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First published online 30 July 2003
doi: 10.1242/jcs.00685

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Interphase and monoastral-mitotic phenotypes of overexpressed MAP4 are modulated by free tubulin concentrations

¹ Department of Molecular Biology, University of Umea, S-901 87 Umea, Sweden
² Department of Medical Biosciences, University of Umea, S-901 87 Umea, Sweden

View larger version (41K): [in a new window]   Fig. 1. Induced ectopic expression of MAP4 results in a mitotic block characterized by monoastral spindles. (A) Immunoblots of cellular lysate, separated by 12% SDS-PAGE, using the indicated antibodies for detection. K562 cell lines harboring pMEP-vector-Co or pMEP-MAP4 were analyzed after various times of Cd2+-induced expression from the hMTIIa promotor. Arbitrary quantification was obtained from serial dilutions of cell lysates, which revealed tenfold increased expression of MAP4 after 24 hours and non-significant alterations in endogenous tubulin levels (see Relative tubulin amount). In the right-hand panel, cell lysates were separated by 8% SDS-PAGE to resolve slowly migrating MAP4 phosphoisomers characteristic of mitotic cells. (B) Transfected cells were Cd2+-induced for 20 hours, fixed and stained with anti--tubulin (green) and propidium iodine. A confocal section of a normal spindle (Vector-Co) and representative monoastral spindles caused by either MAP4 overexpression (MAP4) or by the Eg5 inhibitor monastrol (68 µM, 20 hours) are shown. The distribution of DNA content within transfected or monastrol-treated cell populations is also shown. (C) Cells induced to overexpress MAP4 as in panel B were fixed in methanol and stained with anti--tubulin (green) and anti-pericentrin (red). A confocal section of a representative monoastral spindle observed among MAP4-overexpressing cells is shown (bar, 6 µm). (D) K562, Jurkat and DG75 cell lines harboring pMEP-vector-Co or pMEP-MAP4 were induced for 20 hours with Cd2+ and mitotic figures were evaluated by epifluorescence microscopy with respect to numbers of spindle poles in cells double stained for DNA and MTs (n=450 cells). Data represent mean of two independent determinations.

View larger version (29K): [in a new window]   Fig. 2. Multi-parameter flow cytometry analysis of MT content of G2- and M-phase subpopulations. K562 cell lines harboring pMEP-vector (Control) or pMEP-MAP4 (MAP4) were analyzed after 20 hours of Cd2+-induced expression. One culture of control transfected cells was treated with Paclitaxel (50 ng/ml for 20 hours). Cells were extracted with a MT-stabilizing buffer and fixed according to a protocol designed for determination of MT-specific fluorescence (see Materials and Methods). (A) Cells stained with a histone H3 phosphoepitope antibody were analyzed with respect to propidium iodide-stained DNA and 90° side-scattering properties by dual parameter flow cytometric analysis. This analysis allowed definition of the G1-, G2- and M-phase gates indicated in the dot-plots. (B) Determination of the fractions of histone H3 phosphoepitope-positive cells within the G2- and M-phase gates defined in panel A. (C) Distribution of MT-specific fluorescence within the G2-populations and M-populations (dotted line). Staining of cells with fluorescein-conjugated rabbit anti-mouse immunoglobulin alone gave <1% nonspecific staining (not shown). (D) Mean MT-specific fluorescence intensities of G1-, G2- and M-phase populations derived by gating of cells as depicted in panel A. More than 95% of all cells were included in the acquisition gate and the data are representative of three independent transfection experiments.

View larger version (24K): [in a new window]   Fig. 3. Modulation of MAP4-mediated MT stabilization by two distinct Op18 truncation derivatives in the G2-phase population. (A) Native Op18 is depicted with an N-terminal region (residues 1-45) and an extended -helical region that contain two homologous repeats separated by 51 residues (designated Repeat 1 and Repeat 2), each of which binds an /ß-tubulin heterodimer indicated by open and filled circles (Gigant et al., 2000). Each truncated Op18 derivative is denoted by the numbers within brackets, which indicate the amino acid residues present. As depicted in the figure, the N-terminally truncated Op18(25-149) derivative retains two-site positive binding cooperativity, which facilitates tubulin sequestering, whereas the Op18(1-99) derivative binds single heterodimers with low affinity but still promotes catastrophes via the intact N-terminus (Howell et al., 1999; Larsson et al., 1999). (B) Cotransfected K562 cell lines (DNA ratio 1:2 of MAP4/Vector-Co: truncated Op18/Vector-Co) were induced with Cd2+ for 20 hours. Immunoblots of cellular lysates were probed with anti-SLEEIQ, which recognizes full-length and the two truncated Op18 derivatives with similar efficiency. Mean MAP4-specific fluorescence intensities, as determined by flow cytometry, are given below the autoradiograph. Mean MT-specific fluorescence within the G2 populations was also determined after 20 hours of induced expression and is shown in the bottom panel. The original histogram data, from which the mean fluorescence intensities were derived, included >95% of all cells and revealed a well-defined single peak in all cases (data not shown). All data in this figure are derived from the same transfected cell populations but are representative of at least three independent transfection experiments.

