Kelly A Flanagan et al. (2004) Mid-west Worm Meeting "UNC-82 is a serine/threonine kinase required for thick filament organization in body-wall muscle."

Lawrence A Schriefer, Kelly A Flanagan, Pamela E Hoppe, Johnnie Chau

[Mid-west Worm Meeting, 2004]

To elucidate the mechanisms that guide the assembly of thick filament proteins, we have undertaken an analysis of the unc-82 gene. Mutations in unc-82 cause abnormalities in thick filament morphology and organization (Waterston et al., 1980). A combination of genetic mapping, transformation rescue, and RNAi experiments demonstrate that unc-82 encodes a ~1600 residue protein that contains a serine/threonine kinase active site domain at the N-terminus. No other regions of homology to known proteins have been detected. BLAST analysis identifies putative orthologs in vertebrates, ARK5 and SNARK, and in Drosophila. Sequencing of Yugi Kohara's cDNA clones revealed multiple splice variants that differ in the sequences C-terminal to the kinase domain. The most severe unc-82 allele, e1323 , contains a premature stop codon in the kinase domain, and is likely to represent a null mutation. The allele e1220 contains a missense mutation (E205K) within a conserved residue of the catalytic loop. A rescuing UNC-82: :GFP fusion is detected at or near the M-line in body-wall muscle. The unc-82 phenotype indicates a role for the kinase in regulation of thick filament formation or function. Antibody staining of e1323 embryos suggests that components of the contractile apparatus localize as wild type at the 1.5-fold stage, the time at which muscle contractions begin. However, defects in the localization of thick filament (myosin and paramyosin) and M-line (UNC-89) components are apparent by the 3-fold stage. The organization of the membrane adhesion structures (integrin) and thin filament system (vinculin and actin) appear normal at this stage, supporting the proposal that unc-82 function is required specifically for thick filament organization. Our model is that unc-82 activity is required for reorganization of thick filaments during growth and elongation of the muscle cell, perhaps by regulating filament attachment or mediating filament disassembly. The UNC-82: :GFP fusion protein is expressed in non-muscle tissues, where it localizes to regions containing intermediate filaments. Therefore, UNC-82 may play a role in the organization of cytoskeletal structures in other tissue types.

Pamela E Hoppe et al. (2003) International Worm Meeting "UNC-82 defines a novel class of serine/threonine kinases required for thick filament organization."

Kelly A Flanagan, Lawrence A Schriefer, Pamela E Hoppe, Johnnie Chau

[International Worm Meeting, 2003]

To elucidate the mechanisms that guide the assembly of thick filament proteins into the highly ordered contractile apparatus, we have undertaken an analysis of the unc-82 gene. Mutations in unc-82, which cause a mild uncoordinated phenotype, are associated with abnormalities in thick filament morphology and organization (Waterston et al., 1980). A combination of genetic mapping, transformation rescue, and RNAi experiments demonstrate that unc-82 corresponds to predicted ORF B0496.3. Two transcripts, which differ by alternative inclusion of a single coding exon, encode ~1600 residue proteins that contain a serine/threonine kinase active site domain at the N-terminus. No other regions of homology to known proteins have been detected. BLAST analysis using the UNC-82 kinase domain has identified putative orthologs in Drosophila and vertebrates. Phylogenetic trees based upon comparisons of kinase domain sequences suggest that UNC-82 is a member of a novel class of kinases related to the SNF1 and PAR-1 kinase families. The unc-82 phenotype suggests a role for the kinase in regulation of thick filament formation or function. The more severe allele, e1323, contains a premature stop codon in the kinase domain, and is therefore likely to represent a null. The allele e1220 contains a missense mutation (E202K) within a conserved residue of the catalytic loop. Inactivation of the locus by RNAi produces a phenotype similar to that of these alleles. Potential UNC-82 substrates include the homologous nonhelical tailpiece domains present in myosin and paramyosin (Schriefer and Waterston, 1989). Consistent with this possibility, a rescuing UNC-82::GFP fusion localizes to the M-line in body wall muscle. In current experiments, we are testing the in vivo role of the tailpiece in both myosin and paramyosin, and examining the ability of extracts from unc-82 animals to phosphorylate endogenous paramyosin. The localization of UNC-82::GFP in nonmuscle cells suggests that UNC-82 may function in the organization of other cytoskeletal structures, such as the intermediate filament bundles in the marginal cells of the pharynx.

April Reedy et al. (2007) International Worm Meeting "M-line associated UNC-82 plays a role in maintaining organization of the contractile apparatus of body wall muscle during development."

Pamela Hoppe, April Reedy

[International Worm Meeting, 2007]

Mutations in the unc-82 gene cause defects in thick filament organization in striated muscle (Waterston et al., 1980). UNC-82 is a serine/threonine kinase orthologous to human ARK5 and SNARK. A rescuing UNC-82::GFP fusion localizes at or near the M-line (Chau, Flanagan, Schriefer, and Hoppe, unpublished). In current studies we are using antibody staining of mutant embryos and adults to define the role of UNC-82 in cytoskeletal organization of body-wall muscle cells. All proteins examined showed normal staining patterns during early stages of embryonic elongation. Defects in the localization of thick filament proteins myosin and paramyosin as well as M-line proteins UNC-89/Obscurin and UNC-98 are apparent in all unc-82(e1323) null mutant embryos by the 3-fold stage of embryogenesis. In contrast, dense-body protein vinculin and membrane-proximal proteins such as integrin and UNC-95 appear normal at this stage. Interestingly, actin filament staining is also abnormal in unc-82 mutants by the 3-fold stage. These data suggest that UNC-82 acts in a process independent of the hypodermal-to-muscle-cell signaling required for early patterning of the contractile apparatus. Our current model is that UNC-82 regulates turnover of thick filaments or M-lines during muscle cell growth and body elongation through phosphorylation of an M-line associated protein. Defects in actin filament localization may then occur from physical interaction with disrupted thick filaments.

