Ran-GTP Is Non-essential to Activate NuMA for Mitotic Spindle-Pole Focusing but Dynamically Polarizes HURP Near Chromosomes

•Using AID technology, we developed mitotic depletion assays for the Ran pathway•The pathway is non-essential to activate NuMA spindle-pole focusing•Ran-GTP not required target TPX2 but localize HURP and HSET•The maintains HURP’s polarized spindle localization during metaphase Spindle assembly spatially regulated by a chromosome-derived Ran- GTP gradient. Previous work proposed that Ran-GTP activates factors (SAFs) around chromosomes dissociating inhibitory importins from SAFs. However, it unclear whether gradient equivalently SAFs at distinct regions. In addition, Ran’s dual functions in interphase nucleocytoplasmic transport have made difficult assess its roles somatic cells. Here, using auxin-inducible degron technology human cells, acute dissect systematically separately function. contrast prevailing model, found essential activities occur sites separated chromosomes, including activating focusing or targeting TPX2. On other hand, HSET specifically chromosome-proximal regions set proper length prometaphase. We demonstrated importin-? coordinately promote dynamic microtubule binding-dissociation cycle, which near metaphase. Together, propose acts on independently of cells does regulate all Ran-regulated Ran-dependent likely coupled with additional parallel pathways distantly located chromosomes. During cell division, microtubule-based structure assembled efficiently capture segregate duplicated into daughter cells.1Reber S. Hyman A.A. Emergent properties spindle.Cold Spring Harb. Perspect. Biol. 2015; 7: a015784Crossref PubMed Scopus (27) Google Scholar2Heald R. Khodjakov A. Thirty years search capture: complex simplicity assembly.J. Cell 211: 1103-1111Crossref (88) Scholar dependent guanosine triphosphate (GTP)-bound form (Ran-GTP), surrounds animal cells.3Kalab P. Heald The RanGTP - GPS spindle.J. Sci. 2008; 121: 1577-1586Crossref (205) Scholar4Forbes D.J. Travesa Nord M.S. Bernis C. Reprint “Nuclear factors: global regulation mitosis”.Curr. Opin. 34: 122-134Crossref (11) produced regulator chromosome condensation 1 (RCC1), guanine nucleotide exchange factor (GEF) Ran,5Bischoff F.R. Ponstingl H. Catalysis RCC1.Nature. 1991; 354: 80-82Crossref (527) hydrolyzed Ran-guanosine diphosphate (GDP) RanGAP1, GTPase-activating protein Ran.6Bischoff Klebe Kretschmer J. Wittinghofer RanGAP1 induces GTPase activity nuclear Ras-related Ran.Proc. Natl. Acad. USA. 1994; 91: 2587-2591Crossref (404) As RCC1 mainly cytoplasm, respectively, these opposing enzymes create after envelope breaks down (Figure 1A). After exit, still binds chromatin although localizes envelop cytoplasm. Thus, generate different concentrations nucleus drives interphase.4Forbes has been best characterized Xenopus egg extracts,7Kalab Weis K. Visualization extracts.Science. 2002; 295: 2452-2456Crossref (434) Scholar8Kaláb Pralle Isacoff E.Y. Analysis RanGTP-regulated cells.Nature. 2006; 440: 697-701Crossref (278) also meiotic types.9Dumont Petri Pellegrin F. Terret M.E. Bohnsack M.T. Rassinier Georget V. Kalab Gruss O.J. Verlhac M.H. A centriole- RanGTP-independent meiosis I vertebrate oocytes.J. 2007; 176: 295-305Crossref (170) Scholar, 10Moutinho-Pereira Stuurman N. Afonso O. Hornsveld M. Aguiar Goshima G. Vale R.D. Maiato Genes involved centrosome-independent Drosophila S2 cells.Proc. 2013; 110: 19808-19813Crossref (37) 11Hasegawa Ryu S.J. Kaláb Chromosomal gain promotes formation steep mitosis aneuploid cells.J. 200: 151-161Crossref (30) Recent studies indicate acentrosomal female meiosis,9Dumont Scholar12Holubcová Z. Blayney Elder Schuh Human oocytes. Error-prone chromosome-mediated favors segregation defects oocytes.Science. 