FHOD formin and SRF promote post-embryonic striated muscle growth through separate pathways in C. elegans.

Previous work with cultured cells has shown transcription of muscle genes by serum response factor (SRF) can be stimulated by actin polymerization driven by proteins of the formin family. However, it is not clear if endogenous formins similarly promote SRF-dependent transcription during muscle development in vivo. We tested whether formin activity promotes SRF-dependent transcription in striated muscle in the simple animal model, Caenorhabditis elegans. Our lab has shown FHOD-1 is the only formin that directly promotes sarcomere formation in the worm's striated muscle. We show here FHOD-1 and SRF homolog UNC-120 both support muscle growth and also muscle myosin II heavy chain A expression. However, while a hypomorphic unc-120 allele blunts expression of a set of striated muscle genes, these genes are largely upregulated or unchanged by absence of FHOD-1. Instead, pharmacological inhibition of the proteasome restores myosin protein levels in worms lacking FHOD-1, suggesting elevated proteolysis accounts for their myosin deficit. Interestingly, proteasome inhibition does not restore normal muscle growth to fhod-1(Δ) mutants, suggesting formin contributes to muscle growth by some alternative mechanism. Overall, we find SRF does not depend on formin to promote muscle gene transcription in a simple in vivo system.

Assembly and Maintenance of Myofibrils in Striated Muscle.

In this chapter, we present the current knowledge on de novo assembly, growth, and dynamics of striated myofibrils, the functional architectural elements developed in skeletal and cardiac muscle. The data were obtained in studies of myofibrils formed in cultures of mouse skeletal and quail myotubes, in the somites of living zebrafish embryos, and in mouse neonatal and quail embryonic cardiac cells. The comparative view obtained revealed that the assembly of striated myofibrils is a three-step process progressing from premyofibrils to nascent myofibrils to mature myofibrils. This process is specified by the addition of new structural proteins, the arrangement of myofibrillar components like actin and myosin filaments with their companions into so-called sarcomeres, and in their precise alignment. Accompanying the formation of mature myofibrils is a decrease in the dynamic behavior of the assembling proteins. Proteins are most dynamic in the premyofibrils during the early phase and least dynamic in mature myofibrils in the final stage of myofibrillogenesis. This is probably due to increased interactions between proteins during the maturation process. The dynamic properties of myofibrillar proteins provide a mechanism for the exchange of older proteins or a change in isoforms to take place without disassembling the structural integrity needed for myofibril function. An important aspect of myofibril assembly is the role of actin-nucleating proteins in the formation, maintenance, and sarcomeric arrangement of the myofibrillar actin filaments. This is a very active field of research. We also report on several actin mutations that result in human muscle diseases.

FHOD-1/profilin-mediated actin assembly protects sarcomeres against contraction-induced deformation in C. elegans.

Formin HOmology Domain 2-containing (FHOD) proteins are a subfamily of actin-organizing formins important for striated muscle development in many animals. We showed previously that absence of the sole FHOD protein, FHOD-1, from C. elegans results in thin body-wall muscles with misshapen dense bodies that serve as sarcomere Z-lines. We demonstrate here that actin polymerization by FHOD-1 is required for its function in muscle development, and that FHOD-1 cooperates with profilin PFN-3 for dense body morphogenesis, and profilins PFN-2 and PFN-3 to promote body-wall muscle growth. We further demonstrate dense bodies in fhod-1 and pfn-3 mutants are less stable than in wild type animals, having a higher proportion of dynamic protein, and becoming distorted by prolonged muscle contraction. We also observe accumulation of actin depolymerization factor/cofilin homolog UNC-60B in body-wall muscle of these mutants. Such accumulations may indicate targeted disassembly of thin filaments dislodged from unstable dense bodies, and may account for the abnormally slow growth and reduced strength of body-wall muscle in fhod-1 mutants. Overall, these results show the importance of FHOD protein-mediated actin assembly to forming stable sarcomere Z-lines, and identify profilin as a new contributor to FHOD activity in striated muscle development.

FHOD-1 and profilin protect sarcomeres against contraction-induced deformation> in C. elegans.

