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Proteomic Analysis of the Mammalian Katanin Family of Microtubule-severing Enzymes Defines Katanin p80 subunit B-like 1 (KATNBL1) as a Regulator of Mammalian Katanin Microtubule-severing

2016; Elsevier BV; Volume: 15; Issue: 5 Linguagem: Inglês

10.1074/mcp.m115.056465

1535-9484

Keith Cheung, Silvia Senese, Jiaen Kuang, Ngoc B Bui, Chayanid Ongpipattanakul, Ankur A. Gholkar, Whitaker Cohn, Joseph Capri, Julian P. Whitelegge, Jorge Z. Torres,

Cellular transport and secretion

The Katanin family of microtubule-severing enzymes is critical for remodeling microtubule-based structures that influence cell division, motility, morphogenesis and signaling. Katanin is composed of a catalytic p60 subunit (A subunit, KATNA1) and a regulatory p80 subunit (B subunit, KATNB1). The mammalian genome also encodes two additional A-like subunits (KATNAL1 and KATNAL2) and one additional B-like subunit (KATNBL1) that have remained poorly characterized. To better understand the factors and mechanisms controlling mammalian microtubule-severing, we have taken a mass proteomic approach to define the protein interaction module for each mammalian Katanin subunit and to generate the mammalian Katanin family interaction network (Katan-ome). Further, we have analyzed the function of the KATNBL1 subunit and determined that it associates with KATNA1 and KATNAL1, it localizes to the spindle poles only during mitosis and it regulates Katanin A subunit microtubule-severing activity in vitro. Interestingly, during interphase, KATNBL1 is sequestered in the nucleus through an N-terminal nuclear localization signal. Finally KATNB1 was able to compete the interaction of KATNBL1 with KATNA1 and KATNAL1. These data indicate that KATNBL1 functions as a regulator of Katanin A subunit microtubule-severing activity during mitosis and that it likely coordinates with KATNB1 to perform this function. The Katanin family of microtubule-severing enzymes is critical for remodeling microtubule-based structures that influence cell division, motility, morphogenesis and signaling. Katanin is composed of a catalytic p60 subunit (A subunit, KATNA1) and a regulatory p80 subunit (B subunit, KATNB1). The mammalian genome also encodes two additional A-like subunits (KATNAL1 and KATNAL2) and one additional B-like subunit (KATNBL1) that have remained poorly characterized. To better understand the factors and mechanisms controlling mammalian microtubule-severing, we have taken a mass proteomic approach to define the protein interaction module for each mammalian Katanin subunit and to generate the mammalian Katanin family interaction network (Katan-ome). Further, we have analyzed the function of the KATNBL1 subunit and determined that it associates with KATNA1 and KATNAL1, it localizes to the spindle poles only during mitosis and it regulates Katanin A subunit microtubule-severing activity in vitro. Interestingly, during interphase, KATNBL1 is sequestered in the nucleus through an N-terminal nuclear localization signal. Finally KATNB1 was able to compete the interaction of KATNBL1 with KATNA1 and KATNAL1. These data indicate that KATNBL1 functions as a regulator of Katanin A subunit microtubule-severing activity during mitosis and that it likely coordinates with KATNB1 to perform this function. The remodeling of microtubule structures is important for many aspects of cell physiology including cell division, motility, morphogenesis, and signaling (1Sharp D.J. Ross J.L. Microtubule-severing enzymes at the cutting edge.J. Cell Sci. 2012; 125: 2561-2569Crossref PubMed Scopus (157) Google Scholar). A major group of proteins that remodel microtubules through microtubule-severing activities are the AAA ATPase containing proteins that