Pruyne, David et al. (2015) International Worm Meeting "Roles for formins in the development and function of muscle and myoepithelial cell types."

Hegsted, Anna, Mi-Mi, Lei, Votra, SarahBeth, Pruyne, David, Wright, Forrest

[International Worm Meeting, 2015]

Formins are highly conserved regulators of actin filament dynamics. In vitro, formins stimulate the nucleation of actin filaments from monomers, and frequently enhance the rate of filament elongation, resulting in the formation of long, unbranched actin filaments. Using C. elegans as a model to identify in vivo functions of formin homologs, we have discovered that these proteins contribute to the function of several distinct cell types with contractile activity. The formins encoded by cyk-1 and fhod-1 contribute to the growth of striated body-wall muscle, while the formin encoded by exc-6 is required for the proper behavior of myoepithelial cells of the spermatheca during ovulation. In each of these cases, the formin proteins are physically associated with actin filament-rich contractile structures, but these actin-containing structures are not lost in mutants for these formin genes, suggesting these formins might not simply direct the assembly of contractile structures. Rather, microscopic and biochemical analysis of these mutants suggest some of these formins may play more subtle roles in modulating actin filament dynamics, or possibly even affect the expression of other contraction-related genes.

Pruyne, David et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Exploring Formin Functions in Worm Muscles."

Bretscher, Anthony, Kemphues, Kenneth, Mi-Mi, Lei, Pruyne, David

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

By far, the largest population of actin filaments in most multicellular animals is those that constitute the contractile apparatus of muscle cells, yet little is known of how these filaments are assembled in vivo . Studies of vertebrate cultured muscle cells have implicated a conserved family of cytoskeletal regulatory proteins called Formins as putative muscle actin assembly factors, specifically identifying Formins of the Formin HOmology Domain (FHOD) subfamily as important to this. C. elegans encodes a single FHOD homolog, FHOD-1. We find that this well-conserved FHOD homolog is a muscle-specific Formin, but in surprising contrast to what has been described for cultured cells, loss of FHOD-1 in intact animals results in only modest defects in muscle actin organization in a subset of muscle cells. Despite this mild phenotype, the subcellular distribution of FHOD-1 is consistent with a role for this Formin in helping direct the assembly and organization of contractile actin filaments in worm muscle cells. As one ongoing approach, we are searching for factors that might function redundantly with FHOD-1 in assembly of this large pool of actin filaments.

Mi-Mi, Lei et al. (2013) International Worm Meeting ""Ultrastructure analysis of the sarcomeres in worms that lack Z-line formins"."

Pruyne, David, Mi-Mi, Lei

[International Worm Meeting, 2013]

Even with extensive studies that revolve around actin cytoskeleton, the detailed mechanism as to how actin filaments are recruited into sarcomeres, the smallest units of striated muscle contractile lattice, is yet to be elucidated. The barbed ends of actin filaments anchor at the Z-line structures (dense bodies in C. elegans) that define the sarcomere ends. Net actin polymerization favorably occurs at the barbed end that is known to have unique association with formins, one of the highly conserved and most widespread families of actin nucleating factors. We have shown in our previous study that two nematode formins, CYK-1 and FHOD-1 are Z-line-associated proteins that work together to organize and maintain sarcomeric organization in C. elegans striated muscle - the first evidence in an intact organism. In this study, we have carried out the ultrastructure analysis on sarcomeric structures of worms lacking CYK-1 and/or FHOD-1. Not only do the current electron microscopy results complement our previous conclusions, they also suggest that CYK-1 and/or FHOD-1 may contribute in achieving normal Z-line structures in C. elegans sarcomeric organization.

Hegsted, Anna et al. (2015) International Worm Meeting "The formin EXC-6/INFT-1 is required for normal ovulation."

Pruyne, David, Hegsted, Anna, Wright, Forrest

[International Worm Meeting, 2015]

Actin cytoskeleton organization is important for proper function of the Caenorhabditis elegans gonad, especially contractility of the spermatheca. Aberrant actin organization in the spermatheca has been linked to contractility deficiencies and calcium signaling defects leading to improper ovulation. Formins are actin nucleation factors, and many promote the elongation of unbranched actin filaments. In the absence of the formin EXC-6/INFT-1, worms lay fewer eggs than wild type. These worms also exhibit aberrant ovulation. Further investigation shows that INFT-1 is expressed in the spermatheca and partially colocalizes with actin filament bundles. Due to the role of INFT-1 in proper ovulation and its localization, we hypothesize that INFT-1 is important for spermatheca actin organization and/or spermatheca calcium signaling. .

Refai, Osama M. et al. (2013) International Worm Meeting "Formins Play a Role in the C. elegans Embryonic Elongation."

Pruyne, David, Vanneste, Christopher A., Mains, Paul E., Refai, Osama M.