Formin HOmology Domain 2-containing (FHOD) proteins are a subfamily of actin-organizing formins important for striated muscle development in many animals. We showed previously that absence of the sole FHOD protein, FHOD-1, from Caenorhabditis elegans results in thin body wall muscles with misshapen dense bodies that serve as sarcomere Z-lines. We demonstrate here that mutations predicted to specifically disrupt actin polymerization by FHOD-1 similarly disrupt muscle development, and that FHOD-1 cooperates with profilin PFN-3 for dense body morphogenesis, and with profilins PFN-2 and PFN-3 to promote body wall muscle growth. We further demonstrate that dense bodies in worms lacking FHOD-1 or PFN-2/PFN-3 are less stable than in wild-type animals, having a higher proportion of dynamic protein, and becoming distorted by prolonged muscle contraction. We also observe accumulation of actin and actin depolymerization factor/cofilin homologue UNC-60B in body wall muscle of these mutants. Such accumulations may indicate targeted disassembly of thin filaments dislodged from unstable dense bodies, possibly accounting for the abnormally slow growth and reduced body wall muscle strength in fhod-1 mutants. Overall, these results implicate FHOD protein-mediated actin assembly in forming stable sarcomere Z-lines, and identify profilin as a new contributor to FHOD activity in striated muscle development.

Biochemical and cell biological analysis of actin in the nematode Caenorhabditis elegans.

The nematode Caenorhabditis elegans has long been a useful model organism for muscle research. Its body wall muscle is obliquely striated muscle and exhibits structural similarities with vertebrate striated muscle. Actin is the core component of the muscle thin filaments, which are highly ordered in sarcomeric structures in striated muscle. Genetic studies have identified genes that regulate proper organization and function of actin filaments in C. elegans muscle, and sequence of the worm genome has revealed a number of conserved candidate genes that may regulate actin. To precisely understand the functions of actin-binding proteins, such genetic and genomic studies need to be complemented by biochemical characterization of these actin-binding proteins in vitro. This article describes methods for purification and biochemical characterization of actin from C. elegans. Although rabbit muscle actin is commonly used to characterize actin-binding proteins from many eukaryotic organisms, we detect several quantitative differences between C. elegans actin and rabbit muscle actin, highlighting that use of actin from an appropriate source is important in some cases. Additionally, we describe probes for cell biological analysis of actin in C. elegans.

Formins direct Arp2/3-independent actin filament assembly to polarize cell growth in yeast.

Formins have been implicated in the regulation of cytoskeletal structure in animals and fungi. Here we show that the formins Bni1 and Bnr1 of budding yeast stimulate the assembly of actin filaments that function as precursors to tropomyosin-stabilized cables that direct polarized cell growth. With loss of formin function, cables disassemble,whereas increased formin activity causes the hyperaccumulation of cable-like filaments. Unlike the assembly of cortical actin patches, cable assembly requires profilin but not the Arp2/3 complex. Thus formins control a distinct pathway for assembling actin filaments that organize the overall polarity of the cell.

Formins direct Arp2/3-independent actin filament assembly to polarize cell growth in yeast.

Formins have been implicated in the regulation of cytoskeletal structure in animals and fungi. Here we show that the formins Bni1 and Bnr1 of budding yeast stimulate the assembly of actin filaments that function as precursors to tropomyosin-stabilized cables that direct polarized cell growth. With loss of formin function, cables disassemble, whereas increased formin activity causes the hyperaccumulation of cable-like filaments. Unlike the assembly of cortical actin patches, cable assembly requires profilin but not the Arp2/3 complex. Thus formins control a distinct pathway for assembling actin filaments that organize the overall polarity of the cell.

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(∆) deletion has pleiotropic effects on development. Using a fast acting temperature-sensitive cyk-1(ts) mutant, we show here that neither postembryonic loss nor acute loss of CYK-1 during embryonic sarcomerogenesis cause lasting muscle defects. Furthermore, mosaic expression of CYK-1 in cyk-1(∆) mutants is unable to rescue muscle defects in a cell autonomous manner, suggesting muscle phenotypes caused by cyk-1(∆) 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.

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.

Functional importance of an inverted formin C-terminal tail at morphologically dynamic epithelial junctions.

