Formin-dependent actin assembly is regulated by distinct modes of Rho signaling in yeast

Formins are actin filament nucleators regulated by Rho-GTPases. In budding yeast, the formins Bni1p and Bnr1p direct the assembly of actin cables, which guide polarized secretion and growth. From the six yeast Rho proteins (Cdc42p and Rho1–5p), we have determined that four participate in the regulation of formin activity. We show that the essential function of Rho3p and Rho4p is to activate the formins Bni1p and Bnr1p, and that activated alleles of either formin are able to bypass the requirement for these Rho proteins. Through a separate signaling pathway, Rho1p is necessary for formin activation at elevated temperatures, acting through protein kinase C (Pkc1p), the major effector for Rho1p signaling to the actin cytoskeleton. Although Pkc1p also activates a MAPK pathway, this pathway does not function in formin activation. Formin-dependent cable assembly does not require Cdc42p, but in the absence of Cdc42p function, cable assembly is not properly organized during initiation of bud growth. These results show that formin function is under the control of three distinct, essential Rho signaling pathways

FHOD‐1 is the only formin in Caenorhabditis elegans that promotes striated muscle growth and Z‐line organization in a cell autonomous manner

The striated body wall muscles of Caenorhabditis elegans are a simple model for sarcomere assembly. Previously, we observed deletion mutants for two formin genes, fhod‐1 and cyk‐1, develop thin muscles with abnormal dense bodies (the sarcomere Z‐line analogs). However, this work left in question whether these formins work in a muscle cell autonomous manner, particularly since cyk‐1(Δ) deletion has pleiotropic effects on development. Using a fast acting temperature‐sensitive cyk‐1(ts) mutant, we show here that neither post‐embryonic loss nor acute loss of CYK‐1 during embryonic sarcomerogenesis cause lasting muscle defects. Furthermore, mosaic expression of CYK‐1 in cyk‐1(Δ) mutants is unable to rescue muscle defects in a cell autonomous manner, suggesting muscle phenotypes caused by cyk‐1(Δ) are likely indirect. Conversely, mosaic expression of FHOD‐1 in fhod‐1(Δ) mutants promotes muscle cell growth and proper dense body organization in a muscle cell autonomous manner. As we observe no effect of loss of any other formin on muscle development, we conclude FHOD‐1 is the only worm formin that directly promotes striated muscle development, and the effects on formin loss in C. elegans are surprisingly modest compared to other systems

The mating-specific Gα interacts with a kinesin-14 and regulates pheromone-induced nuclear migration in budding yeast

As a budding yeast cell elongates toward its mating partner, cytoplasmic microtubules connect the nucleus to the cell cortex at the growth tip. The Kar3 kinesin-like motor protein is then thought to stimulate plus-end depolymerization of these microtubules, thus drawing the nucleus closer to the site where cell fusion and karyogamy will occur. Here, we show that pheromone stimulates a microtubule-independent interaction between Kar3 and the mating-specific Gα protein Gpa1 and that Gpa1 affects both microtubule orientation and cortical contact. The membrane localization of Gpa1 was found to polarize early in the mating response, at about the same time that the microtubules begin to attach to the incipient growth site. In the absence of Gpa1, microtubules lose contact with the cortex upon shrinking and Kar3 is improperly localized, suggesting that Gpa1 is a cortical anchor for Kar3. We infer that Gpa1 serves as a positional determinant for Kar3-bound microtubule plus ends during mating. © 2009 by The American Society for Cell Biology

Novel Roles of Formin mDia2 in Lamellipodia and Filopodia Formation in Motile Cells

Actin polymerization-driven protrusion of the leading edge is a key element of cell motility. The important actin nucleators formins and the Arp2/3 complex are believed to have nonoverlapping functions in inducing actin filament bundles in filopodia and dendritic networks in lamellipodia, respectively. We tested this idea by investigating the role of mDia2 formin in leading-edge protrusion by loss-of-function and gain-of-function approaches. Unexpectedly, mDia2 depletion by short interfering RNA (siRNA) severely inhibited lamellipodia. Structural analysis of the actin network in the few remaining lamellipodia suggested an mDia2 role in generation of long filaments. Consistently, constitutively active mDia2 (ΔGBD-mDia2) induced accumulation of long actin filaments in lamellipodia and increased persistence of lamellipodial protrusion. Depletion of mDia2 also inhibited filopodia, whereas expression of ΔGBD-mDia2 promoted their formation. Correlative light and electron microscopy showed that ΔGBD-mDia2–induced filopodia were formed from lamellipodial network through gradual convergence of long lamellipodial filaments into bundles. Efficient filopodia induction required mDia2 targeting to the membrane, likely through a scaffolding protein Abi1. Furthermore, mDia2 and Abi1 interacted through the N-terminal regulatory sequences of mDia2 and the SH3-containing Abi1 sequences. We propose that mDia2 plays an important role in formation of lamellipodia by nucleating and/or protecting from capping lamellipodial actin filaments, which subsequently exhibit high tendency to converge into filopodia

