M Phase Phosphoprotein 1 Is a Human Plus-end-directed Kinesin-related Protein Required for Cytokinesis
2003; Elsevier BV; Volume: 278; Issue: 30 Linguagem: Inglês
10.1074/jbc.m304522200
ISSN1083-351X
AutoresAouatef Abaza, Jean-Marc Soleilhac, J M Westendorf, Matthieu Piel, Isabelle Crevel, Aurélien Roux, Fabienne Pirollet,
Tópico(s)Chromosomal and Genetic Variations
ResumoThe human M phase phosphoprotein 1 (MPP1), previously identified through a screening of a subset of proteins specifically phosphorylated at the G2/M transition (Matsumoto-Taniura, N., Pirollet, F., Monroe, R., Gerace, L., and Westendorf, J. M. (1996) Mol. Biol. Cell 7, 1455–1469), is characterized as a plus-end-directed kinesin-related protein. Recombinant MPP1 exhibits in vitro microtubule-binding and microtubule-bundling properties as well as microtubule-stimulated ATPase activity. In gliding experiments using polarity-marked microtubules, MPP1 is a slow molecular motor that moves toward the microtubule plus-end at a 0.07 μm/s speed. In cycling cells, MPP1 localizes mainly to the nuclei in interphase. During mitosis, MPP1 is diffuse throughout the cytoplasm in metaphase and subsequently localizes to the midzone to further concentrate on the midbody. MPP1 suppression by RNA interference induces failure of cell division late in cytokinesis. We conclude that MPP1 is a new mitotic molecular motor required for completion of cytokinesis. The human M phase phosphoprotein 1 (MPP1), previously identified through a screening of a subset of proteins specifically phosphorylated at the G2/M transition (Matsumoto-Taniura, N., Pirollet, F., Monroe, R., Gerace, L., and Westendorf, J. M. (1996) Mol. Biol. Cell 7, 1455–1469), is characterized as a plus-end-directed kinesin-related protein. Recombinant MPP1 exhibits in vitro microtubule-binding and microtubule-bundling properties as well as microtubule-stimulated ATPase activity. In gliding experiments using polarity-marked microtubules, MPP1 is a slow molecular motor that moves toward the microtubule plus-end at a 0.07 μm/s speed. In cycling cells, MPP1 localizes mainly to the nuclei in interphase. During mitosis, MPP1 is diffuse throughout the cytoplasm in metaphase and subsequently localizes to the midzone to further concentrate on the midbody. MPP1 suppression by RNA interference induces failure of cell division late in cytokinesis. We conclude that MPP1 is a new mitotic molecular motor required for completion of cytokinesis. Eukaryotic cells exhibit dramatic changes of microtubule organization and dynamics as they enter mitosis (2Dustin P.D. Microtubules. 2nd Ed. Springer-Verlag, Berlin1984: 8-18Crossref Google Scholar, 3Kirschner M. Mitchison T. Cell. 1986; 45: 329-342Abstract Full Text PDF PubMed Scopus (983) Google Scholar). These changes are timely and spatially coordinated with nucleus and membranes alterations by the tight control of M phase-promoting factor, whose catalytic component, the p34cdc2 or Cdk1 kinase becomes activated at the G2/M transition (4Murray A.W. Kirschner M.W. Science. 1989; 246: 614-621Crossref PubMed Scopus (512) Google Scholar, 5Lamb N.J. Fernandez A. Watrin A. Labbe J.C. Cavadore J.C. Cell. 1990; 60: 151-165Abstract Full Text PDF PubMed Scopus (107) Google Scholar). 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Cell Biol. 1996; 8: 23-29Crossref PubMed Scopus (104) Google Scholar, 12Heald R. Nat. Cell Biol. 2000; 2: 11-12Crossref PubMed Scopus (15) Google Scholar). Several microtubule-associated proteins have been described to promote tubulin assembly and polymer stabilization or destabilization (13Andersen S.S. Bioessays. 