Interphase and monoastral-mitotic phenotypes of overexpressed MAP4 are modulated by free tubulin concentrations

The microtubule-associated protein 4 (MAP4) has recently been shown to counteract destabilization of interphase microtubules caused by catastrophe promotion but not by tubulin sequestering. To address how MAP4 discriminates between destabilization of microtubules by these two mechanisms, we have evaluated the combined phenotypes of MEAN coexpressed with Op18/stathmin family member derivatives with either catastrophe-promoting or sequestering activities. This approach relies on the finding that overexpression of MAN alone stabilizes microtubules during all phases of the cell cycle in human leukemia cells, and causes a potent mitotic block and a dramatic, previously unobserved, phenotype characterized by large monoastral spindles. Coexpression of either catastrophe-promoting or tubulin-sequestration-specific Op18 derivatives was found to modulate the activity of ectopic MAN during mitosis, but with differential functional outcome. Interestingly, the tubulin-sequestering derivative suppressed the monoastral mitotic phenotype of NUN (i.e. coexpression facilitated the formation of functional spindles). To evaluate whether this phenotypic suppression could be explained by tubulin-sequestration-dependent modulation of MAN activity, a plasma-membrane-targeted, tubulin-sequestering chimera was constructed to decrease the cytosolic free tubulin concentration substantially. This chimera likewise suppressed the monoastral phenotype caused by overexpression of NUN, suggesting a direct downregulation of MAN activity by reduced free tubulin concentrations.