Journal of Science 113, 1623-1633 (2000) 1623 Printed in Great Britain © The Company of Biologists Limited 2000 JCS1028

Two domains of p80 katanin regulate severing and spindle pole targeting by p60 katanin

Karen Perry McNally, Omar A. Bazirgan and Francis J. McNally* Section of Molecular and Cellular Biology, University of California, Davis, CA 95616, USA *Author for correspondence (e-mail: [email protected])

Accepted 19 February; published on WWW 6 April 2000

SUMMARY

The assembly and function of the mitotic spindle requires targeting to spindle poles. The N-terminal WD40 domain the activity of a number of microtubule-binding proteins. of p80 katanin acts as a negative regulator of microtubule Some microtubule-binding proteins bind in disassembly activity and is also required for spindle pole vitro but do not co-localize with microtubules in localization, possibly through interactions with another cells. Instead these proteins associate with specific spindle-pole protein. These results support a model in subregions of the mitotic spindle. Katanin, a heterodimeric which katanin is targeted to spindle poles through a microtubule-severing ATPase, is found localized at mitotic combination of direct microtubule binding by the p60 spindle poles. In this paper we demonstrate that human subunit and through interactions between the WD40 p60 katanin and the C-terminal domain of human p80 domain and an unknown protein. We propose that both katanin both bind microtubules in vitro. Association of domains of p80 are essential in precisely regulating these two proteins results in an increased microtubule katanin’s activity in vivo. affinity and increased microtubule-severing activity in vitro. Association of these subunits in transfected HeLa cells increases microtubule disassembly activity and Key words: Katanin, Microtubule, , Spindle pole,