Asymmetric distribution of Echinoid defines the epidermal leading edge during Drosophila dorsal closure

Upon loss of a binding partner in apposed tissue, the homophilic cell adhesion protein Echinoid adopts a planar polarized localization, which promotes the planar polarized localization of the planar cell polarity protein Bazooka/Par-3 and targets actomyosin cable assembly to the epidermal leading edge, thus establishing the migration direction of the developing epidermis

A Single Molecule Scaffold for the Maize Genome

About 85% of the maize genome consists of highly repetitive sequences that are interspersed by low-copy, gene-coding sequences. The maize community has dealt with this genomic complexity by the construction of an integrated genetic and physical map (iMap), but this resource alone was not sufficient for ensuring the quality of the current sequence build. For this purpose, we constructed a genome-wide, high-resolution optical map of the maize inbred line B73 genome containing >91,000 restriction sites (averaging 1 site/∼23 kb) accrued from mapping genomic DNA molecules. Our optical map comprises 66 contigs, averaging 31.88 Mb in size and spanning 91.5% (2,103.93 Mb/∼2,300 Mb) of the maize genome. A new algorithm was created that considered both optical map and unfinished BAC sequence data for placing 60/66 (2,032.42 Mb) optical map contigs onto the maize iMap. The alignment of optical maps against numerous data sources yielded comprehensive results that proved revealing and productive. For example, gaps were uncovered and characterized within the iMap, the FPC (fingerprinted contigs) map, and the chromosome-wide pseudomolecules. Such alignments also suggested amended placements of FPC contigs on the maize genetic map and proactively guided the assembly of chromosome-wide pseudomolecules, especially within complex genomic regions. Lastly, we think that the full integration of B73 optical maps with the maize iMap would greatly facilitate maize sequence finishing efforts that would make it a valuable reference for comparative studies among cereals, or other maize inbred lines and cultivars

Probing the origins of metazoan formin diversity: Evidence for evolutionary relationships between metazoan and non-metazoan formin subtypes.

Formins are proteins that assist in regulating cytoskeletal organization through interactions with actin filaments and microtubules. Metazoans encode nine distinct formin subtypes based on sequence similarity, potentially allowing for great functional diversity for these proteins. Through the evolution of the eukaryotes, formins are believed to have repeatedly undergone rounds of gene duplications, followed by diversification and domain shuffling, but previous phylogenetic analyses have shed only a little light on the specific origins of different formin subtypes. To improve our understanding of this in the case of the metazoan formins, phylogenetic comparisons were made here of a broad range of metazoan and non-metazoan formin sequences. This analysis suggests a model in which eight of the nine metazoan formin subtypes arose from two ancestral proteins that were present in an ancient unikont ancestor. Additionally, evidence is shown suggesting the common ancestor of unikonts and bikonts was likely to have encoded at least two formins, a canonical Drf-type protein and a formin bearing a PTEN-like domain

Revisiting the Phylogeny of the Animal Formins: Two New Subtypes, Relationships with Multiple Wing Hairs Proteins, and a Lost Human Formin

<div><p>Formins are a widespread family of eukaryotic cytoskeleton-organizing proteins. Many species encode multiple formin isoforms, and for animals, much of this reflects the presence of multiple conserved subtypes. Earlier phylogenetic analyses identified seven major formin subtypes in animals (DAAM, DIAPH, FHOD, FMN, FMNL, INF, and GRID2IP/delphilin), but left a handful of formins, particularly from nematodes, unassigned. In this new analysis drawing from genomic data from a wider range of taxa, nine formin subtypes are identified that encompass all the animal formins analyzed here. Included in this analysis are Multiple Wing Hairs proteins (MWH), which bear homology to formin N-terminal domains. Originally identified in <i>Drosophila melanogaster</i> and other arthropods, MWH-related proteins are also identified here in some nematodes (including <i>Caenorhabditis elegans</i>), and are shown to be related to a novel MWH-related formin (MWHF) subtype. One surprising result of this work is the discovery that a family of pleckstrin homology domain-containing formins (PHCFs) is represented in many vertebrates, but is strikingly absent from placental mammals. Consistent with a relatively recent loss of this formin, the human genome retains fragments of a defunct homologous formin gene.</p></div