include Spastin, Fidgetin, and the Katanins (1Sharp D.J. Ross J.L. Microtubule-severing enzymes at the cutting edge.J. Cell Sci. 2012; 125: 2561-2569Crossref PubMed Scopus (157) Google Scholar, 2McNally F.J. Vale R.D. Identification of katanin, an ATPase that severs and disassembles stable microtubules.Cell. 1993; 75: 419-429Abstract Full Text PDF PubMed Scopus (397) Google Scholar, 3Evans K.J. Gomes E.R. Reisenweber S.M. Gundersen G.G. Lauring B.P. Linking axonal degeneration to microtubule remodeling by Spastin-mediated microtubule severing.J. Cell Biol. 2005; 168: 599-606Crossref PubMed Scopus (180) Google Scholar, 4Zhang D. Rogers G.C. Buster D.W. Sharp D.J. Three microtubule severing enzymes contribute to the "Pacman-flux" machinery that moves chromosomes.J. Cell Biol. 2007; 177: 231-242Crossref PubMed Scopus (141) Google Scholar). Katanins are composed of a catalytic p60 subunit (A subunit, which contains the AAA ATPase domain) and a regulatory p80 subunit (B subunit) (2McNally F.J. Vale R.D. Identification of katanin, an ATPase that severs and disassembles stable microtubules.Cell. 1993; 75: 419-429Abstract Full Text PDF PubMed Scopus (397) Google Scholar). However, in-vitro biochemical studies have shown that the A subunit can form an unstable 14–16 nm hexameric ring structure, which can sever microtubules in the presence or absence of the B subunit (2McNally F.J. Vale R.D. Identification of katanin, an ATPase that severs and disassembles stable microtubules.Cell. 1993; 75: 419-429Abstract Full Text PDF PubMed Scopus (397) Google Scholar, 5McNally F.J. Thomas S. Katanin is responsible for the M-phase microtubule-severing activity in Xenopus eggs.Mol. Biol. Cell. 1998; 9: 1847-1861Crossref PubMed Scopus (92) Google Scholar, 6Hartman J.J. Vale R.D. Microtubule disassembly by ATP-dependent oligomerization of the AAA enzyme katanin.Science. 1999; 286: 782-785Crossref PubMed Scopus (184) Google Scholar), indicating that the A subunit does not require the B subunit for microtubule-severing activity. However, the B subunit has been shown to regulate the rate of microtubule-severing by the A subunit (7Hartman J.J. Mahr J. McNally K. Okawa K. Iwamatsu A. Thomas S. Cheesman S. Heuser J. Vale R.D. McNally F.J. Katanin, a microtubule-severing protein, is a novel AAA ATPase that targets to the centrosome using a WD40-containing subunit.Cell. 1998; 93: 277-287Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 8McNally K.P. Bazirgan O.A. McNally F.J. Two domains of p80 katanin regulate microtubule severing and spindle pole targeting by p60 katanin.J. Cell Sci. 2000; 113: 1623-1633Crossref PubMed Google Scholar), hence its designation as a regulatory subunit. The Katanins have been implicated in the regulation of multiple processes that are important for cellular homeostasis, proliferation, and invasion including the severing of spindle microtubules to generate microtubule density during meiosis and mitosis, the severing of the cilia axoneme microtubules during ciliary resorption, the severing of intercellular bridge microtubules during cytokinesis and the severing of cytoplasmic microtubules to promote cell morphogenesis and migration (5McNally F.J. Thomas S. Katanin is responsible for the M-phase microtubule-severing activity in Xenopus eggs.Mol. Biol. Cell. 1998; 9: 1847-1861Crossref PubMed Scopus (92) Google Scholar, 9Ahmad F.J. Yu W. McNally F.J. Baas P.W. An essential role for katanin in severing microtubules in the neuron.J. Cell Biol. 1999; 145: 305-315Crossref PubMed Scopus (191) Google Scholar, 10Matsuo M. Shimodaira T. Kasama T. Hata Y. Echigo A. Okabe M. Arai K. Makino Y. Niwa S. Saya H. Kishimoto T. Katanin p60 contributes to microtubule instability around the midbody and facilitates cytokinesis in rat cells.PLoS ONE. 