INTRODUCTION γ- that surrounds the only during mitosis does require microtubules (McNally and Thomas, 1998; Within the mitotic spindle, specific interactions between Khodjakov and Rieder, 1999). Like NuMA, the sea urchin microtubules, chromosomes and numerous accessory proteins protein, katanin, is concentrated in a microtubule-dependent allow the generation of spatially regulated forces that bring structure surrounding the γ-tubulin at the poles of embryonic about accurate chromosome segregation. Generation of this spindles (McNally et al., 1996). recognizing proper spatial regulation requires that individual proteins be mammalian homologs of katanin also recognize large targeted to specific substructures within the spindle. For microtubule-dependent structures surrounding γ-tubulin foci at example, the -related protein CENP-E is targeted spindle poles in several cell types and, in addition, recognize specifically to the corona fibers of kinetochores (Yao et al., a smaller microtubule-independent focus at the centrosome in 1997) where its action is required during alignment of some cell types (McNally and Thomas, 1998). chromosomes at the metaphase plate (Schaar et al., 1997; Sea urchin katanin severs microtubules in an ATP-dependent Wood et al., 1997). In contrast, the protein NuMA (nuclear manner in vitro (McNally and Vale, 1993) and is thought to be mitotic antigen) is concentrated at spindle poles where its important in releasing microtubules from their centrosomal activity appears to be required for the focused organization of attachment points in vivo. Indeed, microinjection of antibodies microtubules (Gaglio et al., 1995). recognizing a vertebrate katanin homolog into neurons results The vertebrate mitotic spindle pole is a microtubule- in the accumulation of microtubules attached to the centrosome dependent region surrounding the centrioles and centrosome at (Ahmad et al., 1999), indicating that katanin-mediated each of the two ends of the bipolar mitotic spindle. NuMA, γ- microtubule severing is responsible for the release of tubulin and katanin are proteins whose localization define microtubules from the neuronal centrosome. The concentration the spindle pole. NuMA associates exclusively with the of katanin in mitotic spindle poles (McNally and Thomas, microtubule-dependent spindle pole during mitosis and does 1998) and the specific activation of katanin’s activity in M- not associate with interphase centrosomes (Price and Pettijohn, phase Xenopus extracts (Vale, 1991; McNally and Thomas, 1986; Dionne et al., 1999). In contrast, localization of γ-tubulin 1998) suggest that katanin may also sever microtubules from to the interphase or mitotic centrosome does not require intact their centrosomal attachments during mitosis in non-neuronal microtubules (Khodjakov and Rieder, 1999; Stearns et al., cells. Katanin’s localization in spindle poles may increase the 1991; Zheng et al., 1991). However, localization of additional efficiency of this centrosomal release reaction and thus allow 1624 K. P. McNally and others depolymerization of microtubule minus ends, a force- activator (Damke et al., 1995) were grown in Optimem Media (Life producing process (Waters et al., 1996) that may drive Technologies) supplemented with 10% fetal bovine serum and chromosome movements (Desai et al., 1998). antibiotics. Transfections were carried out with Lipofectamine Plus Katanin is a heterodimer consisting of a 60 kDa subunit that (Life Technologies) on 50% confluent cultures in 60 mm culture utilizes ATP hydrolysis to sever microtubules and an 80 kDa dishes. Transfection efficiencies varied between 10% and 30%. accessory subunit (McNally and Vale, 1993; Hartman et al., Glutathione-Sepharose binding assays 1998). The 60 kDa subunit is composed of an N-terminal In order to monitor association of transfected p80 fragments with p60, domain that binds microtubules (Hartman and Vale, 1999) and HeLa cells were transfected in 60 mm dishes. 24 hours after a C-terminal domain sharing homology with a large family of transfection, cells were washed in PBS then lysed by addition of 1 ml , the AAA family (ATPases associated with many of lysis buffer (100 mM K-HEPES, pH 7.4, 200 mM NaCl, 1 mM cellular activities; Hartman et al., 1998). The 80 kDa subunit EGTA, 10% glycerol, 0.5% Nonidet P40 (NP40; v/v), 200 µg/ml is composed of an N-terminal WD40 repeat domain, a central benzamidine, 5 mM dithiothreitol (DTT), 200 µg/ml Nα-p-tosyl-L- proline-rich domain and a C-terminal domain required for arginine methyl ester (TAME), 100 µg/ml N-tosyl-L-phenylalanine dimerization with the catalytic p60 subunit. The WD40 repeat chloromethyl ketone (TPCK), 0.5 mM phenylmethylsufonyl fluoride domain of a human p80 homolog was previously shown to be (PMSF)). Lysates were cleared by centrifugation at 11,000 g for 20 minutes. Supernatants (1 ml) were added to 100 ul packed sufficient to target green fluorescent protein to interphase glutathione-Sepharose 4B (Pharmacia; Piscataway, NJ) and agitated centrosomes (Hartman et al., 1998). However, in this study, it for 1 hour on a rotary sample mixer at 4¡C. The glutathione-Sepharose was not possible to determine the role of the WD40 repeat was then washed 3 times in lysis buffer by successive centrifugation domain in mitotic spindle pole targeting. and resuspension. GST fusion proteins and associated proteins were In this paper, we demonstrate that purified, recombinant eluted by addition of SDS-Laemmli buffer. Unbound supernatants and human katanin severs microtubules in vitro and disassembles eluted glutathione-binding fractions were then subjected to analysis microtubules in vivo. The effects of p80 katanin domains on by SDS-PAGE and immunoblotting. p60’s microtubule-severing and microtubule-binding activities Protein expression and purification are also described. The requirement for the WD40 repeat For the expression of human katanin subunits, growth of Sf9 cells domain in spindle pole targeting was also tested by generating and infection with baculovirus were as described previously WD40-less katanin heterodimers in vivo and monitoring the (Hartman et al., 1998). The cells were harvested at approximately 72 subcellular localization of these modified katanin molecules. hours post-infection by low speed centrifugation. Cells expressing p60 were suspended in lysis buffer I (50 mM Tris, pH 8.5, 300 mM NaCl, 2 mM MgCl2, 20 mM imidazole, 10 mM 2-mercaptoethanol, MATERIALS AND METHODS 0.05% NP40 (v/v), 0.1 mM ATP, 1 µg/ml aprotinin, 0.1 mM PMSF, 40 µg/ml benzamidine). Cells expressing GST-procon80 were Plasmid constructions suspended in lysis buffer II (50 mM K-HEPES, pH 7.5, 200 mM All glutathione-S-transferase (GST) fusions and untagged p60 and NaCl, 0.1 mM EGTA, 10% glycerol (v/v), 0.05% NP40 (v/v), 0.1 p80 plasmids were constructed in an Epstein Barr Virus-based vector mM DTT, 0.1 mM ATP, 1 µg/ml aprotinin, 0.1 mM PMSF, 40 µg/ml (pFM219) constructed by replacing the RSV promoter and polylinker benzamidine). Suspended cells were frozen in liquid nitrogen and of pEBVHis (Invitrogen; Carlsbad, CA) with the tetracycline stored at −80¡C. repressible (tet) promoter (Gossen and Bujard, 1992) from ptet-tTAK To purify the expressed subunits, frozen cells were thawed and (Life Technologies; Gaithersburg, MD) and a custom polylinker. DNA was sheared by two passes through a 27 1/2 gauge needle. Cell Epitope-tagged p60 was expressed from the vector pCDNA4 debris was removed by centrifugation (100,000 g for 30 minutes). 6- HisMAX (Invitrogen) as were the p60 deletion derivatives used in Fig. histidine tagged subunits were bound in batch to His-Bind Resin 1. Green fluorescent protein (GFP) fusions were constructed in the (Novagen; Madison, WI) and then washed, eluted, and frozen in liquid vectors pEGFP-C1 or pEGFP-N1 (Clontech, Palo Alto). Recombinant nitrogen as described previously (Hartman et al., 1998). GST- baculoviruses were constructed using the Bac to Bac System after procon80 and GST-con80 fusion proteins were bound in batch to subcloning cDNA fragment into pFastBac-HTB (Life Technologies). glutathione-Sepharose 4B, washed three times in lysis buffer II, All katanin derivatives were PCR amplified from the human p60 washed once in GST elution buffer (50 mM K-HEPES, pH 7.5, 50 (McNally and Thomas, 1998) or human p80 (Hartman et al., 1998) mM KCl, 10% glycerol (v/v), 0.1 mM EGTA, 0.1 mM DTT, 0.1 mM cDNAs previously described. ATP) and eluted in GST elution buffer to which glutathione was added to 20 mM. Katanin concentrations were estimated by comparison with p80 derivatives BSA standards using densitometric analysis of Coomassie-stained The WD40 domain of p80 (WD) consisted of amino acids 1-304, WD- SDS-PAGE gels. Heterodimers of p60 katanin and GST-procon80 pro consisted of residues 1-484, pro-con80 consisted of residues 301- were generated by mixing the purified subunits at a ratio of 1:1.5, 655 and con80 consisted of residues 412-655 of human p80 katanin. respectively, and incubating on ice for 10 minutes to 1 hour. Two versions of GST were PCR amplified from the vector pGEX-KG (Guan and Dixon, 1991) for N or C-terminal fusions, respectively. The Assays of microtubule-severing/disassembly activity coding sequence of human p50 dynamitin (Echeverri et al., 1996) was DAPI disassembly assay PCR amplified and inserted into pEGFP-N1 to produce p50-GFP. DAPI (4′,6-diamidino-2-phenylindole) disassembly assays were performed using previously published procedures (Hartman et al., p60 point mutants 1998) except that the reaction buffer was modified (20 mM K-HEPES, Mutations in P loop p60 and DEID p60 were introduced into the full- pH 7.4, 10% glycerol (v/v), 2 mM MgCl2, 0.1 mM EGTA, 1 mM ATP) length human p60 cDNA by site-directed mutagenesis and confirmed because human p60 katanin was inactive in S. purpuratus katanin by DNA sequencing. assay buffer due to the presence of K-glutamate and Triton X-100. Fluorescence intensity was measured by exciting at 360 nm and Cell culture and transfections measuring the emission at 460 nm using a FluoroCount microplate HeLa cells with an integrated tetracycline-regulated transcriptional fluorometer (Packard; Meriden, CT). Spindle pole targeting of katanin 1625