[International Worm Meeting, 2013]

During the C. elegans embryonic development, the embryo undergoes dramatic changes to elongate from a spheroid into a long, thin worm. The epidermis of the embryo provides the driving force for elongation. The epidermal cytoskeleton is a highly organized structure of actin microfilaments, microtubules and intermediate filaments. The C. elegans Rho-binding kinase (LET-502) and myosin phosphatase (MEL-11) are essential regulators for the actomyosin-mediated embryonic elongation and act in parallel with FEM-2/PP2c phosphatase and PAK-1/p21 activated kinase (GALLY et al. 2009; PIEKNY et al. 2000). Actin nucleation is the rate limiting step of actin filament polymerization. We previously identified fhod-1 (formin homology domain), which is highly similar to the human actin nucleators FHOD1 and FHOD3. Our genetic analysis revealed that fhod-1 acts in the mel-11/let-502 pathway, in parallel to fem-2/pak-1. fhod-1 mutants exhibit less than 25% elongation defects, suggesting redundancy with other formins (or other genes) for actin nucleation. Potential candidates include six other C. elegans formins: daam-1, inft-1, inft-2, frl-1, fozi-1 and cyk-1. Only two of these genes, inft-1and cyk-1, appear to have a role in elongation based on let-502 RNAi in mutant backgrounds. Muscle is required for elongation past the 2 fold stage, and perhaps is required redundantly with let-502/mel-11 at earlier stages. However, we found that the ilk-1 Pat (paralyzed at two fold) mutant did not suppress mel-11.

Sundaramurthy, Sumana et al. (2021) International Worm Meeting "Probing formin FHOD-1 contributions to body-wall muscle structure and function"

Sundaramurthy, Sumana, Lesanpezeshki, Leila, Pruyne, David, Littlefield, Ryan, Vanapalli, Siva, Yingling, Curtis

[International Worm Meeting, 2021]

In mammalian striated muscle, six actin-organizing formin proteins have been shown to influence sarcomere organization, but their precise molecular roles in this process remain obscure. In C. elegans, only the formin FHOD-1 works in a muscle cell-autonomous manner to influence sarcomere structure in body-wall muscle (BWM), providing a simpler system to determine the mechanism by which these highly conserved proteins influence the muscle cytoskeleton. Worms bearing a fhod-1 allele that is a putative null for effects on actin organization display modest defects in BWM strength, cell growth and sarcomere assembly, arguing against a primary role in assembling the actin-based sarcomeric thin filaments. However, fhod-1 muscle cells assemble fewer sarcomeres, and the muscle-specific myosin heavy chain MYO-3 is subject to elevated proteasome-dependent proteolysis. Moreover, the dense bodies that serve as thin filament-anchoring sites in BWM are malformed. Comparison of the distribution of immature and mature dense bodies in wild-type and fhod-1 BWM cells, and the spatial relationship of FHOD-1 to those structures, suggests FHOD-1 accumulates at specific foci in the BWM cell to assist the formation of new dense bodies there.

Rocheleau, Simon K. et al. (2011) International Worm Meeting "The roles of rhgf-2/Rho GEF and fhod-1/formin in regulating the actin cytoskeleton during embryonic elongation."

Raharjo, Eko, Mains, Paul E., Steven, Robert, Pruyne, David, Rocheleau, Simon K., Vanneste, Christopher A.

[International Worm Meeting, 2011]

Embryonic elongation, which transforms the roughly spherical embryo into a long, thin vermiform larva, is mediated in part by a smooth muscle-like contraction of an actin/myosin network in the epidermal cells. Here we describe two new players that regulate this process. Previous work has shown that contractile force is generated by two redundant pathways that activate non-muscle myosin. Contraction is triggered by the phosphorylation of MLC-4, which activates the non-muscle myosins NMY-1/NMY-2. This is counteracted by MLC-4 dephosphorylation by MEL-11/myosin phosphatase. One of the two contractile pathways involves the small GTPase RHO-1, which activates LET-502/Rho-binding kinase, which in turn inhibits the MEL-11 brake to contraction. A second, parallel pathway involves FEM-2/protein phosphatase 2c. In a suppressor screen of mel-11, we isolated an allele of a Rho GEF (guanine exchange factor), rhgf-2. As expected, rhgf-2 genetically behaves as an upstream activator of the let-502/Rho-binding kinase branch of the elongation pathway and appears to function in parallel to fem-2. RHGF-2 binds to and acts as a GEF for RHO-1 (See abstract by Tran et al.). Even though circumferential actin filaments are present in all epidermal cells, most of the contractile force is generated by the lateral (seam) cells. The lateral cell microfilaments are qualitatively different than those in their dorsal and ventral neighbours in that they are found further from the apical surface. We have identified a actin nucleator in the formin family that may be in part responsible for these differences. FHOD-1 (formin homology domain) is expressed only in the lateral epidermal cells and mutations cause actin defects only in those cells. Curiously, genetic results indicate that fhod-1 acts downstream of let-502 and in parallel to fem-2, implying that the two pathways may act on different sets of microfilaments.