2013; 8: e80392Crossref PubMed Scopus (21) Google Scholar, 11Rasi M.Q. Parker J.D. Feldman J.L. Marshall W.F. Quarmby L.M. Katanin knockdown supports a role for microtubule severing in release of basal bodies before mitosis in Chlamydomonas.Mol. Biol. Cell. 2009; 20: 379-388Crossref PubMed Scopus (39) Google Scholar, 12Srayko M. Buster D.W. Bazirgan O.A. McNally F.J. Mains P.E. MEI-1/MEI-2 katanin-like microtubule severing activity is required for Caenorhabditis elegans meiosis.Genes Dev. 2000; 14: 1072-1084Crossref PubMed Google Scholar, 13Zhang D. Grode K.D. Stewman S.F. Diaz-Valencia J.D. Liebling E. Rath U. Riera T. Currie J.D. Buster D.W. Asenjo A.B. Sosa H.J. Ross J.L. Ma A. Rogers S.L. Sharp D.J. Drosophila katanin is a microtubule depolymerase that regulates cortical-microtubule plus-end interactions and cell migration.Nat. Cell Biol. 2011; 13: 361-370Crossref PubMed Scopus (86) Google Scholar). For example, in Caenorhabditis elegans, the microtubule-severing activity of the Katanin A subunit MEI-1 is required for regulating spindle length and density during meiotic spindle formation and for stabilizing the association of the meiosis I spindle to the oocyte cortex (14McNally K. Audhya A. Oegema K. McNally F.J. Katanin controls mitotic and meiotic spindle length.J. Cell Biol. 2006; 175: 881-891Crossref PubMed Scopus (218) Google Scholar, 15Srayko M. O'toole E.T. Hyman A.A. Müller-Reichert T. Katanin disrupts the microtubule lattice and increases polymer number in C. elegans meiosis.Curr. Biol. 2006; 16: 1944-1949Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 16Yang H.Y. McNally K. McNally F.J. MEI-1/katanin is required for translocation of the meiosis I spindle to the oocyte cortex in C elegans.Dev. Biol. 2003; 260: 245-259Crossref PubMed Scopus (91) Google Scholar). Additionally, inactivation of a temperature sensitive MEI-1 mutant during metaphase disrupts bipolar spindle assembly and the alignment of chromosomes to the metaphase plate (17McNally K. Berg E. Cortes D.B. Hernandez V. Mains P.E. McNally F.J. Katanin maintains meiotic metaphase chromosome alignment and spindle structure in vivo and has multiple effects on microtubules in vitro.Mol. Biol. Cell. 2014; 25: 1037-1049Crossref PubMed Scopus (35) Google Scholar). In Drosophila melanogaster, the microtubule-severing activity of the Katanin p60-like 1 (Kat-60L1) A subunit is critical for neuronal development (18Lee H.H. Jan L.Y. Jan Y.N. Drosophila IKK-related kinase Ik2 and Katanin p60-like 1 regulate dendrite pruning of sensory neuron during metamorphosis.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 6363-6368Crossref PubMed Scopus (101) Google Scholar). Kat-60L1 mutants exhibit a reduced number and length of dendrites in neurons and a diminished neuronal responsiveness to chemical and thermal stimuli (19Stewart A. Tsubouchi A. Rolls M.M. Tracey W.D. Sherwood N.T. Katanin p60-like1 promotes microtubule growth and terminal dendrite stability in the larval class IV sensory neurons of Drosophila.J. Neurosci. 2012; 32: 11631-11642Crossref PubMed Scopus (53) Google Scholar). Additionally, during mitosis the Katanin p60 (DmKat-60) A subunit localizes to chromosomes and facilitates microtubule depolymerization during chromosome segregation (4Zhang D. Rogers G.C. Buster D.W. Sharp D.J. Three microtubule severing enzymes contribute to the "Pacman-flux" machinery that moves chromosomes.J. Cell Biol. 2007; 177: 231-242Crossref PubMed Scopus (141) Google Scholar). In other organisms such as Tetrahymena thermophila, Chlamydomonas reinhardtii, and Trypanosoma brucei, Katanin A subunits are important for flagellar biogenesis and cell division (11Rasi M.Q. Parker J.D. Feldman J.L. Marshall W.F. Quarmby L.M. Katanin knockdown supports a role for microtubule severing in release of basal bodies before mitosis in Chlamydomonas.Mol. Biol. Cell. 2009; 20: 379-388Crossref PubMed Scopus (39) Google Scholar, 20Benz C. Clucas C. Mottram J.C. Hammarton T.C. Cytokinesis in bloodstream stage Trypanosoma brucei requires a family of katanins and spastin.PLoS ONE. 2012; 7: e30367Crossref PubMed Scopus (28) Google Scholar, 21Casanova M. Crobu L. Blaineau C. Bourgeois N. Bastien P. Pages̀ M. Microtubule-severing proteins are involved in flagellar length control and mitosis in Trypanosomatids.Mol. Microbiol. 