Flow cell severing assay from 4-6 adjacent untransfected cells to yield a ‘fold over-expression’ Microscope-based severing assays have been described previously of p60 or a ‘fold reduction’ in tubulin staining intensity. (McNally and Vale, 1993; McNally and Thomas, 1998; McNally, 1998). To assay human p60/GST-procon80 heterodimers, taxol- Mitotic index measurements stabilized, tetra-methyl rhodamine-labeled microtubules were The mitotic index of transfected cultures was typically less than 0.5% immobilized on coverslips by first perfusing flow cells with a mutant at 24 hours post-transfection compared with 5% in untransfected kinesin that binds strongly to microtubules but is unable to hydrolyze cultures. By 48 hours post-transfection, transfected cultures recovered ATP (McNally and Thomas, 1998). Assays were performed in 20 mM to a 5% mitotic index. Therefore, for measurement of mitotic indexes, HEPES, pH 7.4, 2 mM MgCl2, 1 mM ATP. Images of microtubules transfected cells were trypsinized 24 hours post-transfection and were captured using a Nikon Microphot SA microscope with a ×60 replated on 18 mm coverslips for fixation at 48 and 72 hours. Cells plan Apo 1.4 objective, a Quantix KAF1400 12 bit CCD camera were stained with anti-GST , anti-β-tubulin antibody and (Photometrics; Tucson, AZ) operated with IP Lab Spectrum software DAPI. Prometaphase-like cells were identified as those with (Scanalytics; Fairfax, VA) and a Ludl Electronics (Hawthorne, NY) condensed but disorganized chromatin and distinct microtubule asters. MAC2000 controller and shutter to minimize exposure to light.

Microtubule-binding assays RESULTS Microtubule-binding reactions were carried out in binding buffer (20 mM K-HEPES, pH 7.5, 2 mM MgCl2, 25 mM K-glutamate, 0.1 mM The C-terminal domain of human p80 katanin EGTA, 1 mM ATP, 20 µM taxol, 0.5 mg/ml lysozyme). Recombinant interacts specifically with the N-terminal domain of P loop p60 or an equimolar mixture of P loop p60 and GST-con80 human p60 katanin were diluted to a final concentration of 0.02 µM in binding buffer and pre-spun for 5 minutes at 30,000 rpm in a TLA100 rotor (Beckman Previous studies with in vitro translated deletion derivatives of Inst.) before adding microtubules. Microtubules were assembled from sea urchin p80 katanin indicated that a C-terminal domain of phosphocellulose-purified, MAP-free porcine brain tubulin and p80 is necessary and sufficient for association with sea urchin stabilized with 20 µM taxol before dilution and addition to pre-spun p60 katanin (Hartman et al., 1998). To confirm that the katanin. Binding reactions (50 µl) were incubated for 30 minutes at same interaction occurs between human p80 and human p60, 22¡C, layered onto 50 µl cushions of 30% glycerol in binding buffer, association between human p60 and a glutathione-S- then sedimented at 50,000 rpm for 5 minutes in a Beckman TL100 transferase (GST) fusion to the human p80 C-terminal domain rotor. Supernatants and SDS-resuspended pellets were analyzed by (con80) was examined in lysates of transfected HeLa cells. As immunoblotting utilizing an anti-p60 antibody and chemiluminescent shown in Fig. 1D, full-length human p60 co-purifies with GST- detection. con80 on glutathione-Sepharose (lane 1) but does not co-purify Immunofluorescence microscopy with GST alone (lane 2). To determine the region of p60 Cells grown on coverslips were washed in PBS (137 mM NaCl, 2 that interacts with con80, two deletion derivatives of p60 mM KCl, 5 mM Na2HPO4, 2 mM KH2PO4, pH 7.4), fixed in −20¡C were examined. ∆1-29 p60 is missing the sequence methanol, rehydrated in TBST (150 mM NaCl, 50 mM Tris-Cl, pH AREYALLGNYDS which is highly conserved in p60 7.5, 0.05% Triton X-100 (v/v)), blocked in 4% BSA in TBST, then homologs from sea urchin, human and Chlamydomonas incubated sequentially in primary antibody (usually 0.1-1 µg/ml IgG) (Lohret et al., 1999). ∆1-180 p60 is missing the entire non- followed by fluorescent secondary antibody, both diluted in 4% BSA α α AAA region of human p60. This domain of sea urchin p60 was in TBST. -Tubulin was detected with mouse monoclonal DM1 previously shown to bind microtubules (Hartman and Vale, (Sigma Chemical Co, St Louis, MO). p60 katanin was detected with 1999) but is extremely divergent between p60 homologs. Both an affinity-purified anti-human p60 rabbit polyclonal antibody ∆ ∆ (McNally and Thomas, 1998). Glutathione-S-transferase fusion 1-29 p60 and 1-180 p60 failed to co-purify with GST-con80 proteins were detected with an Alexa488-conjugated rabbit anti-GST (Fig. 1D, lanes 3 and 5) indicating that the conserved N antibody (Molecular Probes; Eugene, OR). NuMA was detected with terminus of p60 is likely to interact directly with the con80 a rabbit polyclonal serum or monoclonal IF1, both provided by domain of p80. Duane Compton (Dartmouth Medical School). Rabbit antibodies were detected either with Oregon Green 488 or with Texas Red-X The in vitro microtubule-severing activity of human secondary antibodies (Molecular Probes) and mouse antibodies were p60 is enhanced by con80 detected with an Alexa594 secondary antibody (Molecular Probes). Previous studies demonstrated that the rate of microtubule For GST/p60 double labeling, cells were first stained with anti-p60 disassembly by sea urchin p60 is enhanced by full-length sea antibody and Texas Red-X secondary antibody, then blocked with 10% rabbit serum before staining with the rabbit anti-GST antibody urchin p80 (Hartman et al., 1998). To determine whether to prevent the anti-GST antibody from binding to the anti-rabbit human p60 severs microtubules in vitro and whether the con80 second antibody. The quadruple labeling in Fig. 8E and F domain is sufficient to stimulate microtubule severing by required the use of Cy5-coupled second antibodies (Jackson human p60, recombinant human katanin subunits were ImmunoResearch; West Grove, PA). Images were acquired as expressed and purified. GST-con80, the longer p80 derivative described above. GST-procon80, 6-histidine-tagged wild-type human p60 and two different 6-histidine-tagged point mutant derivatives of Quantitation of staining intensities p60 were expressed and purified by either glutathione- To quantitate p60 and tubulin staining intensity, exposures resulting Sepharose chromatography or by metal chelate in maximal pixel values of 2000-3500 (4095 maximum) were utilized (typically 0.2-1 seconds). The average pixel intensity was determined chromatography. The mutant derivatives were P loop p60, for irregularly shaped areas of each cell that did not include the which had a substitution of alanine for the lysine at position nucleus or any obvious bright aggregates of second antibody. The 255 of human p60, and DEID p60, which had a double background-subtracted average pixel intensity of each transfected cell substitution of 2 glutamines for the aspartate and glutamate was then divided by the background-subtracted average determined residues at positions 308 and 309. These mutations are similar 1626 K. P. McNally and others