Epithelial cell-cell junctions have dual roles of accommodating morphological changes in an epithelium, while maintaining cohesion during those changes. An abundance of junction proteins has been identified, but many details on how intercellular junctions respond to morphological changes remain unclear. In Caenorhabditis elegans, the spermatheca is an epithelial sac that repeatedly dilates and constricts to allow ovulation. It is thought that the junctions between spermatheca epithelial cells undergo reversible partial unzipping to allow rapid dilation. Previously, we found that EXC-6, a C. elegans protein homolog of the human disease-associated formin INF2, is expressed in the spermatheca and promotes oocyte entry. We show here that EXC-6 localizes toward the apical aspect of the spermatheca epithelial junctions, and that the EXC-6-labeled junction domains "unzip" and dramatically flatten with oocyte entry into the spermatheca. We demonstrate that the C-terminal tail of EXC-6 is necessary and sufficient for junction localization. Moreover, expression of the tail alone worsens ovulation defects, suggesting this region not only mediates EXC-6 localization, but also interacts with other components important for junction remodeling.

Tropomyosin function in yeast.

Tropomyosins were discovered as regulators of actomyosin contractility in muscle cells, making yeasts and other fungi seem unlikely to harbor such proteins. Fungal cells are encased in a rigid cell wall and do not engage in the same sorts of contractile shape changes of animal cells. However, discovery of actin and myosin in yeast raised the possibility for a role for tropomyosin in regulating their interaction. Through a biochemical search, fungal tropomyosins were identified with strong similarities to their animal counterparts in terms ofprotein structure and physical properties. Two particular fungi, the buddingyeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe, have provided powerful genetic systems for studying tropomyosins in nonmetazoans. In these yeasts, tropomyosins associate with subsets ofactin filamentous structures. Mutational studies oftropomyosin genes and biochemical assays of purified proteins point to roles for these proteins as factors that stabilize actin filaments, promote actin-based structures of particular architecture and help maintain distinct biochemical identities among different filament populations. Tropomyosin-enriched filaments are the cytoskeletal structures that promote the major cell shape changes of these organisms: polarized growth and cell division.

Tissue-Specific Functions of fem-2/PP2c Phosphatase and fhod-1/formin During Caenorhabditis elegans Embryonic Morphogenesis.

The cytoskeleton is the basic machinery that drives many morphogenetic events. Elongation of the C. elegans embryo from a spheroid into a long, thin larva initially results from actomyosin contractility, mainly in the lateral epidermal seam cells, while the corresponding dorsal and ventral epidermal cells play a more passive role. This is followed by a later elongation phase involving muscle contraction. Early elongation is mediated by parallel genetic pathways involving LET-502/Rho kinase and MEL-11/MYPT myosin phosphatase in one pathway and FEM-2/PP2c phosphatase and PAK-1/p21 activated kinase in another. While the LET-502/MEL-11 pathway appears to act primarily in the lateral epidermis, here we show that FEM-2 can mediate early elongation when expressed in the dorsal and ventral epidermis. We also investigated the early elongation function of FHOD-1, a member of the formin family of actin nucleators and bundlers. Previous work showed that FHOD-1 acts in the LET-502/MEL-11 branch of the early elongation pathway as well as in muscle for sarcomere organization. Consistent with this, we found that lateral epidermal cell-specific expression of FHOD-1 is sufficient for elongation, and FHOD-1 effects on elongation appear to be independent of its role in muscle. Also, we found that fhod-1 encodes long and short isoforms that differ in the presence of a predicted coiled-coil domain. Based on tissue-specific expression constructions and an isoform-specific CRISPR allele, the two FHOD-1 isoforms show partially specialized epidermal or muscle function. Although fhod-1 shows only impenetrant elongation phenotypes, we were unable to detect redundancy with other C. elegans formin genes.

Stable and dynamic axes of polarity use distinct formin isoforms in budding yeast.

Bud growth in yeast is guided by myosin-driven delivery of secretory vesicles from the mother cell to the bud. We find transport occurs along two sets of actin cables assembled by two formin isoforms. The Bnr1p formin assembles cables that radiate from the bud neck into the mother, providing a stable mother-bud axis. These cables also depend on septins at the neck and are required for efficient transport from the mother to the bud. The Bni1p formin assembles cables that line the bud cortex and target vesicles to varying locations in the bud. Loss of these cables results in morphological defects as vesicles accumulate at the neck. Assembly of these cables depends on continued polarized secretion, suggesting vesicular transport provides a positive feedback signal for Bni1p activation, possibly by rho-proteins. By coupling different formin isoforms to unique cortical landmarks, yeast uses common cytoskeletal elements to maintain stable and dynamic axes in the same cell.