2009; 71: 1353-1370Crossref PubMed Scopus (37) Google Scholar, 22Dymek E.E. Smith E.F. PF19 encodes the p60 catalytic subunit of katanin and is required for assembly of the flagellar central apparatus in Chlamydomonas.J. Cell Sci. 2012; 125: 3357-3366Crossref PubMed Scopus (34) Google Scholar). Finally, the microtubule-severing activity of Katanin A subunits in Arabidopsis thaliana are critical for regulating cell specification, cell growth and cell wall biosynthesis (23Stoppin-Mellet V. Gaillard J. Vantard M. Katanin's severing activity favors bundling of cortical microtubules in plants.Plant J. 2006; 46: 1009-1017Crossref PubMed Scopus (74) Google Scholar, 24Bouquin T. Mattsson O. Naested H. Foster R. Mundy J. The Arabidopsis lue1 mutant defines a katanin p60 ortholog involved in hormonal control of microtubule orientation during cell growth.J. Cell Sci. 2003; 116: 791-801Crossref PubMed Scopus (135) Google Scholar, 25Lin D. Cao L. Zhou Z. Zhu L. Ehrhardt D. Yang Z. Fu Y. Rho GTPase signaling activates microtubule severing to promote microtubule ordering in Arabidopsis.Curr. Biol. 2013; 23: 290-297Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 26Nakamura M. Ehrhardt D.W. Hashimoto T. Microtubule and katanin-dependent dynamics of microtubule nucleation complexes in the acentrosomal Arabidopsis cortical array.Nat. Cell Biol. 2010; 12: 1064-1070Crossref PubMed Scopus (164) Google Scholar, 27Webb M. Jouannic S. Foreman J. Linstead P. Dolan L. Cell specification in the Arabidopsis root epidermis requires the activity of ECTOPIC ROOT HAIR 3–a katanin-p60 protein.Development. 2002; 129: 123-131Crossref PubMed Google Scholar, 28Uyttewaal M. Burian A. Alim K. Landrein B. Borowska-Wykręt D. Dedieu A. Peaucelle A. Ludynia M. Traas J. Boudaoud A. Kwiatkowska D. Hamant O. Mechanical stress acts via katanin to amplify differences in growth rate between adjacent cells in Arabidopsis.Cell. 2012; 149: 439-451Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 29Burk D.H. Liu B. Zhong R. Morrison W.H. Ye Z.H. A katanin-like protein regulates normal cell wall biosynthesis and cell elongation.Plant Cell. 2001; 13: 807-827Crossref PubMed Scopus (311) Google Scholar). Although the majority of Katanin studies have focused on understanding the function of the canonical p60 and p80 subunits in lower organisms, many organisms encode additional p60-like and p80-like proteins known as Katanin-like proteins. For example, the human genome encodes two alternatively spliced isoforms of the canonical p60 A subunit (KATNA1 chromosomal locus 6q25.1, protein IDs NP_001191005.1 and NP_008975.1) and two additional p60-like proteins (KATNAL1 chromosomal locus 13q12.3, protein ID NP_115492.1; KATNAL2 chromosomal locus 18q21.1, protein ID NP_112593.2). Similarly, in addition to the canonical p80 B subunit (KATNB1 chromosomal locus 16q21, protein ID NP_005877.2), the human genome encodes an additional p80-like protein (KATNBL1 chromosomal locus 15q14, protein ID NP_078989.1) (30Roll-Mecak A. McNally F.J. Microtubule-severing enzymes.Curr. Opin. Cell Biol. 2010; 22: 96-103Crossref PubMed Scopus (210) Google Scholar). Interestingly, all human Katanin subunits are ubiquitously expressed across most tissue types including brain, lung, kidney, liver, pancreas, and skin and in established cell lines like HeLa cells (31Rebhan M. Chalifa-Caspi V. Prilusky J. Lancet D. GeneCards: integrating information about genes, proteins and diseases.Trends Genet. 