Work in the laboratory of MB is supported by grants from the Danish Cancer Society Scientific Committee (KBVU; R146\A9322), the Lundbeck Foundation (R215\2015\4081), and the Novo Nordisk Foundation (NNF19OC0058504). transmitted from non\KT\MTs to KT\MTs by the MT couplers HSET and NuMA. Additionally, we found that the MT\flux rate correlates with Gefarnate spindle length, and this correlation depends on the establishment of stable end\on KT\MT attachments. Strikingly, we find that MT\flux is required to regulate spindle length by counteracting kinesin 13/MCAK\dependent MT\depolymerization. Thus, our study unveils the long\sought mechanism of MT\flux in human cells as relying on the coordinated action of four kinesins to compensate for MT\depolymerization and regulate spindle length. embryos (Rogers egg extracts (Gaetz & Kapoor, 2004), while its reduction in human cells either had no effect on spindle length (Ganem (number of cells, number of independent experiments): C bipolar spindles: siControl (49, 5), STLC (31, 3), siKIF15 (39, 3), siKID (32, 3), siKIF4A (28, 3), siKIF2A (36, 3), siCLASPs (21, 2), GSK923295 (44, 3); C monopolar spindles: siControl (35, 3), siKIF15 (44, 3), siKID (38, 3), siKIF4A (44, 3), siKIF2A (38, 3), siCLASPs (12, 2), GSK923295 (33, 3). (number of cells, number of independent experiments): Control (untreated) (23, 3); 1 MOI (33, 3); 2 MOI (26, 3), 4 MOI (9, Gefarnate 1). Gefarnate (number of cells, number of independent experiments): uninduced shKIF4A (48, 4), shKIF4A (60, 4), uninduced KIF4A WT (36, 5), shKIF4A?+?KIF4A WT (43, 5), uninduced KIF4A K94A (42, 7), shKIF4A?+?KIF4A K94A (38, 6), uninduced KIF4A Zip1 (32, 4), and shKIF4A?+?Zip1 (30, 4). (number of cells, number of independent Gefarnate experiments): Caffeine\only control (30, 3) and MUGs (34, 3). (number of cells, number Gefarnate of independent experiments): siControl (49, 5), siPRC1 (45, 3), siNuMA (40, 3), siHSET (37, 3), and siHSET?+?siNuMA (39, 3). (number of cells, number of independent experiments): siControl (23, 3), siHSET 3UTR (33, 3), siHSET 3UTR?+?HSET WT (29, 5), and siHSET 3UTR?+?HSET N593K (30, 4). oocyte spindles assembled (Maddox (number of cells, number of independent experiments): siControl (49, 5), siNDC80 (59, 6), siNDC80?+?siKIF4A (41, 3), siNDC80?+?STLC (bipol.) (28, 3), siNDC80?+?siKIF15 (36, 4), siNDC80?+?siCLASPs (33, 3), siNDC80?+?siKIF2A (38, 4), siNDC80?+?GSK923295 (38, 3), early prometaphase (23, 3), and early prometaphase?+?GSK923295 (28, 3). (number of cells, number of independent experiments): prometaphase (23, 3), metaphase (22, 3). To investigate the molecular mechanism underlying MT\flux before the establishment of stable end\on KT\MT attachments, we further examined the potential contributions of EG5 and KIF15 (Figs?5B and EV4A). Similar to the effect of KIF4A depletion, neither EG5 inhibition nor depletion of KIF15 reduced MT\flux rate after NDC80 RNAi. Since CENP\E inhibition had a negative impact on MT\flux rate exclusively in monopolar spindles, which contain less end\on KT\MT attachments (Kapoor egg extract spindles (Sawin & Mitchison, 1991). This strongly suggests that interpolar MTs overlap over a broader region in early mitosis, but then become more restricted to the spindle equator as cells reach metaphase. The broad distribution of antiparallel MTs in early mitosis coincides with CENP\E localization at laterally attached KTs during prometaphase rosette configuration (Itoh resolution is 70?nm. The expected/described localization of CENP\E-GFP (green) at KTs is shown. In addition, CENP\E-GFP was found associated with interpolar MTs (ipMTs), including regions of overlapping antiparallel MTs, and k\fibers. Chromosomes (confocal mode only) were revealed in the larger panels with DAPI (cyan) and MTs (magenta) were detected with an anti\\tubulin antibody. Arrows indicate examples of clear MT bundles, including regions of overlapping antiparallel MTs. Scale bar in all panels is 5?m. Representative spinning disk confocal live\cell time series of HeLa cells stably expressing CENP\E-GFP. White arrowheads highlight CENP\E-GFP at KTs, white arrows highlight CENP\E-GFP on interpolar MTs. Scale bar, 10?m. Time, h:min. Since earlier work showed that CENP\E localization at the spindle Mouse monoclonal to Alkaline Phosphatase midzone in late anaphase depends on PRC1 (Kurasawa (number of cells, number of independent experiments): siNDC80?+?siPRC1 (37, 3). Immunoblot analyses of the efficiency of knockdown from U2OS cells stably expressing PA\GFP/mCherry\\tubulin treated with control or indicated siRNAs, with \tubulin used as a loading control. Combined action of EG5 and KIF15 support MT\flux driving activities of CENP\E and KIF4A Although we identified CENP\E and KIF4A as drivers of MT\flux in prometaphase and metaphase, respectively, functional inactivation of either of these two motors resulted in only around 50% decrease in MT\flux rate, indicating the presence of other important contributor(s). Therefore, we tested whether EG5 and KIF15 play a synergistic role in driving MT\flux. Importantly, in contrast to their individual functional inactivation, which did not affect MT\flux rates (Fig?1E), inhibition of EG5 in KIF15\depleted cells.