Warren K S, Lin J L, McDermott J P, Lin J J
Department of Biological Sciences, University of Iowa, Iowa City 52242-1324.
J Cell Biol. 1995 May;129(3):697-708. doi: 10.1083/jcb.129.3.697.
Human fibroblasts generate at least eight tropomyosin (TM) isoforms (hTM1, hTM2, hTM3, hTM4, hTM5, hTM5a, hTM5b, and hTMsm alpha) from four distinct genes, and we have previously demonstrated that bacterially produced chimera hTM5/3 exhibits an unusually high affinity for actin filaments and a loss of the salt dependence typical for TM-actin binding (Novy, R.E., J. R. Sellers, L.-F. Liu, and J.J.-C. Lin, 1993. Cell Motil. & Cytoskeleton. 26: 248-261). To examine the functional consequences of expressing this mutant TM isoform in vivo, we have transfected CHO cells with the full-length cDNA for hTM5/3 and compared them to cells transfected with hTM3 and hTM5. Immunofluorescence microscopy reveals that stably transfected CHO cells incorporate force-expressed hTM3 and hTM5 into stress fibers with no significant effect on general cell morphology, microfilament organization or cytokinesis. In stable lines expressing hTM5/3, however, cell division is slow and sometimes incomplete. The doubling time and the incidence of multinucleate cells in the stable hTM5/3 lines roughly parallel expression levels. A closely related chimeric isoform hTM5/2, which differs only in the internal, alternatively spliced exon also produces defects in cytokinesis, suggesting that normal TM function may involve coordination between the amino and carboxy terminal regions. This coordination may be prevented in the chimeric mutants. As bacterially produced hTM5/3 and hTM5/2 can displace hTM3 and hTM5 from actin filaments in vitro, it is likely that CHO-expressed hTM5/3 and hTM5/2 can displace endogenous TMs to act dominantly in vivo. These results support a role for nonmuscle TM isoforms in the fine tuning of microfilament organization during cytokinesis. Additionally, we find that overexpression of TM does not stabilize endogenous microfilaments, rather, the hTM-expressing cells are actually more sensitive to cytochalasin B. This suggests that regulation of microfilament integrity in vivo requires stabilizing factors other than, or in addition to, TM.
人类成纤维细胞从四个不同的基因中产生至少八种原肌球蛋白（TM）异构体（hTM1、hTM2、hTM3、hTM4、hTM5、hTM5a、hTM5b和hTMsmα），并且我们之前已经证明，细菌产生的嵌合体hTM5/3对肌动蛋白丝表现出异常高的亲和力，并且丧失了TM-肌动蛋白结合典型的盐依赖性（诺维，R.E.，J.R.塞勒斯，L.-F.刘，和J.J.-C.林，1993年。《细胞运动与细胞骨架》。26：248-261）。为了研究在体内表达这种突变TM异构体的功能后果，我们用hTM5/3的全长cDNA转染了CHO细胞，并将它们与用hTM3和hTM5转染的细胞进行比较。免疫荧光显微镜检查显示，稳定转染的CHO细胞将强制表达的hTM3和hTM5整合到应力纤维中，对一般细胞形态、微丝组织或胞质分裂没有显著影响。然而，在表达hTM5/3的稳定细胞系中，细胞分裂缓慢，有时不完全。稳定的hTM5/3细胞系中的倍增时间和多核细胞的发生率大致与表达水平平行。一种密切相关的嵌合异构体hTM5/2，仅在内部选择性剪接外显子上有所不同，也会在胞质分裂中产生缺陷，这表明正常的TM功能可能涉及氨基和羧基末端区域之间的协调。这种协调在嵌合突变体中可能会被阻止。由于细菌产生的hTM5/3和hTM5/2可以在体外从肌动蛋白丝上取代hTM3和hTM5，因此CHO表达的hTM5/3和hTM5/2很可能可以取代内源性TMs在体内起主导作用。这些结果支持非肌肉TM异构体在胞质分裂期间微丝组织的微调中发挥作用。此外，我们发现TM的过表达并不能稳定内源性微丝，相反，表达hTM的细胞实际上对细胞松弛素B更敏感。这表明体内微丝完整性的调节需要除TM之外或除TM之外还需要稳定因子。
