Mitosis in the syncytial blastoderm of early Drosophila embryos. Confocal immunofluorescence images of embryos immunostained with an antibody raised against Drosophila embryonic tubulin (yellow) overlaid with the corresponding DNA stain DAPI (blue). (A) Prophase. The chromosomes have begun to condense and centrosomes have separated significantly toward opposite sides of the nucleus. (A, inset) Cross sectional view of a prophase nucleus showing only tubulin immunofluorescence. (B) Metaphase. MTs have entered the nuclear region, and chromosomes have aligned along the metaphase plate. (C) Anaphase A. The sister chromatids have separated to opposite spindle poles but the spindles have not elongated. (D) Anaphase B. The spindles have elongated to an average length of 17.5 μm. Note that the central spindle is no longer fusiform but straight-edged. Bar, 7.6 μm.

The MTs within interpolar MT bundles are parallel near the poles and antiparallel in the midzone. An interzonal MT bundle from a spindle beginning anaphase B was reconstructed from serial cross-sections to determine its MT polarity pattern. (A) The distribution of MT plus-ends in the reconstructed interpolar MT bundle. Individual MTs are represented by horizontal lines. The position of the plus-ends of MTs emanating from the left pole are indicated by open circles and those from the right are indicated as solid diamonds. The MTs from each half spindle are arranged top to bottom in order of increasing length in order to show the extent of possible MT overlap. (B and C) Cross-sectional profiles of the reconstructed bundle near the poles (B) and at the midzone (C). In C, MTs emanating from one pole are marked in the center with a black circle, while those from the other are not. Bar, 70 nm.

Unphospho-KLP61F is excluded from mitotic spindles in Drosophila early embryos. Confocal immunofluorescence images of Drosophila early embryos stained with the unphospho-KLP61F antibody (yellow) and DAPI (blue). Throughout the cell cycle, the localization of unphospho-KLP61F is cytoplasmic and does not appear associated with MTs. (A) Prophase. Before nuclear envelope breakdown, unphospho-KLP61F is specifically excluded from the nuclear region. (B) Metaphase. Once the metaphase spindle has formed, unphospho-KLP61F staining is excluded from the spindle. (C) Anaphase. The exclusion of unphospho-KLP61F from the spindle persists through anaphase. Bar, 12 μm.

Relative distribution of phospho-KLP61F and tubulin in the mitotic spindle during metaphase and anaphase B. Confocal immunofluorescence images showing anti-phospho-KLP61F staining (blue) overlaid on anti β-tubulin (yellow) staining. In these images, yellow indicates the regions of the spindle in which the relative intensity of tubulin immunofluorescence is disproportionately high compared with phospho-KLP61F, white indicates the regions of relatively equal tubulin and phospho-KLP61F concentrations and blue indicates regions in which the intensity of phospho-KLP61F immunofluorescence is disproportionately high compared with tubulin. (A) In metaphase very little phospho-KLP61F colocalizes with astral MTs while the central spindle shows a high degree of overlap. The spindle midzone appears blue indicating a disproportionately high relative concentration of phospho-KLP61F to tubulin. (B) In anaphase B, nearly all of the KLP61F is concentrated in the interzone and very little is detected on chromosomes, spindle poles or astral MTs. Again, some regions of blue are apparent in the midzone indicating that phospho-KLP61F staining is disproportionately higher than tubulin staining. Bar, 5.7 μm.

Antiphospho-KLP61F antibodies associate with electron-dense crossbridges between spindle microtubules. (A) Schematic illustration showing the predicted immunogold labeling pattern in spindles stained with the anti-phospho-KLP61F antibody. Based on the predicted position of the Bim C boxes on a bipolar KLP61F holoenzyme, gold particles should be spaced ∼60 nm apart on electron-dense MT crossbridges. Note that although the antigenic sites are spaced ∼60 nm apart, the crossbridges themselves are longer. Furthermore, the gold should be closely juxtaposed with microtubules, lying, on average, within 10 nm from the microtubule surface, just as we observe, (see Table II). (B) Observed gold labeling pattern in the spindle midzone. (Top) The image shown is the inset from Fig. 8 A with digitally enhanced contrast. (MT, microtubules, red arrowheads point down the long axis gold particle associated crossbridges.) (Bottom) Gallery of gold-labeled crossbridges. Red arrowheads indicate the two ends of the crossbridge adjacent to the associated gold particles. (C) Histogram of cross-link lengths. 115 MT crossbridges with gold particles associated at both ends were identified from two spindles and the length between the center of each gold particle was measured. The spacing ranged between 35 and 95 nm with a mean ∼60-65 nm. Bar: (B, top) 60 nm; (B, bottom) 45 nm.

