Abstract. Mutations in the Caenorhabditis elegans gene unc-89 result in nematodes having disorganized muscle structure in which thick filaments are not organized into A-bands, and there are no M-lines (Waterston, R.H., J.N. Thomson, and S. Brenner. 1980. Dev. Biol. 77:271-302 124:491-506). We propose that the intracellular protein UNC-89 responds to these signals, localizes, and then participates in assembling an M-line.

To further understand the assembly and maintenance of the muscle contractile apparatus, we have identified a new protein, UNC-98, in the muscle of Caenorhabditis elegans. unc-98 mutants display reduced motility and a characteristic defect in muscle structure. We show that the major defect in the mutant muscle is in the M-lines and dense bodies (Z-line analogs). Both functionally and compositionally, nematode M-lines and dense bodies are analogous to focal adhesions of nonmuscle cells. UNC-98 is a novel 310-residue polypeptide consisting of four C2H2 Zn fingers and several possible nuclear localization signal and nuclear export signal sequences. By use of UNC-98 antibodies and green fluorescent protein fusions (to full-length UNC-98 and UNC-98 fragments), we have shown that UNC-98 resides at M-lines, muscle cell nuclei, and possibly at dense bodies. Furthermore, we demonstrated that 1) the N-terminal 106 amino acids are both necessary and sufficient for nuclear localization, and 2) the C-terminal (fourth) Zn finger is required for localization to M-lines and dense bodies. UNC-98 interacts with UNC-97, a C. elegans homolog of PINCH. We propose that UNC-98 is both a structural component of muscle focal adhesions and a nuclear protein that influences gene expression.

Symplostatin 1, an analog of dolastatin 10, was recently isolated from cyanobacteria of the genus Symploca. Symplostatin 1 is a potent inhibitor of cell proliferation with IC 50 values in the low nanomolar range and it exhibits efficacy against a variety of cancer cell types. Symplostatin 1 caused the formation of abnormal mitotic spindles and accumulation of cells in metaphase at concentrations that had only minor effects on interphase microtubules. At higher concentrations, symplostatin 1 caused the loss of interphase microtubules. Cell cycle analysis revealed that symplostatin 1 caused G 2 /M arrest, consistent with its effects on mitotic spindles. Symplostatin 1 initiated the phosphorylation of Bcl-2, formation of micronuclei and activation of caspase 3, indicating induction of apoptosis. The cellular effects of symplostatin 1 are consistent with other antimitotic tubulintargeting drugs. Tubulin polymerization experiments indicated that symplostatin 1 potently inhibits the assembly of purified tubulin, suggesting that tubulin may be its intracellular target. Some microtubule-targeting agents are reported to have antiangiogenic activity and therefore the effects of symplostatin 1 on endothelial cell proliferation and invasion were evaluated. Symplostatin 1 was found to be a potent inhibitor of both endothelial cell proliferation and invasion. Because of its potent and broad activity in vitro, symplostatin 1 was evaluated in vivo. Symplostatin 1 was active against murine colon 38 and murine mammary 16/C; however, it was poorly tolerated and the mice were slow to recover from the toxicity. The data indicate that symplostatin 1 has a mechanism of action similar to dolastatin 10.

To gain further insight into the molecular architecture, assembly, and maintenance of the sarcomere, we have carried out a molecular analysis of the UNC-96 protein in the muscle of Caenorhabditis elegans. By polarized light microscopy of body wall muscle, unc-96 mutants display reduced myofibrillar organization and characteristic birefringent "needles." By immunofluorescent staining of known myofibril components, unc-96 mutants show major defects in the organization of M-lines and in the localization of a major thick filament component, paramyosin. In unc-96 mutants, the birefringent needles, which contain both UNC-98 and paramyosin, can be suppressed by starvation or by exposure to reduced temperature. UNC-96 is a novel ϳ47-kDa polypeptide that has no recognizable domains. Antibodies generated to UNC-96 localize the protein to the M-line, a region of the sarcomere in which thick filaments are cross-linked. By genetic and biochemical criteria, UNC-96 interacts with UNC-98, a previously described component of M-lines, and paramyosin. Additionally, UNC-96 copurifies with native thick filaments. A model is presented in which UNC-96 is required in adult muscle to promote thick filament assembly and/or maintenance. INTRODUCTIONThe myofibril is a complex assemblage of many proteins, with new components being discovered each year. Despite this growing understanding about myofibrillar components, their interactions, and individual functions, we still know little about the assembly of this precise structure (Gregorio and Antin, 2000). Additionally, it is unclear how this structure is maintained in the face of repeated contraction and relaxation. The need to understand the mechanisms in charge of myofibrillar maintenance is underscored by the clinical significance of degenerative muscular atrophies and myopathies. Caenorhabditis elegans is a particularly attractive organism in which to address these problems Moerman and Fire, 1997;Moerman and Williams, 2006). We are able to view the muscle in a whole animal system, allowing us to focus on the true, extracellular matrix (ECM) attachment properties of the muscle cell, an evaluation not possible with tissue culture. Because of the optical transparency of the worm, we are able to visualize the myofibrillar structure by polarized light and localize green fluorescent protein (GFP)-tagged proteins in live worms. A major strength of C. elegans lies in its ability to grow quickly and reproduce in large numbers, allowing genetic analyses in a timely and efficient manner. Last, its usual self-fertilization permits propagation of muscle mutants that would otherwise be unable to mate.In the nematode, most of the muscle is located in the body wall and is used for locomotion. Throughout the muscle cell, the thin filament attachment structures, called dense bodies (analogous to the Z-discs in vertebrate muscle) and the thick filament cross-linking structures, the M-lines, are anchored to the muscle cell membrane. This permits the force of contraction to be transmitted through the cell me...

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