1997; 13: 163Abstract Full Text PDF PubMed Scopus (416) Google Scholar). Human Katanins have important roles in regulating microtubule-dependent processes like mitotic spindle length and structure during cell division and mutation of Katanin subunits has been linked to human disorders like cerebral cortical malformation and male infertility (14McNally K. Audhya A. Oegema K. McNally F.J. Katanin controls mitotic and meiotic spindle length.J. Cell Biol. 2006; 175: 881-891Crossref PubMed Scopus (218) Google Scholar, 32Smith L.B. Milne L. Nelson N. Eddie S. Brown P. Atanassova N. O'Bryan M.K. O'Donnell L. Rhodes D. Wells S. Napper D. Nolan P. Lalanne Z. Cheeseman M. Peters J. KATNAL1 regulation of sertoli cell microtubule dynamics is essential for spermiogenesis and male fertility.PLoS Genet. 2012; 8: e1002697Crossref PubMed Scopus (52) Google Scholar, 33O'Donnell L. Rhodes D. Smith S.J. Merriner D.J. Clark B.J. Borg C. Whittle B. O'Connor A.E. Smith L.B. McNally F.J. de, Kretser D.M. Goodnow C.C. Ormandy C.J. Jamsai D. O'Bryan M.K. An essential role for katanin p80 and microtubule severing in male gamete production.PLoS Genet. 2012; 8: e1002698Crossref PubMed Scopus (69) Google Scholar, 34Mishra-Gorur K. C̨ağlayan A.O. Schaffer A.E. Chabu C. Henegariu O. Vonhoff F. Akgümüş G.T. Nishimura S. Han W. Tu S. Baran B. Gümüş H. Dilber C. Zaki M.S. Hossni H.A. Rivière J.B. Kayserili H. Spencer E.G. Rosti R.Ö. Schroth J. Per H. Çağlar C. Dölen D. Baranoski J.F. Kumandaş S. Minja F.J. Erson-Omay E.Z. Mane S.M. Lifton R.P. Xu T. Keshishian H. Dobyns W.B. Chi N.C. Šestan N. Louvi A. Bilgüvar K. Yasuno K. Gleeson J.G. Günel M. Mutations in KATNB1 cause complex cerebral malformations by disrupting asymmetrically dividing neural progenitors.Neuron. 2014; 84: 1226-1239Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 35Sonbuchner T.M. Rath U. Sharp D.J. KL1 is a novel microtubule severing enzyme that regulates mitotic spindle architecture.Cell Cycle. 2010; 9: 2403-2411Crossref PubMed Scopus (23) Google Scholar). Domain analyses of the human Katanin subunits indicate that KATNA1 and KATNAL1 share a similar domain architecture with an N-terminal microtubule interacting and trafficking domain (MIT) 1The abbreviations used are:MITmicrotubule interacting and trafficking domain. followed by a coiled coil domain (CC), a AAA ATPase domain (AAA) and a C-terminal VPS4 domain (VPS4_C) (36Iwaya N. Akiyama K. Goda N. Tenno T. Fujiwara Y. Hamada D. Ikura T. Shirakawa M. Hiroaki H. Effect of Ca2+ on the microtubule-severing enzyme p60-katanin. Insight into the substrate-dependent activation mechanism.FEBS J. 2012; 279: 1339-1352Crossref PubMed Scopus (8) Google Scholar) (Fig. 1A). In contrast, KATNAL2 only contains the AAA domain, lacks the MIT, CC and VPS_4 domains, and has an N-terminal LisH (LIS1 homology) domain (Fig. 1A). Because KATNAL2 harbors a AAA domain that is required for Katanin microtubule-severing activity, it is predicted to have a role in microtubule-severing, however this has yet to be tested. Additionally, KATNAL2 lacks the MIT domain that is important for microtubule binding in other Katanin A subunits and whether the MIT domain is critical for microtubule-severing activity remains to be determined (37Iwaya N. Kuwahara Y. Fujiwara Y. Goda N. Tenno T. Akiyama K. Mase S. Tochio H. Ikegami T. Shirakawa M. Hiroaki H. A common substrate recognition mode conserved between katanin p60 and VPS4 governs microtubule severing and membrane skeleton reorganization.J. Biol. Chem. 2010; 285: 