THE FUNCTION OF NONMUSCLE MYOSIN II HEAVY CHAINS

非肌肉肌球蛋白重链II-A（NMHC II-A）的截短片段，缺乏氨基酸1-592，已被用来检查NMHC II-A的细胞功能。将绿色荧光蛋白（GFP）融合到全长和截短的NMHC II-A的N-末端，并且融合蛋白在HeLa Tet-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。在HeLa Tet-off细胞中截短的NMHC II-A的表达诱导细胞变圆，肌动蛋白丝重排和粘着斑消失。这些变化被逆转时，截短的NMHC II-A的表达被终止加入强力霉素。表达全长NMHC II-A：GFP融合蛋白的HeLa Tet-off细胞在有或没有强力霉素的情况下均未显示出这种表型。GFP标记的全长和截短的肌球蛋白都定位于肌动蛋白应力纤维。然而，体外试验表明，杆状病毒表达的截短的肌球蛋白不结合肌动蛋白，这表明截短的肌球蛋白被带到肌动蛋白应力纤维的内源性肌球蛋白。免疫荧光研究证实，截短的肌球蛋白与内源性肌球蛋白共定位，通过使用抗体特异性地识别只有内源性肌球蛋白。我们还表明，GFP标记的全长肌球蛋白II-A和II-B，但不是截短的肌球蛋白，在有丝分裂期间被定位到细胞动力学环，这表明肌球蛋白定位到细胞动力学环可能需要功能性运动结构域。- 非肌肉肌球蛋白; HeLa Tet-off;显性阴性

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