range from contractile and protrusive assemblies in muscle and non-muscle cells to actin filament comets propelling vesicles or pathogens through the cytosol. Although nucleation has been extensively studied using purified proteins in vitro, dissection of the process in cells is complicated by the abundance and molecular complexity of actin filament arrays. We here describe the ectopic nucleation of actin filaments on the surface of microtubules, free of endogenous actin and interfering membrane or lipid. All major mechanisms of actin filament nucleation were recapitulated, including filament assembly induced by Arp2/3 complex, formin and Spir. This novel approach allows systematic dissection of actin nucleation in the cytosol of live cells, its genetic re-engineering as well as screening for new modifiers of the process.
2011-10-19T13:44:11Z
2011-10-19T13:44:11Z
2011
Article
Microtubules as platforms for assaying actin polymerization in vivo. 2011, 6 (5):e19931 PLoS ONE
1932-6203
21603613
10.1371/journal.pone.0019931
http://hdl.handle.net/10033/146010
PloS one
en
oai:repository.helmholtz-hzi.de:10033/1963292019-08-30T11:25:43Zcom_10033_6807com_10033_6799col_10033_6884
High-resolution X-ray structure of the trimeric Scar/WAVE-complex precursor Brk1.
Linkner, Joern
Witte, Gregor
Stradal, Theresia
Curth, Ute
Faix, Jan
Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany.
The Scar/WAVE-complex links upstream Rho-GTPase signaling to the activation of the conserved Arp2/3-complex. Scar/WAVE-induced and Arp2/3-complex-mediated actin nucleation is crucial for actin assembly in protruding lamellipodia to drive cell migration. The heteropentameric Scar/WAVE-complex is composed of Scar/WAVE, Abi, Nap, Pir and a small polypeptide Brk1/HSPC300, and recent work suggested that free Brk1 serves as a homooligomeric precursor in the assembly of this complex. Here we characterized the Brk1 trimer from Dictyostelium by analytical ultracentrifugation and gelfiltration. We show for the first time its dissociation at concentrations in the nanomolar range as well as an exchange of subunits within different DdBrk1 containing complexes. Moreover, we determined the three-dimensional structure of DdBrk1 at 1.5 Å resolution by X-ray crystallography. Three chains of DdBrk1 are associated with each other forming a parallel triple coiled-coil bundle. Notably, this structure is highly similar to the heterotrimeric α-helical bundle of HSPC300/WAVE1/Abi2 within the human Scar/WAVE-complex. This finding, together with the fact that Brk1 is collectively sandwiched by the remaining subunits and also constitutes the main subunit connecting the triple-coil domain of the HSPC300/WAVE1/Abi2/ heterotrimer to Sra1(Pir1), implies a critical function of this subunit in the assembly process of the entire Scar/WAVE-complex.
2011-12-07T15:36:57Z
2011-12-07T15:36:57Z
2011
Article
High-resolution X-ray structure of the trimeric Scar/WAVE-complex precursor Brk1. 2011, 6 (6):e21327 PLoS ONE
1932-6203
21701600
10.1371/journal.pone.0021327
http://hdl.handle.net/10033/196329
PloS one
en
oai:repository.helmholtz-hzi.de:10033/2008892019-08-30T11:31:49Zcom_10033_6807com_10033_6799col_10033_6884
Theoretical model for cellular shapes driven by protrusive and adhesive forces.
Kabaso, Doron
Shlomovitz, Roie
Schloen, Kathrin
Stradal, Theresia
Gov, Nir S
Department of Chemical Physics, The Weizmann Institute of Science, Rehovot, Israel.
The forces that arise from the actin cytoskeleton play a crucial role in determining the cell shape. These include protrusive forces due to actin polymerization and adhesion to the external matrix. We present here a theoretical model for the cellular shapes resulting from the feedback between the membrane shape and the forces acting on the membrane, mediated by curvature-sensitive membrane complexes of a convex shape. In previous theoretical studies we have investigated the regimes of linear instability where spontaneous formation of cellular protrusions is initiated. Here we calculate the evolution of a two dimensional cell contour beyond the linear regime and determine the final steady-state shapes arising within the model. We find that shapes driven by adhesion or by actin polymerization (lamellipodia) have very different morphologies, as observed in cells. Furthermore, we find that as the strength of the protrusive forces diminish, the system approaches a stabilization of a periodic pattern of protrusions. This result can provide an explanation for a number of puzzling experimental observations regarding cellular shape dependence on the properties of the extra-cellular matrix.
2012-01-09T10:43:20Z
2012-01-09T10:43:20Z
2011-05
Article
Theoretical model for cellular shapes driven by protrusive and adhesive forces. 2011, 7 (5):e1001127 PLoS Comput. Biol.
1553-7358
21573201
10.1371/journal.pcbi.1001127
http://hdl.handle.net/10033/200889
PLoS computational biology
en
oai:repository.helmholtz-hzi.de:10033/2183772019-08-30T11:34:48Zcom_10033_6807com_10033_6799col_10033_6884
Essential role for Abi1 in embryonic survival and WAVE2 complex integrity.
Dubielecka, Patrycja M
Ladwein, Kathrin I
Xiong, Xiaoling
Migeotte, Isabelle
Chorzalska, Anna
Anderson, Kathryn V
Sawicki, Janet A
Rottner, Klemens
Stradal, Theresia E
Kotula, Leszek
Laboratory of Cell Signaling, New York Blood Center, New York, NY 10065, USA.
Abl interactor 1 (Abi1) plays a critical function in actin cytoskeleton dynamics through participation in the WAVE2 complex. To gain a better understanding of the specific role of Abi1, we generated a conditional Abi1-KO mouse model and MEFs lacking Abi1 expression. Abi1-KO cells displayed defective regulation of the actin cytoskeleton, and this dysregulation was ascribed to altered activity of the WAVE2 complex. Changes in motility of Abi1-KO cells were manifested by a decreased migration rate and distance but increased directional persistence. Although these phenotypes did not correlate with peripheral ruffling, which was unaffected, Abi1-KO cells exhibited decreased dorsal ruffling. Western blotting analysis of Abi1-KO cell lysates indicated reduced levels of the WAVE complex components WAVE1 and WAVE2, Nap1, and Sra-1/PIR121. Although relative Abi2 levels were more than doubled in Abi1-KO cells, the absolute Abi2 expression in these cells amounted only to a fifth of Abi1 levels in the control cell line. This finding suggests that the presence of Abi1 is critical for the integrity and stability of WAVE complex and that Abi2 levels are not sufficiently increased to compensate fully for the loss of Abi1 in KO cells and to restore the integrity and function of the WAVE complex. The essential function of Abi1 in WAVE complexes and their regulation might explain the observed embryonic lethality of Abi1-deficient embryos, which survived until approximately embryonic day 11.5 and displayed malformations in the developing heart and brain. Cells lacking Abi1 and the conditional Abi1-KO mouse will serve as critical models for defining Abi1 function.
2012-04-13T13:42:12Z
2012-04-13T13:42:12Z
2011-04-26
Article
Essential role for Abi1 in embryonic survival and WAVE2 complex integrity. 2011, 108 (17):7022-7 Proc. Natl. Acad. Sci. U.S.A.
1091-6490
21482783
10.1073/pnas.1016811108
http://hdl.handle.net/10033/218377
Proceedings of the National Academy of Sciences of the United States of America
en
Archived with thanks to Proceedings of the National Academy of Sciences of the United States of America