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First published online 31 October 2006
doi: 10.1242/jcs.03235

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Kinesin 6 family member Subito participates in mitotic spindle assembly and interacts with mitotic regulators

Waksman Institute and Department of Genetics, Rutgers, The State University of New Jersey, Piscataway, NJ 08854-8020, USA

View larger version (58K): [in a new window]   Fig. 1. Subito localization in wild-type brains. (A) A whole-mount metaphase cell with the chromosomes aligned in the middle of the spindle. The DNA (blue) is condensed and Subito (red) is present on the microtubules (green). (B) A whole-mount early anaphase cell with Subito on the spindle between the separating chromosomes. (C) A squashed late anaphase cell with Subito on the midzone microtubules and beginning to concentrate on a smaller portion of the spindle than in early anaphase. (D) A whole-mount metaphase cell showing the overlap of Subito (red) and Incenp (green). In some cases, Subito appears to be spreading out along the interpolar microtubules whereas in other cases, it appears in foci. (E) A whole-mount metaphase cell showing Subito (red) present n the microtubules between paired CID foci (blue). The greyscale inset shows DNA staining. Bars, 5 µm. (F,G) Model for the localization pattern of Subito at metaphase. Subito interacts with antiparallel microtubules, which only exist between centromeres. Just before anaphase, the centromeres are pulled in opposite directions, increasing the length of antiparallel microtubules between them.

View larger version (80K): [in a new window]   Fig. 2. Subito is required during anaphase for the localization of Polo and Aurora B but not Fascetto or Pavarotti. All images are stained for DNA (blue) and, except for A and B, tubulin (green). Polo (green) colocalizes with Subito (red) at the midzone in wild-type anaphase (A) but not in sub1/sub131 mutants (B). (C) Aurora B (red) is localized on the chromosomes during metaphase (not shown) and during anaphase it moves off of the chromosomes and localizes on the spindle midzone. (D) In sub1/sub131 mutant anaphase, Aurora B protein is still present on the anaphase chromosomes but not at the midzone. (E,F) Although usually more concentrated near the chromosomes in sub mutant anaphases, Aurora B and Incenp (red) often appeared to have spread out along the microtubules; they were never concentrated in the midzone. In panel E, there was no tubulin staining. Bars, 5 µm.

View larger version (103K): [in a new window]   Fig. 3. (A) Pavarotti (red) is located in the midzone of wild-type late anaphase. (B,C) Pavarotti shows variable midzone staining in sub1/sub131 mutants, which may be related to the degree of microtubule disorganization. (D) Fascetto (FEO, red) localization to the midzone in the wild type. (E,F) Fascetto staining was strong during anaphase and particularly at telophase. Bars, 5 µm.

View larger version (14K): [in a new window]   Fig. 4. Mitotic index in wild-type and mutant brains. The mitotic index was defined as the percentage of cells in mitosis. The number on each bar is the mitotic index and the total number of cells counted is shown in parentheses.

View larger version (72K): [in a new window]   Fig. 5. A variety of mitotic spindle defects and lagging chromosomes in sub1/sub131 mutants stained for DNA (blue) and microtubules (green). Compared with (A) wild-type metaphase, sub mutant metaphase spindles are bipolar but exhibit several defects including (B) frayed microtubules (B', tubulin channel shown with increased levels), (C) asymmetric half spindles and (D) poorly oriented half spindles or absent interpolar microtubules. Although wild-type anaphase (E) (E', DNA channel only) involves the uniform segregation of chromosomes to the poles, in sub mutant anaphases there was an increased frequency of lagging chromosomes (F). A synergistic effect on spindle assembly was observed during (G) metaphase and (H) anaphase in sub1/sub131; polo16-1/+ mutants or metaphase (I) and (J) anaphase spindles in sub1/sub131;Incenp/+ mutants. Bars, 5 µm.

View larger version (23K): [in a new window]   Fig. 6. Subito and Polo co-immunoprecipitate. (A) HA-tagged Subito was immunoprecipitated from mixed stage 0- to 2-hour-old embryos and metaphase-arrested oocytes and then used for western blotting. (B) A control experiment was performed on tissue that did not express the HA-tagged Subito. (C) This experiment was repeated using GFP-tagged Polo (Moutinho-Santos et al., 1999). HA-tagged Subito was immunoprecipitated from mixed stage 0- to 2-hour-old embryos. (D) In the control, using embryos not expressing HA-tagged Subito, GFP-Polo was not precipitated. The lower band is a nonspecific protein in the lysate.

View larger version (83K): [in a new window]   Fig. 7. Metaphase spreads in colchicine-treated swollen cells. (A) A normal karyotype in a wild-type cell. (B) A hyperploid cell in a sub1/sub131 mutant brain. (C) A polyploid cell in a Incenp3747 sub1 /+ sub131 brain. Bar, 5 µm.

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