Fig. 1. The N-terminal 29 residues of human p60 are required for association with the C-terminal domain of human p80. (A) Plasmid constructs used to express human katanin subunits. Full length p80 consists of a WD40 domain (WD), a proline-rich domain (pro) and a C-terminal domain conserved among p80 homologs (con80). GST- con80 is a fusion between glutathione-S-transferase and con80. Human p60 katanin consists of a short N-terminal sequence conserved among p60 homologs (black box), a region that binds microtubules (MT) and an ATPase domain (AAA). ∆1-29 p60 and ∆1-180 p60 lack the N-terminal 29 or 180 amino acids, respectively. (B,C,D) HeLa cells were co-transfected with either GST-con80 (lanes 1, 3, 5) or GST (lanes 2, 4, 6) and deletion derivatives of p60. GST fusion proteins and associated proteins were isolated by glutathione-Sepharose chromatography and analyzed by immunoblotting. (B) Immunoblot analysis of the starting lysates with anti-p60 antibody revealed that full length p60 (lanes 1, 2), ∆1-29 p60 (lanes 3, 4) and ∆1-180 p60 (lanes 5, 6) were all expressed. (C) Immunoblot analysis of glutathione-Sepharose binding fractions with an anti-GST antibody revealed that both GST-con80 and GST bound to the glutathione-Sepharose. (D) Immunoblot analysis of the glutathione-Sepharose binding fractions with anti-p60 antibody Fig. 2. Purified GST-con80 stimulates the microtubule-severing revealed that full length p60 copurifies with GST-con80 (lane 1) but activity of human p60 katanin. (A) Coomassie stained SDS-PAGE of not with GST (lane 2). ∆1-29 p60 and ∆1-180 p60 did not copurify baculovirus-expressed human katanin subunits. Lane 1: 6-histidine- with GST-con80 at all (lanes 3 and 5). Note that endogenous full tagged wild-type human p60; lane 2: 6-histidine-tagged P loop p60; length p60 is not detected with the exposure times shown here. lane 3: GST-procon80; lane 4: GST-con80. (B) DAPI-based microtubule disassembly assays. Taxol-stabilized microtubules incubated without additional protein(᭿) or with 0.2 µM DEID p60 to those characterized in sea urchin p60 (Hartman and Vale, (ٗ) exhibited constant DAPI fluorescence. DAPI-fluorescence 1999) and the AAA enzyme VPS4 (Babst et al., 1998) and are decreased in the presence of wild-type human p60 and ATP (᭡) predicted to prevent ATP hydrolysis by human p60. Full-length indicating microtubule disassembly. The rate and extent of human p80 was unstable in SF9 cells and could not be isolated. microtubule disassembly was increased in the presence of wild-type ᭜ ᭹ The purity of these recombinant proteins is shown in Fig. 2A. p60 and GST-con80 ( ) or GST-procon80 ( ). The average increase The rate of microtubule disassembly by human p60 was first in slope mediated by con80 was 1.7-fold. (C) Time lapse fluorescence images of taxol-stabilized, rhodamine-labeled tested using a DAPI-based assay (Hartman et al., 1998). DAPI microtubules immobilized on glass coverslips with a mutant kinesin fluorescence is greater for polymerized microtubules than for subunit. Exposure to wild-type p60 and ATP resulted in slow unpolymerized tubulin (Heusele et al., 1987). When incubated microtubule severing. Microtubule-severing by a mixture of p60 and with taxol-stabilized microtubules, purified human p60 GST-procon80 proceeded more rapidly and to a greater extent. mediated a decrease in DAPI fluorescence indicative of Numbers indicate time in seconds. Bar, 10 µm. microtubule disassembly only in the presence of ATP (Fig. 2B). No decrease in DAPI fluorescence was observed in containing GST-con80 + p60 or GST-procon80 + p60 (Fig. 2B) reactions containing DEID p60, P loop p60, GST-con80 alone indicating that the rate of microtubule disassembly by p60 is or GST-procon80 alone (Fig. 2B and not shown) indicating that enhanced by the con80 domain of p80. these proteins cannot disassemble microtubules. The rate of To confirm that this microtubule disassembly was due to decrease in DAPI fluorescence was enhanced in reactions microtubule severing, taxol-stabilized, rhodamine-labeled Spindle pole targeting of katanin 1627 microtubules were immobilized on glass coverslips and observed by time lapse fluorescence microscopy after perfusion with different recombinant proteins. Human p60 alone mediated a very slow rate of microtubule severing that could only be observed at very low microtubule concentrations, whereas, GST-con80 + p60 or GST-procon80 + p60 mediated rapid microtubule severing and complete disassembly of glass- immobilized microtubules (Fig. 2C and not shown). In addition to demonstrating that the interaction between con80 and p60 increases microtubule-severing activity, these results represent the first direct demonstration of microtubule-severing activity mediated by any purified homolog of sea urchin katanin. The con80 domain enhances the binding of p60 to microtubules in vitro Because the con80 domain interacts with the N-terminal domain of human p60, and this region has been shown to bind microtubules in sea urchin p60 (Hartman and Vale, 1999), we hypothesized that the con80 domain might enhance p60- mediated microtubule severing by increasing the affinity of p60 for microtubules. To test this hypothesis, microtubule binding by P loop p60 was monitored in the presence or absence of GST-con80 at increasing microtubule concentrations. P loop p60 was used to prevent disassembly of microtubules by p60 and to allow direct comparison with in vivo observations described below. At 0.1-0.5 µM polymerized tubulin, only a fraction of p60 co-sedimented with microtubules (Fig. 3A) whereas nearly all the p60 co-sedimented with microtubules at these concentrations in the presence of GST-con80 (Fig. 3B). While the methods used are inadequate to allow determination of binding constants, these results clearly show an increase in Fig. 3. GST-con80 enhances the affinity of human p60 for µ p60’s microtubule affinity due to the presence of GST-con80. microtubules in vitro. P loop p60 (0.02 M) was incubated with The observed increase in p60’s microtubule affinity might increasing concentrations of taxol-stabilized microtubules. Microtubules and associated proteins (P=pellet) were separated from be mediated by a conformational change induced in p60 by unbound proteins (S=supernatant) by sedimentation through a con80 or it might be due to an increase in valency if con80 glycerol cushion. (A) Immunoblot of reactions containing p60 alone contains an additional microtubule-binding site. To address this probed with anti-p60 antibody. (B) Immunoblot of reactions question, microtubule binding by GST-con80 alone was containing p60 and GST-con80 probed with anti-p60 antibody. monitored. As shown in Fig. 3C, only a fraction of GST-con80 (C) Immunoblot of reactions containing GST-con80 alone probed co-sedimented with 0.1 µM polymerized tubulin but nearly all with anti-p80 antibody. the GST-con80 co-sedimented with higher concentrations of polymerized tubulin (Fig. 3C). These results indicate that GST- untransfected cell. In addition to the reduced average staining con80 may enhance p60’s microtubule affinity by increasing intensity, the microtubules in the p60 over-expressing cell in the number of microtubule binding sites. Fig. 4A are remarkable in that 2 ends can be distinguished for nearly every microtubule. Therefore the length of these Human p60 katanin disassembles microtubules in microtubules can be measured (average length 0.7 µm, range vivo and is both positively and negatively regulated 0.5-1.1 µm). No aster of microtubules could be observed by different p80 katanin domains emanating from a centrosome. This disorganized array of short The contradictory findings that human p60 binds stably to microtubules contrasts sharply with the contiguous array of microtubules in vitro but never co-localizes with microtubules long microtubules shown most clearly in the untransfected cell in vivo (McNally and Thomas, 1998) suggested that katanin in Fig. 4B. Cells with reduced anti-tubulin staining were never might interact transiently with microtubules in vivo. To test this observed in cultures transfected with either DEID p60 or P loop hypothesis, human p60 was over-expressed in HeLa cells by p60 (not shown). The reduced intensity of tubulin staining in transient transfection to determine whether p60 could p60 over-expressing cells demonstrates that katanin promotes disassemble microtubules in vivo. Transfected HeLa cells were microtubule disassembly in vivo and the disorganized array of fixed and stained with anti-p60 antibodies to identify over- short microtubules suggests that this disassembly occurs by a expressing cells and with anti-tubulin antibodies to monitor severing mechanism. effects on the microtubule cytoskeleton. In 40-60% of p60 To determine whether the con80 domain enhances over-expressing cells, a 2-fold to 10-fold reduction in the microtubule-disassembly activity of p60 in vivo as it does in intensity of anti-tubulin staining was observed. In the p60 over- vitro, we determined the minimum p60 over-expression level expressing cell shown in Fig. 4A, the average pixel intensity required to disassemble microtubules in HeLa cells co- of anti-tubulin staining is reduced 5-fold relative to the adjacent transfected with p60 and different p80 derivatives. For each 1628 K. P. McNally and others

