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

ABSTRACT: 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.

SUBMITTER: Steffen A

PROVIDER: S-EPMC1474932 | biostudies-literature | 2006 Jun

REPOSITORIES: biostudies-literature

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

Steffen Anika A Faix Jan J Resch Guenter P GP Linkner Joern J Wehland Juergen J Small J Victor JV Rottner Klemens K Stradal Theresia E B TE

Molecular biology of the cell 20060405 6

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 mode ...[more]

PMID: 16597702

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Project description:Deleting components of the Arp2/3 complex in Candida albicans resulted in a global lack of hyphal specific gene induction. This observation suggests that the failure in hyphal growth of Arp2/3 complex mutants could be a result of failure to activate hyphal specific genes. If the hyphal defect was primarily due to failure to activate gene expression, de-repressing hyphal-specific gene expression by deleting the NRG1 repressor could potentially suppress the defect, as deletion of NRG1 leads to constitutive filamentous growth even in the absence of any hyphal induction signals (Garcia-Sanchez et al., 2005, Kadosh & Johnson, 2005). We therefore created an nrg1Î/Îarp2Î/Î mutant. When grown under non-inducing conditions, nrg1Î/Îarp2Î/Î cells showed the arp2Î/Î mutant morphology of round and swollen cells. When induced for hyphal growth, nrg1Î/Îarp2Î/Î cells also exhibited the arp2Î/Î cell morphology and did not form hyphae even after extended overnight incubation times. To determine if the hyphal-specific genes are de-repressed in the nrg1Î/Îarp2Î/Î mutant, we performed transcript profiling. We compared the nrg1Î/Îarp2Î/Î mutant grown under hyphal conditions to the arp2Î/Î mutant grown under the same conditions (10% serum, 37ï°C, three hours), and found that a significant number of hyphal-specific genes that are normally induced when WT cells are undergoing the yeast to hyphae switch (WT-HY) showed greater expression in the nrg1Î/Îarp2Î/Î mutant compared to arp2Î/Î cells (p-value 4.9x10-9). When we examined the set of NRG1-dependent hyphal-specific genes previously identified (Kadosh & Johnson, 2005), we found that seven of 28 genes (HYR1, SAP5, SAP4, KIP4, ORF19.6079, ALS3 and UME6) showed significantly increased expression (â¥ 2 fold) in nrg1Î/Îarp2Î/Î cells compared to arp2Î/Î cells, while a further four genes (IHD1, CBP1, ORF19.6705 and ALS10) showed moderately increased expression between 1.5 and 2-fold. Thus, while deleting a transcriptional repressor of the filamentation program leads to de-repression of many hyphal genes, the entire regulated gene set is not de-repressed; this presumably reflects the complex interplay that different transcriptional (co-) repressors exert on the yeast-to-hyphae transition (Garcia-Sanchez et al., 2005, Kadosh & Johnson, 2005). We further found that despite the increased induction of some hyphal genes in the nrg1Î/Îarp2Î/Î mutant, a few of those genes are not as highly induced as in WT cells. One gene that was induced in both the ânrg1Î/Îarp2Î/Î vs arp2Î/Îâ and the ânrg1Î/Îarp2Î/Î vs WTâ-comparisons is UME6, a recently identified key regulator of the hyphal program (Banerjee et al., 2008, Zeidler et al., 2009). Interestingly, although constitutive over-expression of UME6 in WT cells resulted in constitutive filamentous growth even in the absence of hyphae signals (Carlisle et al., 2009), the increased expression level of UME6 in the nrg1Î/Îarp2Î/Î mutant is not sufficient to restore filamentation in the absence of a functional Arp2/3 complex. Thus despite partial de-repression of the hyphal program, hyphae do not form, making it likely other roles of the Arp2/3 complex, such as its function in actin patch formation and actin branching, are required for hyphal development.

Dataset Information

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

Publications

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

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