protein required

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 microm/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.


Introduction
Eukaryotic cells exhibit dramatic changes of microtubule organization and dynamics as they enter mitosis (2,3). These changes are timely and spatially coordinated with nucleus and membranes alterations by the tight control of M-phase promoting factor (MPF) 1 , whose catalytic component, the p34 cdc2 or cdk1 kinase becomes activated at the G2-M transition (4,5). Extensive progress has been made to describe the complex circuitry of phosphatases and kinases, which regulates cdk1 activation (6,7) and the downstream molecular pathways (8,9).
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 (10)(11)(12). Several Microtubule-Associated Proteins (MAPs) 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), 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 insure centrosome separation, spindle formation and maintenance, chromosome congression and cytokinesis completion (15)(16)(17)(18)(19).
However, whereas it is well established that the p34 cdc2 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 p34 cdc2 kinase phosphorylates the ubiquitous MAP4 during M-phase (21) and this phosphorylation abolishes MAP4 microtubule stabilizing activity (22). There is evidence that the phosphorylation of the microtubule destabilizing protein Stathmin/Op18 by p34 cdc2 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).

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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 to 50 proteins that become phosphorylated at the G2-M transition (1,(24)(25)(26). 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.

FLAG epitope tagging of MPP1 by mutagenesis
The MPP1 coding sequence starting at base 70 was tagged using mutagenesis technique based on M13-phage ssDNA protocol (Amersham Sculptor Kit). 1C12 ssDNA was obtained using standard procedures (27). A 66-mer oligonucleotide was designed that introduced between bases 69-70, 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.

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Orientation of the mutated Nterminal 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 insects cells
A recombinant full-length MPP1 (rMPP1) and a truncated form (rMC1), both tagged with a N-terminal FLAG epitope, were produced by baculovirus expression following the manufacturer's instructions of the Bac-to-Bac system (Life Technologies). 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 6His 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 MOI=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 MgCl 2 , 5 mM CaCl 2, 1 mM DTT, 0.02%(v/v) Triton X-100, in presence of Complete TM inhibitors (Boehringer). 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 (Pharmacia) equilibrated in 50 mM Tris, pH=7.4, 0.2M NaCl. For biochemical studies, the 6His tag, which induces protein precipitation, was removed by cleavage with the TEV protease as described in the technical information (Life Technologies). The rMPP1 and rMC1 proteins were then concentrated on Ultrafree-4 centrifugal filters (Millipore). 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 MgCl 2 , 0.1mg/ml casein and frozen inserm-00353257, version 1 -21 Jan 2009 without concentration. Protein concentration was determined colorimetrically using BSA as a standard and Bio-Rad Protein Assay (Bio-Rad).

Anti-MPP1 antibody production and purification
A polyclonal anti-MPP1 antibody was raised by Eurogentec using four injections of 100 µg of 6His-rMPP1 proteins in rabbit. The antibody was affinity-purified by three-step positive-negative affinity purification. The antiserum was filtered through 6His-FLAGunrelated protein and 6His-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 6His-rMPP1-affinity column. Purified anti-MPP1 antibody was eluted with Tris 50 mM, MgCl 2 3.5 M, pH=7.5, dialyzed overnight against PBS and stored at 4°C.

Fluorescent microtubule spindown assay
Purification of bovine brain tubulin (28), polymerisation and purification on glycerol cushion of taxol-stabilized microtubules (MTs) were performed using standard procedures.

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 (32). Briefly, ATPase activities were assayed at 30°C in 1 ml reaction volume of 100 mM K-Pipes, pH=6.8, 4 mM MgCl 2 , 1 mM EGTA, 1 mM PEP, 0.3 mM NADH, 40 U of pyruvate kinase and 55 U 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 1/2 MT (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. from Dharmacon (Lafayette, CO) in deprotected and desalted form. As unspecific 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 analyzis or analyzed by Western blot after 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 hours. Cells were fixed in methanol at -20°C for 8 min and processed with primary and secondary antibodies diluted in PBS with 1mg/ml bovine serum albumin. The primary antibodies used were purified anti-MPP1 IgGs (5 µg/ml, this study), a mouse monoclonal anti ß-tubulin 2-3 B11 (1/5,000, a generous gift of Drs A. Giraudel and L. Lafanechère, unpublished results) or mouse monoclonal anti-mitosin 14C10 (1 µg/ml, from GeneTex).

