From the
INSERM U366, Département
Réponse et Dynamique Cellulaires, Laboratoire du Cytosquelette,
Commissariat à l'Energie Atomique-Grenoble, 17 rue des Martyrs, 38 054
Grenoble Cedex 9, France, **Institut Curie, Section
Recherche, UMR 144 du CNRS, 75 248 Paris Cedex 05, France,
Marie Curie Research Institute, Oxted
RH8 OTL, United Kingdom, and
Institut
Curie, Section Recherche, UMR 168 du CNRS, 75 248 Paris Cedex 05, France
Received for publication, April 30, 2003
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ABSTRACT |
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INTRODUCTION |
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Microtubule dynamics are an intrinsic property of the polymer of tubulin and are highly regulated by the balance of the activity of different factors throughout the cell cycle (1012). Several microtubule-associated proteins have been described to promote tubulin assembly and polymer stabilization or destabilization (13, 14). Besides their roles in intracellular trafficking of organelles and vesicles during interphase, dyneins and kinesin-related proteins (KRPs),1 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 (1519).
However, whereas it is well established that the p34cdc2 kinase is centrally involved in the regulation of microtubule dynamics during mitosis (20), only a few Cdc2 substrates with plausible involvement in the control of microtubule dynamics have been identified so far. The p34cdc2 kinase phosphorylates the ubiquitous microtubule-associated protein 4 during M phase (21), and this phosphorylation abolishes microtubule-associated protein 4 microtubule stabilizing activity (22). There is evidence that the phosphorylation of the microtubule destabilizing protein Stathmin/Op18 by p34cdc2 is important for mitotic progression (23). Similar phosphorylation of the mitotic KRP Eg5 is required for Eg5-dependent centrosome migration and bipolar spindle formation in vivo (17). 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 4050 proteins that become phosphorylated at the G2/M transition (1, 2426). 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.
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EXPERIMENTAL PROCEDURES |
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FLAG Epitope Tagging of MPP1 by MutagenesisThe 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 [PDB] single-stranded DNA was obtained using standard procedures (27). 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 [PDB] 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 CellsA 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 CompleteTM inhibitors (Roche Molecular Biochemicals). After sonication, the lysate was cleared by centrifugation at 90,000 x 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 PurificationA 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 AssayPurification of
bovine brain tubulin (28),
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.
(29). 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.
30. 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 2-tubulin antibodies (1:1000; a generous gift of Dr. D.
Job (31)) 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 RatesSteady-state
MT-activated ATPase rates were measured using a pyruvate kinase/lactate
dehydrogenase-linked assay, mainly as described in Ref.
32. 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 -NADH = 6220
M1·cm1. The
kinetic parameters kcat, KMT (the
concentration of MTs required for half-maximal activation), and
Km 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 MicrotubulesA standard motility assay (33) 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 x 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 MPP1The bp 70981 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 CloningHeLa 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 FuGENETM 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 (34).
SiRNA Preparation and TransfectionMPP1-specific small interfering RNA (siRNA) duplexes were designed according to Harborth et al. (35). 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 424446 (siRNA1) or 47824804 (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) (36) or a siRNA1 sequence mutated on two nucleotides (siRNA1m). Annealing and transfection was performed as previously described (37). 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 MicroscopyExponentially growing cells
were plated on glass coverslips and incubated for 2436 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--tubulin 2-3 B11 (1:5000; a generous gift of
Drs. A. Giraudel and L.
Lafanechère),2
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 x 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 AnalysisFor 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 ImagingFor 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 x 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. (38) 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 x 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.
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RESULTS |
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For immunoblot analysis of MPP1 distribution, we used an affinity-purified MPP1 antibody directed against the C2 to tail domains (aa 6511780) 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.
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Recombinant MPP1 Behaves as a Genuine Molecular MotorTo
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 -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)
(15,
41)
(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
kcat and Km(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.
