No abstract

The Caenorhabditis elegans unc-45 locus has been proposed to encode a protein machine for myosin assembly. The UNC-45 protein is predicted to contain an NH2-terminal domain with three tetratricopeptide repeat motifs, a unique central region, and a COOH-terminal domain homologous to CRO1 and She4p. CRO1 and She4p are fungal proteins required for the segregation of other molecules in budding, endocytosis, and septation. Three mutations that lead to temperature-sensitive (ts) alleles have been localized to conserved residues within the CRO1/She4p-like domain, and two lethal alleles were found to result from stop codon mutations in the central region that would prevent translation of the COOH-terminal domain. Electron microscopy shows that thick filament accumulation in vivo is decreased by ∼50% in the CB286 ts mutant grown at the restrictive temperature. The thick filaments that assemble have abnormal structure. Immunofluorescence and immunoelectron microscopy show that myosins A and B are scrambled, in contrast to their assembly into distinct regions at the permissive temperature and in wild type. This abnormal structure correlates with the high degree of instability of the filaments in vitro as reflected by their extremely low yields and shortened lengths upon isolation. These results implicate the UNC-45 CRO1/She4p-like region in the assembly of myosin isoforms in C. elegans and suggest a possible common mechanism for the function of this UCS (UNC-45/CRO1/She4p) protein family.

Microglial activation and oxidative stress are significant components of the pathology of Parkinson's disease (PD), but their exact contributions to disease pathogenesis are unclear. We have developed an in vitro model of nigral injury, in which lipopolysaccharide-induced microglial activation leads to injury of a dopaminergic cell line (MES 23.5 cells) and dopaminergic neurons in primary mesencephalic cell cultures. The microglia are also activated by PD IgGs in the presence of low-dose dopa-quinone- or H(2)O(2)-modified dopaminergic cell membranes but not cholinergic cell membranes. The activation requires the microglial FCgammaR receptor as demonstrated by the lack of activation with PD IgG Fab fragments or microglia from FCgammaR-/- mice. Although microglial activation results in the release of several cytokines and reactive oxygen species, only nitric oxide and H(2)O(2) appear to mediate the microglia-induced dopaminergic cell injury. These studies suggest a significant role for microglia in dopaminergic cell injury and provide a mechanism whereby immune/inflammatory reactions in PD could target oxidative injury relatively specifically to dopaminergic cells.

Myosin isoforms A and B are differentially localized to the central and polar regions, respectively, of thick filaments in body wall muscle cells of Caenorhabditis elegans (Miller, D. M. III, I. Ortiz, G. C. Berliner, and H. F. Epstein, 1983, Cell, 34:477-490). Biochemical and electron microscope studies of KCI-dissociated filaments show that the myosin isoforms occupy a surface domain, paramyosin constitutes an intermediate domain, and a newly identified core structure exists. The diameters of the thick filaments vary significantly from 33.4 nm centrally to 14.0 nm near the ends. The latter value is comparable to the 15.2 nm diameter of the core structures. The internal density of the filament core appears solid medially and hollow at the poles. The differentiation of thick filament structure into supramolecular domains possessing specific substructures of characteristic stabilities suggests a sequential mode for thick filament assembly. In this model, the two myosin isoforms have distinct roles in assembly. The behavior of the myosins, including nucleation of assembly and determination of filament length, depend upon paramyosin and the core structure as well as their intrinsic molecular properties.The body wall muscle cells of the nematode Caenorhabditis elegans contain two chemically and genetically distinct isoforms of myosin heavy chain, A and B. Immunocytochemical localization with both affinity purified and specific monoclonal antibodies shows that the two isoforms coexist in the same muscle cells and sarcomeres (13,17). All isolated thick filaments react with the monoclonal antibodies of either specificity, indicating that the isoforms are contained within the same filaments. However, the A form is localized to the central 1.8 #m of the 9.7-#m-long filaments, whereas the B form is located in the polar region, but is absent in the central 0.9 #m of the filament (17). Fig. 1 is a schematic diagram showing locations of the A and B heavy chains and examples of thick filaments that were reacted with the specific antibodies. The surface of nematode thick filaments is differentiated by myosin isoform content into five zones.The locations of the myosin heavy chain isoforms in this model explain several independent observations regarding nematode body wall myosins. Previous studies of nematode myosins indicate that native molecules are homodimers of either heavy chain isoform; no heterodimers are detected (21,22). The different locations of the heavy chain types imply that the myosin molecules within thick filaments are primarily homodimers. The ratio of B to A heavy chains appears constant, whereas their amounts increase 40-fold during the post-embryonic stages of the nematode life cycle (4). This fixed ratio, ~4:1 (28), is consistent with the distributions of the two forms in the thick filaments shown in Fig. 1.The different locations of myosins A and B have important structural correlations. The central regions that contain only myosin A are the regions in which myosin molecules pack in a bipolar fash...

Abstract. The organization of myosin heavy chains (mhc) A and B and paramyosin (pm) which are the major proteins of thick filaments in adult wild-type Caenorhabditis elegans were studied during embryonic development. As a probe of myosin-paramyosin interaction, the unc-15 mutation e73 which produces a glu3421ys charge change in pm and leads to the formarion of large paracrystalline multi-filament assemblages was compared to wild type. These three proteins colocalized in wild-type embryos from 300 to 550 min of development after first cleavage at 20°C on the basis of immunofluorescence microscopy using specific monoclonal antibodies. Linear structures which were diversely oriented around the muscle cell peripheries appeared at 360 rain and became progressively more aligned parallel to the embryonic long axis until distinct myofibrils were formed at 550 min. In the mutant, mhc A and pm were colocalized in the linear structures, but became progressively separated until they showed no spatial overlap at the myofibril stage. These results indicate that the linear structures represent nascent assemblies containing myosin and pm in which the proteins interact differently than in wild-type thick filaments of myofibrils. In e73, these nascent structures were distinct from the multifilament assemblages. The overlapping of actin and mhc A in the nascent linear structures suggests their possible structural and functional relationship to the "stress fiber-like structures" of cultured vertebrate muscle cells.