The Role of Nonmuscle Myosins in Development

National Heart, Lung, And Blood Institute

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Abstract

Humans with point mutations in MYH9, the gene encoding nonmuscle myosin heavy chain (NMHC) II-A, develop a variety of syndromes including defects in their platelets (macrothrombocytopenia), kidneys (glomerulonephritis) and granulocytes (inclusion bodies). More than 20 different mutations in MYH9 have been reported to date, including both missense and nonsense mutations. The purpose of these studies is to gain insight into the pathological mechanism of the diseases caused by these mutations by creating a mouse model for one of the mutations (R709C) and studying the resultant mouse phenotype. Previous in vitro work has shown that the R709C mutation, which is in the motor domain of NMHC II-A, compromises the MgATPase activity and movement velocity of the myosin. To generate the mutant mouse, we used homologous recombination to alter mouse embryonic stem cells in order to replace the wild-type Myh9 allele with the mutant human R709C II-A myosin heavy chain. We have produced the necessary ES cell clones and have used them to generate chimeric mice using C57BL/6 blastocysts microinjected with the mutant ES cells. We have 7 heterozygous mice. Once the population of the heterozygous NMHC II-A R702C mouse colony is big enough we will begin to analyze the heterozygous and then homozygous mice. In addition to using these mutant mice to study the relation between the nonmuscle myosin II-A mutation and disease, we plan to use various cells derived from these mice to study the effects of the mutation on basic properties of the cell. These include cell-cell and cell matrix adhesion, cell polarity and cell migration. To gain clear insights into the distribution and function of different isoforms of nonmuscle myosin II (NMII) in normal mouse, the enhanced GFP or mCherry sequence has been inserted in front of the start codon of the Myh9 gene in the first coding exon. Embryonic stem cell clones have been screened to verify the correct recombination. Once the mouse model is created, the expression level as well as the distribution of NM II-A during mouse embryonic development will be analyzed. This will shed light on the function of NM II-A in development. Various cell lines derived from the mouse will be used to study the regulation and function of NM II-A in adhesion, cell polarity and cell migration. The purpose of another study is to learn whether one isoform of NM II, specifically NM II-C, can functionally replace a second one, NM II-A, in mice. To replace NM II-A with NM II-C, homologous recombination will be used to inactivate NM II-A by inserting the cDNA for NM II-C-GFP into the first coding exon of the Myh9 gene. We have made the plasmid construct for eletroporation and homologous recombination.