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.

Inverted formins: A subfamily of atypical formins.

Formins are a family of regulators of actin and microtubule dynamics that are present in almost all eukaryotes. These proteins are involved in many cellular processes, including cytokinesis, stress fiber formation, and cell polarization. Here we review one subfamily of formins, the inverted formins. Inverted formins as a group break several formin stereotypes, having atypical biochemical properties and domain organization, and they have been linked to kidney disease and neuropathy in humans. In this review, we will explore recent research on members of the inverted formin sub-family in mammals, zebrafish, fruit flies, and worms.

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

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 Drosophila melanogaster and other arthropods, MWH-related proteins are also identified here in some nematodes (including Caenorhabditis elegans), 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.

INF2- and FHOD-related formins promote ovulation in the somatic gonad of C. elegans.

Formins are regulators of actin filament dynamics. We demonstrate here that two formins, FHOD-1 and EXC-6, are important in the nematode Caenorhabditis elegans for ovulation, during which actomyosin contractions push a maturing oocyte from the gonad arm into a distensible bag-like organ, the spermatheca. EXC-6, a homolog of the disease-associated mammalian formin INF2, is highly expressed in the spermatheca, where it localizes to cell-cell junctions and to circumferential actin filament bundles. Loss of EXC-6 does not noticeably affect the organization the actin filament bundles, and causes only a very modest increase in the population of junction-associated actin filaments. Despite absence of a strong cytoskeletal phenotype, approximately half of ovulations in exc-6 mutants exhibit extreme defects, including failure of the oocyte to enter the spermatheca, or breakage of the oocyte as the distal spermatheca entrance constricts during ovulation. Loss of FHOD-1 alone has little effect, and we cannot detect FHOD-1 in the spermatheca. However, combined loss of these formins in double fhod-1;exc-6 mutants results in profound ovulation defects, with significant slowing of the entry of oocytes into the spermatheca, and failure of nearly 80% of ovulations. We suggest that EXC-6 plays a role directly in the spermatheca, perhaps by modulating the ability of the spermatheca wall to rapidly accommodate an incoming oocyte, while FHOD-1 may play an indirect role relating to its known importance in the growth and function of the egg-laying muscles. © 2016 The Authors. Cytoskeleton Published by Wiley Periodicals, Inc.

Loss of Sarcomere-associated Formins Disrupts Z-line Organization, but does not Prevent Thin Filament Assembly in Caenorhabditis elegans Muscle.

Members of the formin family of actin filament nucleation factors have been implicated in sarcomere formation, but precisely how these proteins affect sarcomere structure remains poorly understood. Of six formins in the simple nematode Caenorhabditis elegans, only FHOD-1 and CYK-1 contribute to sarcomere assembly in the worm's obliquely striated body-wall muscles. We analyze here the ultrastructure of body-wall muscle sarcomeres in worms with putative null fhod-1 and cyk-1 gene mutations. Contrary to a simple model that formins nucleate actin for thin filament assembly, formin mutant sarcomeres contain thin filaments. Rather, formin mutant sarcomeres are narrower and have deformed thin filament-anchoring Z-line structures. Thus, formins affect multiple aspects of sarcomere structure.

The canonical eIF4E isoform of C. elegans regulates growth, embryogenesis, and germline sex-determination.

eIF4E plays a conserved role in initiating protein synthesis, but with multiple eIF4E isoforms present in many organisms, these proteins also adopt specialized functions. Previous RNAi studies showed that ife-3, encoding the sole canonical eIF4E isoform of Caenorhabditis elegans, is essential for viability. Using ife-3 gene mutations, we show here that it is maternal ife-3 function that is essential for embryogenesis, but ife-3 null progeny of heterozygous animals are viable. We find that zygotic ife-3 function promotes body growth and regulates germline development in hermaphrodite worms. Specifically, the normal transition from spermatogenesis to oogenesis in the hermaphrodite germline fails in ife-3 mutants. This failure to switch is reversed by inhibiting expression of the key masculinizing gene, fem-3, suggesting ife-3 resembles a growing number of genes that promote the sperm/oocyte switch by acting genetically as upstream inhibitors of fem-3.