RanBP10 Is a Cytoplasmic Guanine Nucleotide Exchange Factor That Modulates Noncentrosomal Microtubules

Abstract

Microtubule spindle assembly in mitosis is stimulated by Ran⅐GTP, which is generated along condensed chromosomes by the guanine nucleotide exchange factor (GEF) RCC1. This relationship suggests that similar activities might modulate other microtubule structures. Interphase microtubules usually extend from the centrosome, although noncentrosomal microtubules function in some differentiated cells, including megakaryocytes. In these cells, platelet biogenesis requires massive mobilization of microtubules in the cell periphery, where they form proplatelets, the immediate precursors of platelets, in the apparent absence of centrioles. Here we identify a cytoplasmic Ranbinding protein, RanBP10, as a factor that binds ␤-tubulin and associates with megakaryocyte microtubules. Unexpectedly, RanBP10 harbors GEF activity toward Ran. A point mutation in the candidate GEF domain abolishes exchange activity, and our results implicate RanBP10 as a localized cytoplasmic Ran-GEF. RNA interference-mediated loss of RanBP10 in cultured megakaryocytes disrupts microtubule organization. These results lead us to propose that spatiotemporally restricted generation of cytoplasmic Ran⅐GTP may influence organization of the specialized microtubules required in thrombopoiesis and that RanBP10 might serve as a molecular link between Ran and noncentrosomal microtubules.Microtubules (MTs) 3 are essential components of the cytoskeleton in all cells, although specific fiber types and nucleation mechanisms differ according to cell lineage and over the cell cycle. Interphase cells typically contain a single MT-organizing center (MTOC) located within the centrosome. This structure contains two perpendicularly oriented centrioles and the ␥-tubulin ring complex, which initiates assembly of radial MT arrays (1, 2). Centrioles duplicate during S phase and subsequently migrate to opposite cell poles, where they anchor the mitotic spindle and enable chromosome segregation (3-5). After cytokinesis, the centrioles serve again as origins for interphase MTs, which are relatively stable structures. In contrast, the considerably more dynamic MTs in the mitotic spindle are assembled through the activity of the small GTPase Ran (6 -9).Ran controls at least three essential functions at different stages in the cell cycle: nucleocytoplasmic traffic in interphase, spindle formation at mitosis, and nuclear envelope assembly during telophase (10). Each function requires GTP hydrolysis and exchange of GDP for GTP. Small GTPases have low intrinsic rates of nucleotide exchange, which are enhanced over 10 5 -fold by guanine nucleotide exchange factors (GEFs), a diverse group of enzymes that show limited sequence similarity and considerable target protein specificity (11)(12)(13)(14). RCC1, the only known Ran-GEF, is a chromatin-associated nuclear protein that activates Ran by loading GTP (15). Conversely, the cytoplasmic Ran-GTPase-activating protein (GAP) RanGAP1 acts to produce Ran⅐GDP (16), and the resulting interphase Ran⅐GTP concentration gradient drives macromolecul...

Intrinsic impaired proplatelet formation and microtubule coil assembly of megakaryocytes in a mouse model of Bernard-Soulier syndrome

“…Recently, however, a two-hybrid screen of a mouse megakaryocyte library has revealed RanBP10, in similar distribution in the microtubular coil, which could play a role in the process of microtubule mobilization during proplatelet formation. 47 There is to date no indication of a direct or indirect link, through a partner such as Ran BP10, between GPIb and tubulin.…”

Microtubule assembly by the Apc protein is regulated by importin-β—RanGTP

“…This RanGTP gradient directs the localised release of spindle-assembly factors from importin-b and their activation near chromatin (Clarke and Zhang, 2008;Kalab and Heald, 2008). Cytoplasmic areas of elevated RanGTP generated by RanBP10 have also been implicated in reorganising peripheral MTs in interphase (Schulze et al, 2008).…”

Ran on tracks – cytoplasmic roles for a nuclear regulator

“…Finally, shRNA-induced depletion of RanBP10 in megakaryocytes caused disruption of the microtubule cytoskeleton. Thus, the results of Schulze et al (Schulze et al, 2008) suggest that spatiotemporally restricted generation of RanGTP by RanBP10 on the cytoplasmic microtubule cytoskeleton might influence microtubule organization and cytoskeletal dynamics. Notably, we observed concentrates of RanGAP at the growing axon tip in sensory neurons in culture (Yudin et al, 2008).…”

“…Moreover, the fact that RanBP9 is linked to axon-guidance receptors on the one hand (Togashi et al, 2006) and to microtubules on the other (Nakamura et al, 1998) suggests that both of these Ran-binding molecules might directly transduce guidance signaling to cause cytoskeletal responses. (Schulze et al, 2008)], and the lower panels show localization and concentration of RanGAP at the tips of growing sensory-neuron axons [reprinted with permission from Yudin et al (Yudin et al, 2008)]. The juxtaposition of these images (albeit from different cell types) suggests that, if both molecules (or their close homologs) were coexpressed, it might be possible to create localized Ran gradients in the cytoplasm of morphologically complex cells by restricting the amount of GEF to microtubules and that of GAP to other subcellular compartments.…”

“…Schulze et al (Schulze et al, 2008) set out to study the dynamics of microtubules in megakaryocytes, which are large polyploid blood cells that -upon maturation -generate nascent blood platelets within microtubule-based cytoplasmic extensions. This process requires significant mobilization of microtubules that are enriched in β1-tubulin, the form that is required for efficient thrombopoiesis in the cell periphery.…”