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First published online April 3, 2008
doi: 10.1242/10.1242/jcs.017210

Journal of Cell Science 121, 1284-1292 (2008)
Published by The Company of Biologists 2008
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Mob4 plays a role in spindle focusing in Drosophila S2 cells

Matthew A. Trammell1,*, Nicole M. Mahoney2,*, David A. Agard1 and Ronald D. Vale2,{ddagger}

1 The Howard Hughes Medical Institute and the Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94107, USA
2 The Howard Hughes Medical Institute and the Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94107, USA


Figure 1
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  		Fig. 1. Mitotic phenotypes observed after RNAi knockdown of Drosophila Mob4 (Dmob4) in S2 cells. Cells were treated with dsRNA and were fixed and stained with antibodies against tubulin (red) and with DAPI (blue) at day 4 (see Materials and Methods). (A) Untreated cells display bipolar spindles with well-defined centrosomes. (B) Mob4 RNAi cells show abnormally high numbers of monopolar spindles (32%, data not shown) as well as monastral bipolar spindles with splayed kinetochore minus ends (41%, data not shown). This latter defect resembled the frayed spindles that formed after Asp RNAi (D). Bar, 10 µm (A-D). Live imaging of GFP-tubulin revealed further details of the defects in spindle formation arising from Mob4 depletion (E-G). (E) Control cell (no RNA) with two centrosomes forming a bipolar spindle with spindle fibers organized normally at the poles. Time (in minutes and seconds) from the first frame is indicated in the lower left corner of each panel. The rightmost panel shows a cell in early anaphase (same in F and G), indicating the time of anaphase onset. (Full time-lapse movie available in supplementary material Movie 1.) (F) This Mob4-RNAi-treated cell initially formed a monopolar spindle that then converted to a monastral bipolar spindle. The acentrosomal pole on the right never becomes organized, and the left pole loses focus when the centrosome and K fibers begin to dissociate in prometaphase (4th panel). (Full time-lapse movie available in supplementary material Movie 2.) (G) Mob4-RNAi-treated cell with two centrosomes forming a bipolar spindle that becomes unorganized at both poles owing to detachment of K fibers from the centrosome and splaying during prometaphase/metaphase. (Full time-lapse movie available in supplementary material Movie 3.) Anaphase onset in Mob4-depleted cells occurs within the same time range observed in control cells. The K fiber splaying observed after Mob4 depletion resembles that described in live-cell imaging of cells depleted of the Asp protein (data not shown). Bar, 5 µm.

 

Figure 2
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  		Fig. 2. Drosophila Mob4 (Dmob4) has a minor roll in the maintenance of centrosome number and functions in spindle pole organization in the absence of centrosomes. (A) Bars indicate the percentage of mitotic cells with 0, 1, 2, 3, 4 or >=5 centrosomes (Dgrip84-stained foci that nucleate astral microtubules) in untreated cells (tan; n=401) or cells treated with RNAi to Mob4 (green; n=408) or SAK polo-like kinase (blue; n=400). (Error bars indicate s.d. of mean value of two independent experiments.) Depletion of Mob4 increased the proportion of single-centrosome cells relative to untreated cells but did not generate a significant population of acentrosomal cells, as did RNAi of SAK. (B) RNAi of SAK led to loss of centrosomes and astral microtubules from spindle poles, and co-depletion of Mob4 with SAK noticeably exacerbated spindle fiber unfocusing at acentrosomal poles (right). ({alpha}-tubulin, red; Dgrip84, green; DNA, blue; bar, 5 µm.) (C) Histogram showing the distribution of spindle pole focal breadth (yellow line; the distance between the minus ends of the outermost K fibers) at acentrosomal poles in SAK-depleted cells (blue) and in cells depleted of both Mob4 and SAK by double RNAi (red). The mean focal breadth in Mob4-SAK co-depleted cells was 5.0±2.4 µm (n=246), whereas that in cells depleted of SAK alone was 1.9±1.4 µm (n=261; P<0.0001). (Error bars indicate s.d. of mean value of two independent experiments.)