348: 1143-1147Crossref (147) Scholar13Drutovic D. Duan X. Li Solc importin ? function mouse oocytes.EMBO 2020; 39: e101689Crossref (8) significance debated.10Moutinho-Pereira Scholar11Hasegawa Scholar14Furuta Hori T. Fukagawa Chromatin binding important interphase.Mol. Cell. 2016; 27: 371-381Crossref Similar mechanisms transport,4Forbes mitosis, thereby releasing 1A).15Nachury M.V. Maresca T.J. Salmon W.C. Waterman-Storer C.M. Importin beta small assembly.Cell. 2001; 104: 95-106Abstract Full Text PDF (317) 16Wiese Wilde Moore Adam S.A. Merdes Zheng Y. Role importin-beta coupling downstream targets assembly.Science. 291: 653-656Crossref (272) 17Gruss Carazo-Salas R.E. Schatz C.A. Guarguaglini Kast Wilm Le Bot Vernos I. Karsenti E. Mattaj I.W. reversing effect alpha activity.Cell. 83-93Abstract (480) 18Stewart Molecular mechanism import cycle.Nat. Rev. Mol. 8: 195-208Crossref (620) Once activated, most interact microtubules nucleation, dynamics, transport, cross-linking, turn creates specialized local structures spindle.3Kalab For instance, apparatus (NuMA) recognizes minus ends transports cross-links cooperation cytoplasmic dynein focus maintain poles mammalian cells.19Hueschen C.L. Kenny Xu Dumont recruits minus-ends mitosis.eLife. 2017; 6: e29328Crossref (36) 20Okumura Natsume Kanemaki Kiyomitsu Dynein-dynactin-NuMA clusters cortical spindle-pulling forces as multi-arm ensemble.eLife. 2018; e36559Crossref (53) 21Gaglio Saredi Compton D.A. organization aster-like arrays.J. 1995; 131: 693-708Crossref (215) 22Silk A.D. Holland A.J. Cleveland D.W. Requirements maintenance establishment poles.J. 2009; 184: 677-690Crossref (97) Xklp2 (TPX2) pole organization23Wittmann TPX2, novel xenopus MAP organization.J. 2000; 149: 1405-1418Crossref (304) Scholar24Garrett Auer Kapoor T.M. hTPX2 normal morphology centrosome integrity division.Curr. 12: 2055-2059Abstract (117) stimulates nucleation importin-?-regulated manner.25Petry Groen A.C. Ishihara Mitchison Branching extracts mediated augmin TPX2.Cell. 152: 768-777Abstract (208) 26Roostalu Cade N.I. Surrey Complementary chTOG constitute an efficient importin-regulated module.Nat. 17: 1422-1434Crossref (84) 27King M.R. Petry Phase separation enhances coordinates nucleation.Nat. Commun. 11: 270Crossref (46) 28Scrofani Sardon Meunier Microtubule RanGTP-dependent complex.Curr. 25: 131-140Abstract (55) Kinesin-14 spleen, embryo, testes expressed (HSET/XCTK2) both anti-parallel preferentially poles.29Ems-McClung S.C. Walczak C.E. alpha/beta XCTK2 through bipartite signal.Mol. 2004; 15: 46-57Crossref (112) 30Ems-McClung Emch Zhang Mahnoor Weaver L.N. effector ?/? cross-linking.J. 219: e201906045Crossref (4) 31Cai Ems-McClung family proteins HSET/XCTK2 control cross-linking sliding microtubules.Mol. 20: 1348-1359Crossref (133) Hepatoma upregulated (HURP) accumulates bundled kinetochore (k-fibers).32Silljé H.H. Nagel Körner Nigg E.A. Ran-importin beta-regulated stabilizes vicinity chromosomes.Curr. 16: 731-742Abstract (198) Most SAFs, NuMA, HSET, contain sequence/signal (NLS),29Ems-McClung Scholar33Chang C.C. Huang T.L. Shimamoto Tsai S.Y. Hsia K.C. Regulation importin-?.J. 216: 3453-3462Crossref (18) Scholar34Giesecke Stewart Novel (target kinesin-like 2) importin-alpha.J. Chem. 2010; 285: 17628-17635Abstract (65) recognized importin-? some such HURP, are directly 1A).32Silljé was first described Ran-importin-regulated SAF extracts15Nachury Scholar16Wiese 1A) together TPX2.17Gruss now know that, polar cortex, where facilitates astral capture/pulling, respectively.19Hueschen Scholar20Okumura Scholar35Kiyomitsu force-generating machinery: how generated.Curr. 2019; 60: 1-8Crossref (16) Recently, Chang et al.33Chang solved crystal importin-?