View larger version (47K): [in a new window]   Fig. 4. Modulation of MAP4-mediated mitotic arrest and monoastral spindles. The coexpressing cell populations described in Fig. 3 (20 hours of induced expression) were stained with propidium iodide followed by analysis of DNA content by flow cytometry (upper panels). The inserts in two of the upper panels show G2/M block of cells after 24 hours in the presence of paclitaxel (1 µM). Mitotic figures were analyzed with respect to bipolar, small or intermediate-to-large monoastral spindles (see lower panel; bar, 6 µm) by inspection of cells double stained for DNA and MTs. The distribution of different types of mitotic cells represents the mean of duplicate determinations, using independent cell preparations, from one transfection experiment (n=450 cells). All data in this figure are derived from the same transfected cell populations but are representative of at least three independent transfection experiments.

View larger version (27K): [in a new window]   Fig. 5. Decreased cytosolic tubulin concentrations in mitotic cells expressing a plasma-membrane-located high-affinity tubulin-binding chimera. (A) Depiction of the plasma-membrane-targeted CD2-RB3 chimera in complex with two /ß-tubulin heterodimers. It should be noted that RB3 is not inactivated by phosphorylation during mitosis. (B) Transfected K562 cell lines harboring the indicated pMEP-CD2 chimera derivative were Cd2+-induced for 20 hours, directly fixed and co-stained with anti-CD2 (R-phycoerythrin) and anti--tubulin (Alexa Fluor488). A confocal section of a representative CD2-Co and CD2-RB3-expressing mitotic cell is shown (bars, 6 µm). To allow a direct comparison with data from CD2-RB3/MAP4-coexpressing cells, presented in Figs 6 and 7, the transfected DNA was mixed with pMEP-vector Co DNA at a ratio of 1:1. (C) The average fluorescence intensity of non-polymeric -tubulin in confocal sections was determined in the cytosol and at the plasma membrane. The average values of 50 analyzed cells expressing the indicated CD2 chimera is presented. (D) The -tubulin staining fluorescence intensities of individual cells expressing the indicated CD2 chimera are plotted. All data in this figure are derived from the same transfected cell populations but are representative of at least three independent transfection experiments.

View larger version (38K): [in a new window]   Fig. 6. The CD2-RB3 tubulin-sequestering chimera blocks MAP4-mediated MT stabilization. Cotransfected K562 cell lines (DNA ratio 1:1 of MAP4/Vector-Co:CD2-chimera/Vector-Co) were induced with Cd2+ for 20 hours. Total cellular lysates (20 µg/lane) were separated by 12% SDS-PAGE and immunoblots were probed with the antibodies indicated. Mean MAP4-specific fluorescence intensities, as determined by flow cytometry, are given below the autoradiograph. Mean MT-specific fluorescence within the G2 populations was determined after 20 hours of induced expression and is shown in the bottom panel. All data in this figure are derived from the transfected cell populations analyzed in Fig. 5 but are representative of at least three independent transfection experiments.

View larger version (28K): [in a new window]   Fig. 7. The CD2-RB3 tubulin-sequestering chimera counteracts MAP4-mediated mitotic defects. The coexpressing cell populations described in Fig. 6 (20 hours of induced expression) were stained with propidium iodide followed by analysis of DNA content by flow cytometry (upper panels). The inserts in two of the upper panels show G2/M block of cells after 24 hours in the presence of paclitaxel (1 µM). Mitotic figures were analyzed with respect to bipolar, small or intermediate-to-large monoastral spindles (see Fig. 4, lower panel) by inspection of cells double stained for DNA and MTs. The distribution of different types of mitotic cells represents the mean of duplicate determinations, using independent cell preparations, from one transfection experiment (n=450 cells). All data in this figure are derived from the transfected cell populations analyzed in Figs 5 and 6 but are representative of at least three independent transfection experiments.