 

Figure 3
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  		Fig. 3. Double RNAi experiments suggest that the Drosophila Mob4 (Dmob4) phenotype is more similar to Asp than dynein or Ncd. (A) Representative mitotic spindle morphology after RNAi of the genes indicated. ({alpha}-tubulin, red; Dgrip84, green; DNA, blue; bar, 5 µm.) (B) Quantitation of K fiber unfocusing in metaphase spindles. The relative mean width of K fiber minus ends – the distance between the minus ends of the outermost K fibers at each pole (blue line in inset) relative to control cells [average 2.08±0.03 µm (mean±s.d.)] – is shown after RNAi of the gene(s) indicated. RNAi of Mob4, Asp or Ncd induced significant increases in the focal width of K fibers relative to that of control cells, whereas Dhc64C had a minimal effect. Co-depletion of Dhc64C and Ncd had a strong synergistic effect, whereas the synergism in cells co-depleted of Dhc64C and either Asp or Mob4 was minimal. [Error bars indicate the s.d. of the average K fiber unfocusing distance measured in three independent experiments (n>45 spindles for each trial).] (C) Quantitation of centrosome detachment in the same spindles measured in (B). The distance of the gap between the centrosome (Dgrip84-staining foci that nucleate astral microtubules) and the minus end of the K fiber lying closest to the centrosome (green line in inset), relative to control cells [average 1.92±0.07 µm (mean± s.d)] is shown. RNAi of Mob4, Asp or Dhc64C, but not Ncd, caused an increase in centrosome detachment (statistically significant difference for Mob4 RNAi and control cells; P<0.0001). Co-depletion of Dhc64C and either Asp or Mob4 produced a synergistic increase in centrosome detachment, whereas no synergism was observed for co-depletion of Dhc64C and Ncd.

 

Figure 4
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  		Fig. 4. Drosophila Mob4 (Dmob4) is not required for proper localization of Asp. Asp (green) immunolocalizes to centrosomes (indicated by microtubule asters at the poles) and K fiber minus ends (stretching between arrowheads) in controls cells with organized poles (upper panels) as well as in cells depleted of Mob4 (middle panels) or Ncd (lower panels) by RNAi treatment. ({alpha}-tubulin, red; DNA, blue; bar, 5 µm.)

 

Figure 5
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  		Fig. 5. Drosophila Mob4 fused to GFP (Dmob4-GFP) accumulates at mitotic spindle poles and kinetochores. (A) In interphase cells, Mob4-GFP (green in A-D) localizes primarily to the nucleus. ({alpha}-tubulin, red; DNA, blue for A and B.) (B) Mob4-GFP is diffuse throughout the cytoplasm in mitotic cells but accumulates both at spindle poles and at punctate spots immediately adjacent to condensed chromosomes (arrows in Mob4-GFP panel). (C) Mob4-GFP colocalizes with {gamma}-tubulin staining (red) at the centrosome. (DNA, blue.) (D) Mob4-GFP foci adjacent to condensed chromosomes (arrows in Mob4-GFP panel) coincide with immunostaining for the fly CENP-A homolog CID (red). The spindle is oriented diagonally from upper left to lower right. The inset shows a magnified image of the chromosome pair at the upper left in the main panels, taken in an alternative Z section. Bar, 5 µm (2 µm for inset in D). (One CID pair does not appear to have a matching GFP-Mob4 pair because the fluorescent intensity of Rhodamine-stained CID was substantially larger than the GFP signal and bled onto focal planes in which the GFP signal was not visible.)

 

Figure 6
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  		Fig. 6. Time-lapse imaging of mitosis in cells expressing Drosophila Mob4 fused to GFP (Dmob4-GFP). Pairs of GFP foci indicating kinetochores are visible on both mono-oriented chromosomes (left two panels) and on chromosomes aligned at the metaphase plate. In some cases (such as the cell shown), kinetochore fluorescence of mono-oriented chromosomes was stronger before alignment at the metaphase plate than after, but otherwise GFP fluorescence at spindle poles and kinetochores is present throughout mitosis. The time in minutes and seconds from beginning of image acquisition is indicated at the lower right of each panel. Bar, 5 µm. The full time-lapse movie is shown in supplementary material Movie 5.

 

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© The Company of Biologists Ltd 2008