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First published online 15 July 2008
doi: 10.1242/jcs.022996

Journal of Cell Science 121, 2529-2539 (2008)
Published by The Company of Biologists 2008
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Slk19-dependent mid-anaphase pause in kinesin-5-mutated cells

Natalia Movshovich1, Vladimir Fridman2, Adina Gerson-Gurwitz1, Inbal Shumacher1, Irena Gertsberg2, Alexander Fich3,5, M. Andrew Hoyt6, Benjamin Katz2 and Larisa Gheber1,2,4,*

1 Department of Chemistry, Ben-Gurion University of the Negev
2 Department of Clinical Biochemistry, Ben-Gurion University of the Negev
3 Department of Gastroenterology, Ben-Gurion University of the Negev
4 The Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev
5 Soroka Medical Center, Beer-Sheva, Israel
6 Department of Biology, Johns Hopkins University, Baltimore, MD, USA


Figure 1
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  		Fig. 1. Real-time measurement of anaphase kinetics. (A,B) Representative time-lapse sequences (2D projection, combined DIC and green-fluorescence images) of spindle elongation followed by Nuf2-GFP fluorescence. The different anaphase-B phases (fast phase, pause phase and slow phase) are indicated at the bottom of the frames. (A) kip1{Delta} cells; (B) kip1{Delta} cin8-FA cells. The time at which each frame was taken is indicated. Scale bar: 2 µm. (C) Representative real-time spindle-elongation kinetics in cells expressing kinesin-5 mutants. Cells were arrested in S-phase of the cell cycle for 2 hours and then released from arrest. The distance between spindle pole bodies (SPBs), monitored by Nuf2-GFP fluorescence, was measured as a function of time. Time of initiation of spindle elongation was assigned as 0 minutes. `R' represents the rate of spindle elongation. The different anaphase-B phases are indicated as follows: fast phase, diamond; pause, square; slow phase, triangle; post-anaphase spindle elongation or spindle collapse, circle. The genotypes are indicated at the bottom of each plot: (a) wild type; (b) kip1{Delta}; (c) cin8-FA; (d,e) kip1{Delta} cin8-FA.

 

Figure 2
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  		Fig. 2. (A-D) Anaphase spindle morphologies in WT (A), kip1{Delta} (B) cin8-FA (C) and kip1{Delta} cin8-FA (D) cells. Cells expressing tubulin-GFP ware examined by high-resolution fluorescence microscopy (see Materials and Methods). Representative 2D images are shown. Spindle length (µm) is indicated at the bottom of each spindle image. Normal, asymmetric and indistinguishable midzone (ind. midzone) morphologies are indicated at the top. Arrows indicate the location of the midzone region. (E) Percentage of spindles with defects. Left, percentage of intermediate (3-5 µm) asymmetric spindles; right, percentage of anaphase spindles with an indistinguishable midzone region. For each genotype, 80-120 anaphase (>3 µm) spindles were analyzed, half of which were of intermediate length. (F,G) Spindle localization of Ase1 and Slk19 proteins. (G) Images of unsynchronized cells expressing either Ase1-GFP (left) or Slk19-GFP (right) were acquired during anaphase. Representative images (2D projection) of spindles are shown. SPBs are labelled with Nuf2-GFP. Spindle length (µm) is indicated on the bottom of each image. (F) Length of Ase1-GFP signal (left) and Slk19-GFP signal (right), expressed as a percentage of spindle length in intermediate (3-5 µm) and long (>5 µm) spindles. Average and s.d. of 25-35 measurements are shown. Genotypes are as indicated: kip1{Delta}, white; kip1{Delta} cin8-FA, grey.

 

Figure 3
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  		Fig. 3. Effect of SLK19 or SPO12 chromosomal deletion on spindle and nuclear morphology (A), and on cell viability (B). (A) Cells were arrested in S-phase of the cell cycle by hydroxyurea, and were processed for DNA and tubulin immunostaining following release. Representative DNA (left) and spindle (right) morphologies of cells with intermediate or long (4-7 µm) spindles are shown. Genotypes are indicated at the bottom of each panel. Scale bar: 4 µm. (B) Cells were grown overnight, diluted to equal density, plated in serial dilutions and grown at 26°C (left) or 33°C (right) for 3 days. Genotypes are indicated on the left.

 

Figure 4
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  		Fig. 4. Scc1 cleavage as a landmark of anaphase onset. Cells expressing Scc1-HA were arrested in S-phase of the cell cycle by hydroxyurea then processed for tubulin immunostaining and western blot analysis (anti-HA) following release. The Scc1 band (asterisk) and cleavage-product band (arrowhead) are shown. Genotypes are indicated on the top of each panel. The time after release from S-phase arrest are indicated at the top of each lane. The percentage of cells with elongated anaphase spindles (>2.5 µm) are indicated at the bottom of each lane (% ana). Results are representative of three independent experiments. In each experiment, tubulin morphology of 150-200 anaphase cells was examined.

 

Figure 5
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  		Fig. 5. (A) Effect of the overexpression of Cdc20 on spindle elongation kinetics in kip1{Delta} cin8-FA cells. Cells either carrying plasmid containing CDC20 under the control of the MET25 promoter (PMET->CDC20; a,b) or vector only (c,d) were grown to log phase in medium containing methionine. To induce Cdc20 overexpression, cells were placed into methionine-lacking medium (– methionine; b,d). The distance between SPBs, monitored by Nuf2-GFP fluorescence, was measured as a function of time. `R' represents the rate of spindle elongation. The different anaphase-B phases are indicated by the following symbols: fast phase, diamond; pause, square; slow phase, triangle; prior to spindle elongation, post-anaphase spindle elongation or spindle collapse, circle. (B) Representative anaphase DNA morphologies in kip1{Delta} cin8-FA cells overexpressing Cdc20. Normal and deformed DNA morphologies are shown. Arrows point to lagging DNA in cells with two DNA masses separated by more than 5 µm. Scale bar: 5 µm. (C) Percentage of cells with all DNA deformations (left) and percentage of cells with two deformed DNA masses separated by more than 5 µm (right) in anaphase cells. In each genotype, indicated at the bottom, 200-220 cells were analyzed.

 

Figure 6
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  		Fig. 6. A schematic of a possible mechanism for Cin8-mediated anaphase midzone centering. SPBs, iMTs and their plus (+) and minus (–) ends, Cin8, midzone regions, and the regions of iMT plus-end dynamics (double-headed arrows) are indicated. (A) The WT spindle is shown, in which the normal MT-crosslinking function of Cin8 organizes the midzone MTs in a tight bundle. This tight midzone organization restricts the region of iMT plus-end dynamics to a small portion of the iMT length. (B) The Cin8-FA spindle is shown, in which the impaired MT-crosslinking function of Cin8 releases the midzone bundle, allows for the iMTs to be exchangeable throughout a larger portion of their length and uncouples the iMT plus-end dynamics of the two halves of the spindle. This can result in an asymmetric midzone location (bottom).

 

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© The Company of Biologists Ltd 2008