PubMed Health⌕ Search

Biomedical subjects

Klemens Rottner

Publications and source records attributed to Klemens Rottner.

18 recordsLinked to original sources
PubMed2026

Profilin promotes lamellipodium protrusion by tuning the antagonistic activities of capping protein and VASP.

Cell migration frequently employs protrusions termed lamellipodia, constituting the prime model system for generation of branched actin filament networks. Here we utilize genome editing to explore the functional connections between the actin monomer-binding protein profilin (Pfn), the filament nucleating Arp2/3 complex, its co-factor heterodimeric capping protein (CP) and Ena/VASP family polymerases in lamellipodial actin assembly. We show that Pfn counters Ena/VASP but promotes Arp2/3 complex activity, while Ena/VASP and CP mutually antagonize each other. While Pfn promotes Arp2/3 complex activity irrespective of Ena/VASP, sensitivity of CP to Pfn removal vanishes in the absence of Ena/VASP. Our findings establish Pfn as master regulator of Arp2/3 complex-dependent actin network formation, differentially regulating VASP and its antagonizer CP. Mathematical modeling of our data suggest Ena/VASP and CP to compete for recruitment to lamellipodial edges. Our work provides critical insights into the molecular logic of branched actin network assembly in protrusion and force generation.

Profilins↗
PubMed2006

Monomeric red fluorescent protein variants used for imaging studies in different species.

Fluorescent proteins have proven to be excellent tools for live-cell imaging studies. In addition to green fluorescent protein (GFP) and its variants, recent progress was achieved in the development of monomeric red fluorescent proteins (mRFPs) that show improved properties in respect to maturation and intracellular fluorescence. mRFPmars, a red fluorescent protein designed especially for the use in Dictyostelium, has been employed to tag different proteins for live-cell investigations in Dictyostelium. mRFPruby, which differs in sequence from mRFPmars in four amino acids, has a codon usage optimised for the application in mammalian cells. Here, we show that both mRFP variants can also be applied for localisation studies in other organisms. mRFPmars was expressed in Hydra and fused to the Bcl-2 family protein Bax. mRFPruby in combination with histone 2B was expressed in Drosophila S2 cells to monitor mitosis. Using mouse cell lines, mRFPruby fused to beta-actin was assayed with high spatial resolution to study details of actin cytoskeleton dynamics. In addition, we demonstrate that both mRFP variants are also suitable for dual-colour microscopy in the different species.

Animals↗
PubMed2006

Filopodia formation in the absence of functional WAVE- and Arp2/3-complexes.

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.

Actin-Related Protein 2-3 Complex↗
PubMed2005

The making of filopodia.

Filopodia are rod-like cell surface projections filled with bundles of parallel actin filaments. They are found on a variety of cell types and have been ascribed sensory or exploratory functions. Filopodium formation is frequently associated with protrusion of sheet-like actin filament arrays called lamellipodia and membrane ruffles, but, in comparison to these structures, the molecular details underpinning the initiation and maintenance of filopodia are only just beginning to emerge. Recent advances have improved our understanding of the molecular requirements for filopodium protrusion and have yielded insights into the inter-relationships between lamellipodia and filopodia, the two 'sub-compartments' of the protrusive actin cytoskeleton.

Animals↗
PubMed2005

Role of the WASP family proteins for Mycobacterium marinum actin tail formation.

Mycobacterium marinum, a natural pathogen of fish and frogs and an occasional pathogen of humans, is capable of inducing actin tail formation within the cytoplasm of macrophages, leading to actin-based motility and intercellular spread. Actin tail formation by M. marinum is markedly reduced in macrophages deficient in the Wiskott-Aldrich syndrome protein (WASP), which still contain the closely related and ubiquitously expressed protein N-WASP (neuronal WASP). In fibroblasts lacking both WASP and N-WASP, M. marinum is incapable of efficient actin polymerization and of intercellular spread. By reconstituting these cells, we find that M. marinum is able to use either WASP or N-WASP to induce actin polymerization. Inhibition or genetic deletion of tyrosine phosphorylation, Nck, WASP-interacting protein, and Cdc42 does not affect M. marinum actin tail formation, excluding the participation of these molecules as upstream activators of N-WASP in the initiation of actin-based motility. In contrast, deletion of the phosphatidylinositol 4,5-bisphosphate-binding basic motif in N-WASP eliminates M. marinum actin tail formation. Together, these data demonstrate that M. marinum subversion of host actin polymerization is most similar to distantly related Gram-negative organisms but that its mechanism for activating WASP family proteins is unique.

