Biomedical subjects

Jan Faix

Publications and source records attributed to Jan Faix.

13 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

Trix, a novel Rac guanine-nucleotide exchange factor from Dictyostelium discoideum is an actin-binding protein and accumulates at endosomes.

Small Rho family GTPases are involved in regulation of actin cytoskeleton dynamics. These molecular switches are themselves mainly controlled by specific GTPase-activating proteins (GAPs) and guanine-nucleotide exchange factors (GEFs). We have cloned and initially characterized a novel putative RhoGEF from Dictyostelium discoideum. The predicted 135-kDa protein displays a unique domain organization in its N-terminus by harboring two type3 calponin homology (CH) domains followed by a single type1 CH domain. The C-terminal region encompasses a diffuse B-cell lymphoma homology/pleckstrin homology tandem domain that is typically found in RhoGEFs. We therefore refer to this protein as Trix (triple CH-domain array exchange factor). A recombinant N-terminal region of Trix carrying all three CH domains binds to F-actin and bundles actin filaments. Trix-null mutants are viable and display only subtle defects when compared to wild-type cells with the exception of a substantial decrease in exocytosis of a fluid-phase marker. GFP fusions with the full-length protein or the N-terminal part containing all three CH domains revealed that Trix localizes to the cortical region and strongly accumulates on late endosomes. Our results suggest that Trix is specifically involved in a Rho GTPase-signaling pathway that is required for regulation of the actin cytoskeleton during exocytosis.

Actin Cytoskeleton↗

PubMed2006

The bundling activity of vasodilator-stimulated phosphoprotein is required for filopodium formation.

Filopodia are highly dynamic finger-like cell protrusions filled with parallel bundles of actin filaments. Previously we have shown that Diaphanous-related formin dDia2 is involved in the formation of filopodia. Another key player for the formation of filopodia across many species is vasodilator-stimulated phosphoprotein (VASP). It has been proposed that the essential role of VASP for formation of filopodia is its competition with capping proteins for filament barbed-end interaction. To better understand the function of VASP in filopodium formation, we analyzed the in vitro and in vivo properties of Dictyostelium VASP (DdVASP) and extended our findings to human VASP. Recombinant VASP from both species nucleated and bundled actin filaments, but did not compete with capping proteins or block depolymerization from barbed ends. Together with the finding that DdVASP binds to the FH2 domain of dDia2, these data indicate that the crucial role of VASP in filopodium formation is different from uncapping of actin filaments. To identify the activity of DdVASP required in this process, rescue experiments of DdVASP-null cells with mutant DdVASP constructs were performed. Only WT DdVASP, but not a mutant lacking the F-actin bundling activity, could rescue the ability of these cells to form WT-like filopodia. Our data suggest that DdVASP is complexed with dDia2 in filopodial tips and support formin-mediated filament elongation by bundling nascent actin filaments.

Actins↗

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↗

PubMed2006

Staying in shape with formins.

Formins constitute a diverse protein family present in all eukaryotes examined. They are defined by the presence of a formin homology 2 (FH2) domain, which possesses intrinsic and conserved functions regulating cytoskeletal dynamics. Over the past few years, formins have become recognized as potent nucleators of linear actin filaments that control a large variety of cellular and morphogenetic functions. Here, we review the molecular principles of formin-induced cytoskeletal rearrangements and their consequences for a growing number of biological processes.

Actin Cytoskeleton↗

PubMed2006

Generation of multiple knockout mutants using the Cre-loxP system.

Dictyostelium discoideum is an exceptionally powerful system by which to study various aspects of modern cell and developmental biology. The completion of the genome sequencing project together with a high-efficiency of targeted gene disruption have enabled researchers to characterize many specific gene functions. However, as a result of many gene products with overlapping functions, there is great need to produce mutants carrying mutations in multiple genes. We have, therefore, developed a robust system and describe a protocol for the generation of multiple gene mutations in Dictyostelium by recycling the Blasticidin S selectable marker after transient expression of Cre recombinase.

Animals↗

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

The Diaphanous-related formin dDia2 is required for the formation and maintenance of filopodia.

Formins have important roles in the nucleation of actin and the formation of linear actin filaments, but their role in filopodium formation has remained elusive. Dictyostelium discoideum Diaphanous-related formin dDia2 is enriched at the tips of filopodia and interacts with profilin II and Rac1. An FH1FH2 fragment of dDia2 nucleated actin polymerization and removed capping protein from capped filament ends. Genetic studies showed that dDia2 is important for cell migration as well as the formation, elongation and maintenance of filopodia. Here we provide evidence that dDia2 specifically controls filopodial dynamics by regulating actin turnover at the barbed ends of actin filaments.

Actin Cytoskeleton↗

PubMed2005

Dictyostelium LIS1 is a centrosomal protein required for microtubule/cell cortex interactions, nucleus/centrosome linkage, and actin dynamics.