16822-16829Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Although KATNB1 and KATNBL1 both harbor a conserved C-terminal region (con80), only KATNB1 contains additional N-terminal proline-rich (Pro-rich) and WD40 domains that are absent in KATNBL1 (8McNally K.P. Bazirgan O.A. McNally F.J. Two domains of p80 katanin regulate microtubule severing and spindle pole targeting by p60 katanin.J. Cell Sci. 2000; 113: 1623-1633Crossref PubMed Google Scholar, 30Roll-Mecak A. McNally F.J. Microtubule-severing enzymes.Curr. Opin. Cell Biol. 2010; 22: 96-103Crossref PubMed Scopus (210) Google Scholar) (Fig. 1A). Interestingly, full length KATNB1 and the KATNB1 con80 domain alone have been shown to stimulate KATNA1 microtubule-severing activity, whereas the KATNB1 WD40 domain alone inhibits microtubule-severing (7Hartman J.J. Mahr J. McNally K. Okawa K. Iwamatsu A. Thomas S. Cheesman S. Heuser J. Vale R.D. McNally F.J. Katanin, a microtubule-severing protein, is a novel AAA ATPase that targets to the centrosome using a WD40-containing subunit.Cell. 1998; 93: 277-287Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 8McNally K.P. Bazirgan O.A. McNally F.J. Two domains of p80 katanin regulate microtubule severing and spindle pole targeting by p60 katanin.J. Cell Sci. 2000; 113: 1623-1633Crossref PubMed Google Scholar). However, the underlying mechanism of how the B subunits regulate the activity of the A subunits is still unclear. Additionally, outside of KATNA1 and KATNB1, the remaining human Katanin subunits remain poorly characterized. microtubule interacting and trafficking domain. Our previous proteomic analyses of human mitotic microtubule co-purifying proteins identified a then hypothetical p60-like protein KATNAL1 (38Torres J.Z. Summers M.K. Peterson D. Brauer M.J. Lee J. Senese S. Gholkar A.A. Lo Y.C. Lei X. Jung K. Anderson D.C. Davis D.P. Belmont L. Jackson P.K. The STARD9/Kif16a Kinesin Associates with Mitotic Microtubules and Regulates Spindle Pole Assembly.Cell. 2011; 147: 1309-1323Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Tandem affinity purification and mass proteomic analysis of KATNAL1 identified KATNA1, KATNAL1, KATNB1 and a then hypothetical p80-like protein C15orf29 (KATNBL1) as interactors, indicating that in humans multiple Katanins were likely involved in microtubule-severing (39Torres J.Z. Miller J.J. Jackson P.K. High-throughput generation of tagged stable cell lines for proteomic analysis.Proteomics. 2009; 9: 2888-2891Crossref PubMed Scopus (79) Google Scholar). Consistent with this idea, depletion of human KATNA1 or KATNAL1 alone leads to mild changes in spindle size and mitotic defects (35Sonbuchner T.M. Rath U. Sharp D.J. KL1 is a novel microtubule severing enzyme that regulates mitotic spindle architecture.Cell Cycle. 2010; 9: 2403-2411Crossref PubMed Scopus (23) Google Scholar, 40Buster D. McNally K. McNally F.J. Katanin inhibition prevents the redistribution of gamma-tubulin at mitosis.J. Cell Sci. 2002; 115: 1083-1092Crossref PubMed Google Scholar), indicating that multiple Katanins may be involved in regulating spindle size in mammals and/or that other microtubule-severing activities are able to compensate in the absence of either Katanin. Additionally, whether KATNAL2 has microtubule-severing activity that can compensate for the absence of other Katanin A subunits remains to be determined. Finally, the effect, if any, that KATNBL1 has on Katanin A subunit microtubule-severing activity also remains to be determined. To better understand the human Katanins and more broadly the mechanisms controlling mammalian microtubule-severing, we analyzed the human Katanin interactome (Katan-ome) through biochemical tandem affinity purifications and mass proteomic analyses. We further focused on the characterization of