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Suitable Cy3-(Jackson, 1/1,000) or Alexa 488-(Molecular Probes, 1/500) conjugated antibodies were applied as secondary antibodies. DNA was stained with Hoechst 33258 (1 µg/ml) or Topro 3 (1/1,500, 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), which number 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 of Hoechst. Cells were sorted on a FACS Star Plus cytometer (Beckton and Dickinson Co). 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.
Labelling 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 antirabbit or anti-mouse IgGs (1/500, Molecular Probes).

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 (24). Sequence comparison in databases showed that MPP1 belongs to the kinesin superfamily of motor proteins with the characteristic organization into three domains (15), 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 MPP1 motor exhibits two large insertions (186-263, 480-507), which span respectively between alpha helix 2 and β sheet 4 and alpha helix 6 and β sheet 9 when compared to KHC structural data (39,40).
For immunoblot analysis of MPP1 distribution, we used an affinity-purified MPP1 antibody directed against the C2 to tail domains (aa 651 to 1780) of MPP1 (Fig 2). This antibody reacted with a single 200 kDa band in HeLa cell extracts (Fig 2B, lane 3), which comigrates with purified recombinant full-length MPP1 (Fig 2A and 2B, 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 MW 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 inserm-00353257, version 1 -21 Jan 2009 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 (15,41) (Fig 3). Both rMPP1 and rMC1 exhibited a basal ATPase activity, which was activated by addition of MTs (~ 280 or 630-fold for rMPP1 and rMC1, respectively; Fig 3A). The k cat and KmATP values of rMPP1 and rMC1 were close to each other and the MT concentration required for halfmaximal 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 (42)  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 inserm-00353257, version 1 -21 Jan 2009 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 to already described motors (43).

MPP1 distribution during the cell cycle
MPP1 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-labelling, indicating enhanced MPP1 expression in G2. Accordingly, FACS analysis indicated a two to three fold increase of MPP1 expression as cells progress from G1 to G2/M (Fig 4B). During mitosis, in both prophase and metaphase cells, MPP1 staining showed fine punctuations diffuse throughout the cytoplasm (Fig 4C). At anaphase, MPP1 staining accumulated at the mid-plan 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).
We further used GFP-tagged MPP1 to visualize the dynamics of MPP1re-distribution during the cell cycle (Fig 5). We observed extensive cell death during establishment of the then as two dots, four dots and again as two spots. MPP1 concentration at the midbody then decreases asymetricaly, with a spot staying visible in only one of the daughter cell until abscission occurs (Fig 5B). This behavior suggests that the plus-end directed motor activity of MPP1 may play a role in MTs organization during cytokinesis exit.

Knockdown of MPP1 induces apoptosis and cytokinesis defects
We used small interfering RNA (siRNA) duplexes (37) to assess the consequences of MPP1 suppression (Fig 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 Figures 6 and 7. Immunoblot analysis of cell extracts showed extensive depletion of MPP1 24 hours after transfection (Fig 6A). FACS 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 hours following transfection with specific MPP1 oligoduplexes (Fig 6B). We then used phasecontrast videomicroscopy to examine the behavior of the cells over 36h duration, starting 18 hours 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 7A and B). Although a midbody formed, abscission did not occur. Either the midbody regressed with 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 behaviour was observed in 42 % of siRNA treated cells whereas less than 10% 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 for further cell separation.

Discussion
In this study, we find that MPP1, a member of a set of mitotic phosphoproteins specifically recognized by the MPM2 antibody at the G2/M transition (1), is a slow plus-end directed KRP that plays critical roles in cytokinesis.
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.  residues are in bold and the positions of secondary structure elements are indicated according to KHC crystallographic data (39,40). The four conserved motifs, which interact with ATP, are drawn as boxes N1-4 as defined by Sablin et al (78). The open triangle indicates the limit between the KHC motor core domain and its neck region, which consists of β9 and β10 strands and a weak coiled-coil region extending at aa 352 (79). The heptad repeat positions predicted by Coils are underlined. mutant indicates that we selected antibodies, which recognize epitopes present in the C2 to tail domains, the most specific portion of the protein when compared to other KLPs.     blebbing, with non-uniform membranes they were scored as apoptotic. Most of the cells become rounded and a well formed midbody between the two daughter cells appeared indicating that they were undergoing mitosis. When two viable daughter cells were formed, the cells were scored as going through successful mitosis. However, various defects in mitotic exit, daughter cells separation and further appearance of apoptosis were sometimes observed and the cells were then scored as undergoing failure of mitosis. In other cases, either mitosis or apoptosis were observed and we hypothesize that cells may be blocked.     18h00 50h40 49h00 54h20