Actins↗
PubMed2005

Abi1 regulates the activity of N-WASP and WAVE in distinct actin-based processes.

Neural Wiskott-Aldrich syndrome protein (N-WASP) and WAVE are members of a family of proteins that use the Arp2/3 complex to stimulate actin assembly in actin-based motile processes. By entering into distinct macromolecular complexes, they act as convergent nodes of different signalling pathways. The role of WAVE in generating lamellipodial protrusion during cell migration is well established. Conversely, the precise cellular functions of N-WASP have remained elusive. Here, we report that Abi1, an essential component of the WAVE protein complex, also has a critical role in regulating N-WASP-dependent function. Consistently, Abi1 binds to N-WASP with nanomolar affinity and, cooperating with Cdc42, potently induces N-WASP activity in vitro. Molecular genetic approaches demonstrate that Abi1 and WAVE, but not N-WASP, are essential for Rac-dependent membrane protrusion and macropinocytosis. Conversely, Abi1 and N-WASP, but not WAVE, regulate actin-based vesicular transport, epidermal growth factor receptor (EGFR) endocytosis, and EGFR and transferrin receptor (TfR) cell-surface distribution. Thus, Abi1 is a dual regulator of WAVE and N-WASP activities in specific processes that are dependent on actin dynamics.

Actins↗
PubMed2005

Cdc42 is not essential for filopodium formation, directed migration, cell polarization, and mitosis in fibroblastoid cells.

Cdc42 is a small GTPase involved in the regulation of the cytoskeleton and cell polarity. To test whether Cdc42 has an essential role in the formation of filopodia or directed cell migration, we generated Cdc42-deficient fibroblastoid cells by conditional gene inactivation. We report here that loss of Cdc42 did not affect filopodium or lamellipodium formation and had no significant influence on the speed of directed migration nor on mitosis. Cdc42-deficient cells displayed a more elongated cell shape and had a reduced area. Furthermore, directionality during migration and reorientation of the Golgi apparatus into the direction of migration was decreased. However, expression of dominant negative Cdc42 in Cdc42-null cells resulted in strongly reduced directed migration, severely reduced single cell directionality, and complete loss of Golgi polarization and of directionality of protrusion formation toward the wound, as well as membrane blebbing. Thus, our data show that besides Cdc42 additional GTPases of the Rho-family, which share GEFs with Cdc42, are involved in the establishment and maintenance of cell polarity during directed migration.

Cell Adhesion↗
PubMed2005

N-WASP deficiency impairs EGF internalization and actin assembly at clathrin-coated pits.

WASP and WAVE family proteins promote actin polymerization by stimulating Arp2/3-complex-dependent filament nucleation. Unlike WAVE proteins, which are known to drive the formation of protrusions such as lamellipodia and membrane ruffles, vertebrate cell functions of WASP or N-WASP are less well established. Recent work demonstrated that clathrin-coated pit invagination can coincide with assembly of actin filaments and with accumulation of N-WASP and Arp2/3 complex, but the relevance of their recruitment has remained poorly defined. We employed two-colour total internal reflection microscopy to study the recruitment and dynamics of various components of the actin polymerization machinery and the epidermal growth factor receptor signalling machinery during clathrin-coated pit internalization in control cells and cells genetically deficient for functional N-WASP. We found that clathrin-coated pit endocytosis coincides with the recruitment of N-WASP, Arp2/3 complex and associated proteins, but not of WAVE family members. Actin accumulation at clathrin-coated pits requires the Arp2/3 complex, since Arp2/3 complex sequestration in the cytosol abolished any detectable actin assembly. The absence of N-WASP caused a significant reduction in the frequencies of actin and Arp2/3 complex accumulations at sites of clathrin-coated pit invagination and vesicle departure. Although N-WASP was not essential for Arp2/3-complex-mediated actin assembly at these sites or for EGF receptor-mediated endocytosis, N-WASP deficiency caused a marked reduction of EGF internalization. We conclude that the assembly of WASP subfamily proteins and associated factors at sites of clathrin-coated pit invagination amplifies actin accumulations at these sites promoting efficient internalization of ligands via clathrin-mediated endocytosis.

Actin-Related Protein 2-3 Complex↗
PubMed2005

Bacteria-host-cell interactions at the plasma membrane: stories on actin cytoskeleton subversion.