Table 1. Inhibition of the in vivo microtubule-disassembly activity of p60 katanin % of GFP-positive Co-transfected cells with reduced # Cells plasmids microtubule staining counted # Transfections GFP 54±8 552 4 GFP-p60 40±6 625 2 WD-GFP 3.6±1.8 627 2

HeLa cells were triply transfected with epitope tagged p60, GST-con80 and a third plasmid encoding a GFP fusion protein. Reduced microtubule staining indicates that the average anti-tubulin fluorescence intensity was 3- to 10-fold lower than surrounding untransfected cells. Separate coverslips from each transfection were stained with the epitope-tag antibody to monitor co- transfection efficiency which ranged from 50-100%.

disassemble microtubules (Fig. 4C). Immunoblot analysis indicated that full-length p80 was expressed at levels similar to GST-con80 and GST-procon80 (not shown). Transfected full-length p80 co-purified with GST-p60 on glutathione- Sepharose (not shown) indicating that these proteins can dimerize. These results indicated that transfected full-length p80 does not activate p60 as do GST-con80 and GST-procon80. The finding that GST-procon80 activates p60 but that full- length p80 does not could be explained if the WD40 domain of p80 acts as an inhibitor of microtubule severing by p60. To test this hypothesis, we determined whether a green fluorescent protein (GFP) fusion to the WD40 domain (WD-GFP) could inhibit p60 in trans. An epitope-tagged version of p60 and GST-con80 were co-transfected either with GFP or WD-GFP Fig. 4. Overexpression of human p60 katanin in HeLa cells results in and the fraction of bright GFP-positive cells with reduced the disassembly of the interphase microtubule cytoskeleton. (A and B) Anti-tubulin immunofluorescence of a HeLa cell transfected with intensity of anti-tubulin staining (2- to 10-fold reduction in human p60 (double arrows) and untransfected HeLa cells (single average pixel intensity) was determined. Co-transfection with arrows). The average pixel intensity of anti-tubulin staining in this WD-GFP resulted in a 10-fold decrease in the percentage of p60-expressing cell is 20% of that in the surrounding untransfected GFP positive cells with reduced microtubules relative to GFP cells. p60 overexpression was detected by anti-p60 alone or a GFP fusion to wild-type p60 (Table 1). This result immunofluorescence (not shown). Bar, 5 µm (C) Overexpression supported the hypothesis that the WD40 domain of p80 acts as levels of human p60 in transfected cells exhibiting anti-tubulin a negative regulator of p60 activity. staining reduced in intensity by 50% or more. Overexpression is expressed as a ratio of anti-p60 fluorescence intensity in the The con80 domain stimulates spindle pole targeting transfected cell relative to that in adjacent untransfected cells. Each of p60 bar represents an individual cell. Note that p60 is more active when co-transfected with derivatives of p80 that lack the WD40 domain Because the con80 domain enhances microtubule binding by (GST-con80 and GST-pro-con80). p60 and because localization of katanin at mitotic spindle poles requires intact microtubules (McNally and Thomas, 1998), we hypothesized that con80 might influence spindle pole targeting transfection, 25-30 cells exhibiting reduced anti-tubulin of p60. GFP fusions to wild-type and mutant p60 were staining (2- to 10-fold reduction in average pixel intensity) constructed and transfected into HeLa cells. A GFP fusion to were selected and the p60 over-expression level was wild-type p60 exhibited only diffuse cytoplasmic fluorescence determined. The fold over-expression of p60 was determined during mitosis when transfected by itself (Table 2). Co- from the ratio of the average pixel intensity of anti-p60 staining transfection of GFP-wild-type p60 and GST-con80, however, in a transfected cell to that of surrounding untransfected cells. resulted in bright GFP fluorescence at spindle poles (Fig. 5A; The over-expression levels of the 13 cells exhibiting the lowest Table 2), suggesting that GST-con80 stimulates spindle pole levels of p60 over-expression for each co-transfection are localization of GFP-p60. Consistent with this hypothesis, co- displayed in Fig. 4C. In cells co-transfected with p60 and GST, transfection of untagged wild-type p60 and GFP-con80 also reduced tubulin staining was seen in cells over-expressing p60 resulted in bright GFP fluorescence at spindle poles (Fig. 5C; by a minimum of 8-fold (Fig. 4C) and never in cells with lower Table 2) while GFP-con80 by itself exhibited only diffuse expression levels. Co-expression of GST-con80 or GST- fluorescence (Table 2). These results indicate that spindle pole procon80 dramatically reduced the minimum over-expression targeting is promoted by the same con80/p60 interaction level of p60 to 2-fold indicating that the con80 domain that enhances microtubule affinity and microtubule-severing activates p60’s activity in vivo as it did in vitro. Surprisingly, activity. co-expression of full-length p80 with p60 had very little effect In contrast with wild-type p60, which targeted to the spindle on the minimum expression level of p60 required to pole when co-transfected with GST-con80, both inactive p60 Spindle pole targeting of katanin 1629