The widespread LIS1-proteins were originally identified as the target for sporadic mutations causing lissencephaly in humans. Dictyostelium LIS1 (DdLIS1) is a microtubule-associated protein exhibiting 53% identity to human LIS1. It colocalizes with dynein at isolated, microtubule-free centrosomes, suggesting that both are integral centrosomal components. Replacement of the DdLIS1 gene by the hypomorphic D327H allele or overexpression of an MBP-DdLIS1 fusion disrupted various dynein-associated functions. Microtubules lost contact with the cell cortex and were dragged behind an unusually motile centrosome. Previously, this phenotype was observed in cells overexpressing fragments of dynein or the XMAP215-homologue DdCP224. DdLIS1 was coprecipitated with DdCP224, suggesting that both act together in dynein-mediated cortical attachment of microtubules. Furthermore, DdLIS1-D327H mutants showed Golgi dispersal and reduced centrosome/nucleus association. Defects in DdLIS1 function also altered actin dynamics characterized by traveling waves of actin polymerization correlated with a reduced F-actin content. DdLIS1 could be involved in actin dynamics through Rho-GTPases, because DdLIS1 interacted directly with Rac1A in vitro. Our results show that DdLIS1 is required for maintenance of the microtubule cytoskeleton, Golgi apparatus and nucleus/centrosome association, and they suggest that LIS1-dependent alterations of actin dynamics could also contribute to defects in neuronal migration in lissencephaly patients.

Actins↗

PubMed2004

A rapid and efficient method to generate multiple gene disruptions in Dictyostelium discoideum using a single selectable marker and the Cre-loxP system.

Dictyostelium discoideum has proven an exceptionally powerful system for studying numerous aspects of cellular and developmental functions. The relatively small ( approximately 34 Mb) chromosomal genome of Dictyostelium and high efficiency of targeted gene disruption have enabled researchers to characterize many specific gene functions. However, the number of selectable markers in Dictyostelium is restricted, as is the ability to perform effective genetic crosses between strains. Thus, it has been difficult to create multiple mutations within an individual cell to study epistatic relationships among genes or potential redundancies between various pathways. We now describe a robust system for the production of multiple gene mutations in Dictyostelium by recycling a single selectable marker, Blasticidin S resistance, using the Cre-loxP system. We confirm the effectiveness of the system by generating a single cell carrying four separate gene disruptions. Furthermore, the cells remain sensitive to transformation for additional targeted or random mutagenesis requiring Blasticidin selection and for functional expression studies of mutated or tagged proteins using other selectable markers.

Animals↗

PubMed2004

Actin-binding proteins required for reliable chromosome segregation in mitosis.

While studying mitosis in Dictyostelium mutants with deficiencies in actin-binding proteins, we found that two of these proteins, cortexillin and Aip1, are required for the precise segregation of chromosomes. Atypical spindles and nuclei with varying DNA content indicate that mutants lacking cortexillin or Aip1 are genetically unstable. These aberrations are caused by the detachment and irregular reattachment of centrosomes to the nuclear surface. Live imaging showed how coalescing mitotic complexes give rise to a multipolar spindle, and how excess centrosomes can be eliminated by mitotic cleavage between anucleate and nucleated portions of a cell. We hypothesize that mutations in regulatory proteins of the actin network might be one cause of genetic instability of malignant tumor cells.

Actin Cytoskeleton↗

PubMed2003

A Lim protein involved in the progression of cytokinesis and regulation of the mitotic spindle.

DdLimE regulates cell motility and cytokinesis in Dictyostelium. To specify its function, we generated knock-out mutants and analyzed mitosis by marking the mitotic apparatus with GFP-alpha-tubulin. Characteristic of DdLimE-null cells is a late reversal of cytokinesis caused by backward movement of the incipient daughter cells. This process of "retro-cytokinesis" is accompanied by a delay in disassembly of the mitotic spindle. The length of interphase microtubules is increased and their depolymerization at prophase is impaired. These data indicate that DdLimE links the cortical actin network, where it is located, to the microtubule system, whose dynamics it regulates.

Animals↗

PubMed2002

Differential localization of the Dictyostelium kinase DPAKa during cytokinesis and cell migration.

The Dictyostelium kinase DPAKa is a member of the p21-activated kinase (PAK) family, consisting of an N-terminal domain characterized by a coiled-coil region and proline-rich motifs, a Rac-binding CRIB-domain, and a highly conserved C-terminal kinase domain. In this study we show that cells overexpressing a C-terminal DPAKa fragment comprising the kinase domain are significantly impaired in motility and phagocytosis, whereas DPAKa-null cells display no obvious phenotypic change. We analyzed the in vivo localization of full-length and truncated DPAKa tagged with green fluorescent protein (GFP). The N-terminal fragments show a highly dynamic cortical localization without a permanent polarized enrichment, whereas the C-terminal fragment is homogenously distributed throughout the cell. The localization of full-length DPAKa is similar to that of myosin II at the rear end of locomoting cells and at the base of phagocytic cups. During mitosis DPAKa is gradually recruited to the cell cortex starting at metaphase, which also parallels the dynamics of myosin II cortical recruitment. However, in contrast to myosin II, DPAKa does not accumulate in the cleavage furrow but stays uniformly distributed throughout the cell cortex. This finding contrasts with previous work claiming accumulation of DPAKa in the cleavage furrow of dividing cells. Our results suggest that the N-terminus directs DPAKa to the cortex, and the C-terminus is necessary for restricting its localization to the rear of moving cells during chemotaxis. Therefore, DPAKa may play distinct roles in myosin II regulation during cell movement and cell division.

Animals↗