the poorly understood KATNBL1 subunit and its role in microtubule-severing through binding assays, microtubule-severing assays, competition binding assays, and subcellular localization studies. Our results showed that KATNBL1 is uniquely sequestered to the nucleus during interphase and associates with spindle poles in mitosis, is a regulator of KATNAL1 microtubule-severing activity, and competes with KATNB1 for binding to KATNA1 and KATNAL1. These results indicate that in humans microtubule-severing is complex and likely regulated by the concerted action of KATNB1 and KATNBL1. All chemicals were purchased from Thermo Scientific (Waltham, MA) unless otherwise noted. HeLa and HeLa Flp-In T-REx LAP-tagged stable cell lines were grown in F12:DMEM 50:50 medium (Thermo Scientific) with 10% FBS, 2 mm l-glutamine and antibiotics, in 5% CO2 at 37 °C. Cells were induced to express the indicated LAP-tagged proteins by the addition of 0.2 μg/ml doxycycline (Sigma-Aldrich, St. Louis, MO). For synchronization of cells in mitosis, cells were treated with 100 nm Taxol (Sigma-Aldrich) for 16 h. For full-length KATNA1, KATNAL1, KATNAL2, KATNB1, and KATNBL1, or STARD9-START and STARD9-MD expression, cDNA corresponding to the full-length open reading frame of each Katanin or the indicated STARD9 domains was fused to the C terminus of either HA (pCS2-HA-DEST vector), Myc (pCS2-Myc-DEST vector), FLAG (pCDNA3-FLAG-DEST vector), GST (pGEX-6p-1-DEST vector), or EGFP (pGLAP1 vector) using the Gateway cloning system (Invitrogen, Carlsbad, CA) as described previously (38Torres J.Z. Summers M.K. Peterson D. Brauer M.J. Lee J. Senese S. Gholkar A.A. Lo Y.C. Lei X. Jung K. Anderson D.C. Davis D.P. Belmont L. Jackson P.K. The STARD9/Kif16a Kinesin Associates with Mitotic Microtubules and Regulates Spindle Pole Assembly.Cell. 2011; 147: 1309-1323Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The pGLAP1-Katanin vectors were used to generate doxycycline inducible HeLa Flp-In T-REx LAP-KATNA1, KATNAL1, KATNAL2, KATNB1, and KATNBL1 stable cell lines that express the fusion protein from a specific single locus within the genome as described previously (39Torres J.Z. Miller J.J. Jackson P.K. High-throughput generation of tagged stable cell lines for proteomic analysis.Proteomics. 2009; 9: 2888-2891Crossref PubMed Scopus (79) Google Scholar). For KATNBL1 mutations, pGLAP1-KATNBL1 was mutated using the QuickChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA) with primers carrying the desired mutations, as described previously (41Senese S. Cheung K. Lo Y.C. Gholkar A.A. Xia X. Wohlschlegel J.A. Torres J.Z. A unique insertion in STARD9's motor domain regulates its stability.Mol. Biol. Cell. 2015; 26: 440-452Crossref PubMed Scopus (13) Google Scholar), whereas truncation mutants were generated by PCR amplification and cloning into pGLAP1 as described above. All primers were purchased from Fisher Scientific. For a list of primers used see supplemental Table S3. For cell extract immunoprecipitations (IPs), LAP-KATNA1, LAP-KATNAL1, LAP-KATNB1, or LAP-KATNBL1 HeLa stable cells lines were transfected with the indicated HA-tagged Katanin subunit expression vectors for 24 h and whole cell extracts were prepared in LAP150 lysis buffer (50 mm Tris pH 7.4, 150 mm KCl, 1 mm EDTA, 1 mm MgCl2, 10% glycerol) plus 250 μm ATP, 0.3% Nonidet P-40, 0.5 mm DTT and protease and phosphatase inhibitor mixture (Thermo Scientific). Cell extracts were cleared by centrifugation at 15K RPM for 10 min. One hundred forty micrograms of cleared lysate was incubated with 5 μl packed bead volume of anti-HA antibody conjugated magnetic beads (MBL, Woburn, MA) for 1 h at 4 °C. The beads were then washed 3 times with 50 μl of LAP150 lysis buffer and bound proteins were eluted with 20 μl of 