Chiyen Wong et al. (2008) Development & Evolution Meeting "The Role of UNC-82 in Cell-to-Substrate Attachment in the Pharynx and Vulva"

Chiyen Wong, Micheal Tjepkema, Pamela Hoppe

[Development & Evolution Meeting, 2008]

The unc-82 gene was identified by screening for disorganized body-wall muscle cells in mutagenized adults. The unc-82 gene encodes a serine/threonine kinase that is required for proper organization of thick filaments and membrane-distal components of the M-line (Waterston et al., 1980; Chau at al., submitted). In addition to moving more slowly than wild type, unc-82 mutants have decreased pharyngeal pumping and an incompletely penetrant Egl phenotype. A rescuing UNC-82::GFP fusion revealed that UNC-82 is expressed in the pharynx and vulva, as well as in body-wall muscles. Pharyngeal muscles are attached to the cuticle-covered lumen on one side and to the basement membrane surrounding the pharynx organ on the other. TEM of mutant adult pharynges revealed detachment of pharyngeal muscle and marginal cells from the cuticle lining the buccal cavity; attachments to the basement membrane surrounding the organ were unaffected. UNC-82::GFP is found near the pharyngeal muscle-to-cuticle attachment structures, which contain intermediate filaments, Vab10a/plectin and MUP-4. Unlike body-wall muscle staining, UNC-82::GFP is not detected near thick filaments in the pharynx. The muscle detachment in the pharynx suggested that the Egl phenotype might result from detachment of the egg-laying muscles. However, light microscopy and TEM analyses showed that vulva muscles remained attached to the vulval epithelium. Instead, the Egl phenotype is likely due to defects in the epithelial cells of the vulva, which appear damaged in unc-82 mutants. Attachment proteins such as MUP-4 and the intermediate filament MUA-6, are disorganized compared to wild type. Preliminary TEM suggests that the vulval cells detach from the exterior cuticle. The similarity of the cell attachment defects in the vulva and pharynx suggest that UNC-82 is involved in regulation of attachments in these tissues. The pharynx and vulva cells affected contain similar molecular components in their attachment structures, which mediate direct attachment of the cells to the cuticle which they themselves secrete. Our model is that unc-82 regulates proper organization of cell attachment structures at the cuticle-to-membrane interface in the pharynx and vulva.

Jason Kintzele et al. (2008) Development & Evolution Meeting "UNC-82, Which is Required for Myosin Organization in Muscle, is Related to AAK-2 and PAR-1 Kinases, Which Target Myosin"

Pamela Hoppe, Jason Kintzele

[Development & Evolution Meeting, 2008]

The unc-82 gene was previously identified by screening for disrupted muscle cytoskeleton in otherwise apparently normal mutagenized animals (Waterston et al., 1980). The unc-82 locus encodes a serine/threonine kinase required for maintaining organization of thick filaments and membrane-distal components of the M-line during development (Chau et al., submitted). Consistent with these defects, an UNC-82::GFP fusion has been shown to localize at or near the M-line. We propose that activation of UNC-82 kinase during cell elongation promotes detachment and/or disassembly of thick filaments, perhaps through phosphorylation of myosin and paramyosin. Our phylogenetic analysis identified the human UNC-82 orthologs ARK5 and SNARK, as well as anonymous orthologous proteins in Drosophila and zebrafish. The human proteins have been tested for presumed AMP activation, but their roles in normal development or physiology have not been explored. A BLASTp search using the UNC-82 kinase domain identified the most closely related C. elegans proteins as AAK-2, a member of the Snf1/AMP-Activated kinase (AMPK) family, and PAR-1, originally identified for its effect on anterior-posterior polarity in embryos. Kinases within the SNF1/AMPK and PAR-1 families are known to interact with myosin. The C. elegans nonmuscle myosin NMY-2 was identified through its interaction with the PAR-1 protein (Guo and Kemphues, 1996). Drosophila SNF1/AMPK is required during embryogenesis for establishment of epithelial cell polarity, and acts through phosphorylation of myosin regulatory light chain (Lee et al., 2007). The presence of various myosin-associated kinases in this particular branch of the kinome suggests that myosin may have been a substrate of an ancestral kinase prior to the divergence of the UNC-82, AMPK and PAR-1 groups. We suggest that the salient feature shared by these proteins is their substrate specificity, rather than their presumed mode of regulation. To date, we have shown that UNC-82 is required for thick filament organization, but have not identified the UNC-82 target proteins. We are using biochemical approaches to test our hypothesis that UNC-82 phosphorylates myosin and paramyosin. In addition, we are taking two approaches to determine the location of the UNC-82 protein with more precision. First, antibodies have been raised against two UNC-82 epitopes: one adjacent to the N-terminal kinase domain, and one that includes the native C-terminus. These antibodies will be used in immuno-EM experiments to determine the precise location of the UNC-82 protein in muscle and nonmuscle cells. Second, we have created an RFP-tagged UNC-82 construct to enable co-localization of UNC-82 with existing GFP-tagged protein markers in nonmuscle cells.