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First published online March 12, 2004
doi: 10.1242/10.1242/jcs.00993

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The N-terminus of the long MLCK induces a disruption in normal spindle morphology and metaphase arrest

Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA

View larger version (52K): [in a new window]   Fig. 2. Expression of MLCK-GFP fusions in HeLa cells. (A) MLCK truncations. Dark gray boxes represent the Ig motifs, black bars represent the DXR motifs and the light gray box represents GFP. (B) The kinase activities of the immunoprecipitated kinase-dead and wild-type long MLCK-GFP fusions were examined in the presence of Ca2+/calmodulin using the free RLC as a substrate. The wild-type long MLCK phosphorylates the RLC in Ca2+/calmodulin-dependent manner, because phosphorylation was not detected in the presence of EGTA. The kinase-dead MLCK did not phosphorylate the RLC in the presence of Ca2+/calmodulin or Ca2+/calmodulin/EGTA. Symbols are as follows: , wild-type MLCK in the presence of Ca2+/calmodulin; , wild-type MLCK in the presence of Ca2+/calmodulin and 2 mM EGTA; , kinase-dead MLCK in the presence of Ca2+/calmodulin; , kinase-dead MLCK in the presence of Ca2+/calmodulin and 2 mM EGTA. (C) Immunoblot analysis of HeLa whole cell lysates expressing the GFP-kinase-dead-MLCK and the truncated MLCK-GFP fusions. Lane 1 contains the HeLa kinase-dead long MLCK (237 kDa); lane 2 contains the HeLa IgG domain plus five DXRs (138 kDa); lane 3 contains the HeLa IgG domain (121 kDa); lane 4 contains the rabbit five DXR fragment (45 kDa); lane 5 contains GFP alone (27 kDa). The lower band in lane 4 is caused by proteolysis of the five DXRs. The MLCK IgG domain polyclonal antibodies do not cross-react with the five DXRs. The MLCK monoclonal antibody recognizes an epitope from the third and fourth DXR motifs (Blue et al., 2002), and does not react with the IgG domain. The GFP monoclonal antibody detects all four MLCK-GFP fusions. The endogenous long MLCK (210 kDa) can be detected with both the MLCK monoclonal and MLCK IgG domain antibodies at longer exposure. The bottom panel shows ß-tubulin as a loading control. In addition, immunoblot analysis with antibodies to GFP demonstrated that GFP fusions of the full-length chicken skeletal MyBP-H and MyBP-C proteins are expressed in HeLa cells.

View larger version (54K): [in a new window]   Fig. 1. Distribution of endogenous long MLCK during cell division. (A) Immunoblots of HeLa whole cell lysates. Lane 1, incubation with an MLCK monoclonal antibody and lane 2, incubation with a HeLa MLCK IgG domain polyclonal antibody. Molecular weight standards are indicated in kDa. (B) Conventional fluorescence micrographs (left) and deconvolved images (right) of dividing HeLa cells stained with the HeLa MLCK IgG domain antibody. Scale bar, 10 µm. The endogenous long MLCK localizes to the cell cortex (arrows).

View larger version (49K): [in a new window]   Fig. 3. Distribution of MLCK-GFP truncations in interphase and mitotic cells. Fluorescence micrographs of HeLa cells transfected with the HeLa IgG domain plus five DXRs, the HeLa IgG domain, the rabbit five DXRs or GFP. Scale bars, 10 µm. (A) Representative images of intact (top) and Triton-permeabilized (bottom) interphase cells. (B) Representative images of cells in metaphase, early anaphase, late anaphase and telophase. (C) Representative images of Triton-permeabilized cells in anaphase and late telophase.

View larger version (30K): [in a new window]   Fig. 4. Expression of MLCK truncations increases the number of multinucleate cells. (A) HeLa cells were transiently transfected with GFP or with GFP fusions with the rabbit five DXRs, the HeLa IgG domain, the HeLa IgG domain plus five DXRs, MyBP-H or MyBP-C. Cells expressing GFP or GFP fusions were visualized by fluorescence. Values represent the mean and the standard error of the mean for 3000 cells per MLCK construct and 1000 cells per MyBP construct from three independent experiments. (B) Representative field of cells transfected with the IgG domain alone. Cells were fixed at 32 hours after transfection and stained with DAPI for DNA and anti-ß-tubulin antibodies. Cells expressing the IgG domain GFP fusion were visualized by green fluorescence. Arrows indicate cells with multiple nuclei. Scale bar, 10 µm.