1999; 21: 53-60Crossref PubMed Scopus (57) Google Scholar, 14Valiron O. Caudron N. Job D. Cell Mol. Life Sci. 2001; 58: 2069-2084Crossref PubMed Scopus (145) Google Scholar). Besides their roles in intracellular trafficking of organelles and vesicles during interphase, dyneins and kinesin-related proteins (KRPs), 1The abbreviations used are: KRP, kinesin-related protein; MT, microtubule; MPP1, M phase phosphoprotein 1; rMPP1, recombinant MPP1; rMC1, recombinant MC1; GFP, green fluorescent protein; siRNA, small interfering RNA; TRITC, tetramethylrhodamine isothiocyanate; PBS, phosphate-buffered saline; Mes, 2-(N-morpholino)-ethanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid; aa, amino acid(s); FACS, fluorescence-activated cell sorting; AMP-PNP, 5′-adenylyl-β,γ-imidodiphosphate. microtubule-based molecular motors, play important roles in cell division. At each stage of mitosis or meiosis, dyneins and various KRPs interact with microtubules in order to ensure centrosome separation, spindle formation and maintenance, chromosome congression, and cytokinesis completion (15Hirokawa N. Noda Y. Okada Y. Curr. Opin. 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The p34cdc2 kinase phosphorylates the ubiquitous microtubule-associated protein 4 during M phase (21Ookata K. Hisanaga S. Sugita M. Okuyama A. Murofushi H. Kitazawa H. Chari S. Bulinski J.C. Kishimoto T. Biochemistry. 1997; 36: 15873-15883Crossref PubMed Scopus (66) Google Scholar), and this phosphorylation abolishes microtubule-associated protein 4 microtubule stabilizing activity (22Ookata K. Hisanaga S. Bulinski J.C. Murofushi H. Aizawa H. Itoh T.J. Hotani H. Okumura E. Tachibana K. Kishimoto T. J. Cell Biol. 1995; 128: 849-862Crossref PubMed Scopus (240) Google Scholar). There is evidence that the phosphorylation of the microtubule destabilizing protein Stathmin/Op18 by p34cdc2 is important for mitotic progression (23Melander Gradin H. Marklund U. Larsson N. Chatila T.A. Gullberg M. Mol. Cell. Biol. 1997; 17: 3459-3467Crossref PubMed Scopus (126) Google Scholar). Similar phosphorylation of the mitotic KRP Eg5 is required for Eg5-dependent centrosome migration and bipolar spindle formation in vivo (17Blangy A. Lane H.A. d'Herin P. Harper M. Kress M. Nigg E.A. Cell. 1995; 83: 1159-1169Abstract Full Text PDF PubMed Scopus (788) Google Scholar). These data suggest that mitotic kinases regulate microtubule dynamics and organization by phosphorylating various microtubule-interacting proteins, and this has been an incentive for the systematic search of mitotic phosphoproteins. We have recently identified a subset of M phase phosphoproteins by expression library screening using the MPM2 monoclonal antibody, which recognizes a phosphoepitope present on a set of 40–50 proteins that become phosphorylated at the G2/M transition (1Matsumoto-Taniura N. Pirollet F. Monroe R. Gerace L. Westendorf J.M. Mol. Biol. Cell. 1996; 7: 1455-1469Crossref PubMed Scopus (159) Google Scholar, 24Westendorf J.M. Rao P.N. Gerace L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 714-718Crossref PubMed Scopus (239) Google Scholar, 25Davis F.M. Tsao T.Y. Fowler S.K. Rao P.N. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2926-2930Crossref PubMed Scopus (504) Google Scholar, 26Davis F.M. Rao P.N. Schlegel R.A. Halleck M.S. Rao P.N. Molecular Regulation of Nuclear Events in Mitosis and Meiosis. Academic Press, Inc., New York1987: 259-293Crossref Google Scholar). Among the 11 proteins identified, we show here that M phase phosphoprotein 1 (MPP1) has extensive homology with proteins of the kinesin superfamily. We demonstrate that MPP1 is a plus-end-directed molecular motor with an important role in cytokinesis. Cloning and Sequencing of Full-length Human MPP1 cDNA— Human MPP1 cDNAs were obtained by screening two λzapII cDNA libraries made from HeLa, one a generous gift from P. Chambon and the other a Uni-ZAP XR library from Stratagene, following standard procedures (27Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Starting with the 645-bp N-terminal fragment of the previously described partial-length MPP1 cDNA 6-1 (24Westendorf J.M. Rao P.N. Gerace L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 714-718Crossref PubMed Scopus (239) Google Scholar) as a probe, the human MPP1 cDNA, 1L9 (4265 bp), was isolated. The overlapping MPP1 cDNA, 1C12 (2822 bp) was obtained by a second round of screening using an N-terminal fragment