Table 2. Subcellular localization of GFP-katanin fusion proteins Plasmids Spindle poles Filaments Diffuse GFP-p60 - - +++ GFP-DEID p60 - - +++ GFP-P loop p60 + - +++ GFP-p60 + GST-con80 +++ - +++ GFP-DEID p60 + GST-con80 + +++ +++ GFP-Ploop p60 + GST-con80 + +++ +++ GFP-con80 - - +++ GFP-con80 + p60 +++ - +++ GFP-con80 + Ploop p60 - +++ +++ WD-GFP - - +++ GFP-p60 + p80 - - +++

-, never observed; +, dim fluorescence; +++, bright fluorescence.

Table 3. Quantitation of large multinucleate cells among HeLa cells transfected with different katanin subunits 72 hours post-transfection % Large # Cells Plasmids multinucleate counted # Transfections A. GST + GST-con80 2.1±0.1 3327 3 GST + DEID p60 2.2 1142 1 GST + Ploop p60 2.4±0.9 2493 3 GST-con80 + DEID p60 24±5 1515 3 GST-con80 + Ploop p60 36±6 1243 3 B. GST-con80 2.1±0.1 1969 2 Fig. 5. Targeting of wild-type p60 to spindle poles and mutant p60 to GST-con80 + GFP-DEID p60 15±1 572 2 filaments. GFP fluorescence shown in A, C and E. Anti-NuMA GST-con80 + GFP-Ploop p60 30±2 494 2 staining shown in B. Anti-tubulin staining shown in D and F. Co- transfection of GFP-wild-type p60 and GST-con80 results in bright A and B indicate sets of transfections carried out at the same time. GFP fluorescence at spindle poles (A) as indicated by co-localization with NuMA (B). Co-transfection of untagged wild-type p60 with GFP-con80 results in bright GFP fluorescence at spindle poles (C) as indicated by localization at the center of the microtubule aster (D). mutants targeted to filaments when co-expressed with GST- Note that cells in A and C have very small monastral spindles due to con80 (Fig. 5E; Table 2). The filaments nearly always stretched wild-type p60 overexpression. Co-transfection of GFP-DEID p60 across the midbody of dividing cells (not shown) and caused a and GST-con80 results in incorporation of GFP fluorescence into massive increase in the number of extremely large and bundled filaments (E) that do not co-localize with microtubules (F). multinucleate HeLa cells (Table 3) presumably by interfering Bars: 3.5 µm (A, for A,B,C,D; E, for E and F). with the completion of cytokinesis. Filaments were never formed by wild-type p60 or by mutant p60’s in the absence of GST-con80 (Table 2). 80% of GFP-P loop p60/GST-con80 regions of polymer has been observed for the highly expressing cells exhibited long filaments or bundles of cooperative DNA-binding proteins recA and gene 32 protein filaments. Treatment of these cells with 20 µM nocodazole for and has been described in thermodynamic terms 2.5 hours resulted in the complete disappearance of filaments (Kowalczykowski et al., 1986). Spindle pole targeting of greater than 1 µm in length, although 4% of nocodazole-treated katanin may require the cooperative binding of wild-type cells still exhibited short (<1 µm) bundles of filaments. This p60/p80 to a subset of microtubules which are kept short by result indicated that the filaments may represent stable binding microtubule-severing activity. of mutant katanin subunits to a subset of microtubules. Two properties make this microtubule binding highly unusual. First, The WD40 domain of p80 is required for spindle pole in most cells, the filaments are not labeled with anti-tubulin localization antibodies (Fig. 5E,F) suggesting that the mutant katanin The finding that GST-con80, which has no WD40 domain, can subunits completely coat the microtubules and thus exclude promote spindle pole targeting of GFP-p60 might lead to the anti-tubulin antibodies. Second, the majority of microtubules conclusion that the WD40 domain is not required for spindle exhibit no GFP labeling (Fig. 5E,F). A simple hypothesis pole targeting. However, sea urchin katanin forms transient explaining these results is that the con80 domain increases the oligomers when bound to microtubules (Hartman and Vale, affinity of p60 for microtubules in vivo as it does in vitro and 1999) and it is likely that human p60 oligomerizes on that cooperative binding of the mutant p60/GST-con80 dimers microtubules as well. When cells are transfected with GFP-p60 results in a subset of microtubules that are completely coated. and GST-con80, it is likely that some GFP-p60 associates with These coated microtubules remain long because of the lack of endogenous full-length p80. Thus the observed spindle pole severing by mutant p60. This type of extensive coating of long targeting could occur via mixed oligomers of p80/p60 and 1630 K. P. McNally and others

Table 4. Mitotic index measurements of HeLa cells transfected with different katanin subunits 72 hours post- transfection Plasmids % Prometaphase # Cells counted # Transfections GST 1.7±0.5 4926 3 WD-GST 5±1 3480 3 WD-pro-GST 9±2 2928 3

