The chromokinesin Kid is necessary for chromosome arm orientation and oscillation, but not congression, on mitotic spindles

Chromokinesins have been postulated to provide the polar ejection force needed for chromosome congression during mitosis. We have evaluated that possibility by monitoring chromosome movement in vertebrate-cultured cells using time-lapse differential interference contrast microscopy after microinjection with antibodies specific for the chromokinesin Kid. 17.5% of cells injected with Kid-specific antibodies have one or more chromosomes that remain closely opposed to a spindle pole and fail to enter anaphase. In contrast, 82.5% of injected cells align chromosomes in metaphase, progress to anaphase, and display chromosome velocities not significantly different from control cells. However, injected cells lack chromosome oscillations, and chromosome orientation is atypical because chromosome arms extend toward spindle poles during both congression and metaphase. Furthermore, chromosomes cluster into a mass and fail to oscillate when Kid is perturbed in cells containing monopolar spindles. These data indicate that Kid generates the polar ejection force that pushes chromosome arms away from spindle poles in vertebrate-cultured cells. This force increases the efficiency with which chromosomes make bipolar spindle attachments and regulates kinetochore activities necessary for chromosome oscillation, but is not essential for chromosome congression

A Functional Relationship between NuMA and Kid Is Involved in Both Spindle Organization and Chromosome Alignment in Vertebrate Cells

We examined spindle morphology and chromosome alignment in vertebrate cells after simultaneous perturbation of the chromokinesin Kid and either NuMA, CENP-E, or HSET. Spindle morphology and chromosome alignment after simultaneous perturbation of Kid and either HSET or CENP-E were no different from when either HSET or CENP-E was perturbed alone. However, short bipolar spindles with organized poles formed after perturbation of both Kid and NuMA in stark contrast to splayed spindle poles observed after perturbation of NuMA alone. Spindles were disorganized if Kid, NuMA, and HSET were perturbed, indicating that HSET is sufficient for spindle organization in the absence of Kid and NuMA function. In addition, chromosomes failed to align efficiently at the spindle equator after simultaneous perturbation of Kid and NuMA despite appropriate kinetochore-microtubule interactions that generated chromosome movement at normal velocities. These data indicate that a functional relationship between the chromokinesin Kid and the spindle pole organizing protein NuMA influences spindle morphology, and we propose that this occurs because NuMA forms functional linkages between kinetochore and nonkinetochore microtubules at spindle poles. In addition, these data show that both Kid and NuMA contribute to chromosome alignment in mammalian cells

p53 Dimers associate with a head-to-tail response element to repress cyclin B transcription.

DNA damage induced by the topoisomerase I inhibitor SN38 activates cell cycle checkpoints which promote cell cycle arrest. This arrest can be abrogated in p53-defective cells by the Chk1 inhibitor 7-hydroxystaurosporine (UCN-01). Previously, we compared p53 wild-type MCF10A cells with derivatives whose p53 function was inhibited by over-expression of the tetramerization domain (MCF10A/OD) or expression of shRNA against p53 (MCF10A/Δp53). Treatment of SN38-arrested MCF10A/OD cells with UCN-01 abrogated S, but not G2 arrest, while the MCF10A/Δp53 cells abrogated both S and G2 arrest. The MCF10A/OD cells had reduced levels of cyclin B, suggesting that tetramerization of p53 is not required for repression of cyclin B gene expression. In the present study, we analyzed p53 oligomerization status using glutaraldehyde cross-linking. Following SN38 treatment, MCF10A cells contained oligomeric forms of p53 with molecular weights approximating monomers, dimers, trimers, and tetramers. However, MCF10A/OD cells possessed only monomers and dimers suggesting that these complexes may be involved in repression of cyclin B. While genes transcriptionally activated by p53 contain a consensus sequence with elements repeated in a head-to-head orientation, the cyclin B promoter contains similar elements oriented head-to-tail. Chromatin immunoprecipitation (ChIP) assays revealed that p53 associates with this head-to-tail element in both MCF10A and MCF10A/OD. Electrophoretic mobility shift assays (EMSA) using a biotin-labeled probe containing the head-to-tail element showed a shift in mobility consistent with the molecular weight of tetramers and dimers in MCF10A nuclear extract, but only the dimer in MCF10A/OD nuclear extract. Taken together, these results suggest a novel mechanism whereby p53 dimers associate with the head-to-tail element to repress cyclin B transcription

Determination of cyclin B mRNA and protein levels.

<p>MCF10A, MCF10A/OD, and MCF10A/Δp53 cells were incubated with 0–30 ng/ml SN38 for 24 hours. RNA was purified from cell lysates and relative levels of cyclin B mRNA were analyzed using RT-PCR followed by agarose gel electrophoresis. Cell lysates were also analyzed for expression of cyclin B and actin protein using SDS-PAGE followed by immunoblotting with cyclin B- or actin-specific antibodies.</p

Comparison of head-to-head and head-to-tail p53 response elements.

<p>The consensus sequence of the standard p53 response element consists of two half-sites, each with quarter-sites oriented in a head-to-head manner (indicated by arrows). R =  purine, Y =  pyrimidine, W = A/T, and N = A/T/C/G. The p21 promoter contains a standard head-to-head p53 response element, while the MDR1 and cyclin B genes contain p53 response elements with head-to-tail orientations.</p

Analysis of p53 binding to head-to-tail response element using chromatin immunoprecipitation assays.

<p>MCF10A, MCF10A/OD, and MCF10A/Δp53 cells were incubated with either fresh media (untreated) or 10 ng/ml SN38 for 24 hours, followed by cross-linking in 1% formaldehyde. Chromatin was then purified, sonicated, and immunoprecipitated with anti-p53 antibody or negative IgG antibody. Unprecipitated chromatin was used as a positive control (input). DNA was isolated and amplified by PCR using primers flanking the head-to-tail response element in the cyclin B promoter or the RNA Polymerase II binding site from the GAPDH promoter. The amplified PCR fragments were analyzed by agarose gel electrophoresis.</p

Analysis of p53 binding to head-to-tail response element in cyclin B promoter using electrophoretic mobility shift assays.

<p><b>A</b>, MCF10A, MCF10A/OD, and MCF10A/Δp53 cell lines were incubated with 10 ng/ml SN38 for 24 hours (+SN) or were left untreated (–SN). Binding reactions were prepared by incubating nuclear extracts or recombinant p53 with a biotin-labeled probe corresponding to the head-to-tail response element in the cyclin B promoter, in the presence (+) or absence (−) of a 200-fold molar excess of specific DNA (unlabeled probe). All binding reactions were carried out in the presence of a 100-fold molar excess of unlabeled non-specific DNA. Complexes were separated on 4% native polyacrylamide gel electrophoresis, transferred to positively charged nylon membrane, and visualized using a streptavidin-horse radish peroxidase conjugate. <b>B</b>, A parallel gel was transferred to nitrocellulose membrane and immunoblotted with p53-specific antibodies.</p

Determination of p53 oligomerization status using glutaraldehyde cross-linking.

<p><b>A</b>, MCF10A, MCF10A/OD, and MCF10A/Δp53 and cells were incubated with 10 ng/ml SN38 (+SN38) for 24 hours or <b>B</b>, fresh media (untreated) for 24 hours. Cells were harvested in lysis buffer and incubated with the indicated concentrations of glutaraldehyde for 5 minutes. The cross-linked lysates were then analyzed by SDS-Page followed by immunoblotting with p53-specific antibodies. Cell lysates were loaded at 10,000 or 30,000 cells/lane as indicated.</p