of pBS-1L9. Full-length MPP1 plasmid, pBS-MPP1, was obtained by subcloning the 2408-bp N-terminal fragment of the pBS-1C12 in pBS-1L9. The HsMPP1 sequences are available under accession numbers AY282406, AY282407. Sequencing was performed by the Genome Express Company (Grenoble, France). The sequences were analyzed, and data base searches were performed with the GCG, FASTA, and BLAST programs available at NCBI or INFO-BIOGEN resources. Specific motifs were searched using PESTFIND, COIL, PredictNLS, and pSORT programs. FLAG Epitope Tagging of MPP1 by Mutagenesis—The MPP1 coding sequence starting at base 70 was tagged using a mutagenesis technique based on the M13-phage single-stranded DNA protocol (Amersham Sculptor Kit). 1C12 single-stranded DNA was obtained using standard procedures (27Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). A 66-mer oligonucleotide was designed that introduced, between bases 69 and 70, 36 bp containing a NotI restriction site and encoding MDYKDDDDK amino acids, which correspond to the FLAG epitope upstream of the cleavage sequence of enterokinase. After sequencing of the 5′-end of a selected clone, the 3′-end fragment NsiI-BamHI (2484 bp) was replaced by the similar fragment obtained from the original 1C12 to ensure that no other mutations were introduced in plasmid pBS-m1C12. Orientation of the mutated N-terminal EcoRI fragment (484 bp) was inverted in the pBluescript vector, and the 5145-bp NsiI fragment of pBS-MPP1 was subcloned into this plasmid to construct pBS-mMPP1, which encoded full-length MPP1 tagged with the FLAG epitope. Expression and Purification of Recombinant MPP1 Mutants in Insect Cells—A recombinant full-length MPP1 (rMPP1) and a truncated form (rMC1), both tagged with an N-terminal FLAG epitope, were produced by baculovirus expression following the manufacturer's instructions of the Bac-to-Bac system (Invitrogen). The mutated NotI-KpnI fragment of pBS-mMPP1 and the NotI-XhoI of pBS-m1C12 were respectively subcloned into the pFastBac HTb vector in phase with its His6 coding sequence to generate doubly tagged recombinant viruses in Sf9 cells. Recombinant proteins were then expressed in High-Five cells, a generous gift of Dr. B. Goud. Cells were harvested at 48 h after viral infection at a multiplicity of infection of 2, frozen in liquid nitrogen, and stored at –80 °C. Frozen cell pellets were resuspended in ice-cold lysis buffer (50 mm Tris, pH 8, 0.5 m NaCl, 2 mm MgCl2, 5 mm CaCl2, 1 mm dithiothreitol, 0.02% (v/v) Triton X-100, in the presence of Complete™ inhibitors (Roche Molecular Biochemicals). After sonication, the lysate was cleared by centrifugation at 90,000 × g for 45 min at 4 °C and loaded onto an anti-FLAG M2-agarose column (Sigma). After washing, the adsorbed proteins were eluted with 3.5 m MgCl2 and buffer-exchanged on a PD-10 column (Amersham Biosciences) equilibrated in 50 mm Tris, pH 7.4, 0.2 m NaCl. For biochemical studies, the His6 tag, which induces protein precipitation, was removed by cleavage with the TEV protease as described in the technical information (Invitrogen). The rMPP1 and rMC1 proteins were then concentrated on Ultrafree-4 centrifugal filters (Millipore Corp.). The final fractions were aliquoted, frozen in liquid nitrogen, and stored at –80 °C. For gliding assays, the proteins were complemented with 1 mm ATP, 2 mm MgCl2, 0.1 mg/ml casein and frozen without concentration. Protein concentration was determined colorimetrically using bovine serum albumin as a standard and Bio-Rad protein assay. Anti-MPP1 Antibody Production and Purification—A polyclonal anti-MPP1 antibody was raised by Eurogentec using four injections of 100 μg of His6-rMPP1 proteins in a rabbit. The antibody was affinity-purified by three-step positive-negative affinity purification. The antiserum was filtered through His6-FLAG-unrelated protein and His6-rMC1 affinity columns in order to remove any antibodies reacting with tags and conserved motifs present in the MPP1 motor domain. Specific anti-MPP1 antibody, which recognizes epitopes present in the C2 to tail domains, was then purified by passage of the filtrate onto a His6-rMPP1 affinity column. Purified anti-MPP1 antibody was eluted with 50 mm Tris, 3.5 m MgCl2, pH 7.5, dialyzed overnight against PBS, and stored at 4 °C. Fluorescent Microtubule Spindown Assay—Purification of bovine brain tubulin (28Mitchison T. Kirschner M. Nature. 