Table 5. Fraction of transfected prometaphase-like HeLa cells exhibiting different spindle morphologies Plasmid % Bipolar % Monopolar % Multipolar n (cells) GST 88 6 6 98 WD-GST 41 0 59 56 WD-pro-GST 40 5 55 143

Multipolar spindles included those with 3-12 spindle poles as assayed by anti-tubulin or anti-NuMA staining. Large polyploid cells were excluded from this count.

the endogenous p60 katanin). Expression of either GST-con80 or GST-pro-con80 resulted in elimination or reduction in the spindle pole concentration of p60 in 80% of the transfected mitotic cells exhibiting bright GST staining (Fig. 7A,C and not shown). Most often, two small spots of p60 staining were observed (Fig. 7C) that may correspond to the centrosomal katanin previously observed after nocodazole-mediated disruption of the spindle pole (McNally and Thomas, 1998). Fig. 6. Transfected GST-con80 associates with endogenous p60 in Neither expression of the WD repeat domain (WD-GST or HeLa cells. (A) Schematic representation of GST fusion proteins WD-pro-GST) nor expression of GST alone had any effect on expressed in HeLa cells. Top diagram represents the structure of spindle pole localization of endogenous p60 (Fig. 7E and not intact human p80 katanin. Acronyms are described in the legend to shown). These results indicated that p60 dimerized with a Fig. 1. (B) Immunoblot probed with an anti-GST antibody. Each lane WD40-less p80 subunit could not properly target to the spindle was loaded with the glutathione-Sepharose binding fraction from HeLa cells transfected with one fusion protein. Lane numbers pole. correspond to the numbers in A. (C) Immunoblot of the same The failure of WD40-less katanin subunits to target to the fractions probed with an anti-p60 katanin antibody. Note that spindle pole suggests that there is a direct interaction between endogenous p60 copurifies with con80-containing fusions but not the WD40 domain and another component of the spindle pole. with WD-containing fusions. This hypothesis was supported by the finding that over- expression of the WD40 domain, as WD-GST or WDpro-GST, in HeLa cells resulted in an increased mitotic index (Table 4) GST-con80/p60. To test whether the WD40 repeat domain of characterized by a multipolar spindle morphology (Table 5). human p80 katanin is required for spindle pole targeting, we This phenotype could be explained if the WD40 domain binds attempted to generate cells in which the vast majority of p60 to an essential spindle pole component. However, binding of subunits are associated with GST-con80 rather than with the the WD40 domain to a spindle pole protein would have to be endogenous full-length p80 by transient transfection of GST- characterized by a low affinity because the WD40 domain did con80 or GST-procon80 alone. To test whether transfected not target to spindle poles on its own (Table 2). GST-con80 or GST-procon80 effectively associates with endogenous p60, transfected cells were lysed and glutathione- The WD40 domain does not target to the spindle Sepharose binding fractions were analyzed for the presence of pole by direct binding to NuMA endogenous p60 by immunoblotting. As shown in Fig. 6C, NuMA is an attractive candidate for the binding partner of the endogenous p60 associates with GST-con80 and GST-pro- WD40 domain because of the similar localization of NuMA con80 but not with WD-GST, WD-pro-GST, or GST alone. and katanin in the spindle pole (Fig. 8A,B). To test the These results indicated that the WD40-less constructs were hypothesis that the WD40 domain mediates spindle pole indeed dimerizing with endogenous p60 and converting the targeting of p60 by direct binding to NuMA, p60 localization endogenous p60 into WD40-less katanin heterodimers. was monitored in two situations where NuMA localization is To determine whether WD40-less katanin heterodimers are perturbed. When cells were treated with nocodazole for 5 able to properly localize to the spindle pole, HeLa cells minutes, NuMA rapidly moved from the spindle poles to transfected with each construct were fixed and double labeled aggregates associated with the chromosomes as previously with an anti-GST antibody (to identify transfected cells) and a described (Dionne et al., 1999; Fig. 8D) while p60 dispersed human p60 katanin antibody (to determine the localization of into the and showed no co-localization with the Spindle pole targeting of katanin 1631

Fig. 7. Localization of endogenous p60 in HeLa cells expressing different GST fusion proteins. Anti-p60 staining shown in A, C and E. Anti-GST staining shown in B, D and F. (A and B) Prometaphase cell expressing GST-con80 (right cell) next to an untransfected prometaphase cell (left cell). (C and D) Metaphase cell expressing GST-con80. (E and F) Metaphase cell expressing GST. Note the prominent spindle pole staining in E and in the untransfected cell in A. This localization is eliminated in the transfected cell in A and reduced in C due to GST-con80 expression. Bar, 10 µm.

NuMA aggregates (Fig. 8C). Cells were also transfected with mechanism of katanin’s localization at mitotic spindle poles a p50 dynamitin-GFP fusion protein, which would be expected both by determining the effects of transfected katanin subunits to disrupt NuMA localization (Gaglio et al., 1997) by on the localization of endogenous katanin and by monitoring disrupting dynactin-dependent activity (Echeverri et al., the localization of the transfected katanin subunits themselves. 1996). p50-GFP-transfected cells were examined by triple Our results indicate that the WD40 domain and con80 domain label immunofluorescence for GFP-p50 expression, p60 of p80 katanin as well as a wild-type p60 subunit are required localization and NuMA localization. Many cells could be for spindle pole targeting. In the process, we have uncovered found in which p60 localization and NuMA localization were completely dissociated (Fig. 8E,F). In addition, endogenous NuMA never co-purified with transfected WD-GST fusion proteins (not shown). These results indicate that katanin localizes to spindle poles in a NuMA-independent manner.

