The Effect And Mechanism Of Kinesin-12 Depletion On Astrocyte Proliferation And Cell Migration

Objective Kinesin-12(also known as Kif15) is a member of a group of microtubule-dependent motor proteins that are known to play key roles in cell division, contributing to the formation of the mitotic spindle, chromosome separation and cytokinesis. We have found that some of these mitotic motor proteins continue to be expressed in terminally post-mitotic neurons, where they contribute to important events such as migration of young neurons through the brain and the establishment of axons and dendritic arbors. Kinesin-12 is a mitotic motor that participates in all of these events during neuronal development, and appears to do so by regulating microtubule movements and organization. Little is known about the molecular mechanism of Kinesin-12's functions, and even less is known about the roles it might play in other cell types of the nervous system. Here we want to investigate the effect of Kinesin-12 depletion from cultured rat spinal cord astrocytes on cell proliferation and cell migration.Methods We used the Ed U and Transwell methods to evaluate astrocyte proliferation and migration after treated by kinesin-12 si RNA. Co-IP,GST pull-down and si RNA/CO-IP experiments were taken to investigate the directly interacting between Kinesin-12 and Myosin ?B, a non-muscle Myosin ? heavy chain, via their tail domains. We examined the potential co-localization of Kinesin-12 and Myosin ?B in astrocytes by Immunofluorescence analyses and fluorescence resonance energy transfer(FRET).Results 1. In the primary cultured astrocytes, after treated by Kinesin-12, Kinesin-5 and Kinesin-12/ Kinesin-5 si RNA for 3 days, we used the Ed U to investigate the effect of cell proliferation. With Kinesin-12 depletion, the astrocytes showed less frequent cell division, with a 24.7% decrease compared to control si RNA. There was a 32.9% decrease with Kinesin-5 si RNA treatment, and a 46.9% decrease with si RNA to both motor proteins combined. The results of cell cycle analysis indicated Kinesin-12 and Kinesin-5 may have different mechanisms. 2. We then performed the Transwell assay to learn whether depletion of Kinesin-12, Kinesin-5 and Kinesin-12/Kinesin-5 affects cell migration. We found that depletion of Kinesin-12 promoted cell migration notably, with a 162.4% increase compared to the control, but the Kinesin-5 showed no significant promotion of astrocytes migration. 3. CO-IP,GST-pulldown, si RNA/CO-IP experiments confirmed a direct interaction between Kinesin-12(743-1333 aa) and Myosin ?B(1345-1648 aa). 4. Using immunocytochemistry and the fluorescence resonance energy transfer(FRET), we found that Kinesin-12 co-localizes and interacts with Myosin ?B in lamellar region of astrocytes.Conclusions In the primary cultured astrocytes, depletion of Kinesin-12 could promote cell migration and decrease cell proliferation notably. Kinesin-12(743-1333 aa) interacts with Myosin ?B(1345-1648 aa), and co-localizes in lamellar region of astrocytes. We predict that Kinesin-12/Myosin ?B heteropolymer could regulate cytoskeletal rearrangment throgh interacting microtubule and microfilaments.