2009;10:854C865. onset when CDK1 activity is down-regulated. Further studies indicate that cell cycleCregulated membrane association of NuMA underlies anaphase-specific enhancement of cortical NuMA and dynein. By replacing endogenous NuMA with membrane-binding-deficient NuMA, we can specifically reduce the cortical accumulation of NuMA and dynein during anaphase and demonstrate that cortical NuMA and dynein contribute to efficient chromosome separation in mammalian cells. INTRODUCTION Faithful separation of duplicated genetic materials into daughter cells is critical for animal development. It occurs at a specific stage of mitosis known as anaphase. Chromosome separation is driven by microtubules (MTs), which organize into spindle-shaped structure during mitosis (Scholey (Aist and Berns, 1981 ; Aist Partner of Inscuteable (LGN)/nuclear mitotic apparatus (NuMA) ternary complex was shown to recruit and anchor dynein at the cell cortex to direct spindle positioning during mitosis (Nguyen-Ngoc = 50 from each group of cells. * 0.01. (F) Fluorescence images from time-lapse analysis of HeLa cells expressing Venus-NuMA1981-2060. Time points are shown as seconds. Time point zero indicates the time when calcium (1 mM final concentration) and ionomycin (10 M final concentration) were added to the medium. The membrane association of NuMA MBD is cell cycle regulated during mitosis NuMA is a nuclear protein (Van Ness and Pettijohn, 1983 ). The identified membrane association of NuMA could happen only during mitosis when the nuclear envelope is broken down. We observed mitotic Cos 7 cells expressing Venus-NuMA1981-CT, which contains the membrane-binding domain. Surprisingly, unlike in interphase cells, the expressed protein did not exhibit obvious membrane association in prophase and metaphase cells (Figure?4A; similar results were obtained in 100% of cells observed, 50). The membrane association of Venus-NuMA 1981-CT, however, was evident when cells entered anaphase (Figure?4A; similar results were obtained in 100% of cells observed, 50), suggesting that the membrane association of NuMA MBD could be cell cycle regulated. Open in a separate window FIGURE 4: Cell cycleCregulated membrane Doxycycline association of NuMA MBD. (A) The membrane association of NuMA1981-CT is evident only in anaphase cells. Representative images of mitotic Cos 7 cells expressing Venus-NuMA1981-CT. Cells were transfected with plasmids expressing Venus-NuMA1981-CT. At 24 h later, cells were fixed and stained with DNA dye. Left, prophase and metaphase cells; right, anaphase cells. (B) Amino acid sequence of the membrane-binding domain of NuMA (aa 1981C2060). The paired, positively charged lysine and arginine residues are highlighted in italic; Thr-2041 is highlighted in bold. (C) Phosphorylation of Thr-2041 regulates membrane association of NuMA during mitosis. Representative images of mitotic Cos 7 cells expressing Venus-NuMA1981-CT-T2041A (left), Venus-NuMA1981-CT-T2041E (middle), or Venus-NuMA1981-2040 (right). Cells were transfected with plasmids expressing the indicated proteins, fixed, and stained as in A. (D) Phosphorylation of Thr-2041 affects membrane association of NuMA MBD in interphase cells. Representative single-layer confocal images of HeLa cells expressing Venus-NuMA1981-2060 (WT; left), Venus-NuMA1981-2060-T2041A (middle), or Venus-NuMA1981-2060-T2041E (right). (E) Quantification of membrane-to-cytosol fluorescence intensity ratio from images in D. Results are from three independent experiments. Error bars, SD. = 50 for each group of cells. * 0.01. Bars, 10 m. The phosphorylation state of T2041 is critical for the membrane association of NuMA MBD What could be preventing the membrane association of NuMA MBD during prophase and metaphase? A clue came from a previous study from the Compton lab showing that when Rabbit Polyclonal to Cyclin E1 (phospho-Thr395) several putative CDK1 phosphorylation sites at the C-terminal of NuMA were mutated, the mutant proteins mislocalized during mitosis (Compton and Luo, 1995 ). In particular, when Thr-2041 (T2041, Figure?4B; originally described as T2040 in the Compton article and as T2055 when another human NuMA cDNA [“type”:”entrez-protein”,”attrs”:”text”:”NP_006176″,”term_id”:”71361682″,”term_text”:”NP_006176″NP_006176] was used) was mutated to alanine, the Doxycycline mutant protein appeared Doxycycline to localize to the plasma membrane during mitosis (Compton and Luo, 1995 ). The underlying mechanism for this altered localization is not known. T2041 locates within the identified NuMA MBD (Figure?4B). It was confirmed to.
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