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Microinjection of intact MAP-4 and fragments induces changes of the cytoskeleton in PtK2 cells
T Yoshida1, K Imanaka-Yoshida, H Murofushi
1Department of Pathology, Mie University School of Medicine, Japan.
Abstract:
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.
Insights
Microtubule-associated protein (MAP)-4 and its PA4 fragment promote microtubule assembly and stability both in vitro and in vivo by binding to microtubules. MAP-4 also indirectly affects stress fibers.
Area of Science:
- Cell Biology
- Cytoskeleton Dynamics
- Protein Function Analysis
Background:
- Microtubule-associated protein (MAP)-4 is known to influence microtubule assembly and stability in vitro.
- The in vivo function of MAP-4 within the cytoskeleton remains largely undetermined.
- MAP-4 possesses microtubule-binding repeats near its C-terminus and a projection domain near its N-terminus.
Purpose of the Study:
- To investigate the in vivo and in vitro functions of MAP-4 in the cytoskeleton.
- To determine the roles of intact MAP-4 and its N-terminal (NR) and C-terminal (PA4) fragments in microtubule dynamics.
- To elucidate the interaction of MAP-4 with microtubules and F-actin.
Main Methods:
- Preparation of intact MAP-4 from bovine adrenal cortex.
- Expression and isolation of truncated NR and PA4 fragments in E. coli.
- In vitro binding assays with microtubules (MT) and F-actin.
- Microinjection of MAP-4, PA4, and NR fragments into PtK2 cells.
- Assessment of MT array changes and drug stability post-injection.
Main Results:
- Intact MAP-4 and the PA4 fragment bound to MT in vitro, while NR did not.
- Microinjection of intact MAP-4 or PA4 increased cytoplasmic MT number and induced MT bundling in PtK2 cells.
- MAP-4 and PA4 stabilized MT against nocodazole treatment and were associated with increased MT.
- PA4 fragment injection increased stress fibers, suggesting an indirect effect of MAP-4.
Conclusions:
- Intact MAP-4 and the PA4 fragment promote MT assembly and stabilization through direct MT binding, both in vitro and in vivo.
- MAP-4 exerts an indirect influence on stress fibers, rather than a direct interaction.
- The C-terminal PA4 fragment contains key domains for MAP-4's MT-binding and regulatory functions.