Spatial signals link exit from mitosis to spindle position
- Howard Hughes Medical Institute, Massachusetts Institute of Technology, United States
- Indiana University, United States
- The University of North Carolina at Chapel Hill, United States
Figures
Figure 1
A system to induce spindle misposition.
(A) Zone model of exit from mitosis. Yeast cells are partitioned into two zones: a MEN inhibitory zone in the mother cell compartment (red) and a MEN activating zone in the bud cell compartment …
Figure 2
Analysis of cytoplasmic microtubules in the bud neck.
Cells harboring osTIR1 DYN-AID kar9Δ and expressing GFP-labeled tubulin (A35707) were grown and imaged as descried in Figure 1E–I. A table summarizing cMT-bud neck contact for cells that contained a …
Figure 3
cMT laser ablation does not promote exit from mitosis in cells with mispositioned spindles.
(A-B) Cells harboring the osTIR1 DYN-AID kar9Δ constructs that also expressed GFP-tagged α-tubulin, mCherry-tagged Cdc3 and an NLS-mCherry (A35143) were grown overnight to mid-log in YEPD at 25°C. …
Figure 4 with 4 supplements
Cdc14 Release from the nucleolus precedes cMT retraction in cells that inappropriately breakdown their anaphase spindles in the mother cell compartment.
(A–D) A diploid strain (A37463) homozygous for osTIR1 dyn1-AID kar9Δ and heterozygous for GFP-Tub1 and Cdc14-tdTomato was grown to midlog in Synthetic Complete medium. Cycling cells were imaged on a …
Figure 4—figure supplement 1
Analysis of the CDC14-tdTomato allele.
(A) Cells homozygous for osTIR1 dyn1-AID kar9Δ and heterozygous for either GFP-Tub1 (A38232) or GFP-Tub1 and Cdc14-td Tomato (A37464) were grown overnight in YPD to mid-log and then loaded onto a …
Figure 4—figure supplement 2
Analysis of Cdc14 Release in cells with mis-positioned spindles.
The diploid strain (A37463) homozygous for osTIR1 dyn1-AID kar9Δ and heterozygous for GFP-Tub1 and Cdc14-tdTomato was grown to midlog in synthetic complete medium. Cycling cells were imaged as in Fig…
Figure 4—figure supplement 3
Montage of Cdc14 Release in cells with mis-positioned spindles containing the nucleolar marker Cfi1.
The diploid strain (A38233) homozygous for osTIR1 dyn1-AID and kar9Δ and heterozygous for GFP-Tub1, Cfi1-GFP and Cdc14-tdTomato was grown overnight in synthetic complete medium and then imaged as in …
Figure 4—figure supplement 4
Montage of Cdc14 Release in cells with mis-positioned spindles containing the nucleolar marker Cfi1.
The diploid strain (A38233) homozygous for osTIR1 dyn1-AID and kar9Δ and heterozygous for GFP-Tub1, Cfi1-GFP and Cdc14-tdTomato was grown overnight in synthetic complete medium and then imaged as in …
Figure 5
Inhibition of Cdc14, the MEN and the FEAR Network prevents Spindle Breakdown in the mother cell compartment.
(A) osTIR1 dyn1-AID kar9Δ (A35707) and osTIR1 dyn1-AID kar9Δ cdc14-1-AID (A37895) cells harboring GFP-tagged α-tubulin were grown as described in Figure 1E–I. Cells were monitored by live cell …
Figure 6
Analysis of Exit from Mitosis in prm3Δ heterokaryons.
(A) Cartoon of the prm3Δ heterokaryon system showing the two main classes of heterokaryons obtained. (B–F) Heterokaryons were obtained by mating cells lacking PRM3 (see Materials and methods for …
Figure 7
Exit from Mitosis in a prm3Δ heterokaryon with one aligned anaphase spindle and one metaphase spindle.
Montage of binucleate zygotes created by mating homozygous for prm3Δ, osTIR1, DYN-AID, kar9Δ and heterozygous for mCherry-labeled Cdc3 and GFP-labeled α-tubulin (A35570 x A35571). The montage …
Figure 8
Analysis of Exit from Mitosis in swe1Δ heterokaryons.
(A) Cartoon of swe1Δ return to growth heterokaryon system and depictions of cell type classes that were analyzed. Briefly, diploid cells lacking SWE1, also harboring the meiotic prophase marker …
Videos
Video 1
A cell with a mispositioned spindle that has a cMT in the bud neck.
osTIR1 DYN-AID kar9Δ cells expressing GFP-labeled α-tubulin (A35707) were grown and imaged as described in Figure 1E-I. Depicted is a cell that mis-positions its spindle and displays cMTs that …
Video 2
A cell with a mispositioned spindle that inappropriately exits mitosis in the mother cell compartment.
osTIR1 DYN-AID kar9Δ cells expressing GFP-labeled α-tubulin (A35707) were grown and imaged as described in Figure 1E-I. Depicted is a cell that mis-positions its spindle and then inappropriately …
Video 3
A cell with a mispositioned spindle that inappropriately exits mitosis but does not complete spindle disassembly or cytokinesis.
osTIR1 DYN-AID kar9Δ cells expressing GFP-labeled α-tubulin (A35707) were grown and imaged as described in Figure 1E-I. Depicted is a cell that mispositions its spindle and then inappropriately …
Video 4
A cell with a mispositioned spindle that lacks cMT-bud neck interactions but arrests in anaphase.
osTIR1 DYN-AID kar9Δ cells expressing GFP-labeled α-tubulin (A35707) were grown and imaged as described in Figure 1E-I. Depicted is a cell that arrests in late anaphase with a mispositioned spindle. …
Additional files
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Supplementary file 1
Yeast Strain Table.
The strains listed in Supplementary file 1 are all derivatives of W303. The table shows the relevant genotype for each yeast strain and the associated strain number.
- https://doi.org/10.7554/eLife.14036.019