1984; 312: 232-237Crossref PubMed Scopus (571) Google Scholar), polymerization, and purification on a glycerol cushion of taxol-stabilized microtubules (MTs) were performed using standard procedures. MT concentration (i.e. concentration of tubulin dimer in MT polymer) was determined according to Desai et al. (29Desai A. Verma S. Mitchison T.J. Walczak C.E. Cell. 1999; 96: 69-78Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar). Recombinant MPP1 proteins, routinely 0.1 μm, were cleared by ultracentrifugation and mixed with taxol-MTs (1 μm) in 100 μl of BRB80 buffer supplemented with 10 μm taxol. After incubation for 15 min at 25 °C, reaction was fixed with 1 ml of fixative buffer (100 mm Mes, pH 6.75, 1 mm EGTA, 1 mm MgCl2 plus 50% sucrose and 1% glutaraldehyde). MTs were diluted and sedimented through a glycerol cushion onto coverslips as described in Ref. 30Pirollet F. Job D. Margolis R.L. Garel J.R. EMBO J. 1987; 6: 3247-3252Crossref PubMed Scopus (59) Google Scholar. Coverslips were postfixed in –20 °C methanol for 6 min and washed three times for 10 min in PBS containing 0.1% NaBH4 to prevent glutaraldehyde autofluorescence. MTs and recombinant proteins were stained with rabbit anti-Glu and Δ2-tubulin antibodies (1:1000; a generous gift of Dr. D. Job (31Paturle-Lafanechère L. Manier M. Trigault N. Pirollet F. Mazarguil H. Job D. J. Cell Sci. 1994; 107: 1529-1543Crossref PubMed Google Scholar)) and a mouse anti-FLAG M2 antibody (1:500; Sigma). Fluorescent labeling was performed with Alexa 488-labeled anti-rabbit (1:4000) and TRITC-labeled anti-mouse (1:1000) IgG antibodies from Molecular Probes, Inc. (Eugene, OR) and Jackson. Measurement of Steady State ATPase Rates—Steady-state MT-activated ATPase rates were measured using a pyruvate kinase/lactate dehydrogenase-linked assay, mainly as described in Ref. 32Huang T.G. Hackney D.D. J. Biol. Chem. 1994; 269: 16493-16501Abstract Full Text PDF PubMed Google Scholar. Briefly, ATPase activities were assayed at 30 °C in a 1-ml reaction volume of 100 mm K-Pipes, pH 6.8, 4 mm MgCl2, 1 mm EGTA, 1 mm phosphoenol-pyruvate, 0.3 mm NADH, 40 units of pyruvate kinase, and 55 units of lactate dehydrogenase. NADH oxidation was followed at 340 nm in a temperature-controlled UVIKON 923 spectrophotometer. Rates were determined during the linear phase after 5 min for attainment of steady state, using ϵ-NADH = 6220 m–1·cm–1. The kinetic parameters k cat, K MT (the concentration of MTs required for half-maximal activation), and K m for ATP were obtained by least squares fitting the MT activation or ATP dependent data to rectangular hyperbolae using Sigmaplot. Motility Assay with Polarity-marked Microtubules—A standard motility assay (33Kapoor T.M. Mitchison T.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9106-9111Crossref PubMed Scopus (53) Google Scholar) was performed with recombinant MPP1 proteins using the fluorescence-based kinesin motility kit from Cytoskeleton. Polarity-marked MTs with a bright seed and a dim elongated segment at their plus-end were prepared by inclusion of N-ethylmaleimide-treated tubulin, according to previously described protocols (see the "Methods" page at the kinesin Web site at www.proweb.org/kinesin). Briefly, acid-washed flow cells were coated with motor protein (typically 0.15 and 0.3 μm for rMPP1 and rMC1, respectively) in motility assay buffer (BRB80 buffer supplemented with 0.1 mg/ml casein, 1 mm ATP, 20 μm taxol, and an oxygen scavenging mix). After 5 min at room temperature, nonadsorbed motor was washed out with two flow cell volumes of motility assay buffer. Asymmetrically labeled MTs (0.2 μm) were then flowed through the cell