Yoshida T, Imanaka-Yoshida K, Murofushi H, Tanaka J, Ito H, Inagaki M
Department of Pathology, Mie University School of Medicine, Japan.
Cell Motil Cytoskeleton. 1996;33(4):252-62. doi: 10.1002/(SICI)1097-0169(1996)33:4<252::AID-CM2>3.0.CO;2-B.
The molecular cloning and sequencing of microtubule-associated protein (MAP)-4 identified microtubule-binding repeats near the C-terminus and a projection domain near the N-terminus. Although it is well known that MAP-4 stimulates the assembly of and stabilizes microtubules (MT) in vitro, the function of MAP-4 in vivo is still unclear. In this study, we examined the function of MAP-4 in the cytoskeleton both in vitro and in vivo. Intact MAP-4 was prepared from bovine adrenal cortex, and the truncated fragments of the N- and the C-terminal halves (named NR and PA4 fragments, respectively) were expressed in Escherichia coli and isolated. In vitro studies demonstrated that in a solution containing a physiological concentration of NaCl, intact MAP-4 and the PA4 fragment were bound to MT, but not to F-actin. The NR fragment was not bound to MT or to F-actin. We also examined the MT changes in PtK2 cells after they had been microinjected with intact MAP-4 and the truncated fragments of PA4 and NR. The injection of intact MAP-4 or PA4 into the cells induced an increase in the number of cytoplasmic MT, as well as MT bundling. The NR fragment did not affect the MT array. Injected MAP-4 and PA4 were associated with the increased MT. In addition, injection with MAP-4 and PA4 stabilized MT in relation to treatment with the MT-disrupting drug, nocodazole. These results indicated that intact MAP-4 and the PA4 fragment promoted MT assembly and stabilized MT, by binding to MT, in vivo as well as in vitro. Further, the injection of the PA4 fragment induced an increase in stress fibers. However, these proteins did not show any association with the stress fibers. Our results suggest that there is an indirect effect of MAP-4 on stress fibers rather than a direct interaction between MAP-4 and stress fibers.
微管相关蛋白（MAP）-4的分子克隆和测序确定了其C末端附近的微管结合重复序列以及N末端附近的突出结构域。尽管众所周知MAP-4在体外能刺激微管（MT）的组装并使其稳定，但MAP-4在体内的功能仍不清楚。在本研究中，我们在体外和体内研究了MAP-4在细胞骨架中的功能。从牛肾上腺皮质制备完整的MAP-4，并在大肠杆菌中表达并分离其N端和C端半段的截短片段（分别命名为NR和PA4片段）。体外研究表明，在含有生理浓度NaCl的溶液中，完整的MAP-4和PA4片段与MT结合，但不与F-肌动蛋白结合。NR片段不与MT或F-肌动蛋白结合。我们还检测了向PtK2细胞显微注射完整的MAP-4以及PA4和NR截短片段后MT的变化。向细胞中注射完整的MAP-4或PA4会导致细胞质MT数量增加以及MT成束。NR片段不影响MT阵列。注射的MAP-4和PA4与增加的MT相关。此外，与用微管破坏药物诺考达唑处理相比，注射MAP-4和PA4可使MT稳定。这些结果表明，完整的MAP-4和PA4片段通过在体内和体外与MT结合来促进MT组装并使其稳定。此外，注射PA4片段会导致应力纤维增加。然而，这些蛋白质与应力纤维没有任何关联。我们的结果表明，MAP-4对应力纤维有间接作用，而不是MAP-4与应力纤维之间的直接相互作用。
