RanBP10 Is a Cytoplasmic GDP/GTP Exchange Factor That Modulates Microtubule Dynamics in Late Megakaryocytes.

Megakaryocytes are large cells within the bone marrow that undergo complex fragmentation to release up to thousands of virtually identical blood platelets into the periphery. Each platelet contains a characterisitic microtubule (MT) marginal band that is derived from MT filaments present in long protrusion-like intermediate structures, designated proplatelets, that are immediate precursors of platelets. These MT filaments are generated in the MK periphery, where they require massive mobilization that is supposed to be different from either normal interphase MT nucleation that commonly depends on γ-tubulin in the MT-organizing center. or from MTs in the mitotic spindle that require Ran·GTP, which is generated along condensed chromosomes by the chromatin-asociated guanine nucleotide exchange factor (GEF) RCC1. We first demonstrated that γ-tubulin is absent in most of the mature or proplatelet-forming MKs, where it is therefore unlikely to attribute to the total MT nucleation. MTs are tubular cytoskeletal structures that contain polymerized α- or β-tubulin subunits. Mammalian genomes share 5–6 β-tubulin isotypes of which β1-tubulin is the most divergent, especially in its C-terminal domain. β1-tubulin expression is restricted to late MKs and platelets, where it accounts for most of the β-tubulin in MT filaments. Its ablation in the mouse results in thrombocytopenia, spherocytosis and attenuated platelet function. We therefore sought to identify proteins that bind to β1-tubulin and performed a yeast two-hybrid screen using a MK-derived cDNA library. We identified a cytoplasmic Ran-binding protein, RanBP10, as a factor that associates with cellular MTs and unexpectedly harbors GEF activity toward Ran. Loss of RanBP10 in cultured MKs disrupts MT organization and its overexpression drives accumulation of extranuclear Ran and assembly of thick and abnormally long MTs. RanBP10 thus functions as a localized β-tubulin binding protein that harbors GEF activity toward Ran in the cytoplasm, much like RCC1 in the nucleus. Our results suggest that spatiotemporally restricted generation of Ran·GTP in the cytoplasm organizes specialized MTs required for thrombopoiesis and that RanBP10 provides a molecular link between Ran and non-centrosomal MTs.