Slow myosin heavy chain 1 is required for slow myofibril and muscle fibre growth but not for myofibril initiation.

Slow myosin heavy chain 1 (Smyhc1) is the major sarcomeric myosin driving early contraction by slow skeletal muscle fibres in zebrafish. New mutant alleles lacking a functional smyhc1 gene move poorly, but recover motility as the later-formed fast muscle fibres of the segmental myotomes mature, and are adult viable. By motility analysis and inhibiting fast muscle contraction pharmacologically, we show that a slow muscle motility defect persists in mutants until about 1 month of age. Breeding onto a genetic background marking slow muscle fibres with EGFP revealed that mutant slow fibres undergo terminal differentiation, migration and fibre formation indistinguishable from wild type but fail to generate large myofibrils and maintain cellular orientation and attachments. In mutants, initial myofibrillar structures with 1.67 â€‹µm periodic actin bands fail to mature into the 1.96 â€‹µm sarcomeres observed in wild type, despite the presence of alternative myosin heavy chain molecules. The poorly-contractile mutant slow muscle cells generate numerous cytoplasmic organelles, but fail to grow and bundle myofibrils or to increase in cytoplasmic volume despite passive movements imposed by fast muscle. The data show that both slow myofibril maturation and cellular volume increase depend on the function of a specific myosin isoform and suggest that appropriate force production regulates muscle fibre growth.

Maternal Larp6 controls oocyte development, chorion formation and elevation.

La-related protein 6 (Larp6) is a conserved RNA-binding protein found across eukaryotes that has been suggested to regulate collagen biogenesis, muscle development, ciliogenesis, and various aspects of cell proliferation and migration. Zebrafish have two Larp6 family genes: larp6a and larp6b Viable and fertile single and double homozygous larp6a and larp6b zygotic mutants revealed no defects in muscle structure, and were indistinguishable from heterozygous or wild-type siblings. However, larp6a mutant females produced eggs with chorions that failed to elevate fully and were fragile. Eggs from larp6b single mutant females showed minor chorion defects, but chorions from eggs laid by larp6a;larp6b double mutant females were more defective than those from larp6a single mutants. Electron microscopy revealed defective chorionogenesis during oocyte development. Despite this, maternal zygotic single and double mutants were viable and fertile. Mass spectrometry analysis provided a description of chorion protein composition and revealed significant reductions in a subset of zona pellucida and lectin-type proteins between wild-type and mutant chorions that paralleled the severity of the phenotype. We conclude that Larp6 proteins are required for normal oocyte development, chorion formation and egg activation.

Structural and functional insights into asymmetric enzymatic dehydration of alkenols.

The asymmetric dehydration of alcohols is an important process for the direct synthesis of alkenes. We report the structure and substrate specificity of the bifunctional linalool dehydratase isomerase (LinD) from the bacterium Castellaniella defragrans that catalyzes in nature the hydration of ß-myrcene to linalool and the subsequent isomerization to geraniol. Enzymatic kinetic resolutions of truncated and elongated aromatic and aliphatic tertiary alcohols (C5-C15) that contain a specific signature motif demonstrate the broad substrate specificity of LinD. The three-dimensional structure of LinD from Castellaniella defragrans revealed a pentamer with active sites at the protomer interfaces. Furthermore, the structure of LinD in complex with the product geraniol provides initial mechanistic insights into this bifunctional enzyme. Site-directed mutagenesis confirmed active site amino acid residues essential for its dehydration and isomerization activity. These structural and mechanistic insights facilitate the development of hydrating catalysts, enriching the toolbox for novel bond-forming biocatalysis.