Depletion of kinesin-12, a myosin-IIB-interacting protein, promotes migration of cortical astrocytes

Abstract

Kinesin-12 (also named Kif15) participates in important events during neuronal development, such as cell division of neuronal precursors, migration of young neurons and establishment of axons and dendritic arbors, by regulating microtubule organization. Little is known about the molecular mechanisms behind the functions of kinesin-12, and even less is known about its roles in other cell types of the nervous system. Here, we show that kinesin-12 depletion from cultured rat cortical astrocytes decreases cell proliferation but increases migration. Co-immunoprecipitation, GST pulldown and small interfering RNA (siRNA) experiments indicated that kinesin-12 directly interacts with myosin-IIB through their tail domains. Immunofluorescence analyses indicated that kinesin-12 and myosin-IIB colocalize in the lamellar region of astrocytes, and fluorescence resonance energy transfer analyses revealed an interaction between the two. The phosphorylation at Thr1142 of kinesin-12 was vital for their interaction. Loss of their interaction through expression of a phosphorylation mutant of kinesin-12 promoted astrocyte migration. We suggest that kinesin-12 and myosin-IIB can form a hetero-oligomer that generates force to integrate microtubules and actin filaments in certain regions of cells, and in the case of astrocytes, that this interaction can modulate their migration.

Fidgetin regulates cultured astrocyte migration by severing tyrosinated microtubules at the leading edge

“…To probe cell migration, we performed Transwell assays as in our previous study ( Feng et al. , 2016 ).…”

“…, 2015 ), we found that Fign depletion reduced astrocyte migration, with a 47.5% decrease compared with the control (migrated cells were stained with crystal violet in red; Figure 1C ). Considering that cell proliferation is the other potential variable in this assay, we applied the 5-ethynyl-2’-deoxyuridine (EdU) incorporation DNA assay to cultured astrocytes depleted of Fign with siRNA as in our previous study ( Feng et al. , 2016 ).…”

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9‐mediated kif15 mutations accelerate axonal outgrowth during neuronal development and regeneration in zebrafish

“…Our previous data showed that kif15 is strongly expressed in developing nervous system in zebrafish with the expression gradually decreasing as the neurons mature 14 , which is similar to the case in rodent neurons 11 . Depletion of KIF15 from cultured rodent neurons results in longer axons with fewer branches 11 , while its depletion from cultured astrocytes results in increased migration 21 . In the present study, we applied CRISPR/Cas9-based knockout technology to create kif15 mutants, and labeled neurons by hybridizing with Tg(mnx1:GFP) zebrafish or by injecting plasmid pDestTol2-elavl3:EGFP-alpha tubulin.…”

“…The KIF15 tetramer may behave as a brake via its cross-linking function, with our data showing accelerated axon growth explicable on the basis of releasing the KIF15 brake. Studies to date indicate that KIF15 can do the same sorts of things as KIF11, in terms of regulating microtubule sliding, but can also regulate microtubule-actin interactions, via KIF15’s unique ability to interact with Myosin-IIB, a property not shared by KIF11 21,29 . We believe the braking effect of KIF15 is at least partly the effect of KIF15-Myosin-IIB complex.…”

“…Drugs could potentially be developed that attenuate KIF15’s microtubule-microtubule functions while not affecting its microtubule-actin functions, or vice versa. Finally, it is relevant to note that KIF15 inhibition may have beneficial effects on other cell types relevant to nerve regeneration, such as the cells that form the glial scar 21 . The cancer community is already at work developing drugs against KIF15, and hence the time is ripe for the nerve regeneration community to participate.…”