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First published online 22 January 2008
doi: 10.1242/jcs.015172

Journal of Cell Science 121, 466-476 (2008)
Published by The Company of Biologists 2008
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Dynein-dependent nuclear dynamics affect morphogenesis in Candida albicans by means of the Bub2p spindle checkpoint

Kenneth R. Finley1, Kelly J. Bouchonville1, Aaron Quick1 and Judith Berman1,2,*

1 Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, Minnesota, 6-160 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455, USA
2 Department of Microbiology, University of Minnesota, Minneapolis, Minnesota, 6-160 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455, USA


Figure 1
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  		Fig. 1. Nuclear dynamics in dyn1{Delta}/{Delta} hyphae. Time-lapse microscopy was performed as described in Materials and Methods. Images were acquired at 5-minute intervals. Time, minutes. (A) Spindle dynamics in wild-type hyphae. The bipolar spindle moves into the germ tube (56 minutes) before anaphase (101 minutes). The septum becomes evident after spindle disassembly (arrow, 114 minutes). (B) In a dyn1{Delta}/{Delta} hypha, a bipolar spindle moves into the germ tube (47 minutes) and reaches the presumptum (96 minutes) before anaphase (106 minutes). The septum is evident after spindle disassembly (arrow, 121 minutes). (C) Spindle migration fails in a dyn1{Delta}/{Delta} hypha. Anaphase begins with the bipolar spindle in the basal cell (109 minutes). The dSPB does not cross the presumptum before septation (167 minutes). After septation, the hyphal tip swelled slightly before elongation arrested. Arrowheads, SPBs after septation. Bar, 10 µm.

 

Figure 2
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  		Fig. 2. MT dynamics during spindle movement in dyn1{Delta}/{Delta} hyphae. (A) A bipolar spindle in a wild-type hypha migrates into the germ tube (angled line) even as the dSPB-bound MT grows longer (arrowheads). Time, seconds. (B) A bipolar spindle in a dyn1{Delta}/{Delta} hypha remains at the bud neck (straight line) despite repeated MT growth, shrinkage and interaction with the cortex. Arrows, SPBs; arrowheads, MT plus-ends; time, seconds. (C) Bipolar spindle movement captured in a dyn1{Delta}/{Delta} hypha at 15-second intervals. Time, minutes. Bar, 10 µm. (D) An example of spindle position (y-axis) plotted against time (x-axis) used to indirectly calculate rates of spindle movement in dyn1{Delta}/{Delta} hyphae (black triangles). An example of spindle position versus time in a wild-type hypha is also shown for comparison (grey squares).

 

Figure 3
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  		Fig. 3. Dynein contributes to nuclear migration during cell cycle arrest. (A) Wild-type (YJB6284) and dyn1{Delta}/{Delta} (YJB7812) strains were grown to stationary phase overnight in YPD at 30°C. They were then diluted 1:100 into fresh YPD containing 100 mM HU and grown at 30°C for 6 hours before being fixed with ethanol and stained with DAPI for imaging. Bar, 10 µm. (B) Scatter plot of nuclear position with respect to the bud tip (y-axis) plotted against bud length (x-axis). Strains: wild-type, BWP17; dyn1{Delta}/{Delta}, YJB7812.

 

Figure 4
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  		Fig. 4. Colony and cellular morphology of dynein-dynactin mutants. (A-C) Colony morphology of wild-type (A), dyn1{Delta}/{Delta} (B) and nip100{Delta}/{Delta} (C) colonies after 2 days of growth on YPD-agar at 30°C. (D-F) Cellular morphology of wild-type (D), dyn1{Delta}/{Delta} (E) and nip100{Delta}/{Delta} (F) cells diluted to OD600=1.0 in YPD liquid medium and grown at 30°C for 4 hours. Cells were collected, fixed with ethanol and stained with DAPI for simultaneous DIC-DAPI microscopy. Arrowheads, yeast cells with a single nucleus; arrows, binucleate cells; carets, anucleate cells. Bar, 10 µm.

 