DISCUSSION

Katanin is a microtubule-severing protein activated in mitotic Xenopus extracts (Vale, 1991; McNally and Thomas, 1998) and concentrated at mitotic spindle poles. Activation of the microtubule-severing activity of katanin by increased local concentration at spindle poles during mitosis may be important for proper spindle function. In this study we have examined the

Fig. 8. Co-localization of p60 and NuMA is disrupted by nocodazole or p50, dynamitin. Immunofluorescence of mitotic HeLa cells with anti-p60 antibody (A,C,E) or anti-NuMA antibody (B,D,F). p60 and NuMA co-localize at spindle poles in an untreated cell (A,B). Brief (5 minute) treatment with 20 µm nocodazole (C,D) causes aggregation of NuMA (D) but dispersal of p60 (C) with no association of p60 with the NuMA aggregates. (E and F) Expression of a p50 dynamitin-GFP fusion protein reveals differences between p60 and NuMA. In the p50-GFP expressing HeLa cell in E and F, a bright focus of p60 is found only on the left spindle pole where no NuMA is present. In contrast, NuMA can be seen spreading from the right spindle pole onto the microtubules but p60 is absent from this spindle pole. Bar, 3.5 µm. 1632 K. P. McNally and others a novel role for the WD40 domain as an inhibitor of the oligomers with GST-con80/p60 dimers. These mixed oligomers microtubule-severing activity of p60 katanin. could target to spindle poles because they contain some WD40 domains. When only GST-con80 is transfected, it is likely to be The WD40 repeat domain of p80 katanin is essential present at a molar excess over endogenous p60 thus precluding for spindle pole targeting the formation of p60 dimers with endogenous full length p80 By expressing WD40-less p80 katanin fusion proteins in HeLa and preventing spindle pole targeting of p60. cells, we have been able to cause the mislocalization of A model for spindle pole targeting that requires microtubule endogenous p60 katanin from the spindle poles to the binding by con80/p60 complexes and WD40 interaction with cytoplasm. We propose that this occurs because the another spindle pole protein predicts that co-transfection of full endogenous p60 molecules preferentially dimerize with the length p80 and GFP-p60 should result in spindle pole targeting. GST fusion proteins, which are present at much higher Indeed, transfection of GFP-p60 alone should result in concentrations than the endogenous p80 subunits. The dimerization with endogenous p80 and spindle pole targeting. hypothesis that mislocalization is caused by the formation of Neither situation results in detectable GFP fluorescence at WD40-less heterodimers is supported by the finding that spindle poles (Table 2). One possibility is that the observed mislocalization was caused only by constructs that dimerized targeting of GFP to spindle poles represents loading of ‘extra’ with the endogenous p60 (GST-con80 and GST-pro-con80) katanin at spindle poles with ‘normal’ levels of katanin being and not by constructs that did not dimerize with p60 (WD-GST, undetectable by GFP fluorescence. In cells transfected with WD-pro-GST and GST). These results provide convincing GFP-p60 alone or p80 + GFP-p60, each WD40 domain of a evidence that the WD40 repeat domain of p80 katanin is GFP-p60/p80 dimer could target only one GFP molecule to the required for spindle pole localization of the katanin spindle pole. In cells transfected with GST-con80 + GFP-p60, heterodimer. a mixed dodecamer with one p80/GFP-p60 dimer and five The finding that the WD40 domain is essential for spindle GST-con80/GFP-p60 dimers could form. In this mixed pole targeting suggested that the WD40 domain may bind dodecamer, a single WD40 domain could target six GFP directly to another spindle pole protein. This model was molecules to the spindle pole. If the proposed WD40-binding supported by the finding that transfection of WD-GST or sites in the spindle pole are limiting in number, then WDpro-GST caused HeLa cells to delay in mitosis with a transfection of GST-con80 + GFP-p60 could result in six times multipolar spindle morphology. The binding partner for the more GFP at spindle poles than transfection of p80 + GFP-p60. WD40 domain is not NuMA because co-localization of katanin In support of this model, very dim GFP-fluorescence was and NuMA can be disrupted by nocodazole treatment or observed at spindle poles when cells were transfected with dynamitin expression. Binding of the WD40 domain to an GFP-P loop p60 alone (Table 2). unknown spindle pole component may be characterized by a An alternative explanation for the promotion of spindle pole low affinity since the WD40 domain was not sufficient for targeting by a WD40-less p80 subunit does not require the targeting GFP to the spindle pole by itself. existence of hetero-oligomers. Since GST-con80/mutant p60 forms unusual filaments never seen naturally, it is possible that Cooperative microtubule binding by p60 is required GST-con80/wild-type p60 forms structures that assemble at the for spindle pole targeting spindle pole by a mechanism different than that used by Consistent with the finding that the WD40 domain is not endogenous katanin. In this scenario, it is not possible to detect sufficient for spindle pole targeting, we have found that a wild- normal spindle pole targeting of transfected subunits by GFP type p60 subunit is also required for spindle pole targeting. fluorescence. Wild-type or mutant p60 subunits exhibited only diffuse localization when transfected alone. Co-transfection with GST- The WD40 domain is a negative regulator of con80, however, allowed transfected wild-type p60 to target to microtubule severing by p60 spindle poles and transfected mutant p60’s to target to The finding that p60/GST-con80 had more potent microtubule filaments. Since we have demonstrated that GST-con80 disassembly activity than p60/p80 indicated that the WD40 enhances p60’s microtubule affinity in vitro, we propose that domain of p80 might inhibit the microtubule-severing activity spindle pole association by GST-con80/p60 involves direct of p60. While it is possible that transfected full-length p80 does binding to microtubules which are kept short by microtubule- not fold properly, the WD40-inhibition hypothesis was severing activity. The weak interaction between the WD40 supported by the result that a WD40-GFP fusion protein could domain and an unknown spindle pole component would keep inhibit microtubule disassembly by p60/GST-con80 in vivo. the short microtubules focused at the spindle pole. This result was unexpected and indicates that the WD40 Enhancement of the microtubule affinity of mutant p60 by domain of p80 has multiple functions. If the WD40 domain GST-con80 results in stable binding to microtubules which inhibits microtubule disassembly by preventing microtubule remain long because of a lack of severing. binding by p60, then the WD40 domain might function in This model requires that GST-con80, which has no WD40 limiting the amount of p60 that can be loaded at the spindle domain, can stimulate WD40-dependent targeting of p60. This pole. paradox is readily explained when one considers that p60 katanin assembles into transient hexamers when bound to microtubules We thank Duane Compton for providing anti-NuMA antibodies, (Hartman and Vale, 1999; reviewed by McNally, 2000). When Dan Buster, Jodi Nunnari, Lesilee Rose, Jon Scholey and Dave Sharp p60 and GST-con80 are both transfected, some cells are likely for critical reading of the manuscript and Alexis Madrid for technical to have a molar excess of p60 relative to GST-con80. In these assistance. This work was supported by grant GM53060 from the cells, dimers of p60 and endogenous full-length p80 could form National Institutes of Health to F.J.M. Spindle pole targeting of katanin 1633

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