Detailed ultrastructure of MT bundles and spindle poles during mitosis. (A) Kinetochore MT bundles. (B) Interpolar MT bundles during metaphase. (C) Interpolar MT bundles during anaphase. D–G show the organization and structure of centrioles throughout mitosis. (D) Spindle poles contain two orthogonally positioned centrioles. The image shown is from the pole of a metaphase spindle. (E) During prophase, each centriole consists of nine singlet MTs around a central singlet MT. (F) by metaphase, the centrioles become much more electron dense and some of the outer MTs begin to form doublets. (G) In anaphase, doublets continue to form on the outer MTs. The standard organization of centrioles, nine outer triplets offset in a barrel shape, is never observed in Drosophila early embryos. Bar: (A) 1.3 μm; (B) 1.5 μm; (C) 1.1 μm; (D) 1.2 μm; (E–G) 0.3 μm.

Characterization of the unphospho- and phospho-KLP61F antibodies. (A) Western blot of Drosophila 0–2-h embryonic cytosol probed with the anti-BCB and p-BCB antibodies. Both antibodies react specifically with the KLP61F polypeptide in Drosophila syncytial blastoderm cytosol, however, at equivalent antibody concentrations, the p-BCB antibody shows more intense staining. (B) Western blots of bacterially expressed, recombinant KLP61F before and after phosphorylation by incubation with Xenopus M-Phase extracts, probed with the BCB and p-BCB antibodies. While the BCB antibody reacts with untreated recombinant KLP61F (rKLP61F), the p-BCB antibody only reacts specifically with rKLP61F after it has been phosphorylated in M-phase extracts (lane M) and does not react with untreated KLP61F (lane U) or with recombinant KLP61F treated with matched interphase extracts (lane I).

Phospho-KLP61F is sequestered in the nucleus during interphase and prophase and it associates with the mitotic spindle during metaphase and anaphase. Confocal immunofluorescence images of Drosophila early embryos stained with the anti-phospho-KLP61F antibody (yellow) and DAPI (blue). White indicates high overlap. (A) In interphase, before mitosis, phospho-KLP61F is concentrated in nuclei and overlaps significantly with DNA. (B) In prophase, before nuclear envelope breakdown, phospho-KLP61F is still concentrated in the nuclei. At this point, some staining is also detectable in the cytoplasm and perhaps on centrosomes but not on cytoplasmic microtubules. (C) In metaphase, phospho-KLP61F is now concentrated on the half spindles and on dense filaments bridging the half spindles (arrow). (D) In anaphase A, phospho-KLP61F is still concentrated on the spindles as well as on filaments bridging the half spindles. (E) In anaphase B, nearly all of the phospho-KLP61F is now concentrated in the spindle interzone, while the spindle poles and astral MTs show very low phospho-KLP61F staining. (F) In telophase, phospho-KLP61F is concentrated at two distinct punctae oriented between the daughter nuclei. Often, smaller punctae can be seen in the reforming daughter nuclei, as well. Bar, 10 μm.

Visualization of phospho-KLP61F associated with interpolar MT bundles in metaphase and anaphase spindles. EMs of thin sections of Drosophila syncytial blastoderms preserved by HPF/FS and immunostained with the anti p-BCB antibody and gold conjugated secondary antibodies. (A and E) Phospho-KLP61F staining pattern on whole metaphase and anaphase spindles, respectively, from embryos embedded in LR-White and labeled with secondary antibodies conjugated to 10 nm gold (P, spindle poles; MB, interpolar MT bundle; N, separating daughter nuclei). (A inset) Higher magnification of the interpolar bundle (MB) indicated by the arrow and shown in digitally enhanced contrast in Fig. 9. When this region is enhanced for contrast, the gold can be seen to associate with electron-dense crossbridges between microtubules (Fig. 9). (B and C) Higher magnification of the immunostaining pattern in metaphase spindle midzones. (D and F) Phospho-KLP61F immunostaining pattern on interpolar MT bundles during metaphase and anaphase B, respectively, from embryos embedded in Eponate 12/Araldite and stained with secondary antibodies conjugated to 5 nm gold to increase the precision with which the proximity of the gold to the MT can be determined. Gold particles are closely associated with the surface of MTs within these bundles. Bar: (A and E) 1.3 μm; (B) 320 nm; (C) 160 nm; (D and F) 100 nm.

Model for KLP61F function. (A) As the nuclear envelope fenestrates and MTs enter the nuclear region, KLP61F cross-links spindle MTs regardless of orientation and rides toward their plus ends thereby becoming concentrated in the midzone. (B) If KLP61F cross-links parallel MTs they will be organized into bundles but no MT-MT sliding will be generated. (C) If KLP61F cross-links antiparallel MTs, the force generated by KLP61F will push the MTs apart. This results in isometric forces that hold the poles apart during metaphase and anaphase A, and isotonic forces that drive spindle elongation during anaphase B.