| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2004: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2003: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
Myosin is a functional protein associated with cellular movement, cell division, muscle contraction and other functions. Members of the myosin super-family are distinguished from the myosin heavy chains that play crucial roles in cellular processes. Although there are many studies of myosin heavy chain in this family, there are few on non-muscle myosin heavy chain than of muscle myosin heavy chain. Myosin is classified as type I (myosin I) or type II (myosin II). Myosin I, called unconventional myosin or mini-myosin, has one head, while myosin II, called conventional myosin, has two heads. We transfected non-muscle myosin heavy chain, myosin II, myosin heavy polypeptide 9 (MYH9), into HeLa cells as fusion protein with enhanced green fluorescent protein (EGFP). We analyzed in HeLa cells, MYH9 localization and distribution comparatively with actin or tubulin presence. To study the cellular localization of MYH9, we transfected mammalian expression plasmid vector, pEGFP-N3-MYH9 or pEGFP-N3
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, into HeLa cells with cellfectin reagent. When only EGFP was expressed in HeLa cells, there was no regulation of its expression. However, when MYH9-EGFP was overexpressed in HeLa cells, it existed with actin stress fibers. Thus, while MYH9 colocalized with actin stress fibers, it did not colocalize with tubulin as an intermediate filament. When cytochalasin D was added to MYH9-EGFP transfectants, cells lost actin stress fibers. However, there were no changes in localization of tubulin whether cytochalasin D was added or not, indicating that MYH9-EGFP existed with actin, but did not affect the form of tubulin expression. Recently, it has been clearly shown by genetic analysis that autosomal dominant giant platelet syndromes are MYH9-related disorders. Our development of transfected EGFP-MYH9 might be useful for predicting the association between the function of actin polymerization, MYH9 motor domain, and these disorders. In the future, we hope to find cofactors of MYH9 using MYH9-EGFP as the tag and we are trying to decide what is downstream. Our subjects are application clinically with using this factor as target. Less
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