Schein JE et al. (1997) International C. elegans Meeting "POSITIONING OF ESSENTIAL GENES IN THE lin-3 - let-60 INTERVAL"

Franz NW, Rose AM, Janke DL, Bilkhu N, Baillie DL, Schein JE

[International C. elegans Meeting, 1997]

The lin-3 - let-60 interval of the right arm of chromosome IV contains thirteen essential gene mutations and 22 overlapping sequenced cosmids. The Sanger Center has nearly finished sequencing the entire interval. Analysis of cosmid sequences shows over one hundred potential coding regions. Of the coding regions with proposed protein function, more than 50% have strong homology to human genes. Most notably, the putative genes include three phosphatases, four protein kinases, a kinesin, and a cluster of histones. C. elegans strains carrying sequenced cosmids (in extrachromosomal arrays) have been constructed (see abstract for D. Janke et al., this meeting) and will be used in rescue experiments involving the essential genes in this interval. lin-3 and let-60(ras), the two genes bracketing this interval, were previously placed on the physical map by others. One of the remaining eleven essential genes has been already positioned to a cosmid: let-70(ubc2) (in collaboration with Mei Zhen and Peter Candido). By the time of the meeting, we anticipate that several of the remaining mutations will have been positioned to cosmids. This work is being funded by a grant from the Medical Research Council to Ann Rose and David Baillie and by a grant from the Natural Sciences & Engineering Research Council to David Baillie.

Stewart HI et al. (1996) West Coast Worm Meeting "Rescue of New Essential Genes on LGIII(left), in the Nematode Caenorhabditis elegans."

Stewart HI, Franz NW, Barbazuk BW, Baillie DL, Ha TT

[West Coast Worm Meeting, 1996]

We utilized trangenic strains containing cosmids, sequenced by the sequencing consortium, to rescue the lethal phenotype of several essential genes (See presentation by Diana Janke et al, this session). The rescued genes are positioned on LGIII(left) balanced by the duplication sDp3(see poster; Stewart et al, this session). A set of overlapping deficiencies was utilized to define areas within which to focus our rescue attempts). PCR analysis was utilized to further define the end points of these deficiencies, facilitating our analysis ( see poster ; Norm Franz et al, this session). We concentrated our analysis on areas to the right of dpy-17, in the interval between dpy-17 to dpy-19. Lethals mapping to sDf125 that were not rescued by sDp8 are presented in a poster by Nigel O'Niel et al, this session. We have rescued several essential genes. Our rescues will facilitate the functional analysis of these new genes. These rescues have also helped us to align and correlate the physical and genetic maps. Our estimates are that there will be more than two essential genes per cosmid. A total of 202 recessive lethal mutations have been generated, using EMS as a mutagen. We intend to make molecular assignments of all of these elements, through our cosmid rescue experiments. As we have estimated that there is only a 28% saturation of the area for essential genes, we will be generating more recessive lethal genes, to facilitate this analysis. This work has been funded by grants from the National Institute of Health (N.I.H.), U. S.A. to Ann Rose; D. Riddle and David Baillie and Canadian Genome and Advanced Technologies (C.G.A.T.), Canada to Ann Rose and David Baillie.

O'Neil NJ et al. (1996) West Coast Worm Meeting "SYSTEMATIC HIGH RESOLUTION MAPPING OF ESSENTIAL GENES IN THE sDf125 REGION BY TRANSGENIC RESCUE."

Kuervers LM, Baillie DL, Vatcher GP, O'Neil NJ

[West Coast Worm Meeting, 1996]

We have demonstrated that transgenic rescue is an effective method for high resolution mapping of essential genes. Our lab is constructing a map of essential genes on LGIII left. We have divided the left end of the central cluster into manageable regions which are defined by overlapping deficiencies and duplications. These regions are determined physically by PCR testing the deficiencies to determine endpoints (see N.Franz et al. abstract, this meeting). Into these regions, we have mapped a set of EMS induced lethals and maternal effect lethals. (see abstracts by H. Stewart et al. and G. Vatcher and D. Baillie, this meeting). One such region is defined by sDf125 and sDp8. This region spans 340 000 base pairs and is completely covered by nine cosmids. Within this region there are 75 putative genes predicted by Genefinder. We have used a sytematic approach to rescue essential genes within the sDf125 region with transgenic cosmids from the library created by J. Schein, D. Janke and The Ha as part of a CGAT grant to Ann Rose and David Baillie (see abstracts by J.Schein et al. and D. Janke et al., this meeting) To date this method has proven very successful. At least 8 genes have been completely rescued in this region by this approach. These transgenic rescues map these genes to 40 kB (one cosmid) resolution as opposed to the 340 kB resolution of the deficiency and duplication data. This is almost a ten-fold increase in resolution and reduces the number of ORFs which may be responsible for the gene function. The next step will be the subcloning of cosmids with which we have rescued essential genes. This will directly corellate the lethal or maternal effect lethal phenotype to the appropriate ORF. We have already begun the subcloning of cosmid C05D11 (see G.Vatcher and D.Baillie abstract, this meeting). This project is funded by a CGAT grant to Ann Rose and David Baillie.