Figure 5
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  		Fig. 5. Nuclear dynamics in wild-type and dyn1{Delta}/{Delta} yeast and pseudohyphae. Time-lapse microscopy images were acquired at ~5-minute intervals. Time, minutes. (A) In wild-type yeast cells, segregation of the daughter nucleus (Nop1) occurs within 5 minutes of nuclear segregation (caret, 42 minutes). During anaphase, the spindle is oriented parallel to the mother-bud axis (Tub2, 56-66 minutes). The dSPB penetrates the bud within 5 minutes of onset of anaphase (61 minutes). Septation, which is detected as a darkening of the bud neck (arrows), occurs after nuclear segregation (Nop1, 52 minutes) and spindle disassembly (Tub2, 76 minutes). (B) In wild-type pseudohyphae grown at 35°C, nuclear dynamics observed with Nop1-YFP are similar to those of yeast cells. Tub2-GFP is not shown because of a temperature-sensitive phenotype at 35°C. (C) In dyn1{Delta}/{Delta} yeast cells, mother cells are transiently binucleate (Nop1, 46 minutes, asterisk). Bipolar spindles are positioned away from the bud neck (Tub2, 40 minutes) and oriented oblique to the mother-bud axis during anaphase (45-50 minutes). The spindle remains intact until the dSPB penetrates the bud (60 minutes). Septation occurs after nuclear segregation (Nop1 arrow, 78 minutes) and spindle disassembly (Tub2, 80 minutes, arrow). (D) In dyn1{Delta}/{Delta} pseudohyphal cells, undivided nuclei (Nop1, 103 minutes) and bipolar spindles (Tub2, 95 minutes) migrate into the elongating bud. Nuclei separate (Nop1, 157 minutes) as the spindle elongates entirely within the bud (Tub2, 100 minutes). The spindle disassembles without the mSPB returning to the mother cell (Tub2, 115 minutes). Septation occurs before the mother nucleus returns to the mother cell (Nop1, 182 minutes). Arrowheads, SPBs; bar, 10 µm. Strains: (A) wild-type Nop1-YFP, YJB7062; Tub2-GFP YJB8953; (B) wild-type Nop1-YFP, YJB7062; (C,D) dyn1{Delta}/{Delta} Nop1-YFP, YJB9430; dyn1{Delta}/{Delta} Tub2-GFP, YJB8973.

 

Figure 6
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  		Fig. 6. Bub2p affects cell cycle progression and morphogenesis in dynein-depleted yeast cells. (A) Immunoblots of PMET3-GFP-Dyn1p expression (left) or repression (right). Strains YJB6755 (PMET3-GFP-CDC3/CDC3, lanes 1 and 4), YJB7944 (DYN1-GFP/DYN1, lanes 2 and 5) and YJB8046 (PMET3-GFP-DYN1/dyn1{Delta}, lanes 3 and 6) were grown overnight in PMET3 ON conditions, diluted to OD600=0.01 in fresh SDC medium containing 10 mM methionine and 2 mM cysteine (PMET3 OFF) or lacking methionine and cysteine (PMET3 ON) and incubated at 30°C for 14 hours. Immunoblots were probed with antibodies against GFP or histone H4, as described in Materials and Methods. Dyn1-GFP in YJB7944 was not detectable under these conditions, presumably owing to low expression from the native promoter; it was detectable by microscopy (supplementary material Fig. S1). (B) Bub2p monitors two steps of anaphase in dynein-depleted cells. The strains indicated were grown as in A. The number of DAPI-stained nuclei per cell is plotted as a histogram at the side of each micrograph. Strains: wild-type, BWP17; bub2{Delta}/{Delta}, CB432; PMET3-DYN1/dyn1{Delta}, YJB8039; PMET3-DYN1/dyn1{Delta} bub2{Delta}/{Delta}, YJB9993. (C) Bub2p inhibits disassembly of the mitotic spindle in dynein-deficient cells. Strain YJB10232 (PMET3-DYN1/{Delta} bub2{Delta}/{Delta} TUB2-GFP:NAT1) was cultured as in A. When PMET3-DYN1 was expressed (left), mitotic spindles remained intact until after the dSPB penetrated the bud; when PMET3-DYN1 was repressed (right), mitotic spindles disassembled prematurely. DIC and fluorescent images were merged in B and C. Bars, 5 µm.

 

Figure 7
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  		Fig. 7. Bub2p mediates cell cycle progression and morphogenesis in dynein-depleted hyphae. Strains were grown to stationary phase overnight at 30°C in either PMET3 ON or PMET3 OFF conditions and then diluted 1:20 into the same medium containing 10% serum and prewarmed to 37°C. They were grown at 37°C for 2 hours, fixed with ethanol and stained with DAPI. Strains: wild-type, YJB6284 (Bensen et al., 2002Go), PMET3-DYN1/dyn1{Delta}, YJB10155, bub2{Delta}/{Delta}, CB432 (Bachewich et al., 2005Go), PMET3-DYN1/dyn1{Delta} bub2{Delta}/{Delta}, YJB9993.

 

Figure 8
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  		Fig. 8. Model for the role of C. albicans Bub2p in regulating nuclear position. (A) In unperturbed wild-type cells, the bipolar spindle (yellow bar) migrates to the bud neck (orange) and elongates across the bud neck. After the dSPB/daughter nucleus (blue) resides in the bud, the spindle disassembles (dashed yellow line) and septation ensues (red). (B) Yeast cells lacking dynein-dynactin function undergo anaphase within the mother cell. Bub2p inhibits spindle disassembly and septation until the dSPB/daughter nucleus has moved into the bud. (C) Bub2p inhibits onset of anaphase and stimulates polarized growth in dyn1{Delta}/{Delta} cells. In pseudohyphal-like cells lacking dynein, the bipolar spindle migrates into the elongating bud in a dynein-independent, bud length-dependent, fashion that might be mediated by interactions between the spindle and the bud tip. Spindle elongation eventually ensues. Spindle disassembly is not inhibited because the dSPB is already within the bud. Septation can occur before the mSPB/mother nucleus returns to the mother cell because no checkpoint is activated.

 

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© The Company of Biologists Ltd 2008