MKlp2 is required for normal cell division. (A) HeLa S3 cells were treated with the MKlp2 siRNA duplex for 36 h and the lamin A siRNA duplex for 72 h, arrested with 90 ng/ml nocodazole for 12 h to enrich for mitotic cells that express MKlp2, and then lysed in sample buffer. Western blot analysis of 25 μg protein was performed with antibodies to MKlp2, lamin A, MKlp1, Plk1, and α-tubulin. (B) The number of binucleated or multinucleated cells as a percentage of the total cell population was quantitated in HeLa S3 cells treated with lamin A or MKlp2 siRNA duplexes for the times indicated (n = 400 cells). (C) Live-cell imaging of HeLa S3 cells treated with control lamin A or (D) MKlp2 siRNA duplexes for 24 h. Representative frames reflecting the different stages of mitosis in these cells are shown, and times are indicated in h:min. Arrows indicate the position of the cleavage furrow; note the partial and asymmetric furrow ingression in MKlp2-depleted cells. Bar, 10 μM.

Microtubules regulate the phosphorylation of MKlp2 by Plk1. (A) Microtubule-binding assays were performed with 5 pmol Plk1, MKlp2, and survivin. Samples were then analyzed by Western blotting and detection with antibodies to the hexahistidine tag, and on Coomassie brilliant blue–stained SDS-PAGE 10% minigels (right). (B) Microtubule-binding assays were performed with 5 pmol GST-tagged Plk1 amino acids 1–305 and 305–603. Samples were then analyzed by Western blotting and detection with antibodies to GST. (C) Constructs corresponding to the full-length myc-tagged human Plk1, amino acids 1–305, and 305–603 were expressed in HeLa S3 cells for 24 h using transient transfection. Cells were fixed with methanol and stained with antibodies to the myc-epitope and α-tubulin. The localization of the exogenously expressed Plk1 in telophase cells is shown; arrows mark the position of the microtubule bridge, central spindle structure. Bar, 10 μM. (D) Phosphorylation assays were performed with 1 pmol purified histone H1, PRC1, MKlp1, and MKlp2 using 50 ng recombinant cdk1–cyclin B kinase, and with 1 pmol purified casein, PRC1, MKlp1, and MKlp2 using 50 ng recombinant Plk1. Autoradiographs of samples separated by SDS-PAGE on 12% minigels are shown. The asterisk marks phosphorylated cyclin B. (E) Phosphorylation assays were performed with 1 pmol purified casein and MKlp2 using 10 ng recombinant Plk1 in the presence and absence of microtubules. Autoradiographs of samples separated by SDS-PAGE on 12% minigels are shown. The double asterisk marks a nonstoichiometric phosphorylation of tubulin by Plk1.

Plk1 regulates the microtubule-bundling activity of MKlp2. Microtubules labeled with fluorescein were incubated with buffer alone, MKlp2, or MKlp2^S528A in the presence and absence of 1 mM ATP, Plk1, MKlp2 or MKlp2^S528A treated with Plk1 in the presence of 1 mM ATP, and PRC1 treated with 1 mM ATP or Plk1 and 1 mM ATP. Bundling was observed in the presence of MKlp2 and PRC1, but not Plk1 or with microtubules alone. Exposure time was 500 msec for all images, and representative images from 14 independent experiments are shown. Bar, 10 μM.

A phosphorylation site mutant of MKlp2 fails to rescue targeting of Plk1 to the central spindle in MKlp2-depleted cells. HeLa S3 cells were treated with the MKlp2 siRNA duplex for 18 h and were then transfected with siRNA-resistant GFP-tagged MKlp2 or a phosphorylation site mutant for 12 h. Cells were fixed with methanol and then stained with antibodies to Plk1, GFP was directly visualized, and DNA was stained with DAPI. Bar, 10 μM.

Plk1 and MKlp2 form a microtubule-sensing device at the central spindle. Early in anaphase as the chromosomes (blue) are partitioned, MKlp2 is recruited to the forming microtubule bundles that will give rise to the central spindle. Plk1, although able to weakly associate with microtubules (green), is not present on all microtubule structures, and therefore must have additional specificity factors in order to localize properly; MKlp2 appears to be one such factor. Plk1 associates with microtubules and phosphorylates MKlp2, to which it can then bind and thus maintain a stable association with the central spindle. We propose that this MKlp2-associated and thus spatially restricted pool of Plk1 can then phosphorylate other targets at the central spindle and cleavage furrow to control cleavage furrow ingression and cytokinesis. Red arrows indicate phosphorylation events.