and allowed to interact with the motor for 5 min at room temperature. Finally, unbound MTs were washed out with two flow cell volumes of motility assay buffer. Video images of MTs were acquired in a thermostated room with a Princeton CCD Micromax RTE 1317K1 camera on a Zeiss Axioscop with a 100 × 1.3 numerical aperture Plan-Neofluar lens using IPLab software (Ropper Scientific). Measurement of MT velocities was performed using the RETRAC program (available on the World Wide Web at mc11.mcri.ac.uk). Preparation of a GFP-fused Mutant of MPP1—The bp 70–981 portion of MPP1 cDNA was amplified by recombinant PCR using the TA-cloning kit (Invitrogen). This fragment was cloned into the XhoI site of pEGFP-C2 eukaryotic expression vector (Clontech), using an XhoI restriction site introduced upstream of the initiation codon. The pEGFP-sM plasmid obtained encodes the GFP protein fused to the N terminus region of MPP1 extending from aa 1 to 304. The C-terminal HindIII-KpnI fragment of pBS-MPP1 was subcloned into this plasmid to construct pEGFP-MPP1-FL, which encodes the full-length fusion protein GFP-MPP1. Cell Culture and Cloning—HeLa and HCT116 cells, a generous gift from Dr. R. L. Margolis, were grown in RPMI 1640 and McCoy medium (Invitrogen) supplemented with 10% fetal calf serum, respectively. HeLa cells were transfected with pEGFP-MPP1-FL plasmid using the FuGENE™ 6 transfection reagent as described by the manufacturer (Roche Molecular Biochemicals). Stable clones expressing the GFP-MPP1 fusion protein were then isolated, by using the limited dilution method in the presence of 500 μg/ml G418. In some instances, cells were analyzed by treatment with 250 nm trichostatin A overnight to increase expression (34Condreay J.P. Witherspoon S.M. Clay W.C. Kost T.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 127-132Crossref PubMed Scopus (368) Google Scholar). SiRNA Preparation and Transfection—MPP1-specific small interfering RNA (siRNA) duplexes were designed according to Harborth et al. (35Harborth J. Elbashir S.M. Bechert K. Tuschl T. Weber K. J. Cell Sci. 2001; 114: 4557-4565Crossref PubMed Google Scholar). Sequences of the type AA(N19)UU (where N represents any nucleotide) were searched in the open reading frame of MPP1-mRNA and submitted to a BLAST search to ensure their specificity. Selected 21-nucleotide sense and 21-nucleotide antisense oligonucleotides targeting MPP1 from positions 424–446 (siRNA1) or 4782–4804 (siRNA2) relative to the start codon were purchased from Dharmacon (Lafayette, CO) in deprotected and desalted form. As nonspecific siRNA controls, we used an unrelated sequence that failed to target p160ROCK mRNA (siRNAU) (36Chevrier V. Piel M. Collomb N. Saoudi Y. Frank R. Paintrand M. Narumiya S. Bornens M. Job D. J. Cell Biol. 2002; 157: 807-817Crossref PubMed Scopus (117) Google Scholar) or a siRNA1 sequence mutated on two nucleotides (siRNA1m). Annealing and transfection was performed as previously described (37Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8186) Google Scholar). HCT116 cells were transfected with siRNAs using Oligofectamine (Invitrogen). Mock transfections were also performed using control buffer instead of oligonucleotides. At different time points after transfection, cells were harvested and either fixed and processed for FACS analysis or analyzed by Western blot after the addition of SDS-PAGE sample buffer. Time-lapse imaging was also performed. Immunofluorescence Microscopy—Exponentially growing cells were plated on glass coverslips and incubated for 24–36 h. Cells were fixed in methanol at –20 °C for 8 min and processed with primary and secondary antibodies diluted in PBS with 1 mg/ml bovine serum albumin. The primary antibodies used were purified anti-MPP1 IgGs (5 μg/ml, this study), a mouse monoclonal anti-β-tubulin 2-3 B11 (1:5000; a generous gift of Drs. A. Giraudel and L. Lafanechère), 2A. Giraudel and L. Lafanechère, unpublished results. or mouse monoclonal anti-mitosin 14C10 (1 μg/ml, from GeneTex). Suitable Cy3-conjugated (Jackson; 1:1000) or Alexa 488-conjugated (Molecular Probes; 1:500) antibodies were applied as secondary antibodies. DNA was stained with Hoechst 33258 (1 μg/ml) or Topro 3 (1:1500; Molecular Probes). The coverslips were examined on a Zeiss microscope by using a 100 × 1.4 oil immersion objective. Confocal images were obtained on a TCS-SP2 Leica laser-scanning microscope. Z series were collected, and displayed images correspond to projections of optical sections (0.2 μm thick), the number of which varied in relation to the cell depth. Flow Cytometric Analysis—For standard analysis of DNA content, cells were washed once with PBS, trypsinized, fixed with 4% paraformaldehyde in PBS for 10 min, and permeabilized with 0.2% Triton X-100 in PBS. DNA was stained overnight at 4 °C with 2 μg/ml Hoechst. Cells were sorted on a FACS Star Plus cytometer (BD Biosciences). After collection of 20,000 events, results were analyzed with CellQuest software, and aggregated cells were gated out. For double staining of DNA and specific antigen, cells were fixed with ice-cold 70% ethanol. Labeling of MPP1 or mitosin was performed before the DNA counterstaining step by incubation with anti-MPP1 or anti-mitosin antibodies followed by Alexa 488-conjugated anti-rabbit or anti-mouse IgGs (1:500; Molecular Probes). Time Lapse Imaging—For phase-contrast imaging, control or MPP1 siRNA-transfected cells were trypsinized 6 h after transfection and transferred into a multiple well chamber of a polydimethyl siloxane gel fixed on a glass chamber coated with collagen and fibronectin. Sequential phase-contrast images of the various samples were recorded on a Leica DMIRBE microscope controlled by Metamorph software (Universal Imaging) for 66 h. The microscope was equipped with an open chamber equilibrated in 5% CO2 and maintained at 37 °C, and images were taken with a × 20 objective using a cooled MicroMax 1-MHz CCD camera (Roper Scientific). For GFP-MPP1 imaging during mitosis, stably transfected cells were plated on coated coverslips and maintained at 37 °C in sealed chambers containing complete phenol red-free RPMI medium supplemented with 20 mm Hepes. Rounded cells were searched and time lapse Z-sequences were collected as described by Savino et al. (38Savino T.M. Gebrane-Younes J. De Mey J. Sibarita J.B. HernandezVerdun D. J. Cell Biol. 2001; 153: 1097-1110Crossref PubMed Scopus (146) Google Scholar) on a Leica DMIRBE microscope controlled by Metamorph software (Universal Imaging). This microscope was equipped with a piezoelectric device for rapid and reproducible focal changes, a 100 × 1.4 numerical aperture Plan Apo lens, a cooled CCD camera (Micromax, 5 MHz; Roper Scientific), and a DG4 illumination device. Z-stacks were deconvoluted and maximal intensity projections were constructed. Identification of MPP1 as a Kinesin-related Protein Present in Several Human Tissues—The entire human MPP1 cDNA (6325 bp) was cloned using two rounds of conventional cDNA library screening, starting with the previously obtained partial clone 6-1 (24Westendorf J.M. Rao P.N. Gerace L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 714-718Crossref PubMed Scopus (239) Google Scholar). Sequence comparison in data bases showed that MPP1 belongs to the kinesin superfamily of motor proteins with the characteristic organization into three domains (15Hirokawa N. Noda Y. Okada Y. Curr. Opin. Cell Biol. 1998; 10: 60-73Crossref PubMed Scopus (275) Google Scholar), as detailed in Fig 1A. A search of genome resources indicated that a unique human MPP1 gene located on chromosome 10 in the 10q23.31 region spreads at least 73 kb and consists of 33 exons. A mouse ortholog (82% similarity) encoded by a conserved syntheny was found on murine chromosome 19. Alignment of the conserved motor domains of MPP1 and conventional kinesin heavy chain, KHC (Fig. 1B) showed that the MPP1 motor exhibits two large insertions (186–263 and 480–507), which span between α-helix 2 and β-sheet 4 and α-helix 6 and β-sheet 9, respectively, when compared with KHC structural data (39Kozielski F. Sack S. Marx A. Thormahlen M. Schonbrunn E. Biou V. Thompson A. Mandelkow E.M. Mandelkow E. Cell. 1997; 91: 985-994Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, 40Kull F.J. Sablin E.P. Lau R. Fletterick R.J. Vale R.D. Nature. 