-NuMA-NLS complex, illustrating NuMA’s microtubule-binding inhibited steric blockage vitro. rigorously examined. To understand Ran-based critical separate achieve this, combining drugs (AID) technology,36Natsume Saga Rapid tagging short homology donors.Cell Rep. 210-218Abstract (245) allows us degrade mAID-tag fusion half-life 20 min. RCC1, importin-?, even substantially affect poles. Overall, our two domains (MTBDs) C-terminal region33Chang Scholar37Gallini Carminati De Mattia Pirovano L. Martini Oldani Asteriti I.A. Mapelli phosphorylation Aurora-A orchestrates orientation.Curr. 26: 458-469Abstract (43) 1B) focusing.19Hueschen domain focusing, replaced endogenous truncation mutants 1B). Endogenous fused mAID-mClover-FLAG (mACF) tag20Okumura S1A) depleted system following doxycycline (Dox) indole-3-acetic acid (IAA) treatment.20Okumura Scholar36Natsume parallel, mCherry-tagged were Rosa 26 locus Dox treatment (Figures 1B, 1C, S1B, S1C).20Okumura Equivalent wild type (WT) accumulated nuclei S1C) 1C) able rescue pole-focusing caused 1C 1D).20Okumura NuMA-?NLS unable S1C), accumulate 1D). expected, ?C-ter mutants, lack MTBDs, diffused cytoplasm 1D).19Hueschen Similarly, ?ex24 NLS part MTBD1, fully defects, despite localizing Although this appears be slightly reduced 1C), fluorescence intensities significantly between ?NLS either S1D S1E). contrast, ?(NLS+MTBD2) centrosomes, 1D, S1C). These results containing well-conserved NuMA-Lin5-Mud (NLM) motif S1F),38Siller K.H. Cabernard Doe C.Q. NuMA-related Mud Pins regulates orientation neuroblasts.Nat. 594-600Crossref (247) Scholar39Du Q. Stukenberg P.T. Macara I.G. Partner inscuteable organization.Nat. 3: 1069-1075Crossref (220) similar fibroblasts.22Silk MTBD1 adjacent NLS, 1A, S1F).33Chang recent vitro study sterically importin-?/? releases allowing 1A).33Chang test model (RanGEF) integrating mAID-mClover (mAC) tag gene S1A S2A; hereafter, PCR validation established lines summarized Figure S7).36Natsume Surprisingly, did 2A). RCC1-depleted 2B), relative increased 2C) result reduction 2A S2B). shortened 2D) delayed progression S2C–S2E), focused normally eventually exited reported chicken DT40 cells.14Furuta aspects assembly, further analyze next co-depleted NuMA. Following IAA, RCC1-mAC NuMA-mAID-mCherry 2E), unfocused spindles frequently observed 2E 2F). completely collapsed bi-polar 2E, bottom, Unexpectedly, co-depletion diminished tubulin 2G S2F). suggest functional absence stabilize microtubules. note frequency 2F; ~20%) lower than single-depleted 1D; ~74%). milder phenotype might reflect smaller pole-splitting exerted shorter dispensable function, may cause overactivation 1A), resulting defects. 2H–2K S2G–S2J). 2H 2I), length, duration S2H). Importin-? 2J 2K), delay S2J). contribution activation, ?IBB importin-?-binding (IBB) S2K). Importin-? insensitive due IBB partially inhibit S2K).33Chang similarly WT, neither affected co-localized experimental conditions S2L S2M). traditional activation cultured above experiments, asynchronous culture. given nuclear-cytoplasmic unknown secondary effects induced their phenotypes. because maintained S2E; t = ?0:10), majority already liberated pre-existing exist active nucleus, producing no aberrant phenotypes mitosis. exclude possibilities, nocodazole-arrested analyzed behavior nocodazole washout 3A). arrest 3B; ?90), rapidly masses 10; Video S1). displayed punctate signals throughout 3B, 10, S3A), SiR-tubulin S3B). dots disappeared localized within 60 min 60). 85). https://www.cell.com/cms/asset/1cc5d503-20ad-4ba9-8713-9f7f0112e298/mmc2.mp4Loading ... Download .mp4 (0.4 MB) Help files S1. Time-Lapse Movie Control RCC1-Positive Cells Showing NuMA-mCh, Related 3BIAA added ?90, removed 0