1× Laemmli SDS sample buffer (Bio-Rad, Irvine, CA). Six percent of the sample inputs, 6% of the unbound fractions and the entire eluates from the immunoprecipitations were resolved on a 10% Tris gel (Bio-Rad) with Tris-Glycine SDS running buffer, transferred to a Immobilon PVDF membrane (EMD Millipore, Billerica, MA), immunoblotted with the indicated antibodies, and imaged with a LI-COR Odyssey imager (LI-COR Biosciences, Lincoln, NE). For in vitro binding assays, HA, Myc, or FLAG-tagged KATNA1, KATNAL1, KATNAL2, KATNB1, or KATNBL1 were in vitro transcribed and translated (TnT® Quick Coupled Transcription/Translation System, Promega, Madison, WI) in 50 μl reactions. Two different Katanin reactions were combined and incubated with 5 μl packed bead volume of anti-HA antibody conjugated magnetic beads (MBL) for 1 h. Beads were washed four times with a wash buffer containing 10 mm Tris pH 7.4, 100 mm NaCl, and 0.1% Nonidet P-40. The beads were then boiled in 20 μl of 1X Laemmli SDS sample buffer (Bio-Rad). 6% of the sample inputs, 6% of the unbound fractions (where indicated), and the entire eluates from the immunoprecipitations were resolved on a 10% Tris gel (Bio-Rad) with Tris-Glycine SDS running buffer, transferred to a Immobilon PVDF membrane (EMD Millipore), and binding was monitored by radiometric analysis with a PharosFX Plus molecular imaging system (Bio-Rad). The LAP-KATNA1, KATNAL1, KATNAL2, KATNB1, and KATNBL1 inducible stable cell lines were grown in roller bottles and induced with .2 μg/ml Dox for 16 h in the presence of 100 nm Taxol prior to harvesting cells, as described in (39Torres J.Z. Miller J.J. Jackson P.K. High-throughput generation of tagged stable cell lines for proteomic analysis.Proteomics. 2009; 9: 2888-2891Crossref PubMed Scopus (79) Google Scholar). Mitotic cells were then harvested in the presence of protease (Thermo Scientific), phosphatase (Thermo Scientific), and proteasome inhibitors (MG132, Enzo Lifesciences, Farmingdale, NY). LAP-KATNA1, KATNAL1, KATNAL2, KATNB1, and KATNBL1 were purified from cleared extracts using a previously established tandem affinity purification protocol (39Torres J.Z. Miller J.J. Jackson P.K. High-throughput generation of tagged stable cell lines for proteomic analysis.Proteomics. 2009; 9: 2888-2891Crossref PubMed Scopus (79) Google Scholar). Sample lanes from SDS-PAGE were sliced into six pieces and placed into individual micro-centrifuge tubes. Each gel slice was dehydrated with 100% acetonitrile for 30 min. Cysteines were reduced with 100 mm dithiothreitol in 50 mm ammonium bicarbonate for 60 min at 37°C, followed by the removal of buffer, and subsequently alkylated with 55 mm iodoacetamide in 50 mm ammonium bicarbonate for 45 min at room temperature in the dark. Buffer was decanted and gel slices were dehydrated with 100% acetonitrile followed by rehydration with 50 mm ammonium bicarbonate, repeated twice, except swelling in 5 ng/μl trypsin with the second 50 mm ammonium bicarbonate rehydration step on ice for 45 min. Trypsin solution was decanted and samples were incubated at 37°C overnight. Peptides were extracted from the gel slices using 100 μl of 50% acetonitrile for 20 min using water-bath sonication, repeated twice. Extracted peptides were dried using SpeedVac and reconstituted in 80 μl of 3% acetonitrile with 0.1% formic acid. Peptides were desalted using C18 StageTips as previously described (42Rappsilber J. Mann M. Ishihama Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips.Nat. Protoc. 2007; 2: 1896-1906Crossref PubMed Scopus (2569) Google Scholar). Nano-LC-MS/MS with collision-induced dissociation was performed on an Orbitrap XL (Thermo Scientific) integrated with an Eksigent 2D nano-LC instrument. A laser-pulled reverse-phase column, 75 μm ×