1996; 380: 550-555Crossref PubMed Scopus (581) Google Scholar). For immunoblot analysis of MPP1 distribution, we used an affinity-purified MPP1 antibody directed against the C2 to tail domains (aa 651–1780) of MPP1 (Fig. 2). This antibody reacted with a single 200-kDa band in HeLa cell extracts (Fig. 2B, lane 3), which co-migrates with purified recombinant full-length MPP1 (Fig. 2, A and B, lane 1). MPP1 was detected in several human tissues, including brain, ovary, and kidney (Fig. 2B). In the testis extract, a strong signal corresponding to a slightly lower molecular weight band was detected and may correspond to a testis-specific splicing variant of MPP1. Recombinant MPP1 Behaves as a Genuine Molecular Motor—To assay MPP1 motor activity, we used recombinant proteins corresponding either to the complete MPP1 (rMPP1) or to a deletion mutant of the protein containing the putative motor domain and the first α-helical domain (rMC1) (Fig. 2A). The proteins were assayed for the characteristic activities of genuine KRP (i.e. regulated ATPase activity, binding to MTs, and ability to induce microtubule gliding on motor-coated coverslips) (15Hirokawa N. Noda Y. Okada Y. Curr. Opin. Cell Biol. 1998; 10: 60-73Crossref PubMed Scopus (275) Google Scholar, 41Vale R.D. Fletterick R.J. Annu. Rev. Cell Dev. Biol. 1997; 13: 745-777Crossref PubMed Scopus (402) Google Scholar) (Fig. 3). Both rMPP1 and rMC1 exhibited a basal ATPase activity, which was activated by the addition of MTs (∼280- or 630-fold for rMPP1 and rMC1, respectively; Fig. 3A). The k cat and K m(ATP) values of rMPP1 and rMC1 were close to each other, and the MT concentration required for half-maximal activation was ∼6-fold higher for rMC1 mutant than for rMPP1. The microtubule binding activity of rMPP1 and rMC1 could not be assayed by conventional MT pelleting assays (42Lockhart A. Crevel I.M. Cross R.A. J. Mol. Biol. 1995; 249: 763-771Crossref PubMed Scopus (67) Google Scholar) due to partial insolubility of the unbound proteins. For visualization of microtubule binding, control microtubules or microtubules incubated with rMPP1 or with rMC1 were centrifuged on coverslips and subsequently double-stained with tubulin and FLAG antibodies (Fig. 3B). In control samples, short individual MTs were observed. Incubation with rMPP1 or rMC1 prior to centrifugation induced extensive MT cross-linking. Recombinant proteins were associated with microtubule bundles, while being undetectable on single polymers. When ATP was added in the incubation medium, microtubule bundling was inhibited in a dose-dependent way, and many individual MTs could be observed on the coverslip (Fig. 3B and data not shown). These results indicate that both rMPP1 and rMC1 bind to MTs in an ATP-dependent way and induce microtubule bundling in vitro. To test the force-producing capability of MPP1, we used a multiple-motor assay using polarity-marked MTs. Protein rMC1 induced MT motility with the minus-end leading, and most of the MTs (>90%) were seen gliding (Fig. 3C). MT gliding was also observed with rMPP1, but, curiously, only a subset of relatively short microtubules (1–5 μm in length) was seen moving (data not shown). In both cases, gliding was abolished in the presence of 1 mm AMP-PNP (data not shown). The average velocity of microtubule gliding was 0.07 ± 0.01 μm/s and 0.071 ± 0.007 μm/s for rMPP1 and rMC1, respectively. These data demonstrate that MPP1 is a slow plus-end-directed KRP, when compared with already described motors (43Woehlke G. Schliwa M. Nat. Rev. Mol. Cell. Biol. 2000; 1: 50-58Crossref PubMed Scopus (119) Google Scholar). MPP1 Distribution during the Cell Cycle—MPP1 expression and localization during the cell cycle
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