MICROINJECTION OF INTACT MAP-4 AND FRAGMENTS INDUCES CHANGES OF THE CYTOSKELETON IN PTK2 CELLS

The molecular cloning and sequencing of microtubule-associated protein (MAP)-4 identified microtubule-binding repeats near the C-terminus an d a projection domain near the N-terminus. Although it is well known t hat MAP-4 stimulates the assembly of and stabilizes microtubules (MT) in vitro, the function of MAP-4 in vivo is still unclear. In this stud y, 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, an d the truncated fragments of the N- and the C-terminal halves (named N R and PA(4) fragments, respectively) were expressed in Escherichia col i and isolated. In vitro studies demonstrated that in a solution conta ining a physiological concentration of NaCl, intact MAP-4 and the PA(4 ) fragment were bound to MT, but not to F-actin. The NR fragment was n ot 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 trun cated fragments of PA(4) and NR. The injection of intact MAP-4 or PA(4 ) 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. I njected MAP-4 and PA(4) were associated with the increased MT. In addi tion, injection with MAP-4 and PA(4) stabilized MT in relation to trea tment with the MT-disrupting drug, nocodazole. These results indicated that intact MAP-4 and the PA(4) fragment promoted MT assembly and sta bilized MT, by binding to MT, in vivo as well as in vitro. Further, th e injection of the PA4 fragment induced an increase in stress fibers. However, these proteins did not show any association with the stress f ibers. Our results suggest that there is an indirect effect of MAP-4 o n stress fibers rather than a direct interaction between MAP-4 and str ess fibers. (C) 1996 Wiley-Liss, Inc.