The Function Of Nonmuscle Myosin II Heavy Chains

Heart, Lung, And Blood Institute

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Abstract

We have been studying the effects of Pitx2a expression on the actin-myosin cytoskeleton organization in HeLa cells. Ectopic expression of Pitx2a induced actin-myosin reorganization, leading to increased cell spreading, suppression of cell migration, and the strengthening of cell-cell adhesion, marked by the accumulation and localization of beta-catenin and N-cadherin to the sites of cell-cell contacts. Moreover, Pitx2a expression resulted in activation of the Rho GTPases Rac1 and RhoA, and the dominant negative Rac1 mutant N17Rac1 inhibited cell spreading and disrupted localization of beta-catenin to the sites of cell-cell contacts. Both reorganization of actin-myosin and cell spreading require phosphatidylinositol 3-kinase activity, which is also necessary for activation of the Rho GTPase proteins. At the same time, we are also interested in understanding the functions of nonmuscle myosin heavy chain II-B using a mouse model. Previous reports (Tullio AN et al., PNAS, 94:12407, 1997; Tullio AN et al., J. Comparative Neurology, 433:62, 2001) demonstrated that ubiquitous ablation of NMHC II-B in mice resulted in defects in both brain and heart, leading to embryonic lethality between E14.5 and E18.5. However, the presence of both severe cardiac and brain defects in B-/B- mice raised the possibility that at least some of the abnormalities found in these organs could be secondary to the malfunction of the other organ rather than due to the absence of NMHC II-B. Therefore, we utilized the loxP/Cre strategy to specifically delete exon II of NMHC II-B in a tissue-specific manner. A targeting vector was generated and electroporated into embryonic stem (ES) cells, so that exon II of NMHC II-B was flanked by loxP sites. The ES cells with the modified NMHC II-B locus were then used to establish a mouse line B-loxP/loxP. The homozygous B-loxP/loxP mice are normal and fertile. NesCre, a transgenic mouse line harboring a Cre expression cassette under control of the nestin promoter, was crossed to B-loxP/loxP mice to obtain B-loxP/loxP NesCre, which should allow for removal of NMHC II-B in the brain, but not the heart. The B-loxP/loxP NesCre mice are smaller than wild-type littermates and develop hydrocephalus. Although some of the B-loxP/loxP NesCre mice die by postnatal day 12, others can live at least up to postnatal day 18. We are presently characterizing the defects to see whether they differ from those found in B-/B- mice. In addition, by crossing B-loxP/loxP mice with a cardiac-specific Cre transgenic mouse line, we hope to generate mice with pure cardiac defects, possibly free of neuronal abnormalities.