Loss of Plk1 from the central spindle in MKlp2-depleted cells. (A) HeLa cells fixed with PFA were stained with antibodies to Plk1 (red) and MKlp2 (green), and DNA was stained with DAPI (blue). Colocalization of Plk1 and MKlp2 was observed from early anaphase until cytokinesis, with the difference that MKlp2 was absent from kinetochores, unlike Plk1 (see the contrast-enhanced boxed area). (B–D) HeLa S3 cells treated with control lamin A and MKlp2 siRNA duplexes were fixed with methanol and then stained with antibodies to MKlp2, MKlp1, α-tubulin, Plk1, and anillin. Bar, 10 μM.

Analysis of the sites on human MKlp2 phosphorylated in vivo and in vitro by Plk1. (A) MKlp2, MKlp2 treated with Plk1 in vitro, and MKlp2 immune precipitated from 2.5 mg extract prepared from asynchronous cells (Int) and cells arrested with nocodazole and released for 2 h (Mit) were digested with trypsin and then analyzed by mass spectrometry. Note that as expected, MKlp2 is highly enriched in the mitotic cells. A region of the spectra showing phosphorylation of a peptide from 526 to 537 in Plk1-treated and in vivo MKlp2 is shown. Fragmentation of this peptide showed that the serine at 528 is phosphorylated by Plk1 and in vivo. (B) MKlp2 and MKlp2^S528A (1 μg) were treated with the indicated amounts of Plk1 for 1 h at 30°C, and were then analyzed by gel electrophoresis and autoradiography. (C) Microtubule-binding assays were performed with 5 pmol MKlp2 and MKlp2^5528A treated with buffer or Plk1. Samples were then analyzed by Western blotting and detection with antibodies to the hexahistidine tag.

The Plk1 polo box domain binds to phosphorylated MKlp2. (A) HeLa S3 cells grown in suspension were arrested with 90 ng/ml nocodazole for 20 h, and then released for 2 h. Immune precipitations with affinity-purified antibodies to MKlp2, Plk1, and the nonspecific control GFP were performed from 100-μg soluble extracts prepared from these cells. Western blot analysis was performed on 20 μg cell extract, and one fifth of the immune precipitated material. (B) Ligand blots were performed with Plk1 amino acids 305–603 as the probe, and Plk1, cdk1/cyclin B, PRC1, MKlp2, treated with Plk1 or cdk1/cyclin B as targets. (C) Ligand blots were performed with Plk1 amino acids 305–603 as the probe, and MKlp2 or MKlp2^S528A treated with Plk1 as targets. The asterisks mark a truncation product in the MKlp2 preparations.

Antibodies to the neck and tail region of MKlp2 prevent phosphorylation by Plk1 and cytokinesis. (A) HeLa cells were injected with affinity-purified antibodies to the MKlp2 neck region and control affinity-purified antibodies to GFP. Cells were fixed with methanol 24 h after injection and then stained with a CY2-conjugated secondary antibody to detect the injected antibody (green) and with DAPI for the DNA (blue). The number of binucleated cells was counted and is expressed as a percentage of total injected cells detected (n = 3). Bar, 10 μM. (B) 1 μg MKlp2 preincubated with buffer, affinity-purified antibodies to MKlp2 or GFP was treated for 1 h at 30°C with 125 ng Plk1, itself preincubated with affinity-purified antibodies to Plk1 or GFP, then analyzed by gel electrophoresis and autoradiography. (C) 1 μg MKlp2 preincubated with buffer, affinity-purified antibodies to MKlp2 or GFP was treated with 250 ng Plk1, and then was used as a target in a ligand blot with Plk1 amino acids 305–603 as the probe. Two strips containing MKlp2 phosphorylated by Plk1 were incubated with 2 μg affinity-purified antibodies to MKlp2 or GFP before probing with the Plk1 polo box domain. (D) Microtubule-binding assays were performed with 5 pmol MKlp2 and MKlp2 preincubated on ice for 60 min with 1 μg affinity-purified antibodies to MKlp2 or GFP. Samples were then analyzed by Western blotting and detection with antibodies to the hexahistidine tag. (E) Microtubules labeled with fluorescein were incubated with buffer alone, MKlp2, or MKlp2 preincubated for 60 min on ice with 1 μg affinity-purified antibodies to MKlp2 or GFP. Exposure time was 1,000 msec for all images, and representative images from five independent experiments are shown. Samples were renumbered and scored for bundling activity by two different people. Bar, 10 μM.