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The microtubule binding activity of rMPP1 and rMC1 could not be assayed by conventional MT pelleting assays (42) 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 (15 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 (43).
MPP1 Distribution during the Cell CycleMPP1 expression and localization during the cell cycle was investigated by immunofluorescence analysis of fixed HeLa cells (Fig. 4). In interphase cells double stained with MPP1/tubulin antibodies, MPP1 was mainly localized in the nucleus. MPP1 was also detected in the cytoplasm as a punctuated pattern without clear association with microtubules (Fig. 4A). The nuclear staining varied from cell to cell, suggesting a cell cycle-dependent expression of the protein MPP1. This possibility was tested using indirect immunofluorescence and FACS analysis of HeLa cells double stained for MPP1 and mitosin, a centromere-associated protein whose expression is strongly enhanced in G2 cells (44) (Fig. 4B). The strongest MPP1 staining was observed in cells with bright mitosin labeling, indicating enhanced MPP1 expression in G2. Accordingly, FACS analysis indicated a 23-fold increase of MPP1 expression as cells progressed from G1 to G2/M (Fig. 4B). During mitosis, in both prophase and metaphase cells, MPP1 staining showed fine punctations diffuse throughout the cytoplasm (Fig. 4C). At anaphase, MPP1 staining accumulated at the midplan of the cell and formed a distinct band extending across the spindle midzone (Fig. 4C). In telophase cells, MPP1 was sharply concentrated in the midbody (Fig. 4C).
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We further used GFP-tagged MPP1 to visualize the dynamics of MPP1 redistribution during the cell cycle (Fig. 5). We observed extensive cell death during establishment of the cell line, suggesting a toxic effect of MPP1 overexpression. In cells with a GFP-MPP1 level apparently similar to that of the endogenous protein (Fig. 5A), the pattern of expression was similar to that of the endogenous protein (data not shown). During cytokinesis, time lapse imaging of GFP-tagged MPP1 showed MPP1 in the midbody first as a single spot and then as two dots, four dots and again as two spots. MPP1 concentration at the midbody then decreases asymmetrically, with a spot staying visible in only one of the daughter cells until abscission occurs (Fig. 5B). This behavior suggests that the plus-end-directed motor activity of MPP1 may play a role in MT organization during cytokinesis exit.
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Knockdown of MPP1 Induces Apoptosis and Cytokinesis DefectsWe used siRNA duplexes (37) to assess the consequences of MPP1 suppression (Figs. 6 and 7). Human HCT116 epithelial cells were transfected with two independent MPP1-specific siRNAs. Results were similar with both siRNAs and are shown in the case of siRNA1 in Figs. 6 and 7. Immunoblot analysis of cell extracts showed extensive depletion of MPP1 24 h after transfection (Fig. 6A). Fluorescence-activated cell sorting analysis showed no clear modification at this time point, but after 48h, a hypodiploid peak appeared, indicating accumulation of apoptotic cells. Cell apoptosis was further enhanced at the point 72 h following transfection with specific MPP1 oligoduplexes (Fig. 6B). We then used phase-contrast videomicroscopy to examine the behavior of the cells over 36-h duration, starting 18 h after transfection (Fig. 7). Over this period of time, most cells, which have lost MPP1 (Fig. 7C), underwent at least one M phase (Fig. 7A). In MPP1-siRNA1-treated cells, a high proportion of cytokinesis failure was observed (Fig. 7, A and B). Although a midbody formed, abscission did not occur. Either the midbody regressed with the appearance of a binucleated cell, which further underwent apoptosis during a second round of mitosis, or the midbody persisted, and the two ill separated daughter cells finally underwent apoptosis. Such behavior was observed in 42% of siRNA-treated cells, whereas less than 10% of control cells showed similar cytokinesis defects.
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These data indicate that MPP1 is important for cell growth and completion of cytokinesis. Whereas it does not appear to be necessary for initiation of furrowing and cleavage furrow ingression, it seems to play an important function in further cell separation.