Exploitation of the host-cell actin cytoskeleton is pivotal for many microbial pathogens to enter cells, to disseminate within and between infected tissues, to prevent their uptake by phagocytic cells, or to promote intimate attachment to the cell surface. To accomplish this, these pathogens have evolved common as well as unique strategies to modulate actin dynamics at the plasma membrane, which will be discussed here, exemplified by a number of well-studied bacterial pathogens.

Actins↗
PubMed2004

Xin repeats define a novel actin-binding motif.

Xin is a protein that is expressed during early developmental stages of cardiac and skeletal muscles. Immunolocalization studies indicated a peripheral localization in embryonic mouse heart, where Xin localizes with beta-catenin and N-cadherin. In adult tissues, Xin is found primarily in the intercalated discs of cardiomyocytes and the myotendinous junctions of skeletal muscle cells, both specialized attachment sites of the myofibrillar ends to the sarcolemma. A large part of the Xin protein consists of unique 16 amino acid repeats with unknown function. We have investigated the characteristics of the Xin repeats by transfection experiments and actin-binding assays and ascertained that, upon expression in cultured cells, these repeats bind to and stabilize the actin-based cytoskeleton. In vitro co-sedimentation assays with skeletal muscle actin indicated that they not only directly bind actin filaments, but also have the capability of arranging microfilaments into networks that sediment upon low-speed centrifugation. Very similar repeats were also found in 'Xin-repeat protein 2' (XIRP2), a novel protein that seems to be expressed mainly in striated muscles. Human XIRP2 contains 28 Xin repeats with properties identical to those of Xin. We conclude that the Xin repeats define a novel, repetitive actin-binding motif present in at least two different muscle proteins. These Xin-repeat proteins therefore constitute the first two members of a novel family of actin-binding proteins.

Actins↗
PubMed2004

Sra-1 and Nap1 link Rac to actin assembly driving lamellipodia formation.

The Rho-GTPase Rac1 stimulates actin remodelling at the cell periphery by relaying signals to Scar/WAVE proteins leading to activation of Arp2/3-mediated actin polymerization. Scar/WAVE proteins do not interact with Rac1 directly, but instead assemble into multiprotein complexes, which was shown to regulate their activity in vitro. However, little information is available on how these complexes function in vivo. Here we show that the specifically Rac1-associated protein-1 (Sra-1) and Nck-associated protein 1 (Nap1) interact with WAVE2 and Abi-1 (e3B1) in resting cells or upon Rac activation. Consistently, Sra-1, Nap1, WAVE2 and Abi-1 translocated to the tips of membrane protrusions after microinjection of constitutively active Rac. Moreover, removal of Sra-1 or Nap1 by RNA interference abrogated the formation of Rac-dependent lamellipodia induced by growth factor stimulation or aluminium fluoride treatment. Finally, microinjection of an activated Rac failed to restore lamellipodia protrusion in cells lacking either protein. Thus, Sra-1 and Nap1 are constitutive and essential components of a WAVE2- and Abi-1-containing complex linking Rac to site-directed actin assembly.

3T3 Cells↗
PubMed2004

Pathogen-induced actin filament rearrangement in infectious diseases.

Host defence mechanisms involve the establishment and maintenance of numerous barriers to infectious microbes, including skin and mucosal surfaces, connective tissues, and a sophisticated immune system to detect and destroy invaders. Defeating these defence mechanisms and breaching the cell membrane barrier is the ultimate challenge for most pathogens. By invading the host and, moreover, by penetrating into individual host cells, pathogens gain access to a protective niche, not only to avoid immune clearance, but also to replicate and to disseminate from cell to cell within the infected host. Many pathogens are accomplishing these challenges by exploiting the actin cytoskeleton in a highly sophisticated manner as a result of having evolved common as well as unique strategies.

Actins↗
PubMed2004

Enterohaemorrhagic and enteropathogenic Escherichia coli use different mechanisms for actin pedestal formation that converge on N-WASP.

Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC), two closely related diarrhoeagenic pathogens, induce actin rearrangements at the surface of infected host cells resulting in the formation of pseudopod-like structures termed pedestals beneath intimately attached bacteria. We have shown previously that N-WASP, a key integrator of signalling pathways that regulate actin polymerization via the Arp2/3 complex, is essential for pedestal formation induced by EPEC using N-WASP-defective cell lines. Here we show that actin pedestal formation initiated by EHEC also depends on N-WASP. Amino acid residues 226-274 of N-WASP are both necessary and sufficient to target N-WASP to sites of EHEC attachment. The recruitment mechanism thus differs from that used by EPEC, in which amino-terminal sequences of N-WASP mediate recruitment. For EPEC, recruitment of N-WASP downstream of Nck has been postulated to be mediated by WIP. However, we find a direct interaction of N-WASP with WIP to be dispensable for EPEC-induced pedestal formation and present data supporting an F-actin-dependent localization of WIP to actin pedestals induced by both EPEC and EHEC. In summary, our data show that EPEC and EHEC use different mechanisms to recruit N-WASP, which is essential for actin pedestal formation induced by both pathogens.

Actins↗
PubMed2003

Actin-based motility of Burkholderia pseudomallei involves the Arp 2/3 complex, but not N-WASP and Ena/VASP proteins.

The facultative intracellular bacterium Burkholderia pseudomallei induces actin rearrangement within infected host cells leading to formation of actin tails and membrane protrusions. To investigate the underlying mechanism we analysed the contribution of cytoskeletal proteins to B. pseudomallei-induced actin tail assembly. By using green fluorescent protein (GFP)-fusion constructs, the recruitment of the Arp2/3 complex, vasodilator-stimulated phosphoprotein (VASP), Neural Wiskott-Aldrich syndrome protein (N-WASP), zyxin, vinculin, paxillin and alpha-actinin to the surface of B. pseudomallei and into corresponding actin tails was studied. In addition, antibodies against the same panel of proteins were used for immunolocalization. Whereas the Arp2/3 complex and alpha-actinin were incorporated into B. pseudomallei-induced actin tails, none of the other proteins were detected in these structures. The overexpression of an Arp2/3 binding fragment of the Scar1 protein, shown previously to block actin-based motility of Listeria, had no effect on B. pseudomallei tail formation. Infections of either N-WASP- or Ena/VASP-defective cells showed that these proteins are not essential for B. pseudomallei-induced actin polymerization. In conclusion, our results suggest that B. pseudomallei induces actin polymerization through a mechanism that differs from those evolved by Listeria, Shigella, Rickettsia or vaccinia virus.

Actin-Related Protein 2↗
PubMed2002

Phosphatidylinositol 4,5-biphosphate (PIP2)-induced vesicle movement depends on N-WASP and involves Nck, WIP, and Grb2.

Wiskott-Aldrich syndrome protein (WASP)/Scar family proteins promote actin polymerization by stimulating the actin-nucleating activity of the Arp2/3 complex. While Scar/WAVE proteins are thought to be involved in lamellipodia protrusion, the hematopoietic WASP has been implicated in various actin-based processes such as chemotaxis, podosome formation, and phagocytosis. Here we show that the ubiquitously expressed N-WASP is essential for actin assembly at the surface of endomembranes induced as a consequence of increased phosphatidylinositol 4,5-biphosphate (PIP2) levels. This process resulting in the motility of intracellular vesicles at the tips of actin comets involved the recruitment of the Src homology 3 (SH3)-SH2 adaptor proteins Nck and Grb2 as well as of WASP interacting protein (WIP). Reconstitution of vesicle movement in N-WASP-defective cells by expression of various N-WASP mutant proteins revealed three independent domains capable of interaction with the vesicle surface, of which both the WH1 and the polyproline domains contributed significantly to N-WASP recruitment and/or activation. In contrast, the direct interaction of N-WASP with the Rho-GTPase Cdc42 was not required for reconstitution of vesicle motility. Our data reveal a distinct cellular phenotype for N-WASP loss of function, which adds to accumulating evidence that the proposed link between actin and membrane dynamics may, at least partially, be reflected by the actin-based movement of vesicles through the cytoplasm.

Adaptor Proteins, Signal Transducing↗
PubMed2002

The lamellipodium: where motility begins.

Lamellipodia, filopodia and membrane ruffles are essential for cell motility, the organization of membrane domains, phagocytosis and the development of substrate adhesions. Their formation relies on the regulated recruitment of molecular scaffolds to their tips (to harness and localize actin polymerization), coupled to the coordinated organization of actin filaments into lamella networks and bundled arrays. Their turnover requires further molecular complexes for the disassembly and recycling of lamellipodium components. Here, we give a spatial inventory of the many molecular players in this dynamic domain of the actin cytoskeleton in order to highlight the open questions and the challenges ahead.

Actins↗