THE FUNCTION OF NONMUSCLE MYOSIN II HEAVY CHAINS

缺乏氨基酸1-592的非肌球蛋白重链II-A （NMHC II-A）的截断片段已被用于检测NMHC II-A的细胞功能。将绿色荧光蛋白（GFP）融合到全长和截断的NMHC II- a的n端，融合蛋白在HeLa et-off细胞系（Clontech, Palo Alto， CA）中稳定表达，该细胞系表达NMHC II- a，但不表达NMHC II- b。截断的NMHC II- a在HeLa et-off细胞中的表达诱导细胞圆融，肌动蛋白丝重排，局灶粘连消失。当添加强力霉素终止截断的NMHC II-A的表达时，这些变化被逆转。表达全长NMHC II-A:GFP融合蛋白的HeLa t-off细胞在使用强力霉素或不使用强力霉素时均未表现出这种表型。GFP标记的全长和截断的肌球蛋白都定位于肌动蛋白应激纤维。然而，体外实验表明杆状病毒表达的截短肌球蛋白不能与肌动蛋白结合，这表明截短的肌动蛋白是由内源性肌动蛋白带入肌动蛋白应激纤维的。免疫荧光研究证实，截断的肌球蛋白与内源性肌球蛋白共定位使用抗体特异性识别内源性肌球蛋白。我们还证明了gfp标记的全长肌球蛋白II-A和II-B，而不是截断的肌球蛋白，在有丝分裂期间定位于细胞动力学环，这表明肌球蛋白可能需要功能性运动结构域定位于细胞动力学环。-非肌球蛋白；海拉Tet-off;主要负

A truncated fragment of nonmuscle myosin heavy chain II-A (NMHC II-A), lacking amino acids 1-592, has been used to examine the cellular functions of NMHC II-A. Green fluorescent protein (GFP) was fused to the N-terminal end of both full-length and truncated NMHC II-A and the fusion proteins were stably expressed in the HeLa Tet-off cell line (Clontech, Palo Alto, CA), which expresses NMHC II-A, but not NMHC II- B. The expression of truncated NMHC II-A in HeLa Tet-off cells induced cell rounding with rearrangements of actin filaments and disappearance of focal adhesions. These changes were reversed when the expression of truncated NMHC II-A was terminated by addition of doxycycline. HeLa Tet-off cells expressing the full-length NMHC II-A:GFP fusion protein did not show this phenotype with or without doxycycline. Both GFP- tagged full-length and truncated myosin was localized to actin stress fibers. However, in vitro assays showed that the baculovirus-expressed truncated myosin did not bind to actin, suggesting that the truncated myosin was brought to actin stress fibers by the endogenous myosin. Immunofluorescent studies confirmed that the truncated myosin colocalized with the endogenous myosin by using an antibody specifically recognizing only the endogenous myosin. We also demonstrated that GFP-tagged full-length myosin II-A and II-B, but not the truncated myosin, were localized to the cytokinetic ring during mitosis, indicating that the functional motor domain might be required for myosins to localize to the cytokinetic ring. - nonmuscle myosin; HeLa Tet-off; dominant negative