View larger version (143K): [in a new window]   Fig. 5. Time-lapse series of phase and fluorescence micrographs of mitotic HeLa cells expressing GFP or GFP fusions of the IgG domain, IgG domain plus five DXRs or kinase-dead MLCK. Scale bar, 10 µm. (A) A cell expressing GFP. (B,C) Cells expressing the IgG domain plus five DXRs. (D,E) Cells expressing the IgG domain. Arrowheads indicate an actively growing lamellipodia of a cell that exits M phase. (F) Cell expressing the kinase-dead MLCK. Arrows indicate blebs that form in arrested cells.

View larger version (82K): [in a new window]   Fig. 6. Expression of the N-terminus of the long MLCK disrupts normal spindle morphology during mitosis and inhibits chromosome alignment. Scale bars, 10 µm. (A) Metaphase arrested cells were identified, followed for approximately 3 hours, fixed and stained for microtubules with antibodies against ß-tubulin (top) and for DNA with DAPI (middle). Merged images of microtubules (red) and DNA (blue) (bottom). In GFP controls and cells expressing the five DXRs, alignment of the chromosomes and well-organized bipolar spindles were observed (g,h). Cells expressing the IgG domain or IgG domain plus five DXRs frequently showed abnormal spindle morphology (a-f). The arrows indicate extended, curving astral microtubules (a,b,d,f). In some cases, spindle pole fragmentation was observed (a,c). Chromosome alignment was also abnormal in metaphase-arrested cells. Several mono-oriented chromosomes were observed close to the spindle pole (b,d) and sometimes the chromosomes aligned along the length of the spindle (a,e). (B) Transfected cells were fixed and stained for pericentrosomal material with antibodies against -tubulin and DNA with DAPI. -Tubulin staining showed that cells expressing N-terminal fragments of MLCK possessed two morphologically normal centrosomes. (C) Transfected cells were chilled to depolymerize the labile non-kinetochore-associated microtubules and the morphology of the remaining spindle microtubules evaluated by ß-tubulin staining (bottom). The cells were also stained with antibodies against CENP-E, which localized to kinetochores (middle). In cells expressing the IgG domain or IgG domain plus five DXRs (a,b), the spindle microtubules were bundled, instead of containing well-organized kinetochore fibers (c,d). (D) Cells expressing N-terminal fragments of MLCK display abnormalities in chromosome alignment. Indirect immunofluorescence with the centromere marker ACA was used to monitor the position of the kinetochores (top) and DAPI to visualize the chromosomes (middle) in metaphase transfected cells. Merged images of centromeres (red) and DNA (blue) (bottom). In 60% of the cells expressing the IgG domain or IgG domain plus five DXRs, there were uncongressed chromosomes close to one of the spindle poles or between the equator and one spindle pole (a,b). In cells expressing GFP or the five DXRs, less than 10% of mitotic cells with visible metaphase chromosome alignment had uncongressed chromosomes.

View larger version (77K): [in a new window]   Fig. 7. Localization of the 3F3/2 phosphoepitope and checkpoint proteins in cells expressing N-terminal fragments of MLCK. Scale bars, 10 µm. (A) Immunostaining with the 3F3/2 antibody was used to examine the presence of unattached or mono-oriented chromosomes in transfected cells (left). DNA was visualized with DAPI (right). In cells expressing the IgG domain or IgG domain plus five DXRs, 3F3/2 staining was observed in more than 50% of cells with a visible metaphase plate, typically on misaligned/uncongressed chromosomes. In untransfected metaphase cells or cells expressing GFP or the five DXRs, 3F3/2 fluorescence was not detected. In transfected cells, the Bub3 (B) and Mad1 (C) checkpoint proteins (top) localized to unattached kinetochores during prophase and prometaphase (a,c,e). In cells expressing the IgG domain or IgG domain plus five DXRs, bright Bub3 and Mad1 staining was observed on the kinetochores of misaligned chromosomes near spindle poles or uncongressed chromosomes in metaphase cells (arrows), whereas only residual staining was detected on the kinetochores of aligned chromosomes. Chromosomes and nuclei were stained with DAPI (middle). Merged images of checkpoint proteins (red) and DNA (blue) (bottom).

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