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DISCUSSION |
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As other KRPs, MPP1 exhibits MT-activated ATPase activity and binds to MTs (43). Additionally, MPP1 apparently bundles microtubules in vitro and behaves as a slow molecular motor in gliding assays. Phylogenic analysis of KRP motor domains (45, 46)3 shows that MPP1 belongs to the N6 class of KRPs, which includes RB6K (18, 47, 48) and orthologs of MKLP1 (4952). As these KRPs, MPP1 displays an insertion in the N1-N2 sequence landmark of the motor domain when compared with conventional KHC (53). MPP1 insertion (77 aa) is the longest, and this determines a N1-N2 spacing of 190 aa compared with less than 132 in other KRP classes (53). MPP1 also displays an insertion (28 aa) at the end of the core motor domain, suggesting an extended neck linker region in the protein. Both MKLP1 and RB6K display similar insertions. Interestingly, motor domain and neck linker insertions concern loops (L6 and neck linker loop, respectively) whose interaction is important for motility behavior (54), and this suggests concerted evolution of the two loop domains. As MPP1, MKLP1 cross-bridges microtubules in vitro and induces a slow (0.066 µm/s) plus-end movement of the tubulin polymers in gliding assays (50). These common functional features may be related to the structural characteristics shared by the two proteins.
MPP1 has a specific pattern of localization and expression during the cell cycle, being mostly nuclear in interphase cells with a sharp increase in expression in G2, and diffuse in metaphase cells, with subsequent association to the central spindle and the midbody at the end of mitosis. Motors with mitotic functions show similar cell cycle-regulated localization on mitotic structures and subsequent concentration in the midbody during cytokinesis (18, 48, 50, 5557). The midbody localization of MPP1 was not evident in previous works (46, 58), but it seems to be a consistent property of MPP1 in different localization assays (immunofluorescence, GFP-MPP1 distribution in vivo) and different cell lines: HeLa, HCT116 (this work), and human umbilical vein endothelial cells (data not shown). This new localization of MPP1 seems important in light of our siRNA studies indicating a vital role of the protein in the correct completion of cytokinesis.
Cytokinesis depends on the presence of a number of proteins (5961) including the other N6-class KRPs, RB6K and MKLP1 orthologs (18, 19, 48, 49, 51). Given their properties in vitro, these KRPs may be involved in both midzone microtubule bundling and subsequent microtubule sliding, which are required for successful completion of cell cleavage (62). It is remarkable that MPP1, RB6K, and MKLP1 are all independently required for cytokinesis, despite apparent functional and structural redundancy. Perhaps the rational for this multiplicity of motors will be revealed by detailed studies of molecular interactions between these motors themselves and between these motors and other major components of the midbody such as INCENP (63), survivin (64, 65), TD60 (66), and PRC1 (67). The three motors may also be included in different signaling pathways, involving different GTPases (6871) and kinases (7274).
In conclusion, our data show that MPP1 is a novel KRP whose activity is required for proper progression of cytokinesis in human cells. Further work is needed to see if this newly discovered function of MPP1 is related to its mitotic hyperphosphorylation.
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FOOTNOTES |
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Recipient of French Research Ministry and Fondation pour la Recherche
Médicale fellowships.
¶ These two authors contributed equally to this work.
|| Supported by fellowships from Association pour la Recherche contre le
Cancer and Fondation pour la Recherche Médicale.
¶¶ To whom correspondence should be addressed. Tel. 33-4-38-78-54-82; Fax: 33-4-38-78-50-57; E-mail: fpirollet{at}cea.fr.
1 The 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-,
-imidodiphosphate.
2 A. Giraudel and L. Lafanechère, unpublished results.
3 F. Pirollet, unpublished results.
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ACKNOWLEDGMENTS |
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REFERENCES |
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