Kinetochore protein depletion underlies cytokinesis failure and somatic polyploidization in the moss Physcomitrella patens
- Nagoya University, Japan
Figures
Figure 1 with 7 supplements
Unconventional localization of kinetochore proteins in P. patens.
(A) Live imaging of P. patens caulonemal apical cells expressing mCherry-tubulin and selected kinetochore proteins: Citrine-CENP-A; Citrine-CENP-C; Citrine-CENP-S; KNL1-Citrine; Ndc80-Citrine and …
Figure 1—figure supplement 1
Summary of kinetochore protein tagging and disruption/knockdown in P. patens.
(Left) Maximum-likelihood phylogenetic trees of conserved centromere/kinetochore proteins in Physcomitrella patens, Arabidopsis thaliana and Homo sapiens. Numbers represent bootstrapping values …
Figure 1—figure supplement 2
Localization of CENP-A and KNL2/MIS18BP1 during cell division.
Live imaging of P. patens protonemal apical cells expressing mCherry-tubulin (magenta) and Citrine-CENP-A (A) or KNL2-Citrine (B). Citrine-CENP-A data is an expanded version of Figure 1. …
Figure 1—figure supplement 3
Localization of CCAN proteins during cell division.
Live imaging of P. patens protonemal apical cells expressing mCherry-tubulin (magenta) and Citrine-tagged (green) CENP-C (A), CENP-O (B), CENP-X (C), CENP-S (D) and CENP-S-like protein Taf9 (E). …
Figure 1—figure supplement 4
CENP-C is not a constitutive centromeric protein in P. patens.
Citrine-CENP-A (A) and Citrine-CENP-C (B) localization starting from NEBD. At each time point, 10 z-sections were acquired (separated by 1 µm). Merged images of mCherry-tubulin (single focal plane) …
Figure 1—figure supplement 5
Localization of Mis12, Nnf1 and KNL1 during cell division.
Live imaging of P. patens protonemal apical cells expressing GFP-tubulin and Mis12-mCherry (A) or mCherry-tubulin and Nnf1-Citrine(B) or KNL1-Citrine (C) KNL1-Citrine data is an expanded version of F…
Figure 1—figure supplement 6
Localization of outer kinetochore proteins during cell division.
Live imaging in P. patens protonemal apical cells expressing mCherry-tubulin (magenta) and Citrine-tagged (green) Ndc80 (A), Nuf2 (B), Spc25 (C), SKA1 (D) and SKA2 (E). Ndc80-Citrine and …
Figure 1—figure supplement 7
Localization of CPC and SAC proteins during cell division.
Live imaging of P. patens protonemal apical cells expressing mCherry-tubulin (magenta) and Citrine-tagged (green) Borealin (A), Mps1 (B), BubR1(C) and Mad2 (D, E). Red arrowheads indicate punctate …
Figure 2 with 2 supplements
Lagging chromosomes in anaphase induce cytokinesis failure.
(A, B) Lagging chromosomes (yellow arrowheads) present for several minutes in the midzone between separated chromatids cause cytokinesis failure in CENP-A, CENP-X and SKA1 RNAi lines. GH represents …
-
Figure 2—source data 1
Dataset used for Fisher’s test in Figure 2C.
- https://doi.org/10.7554/eLife.43652.018
Figure 2—figure supplement 1
Chromosome segregation defects following depletion of CENP-A, CENP-X or SKA1.
(A) Representative mitotic progression and chromosome missegregation caused by depletion of CENP-A, CENP-X or SKA1. ‘GH’ is the control line. Bar, 5 µm. (B) Duration of mitosis (from NEBD to …
Figure 2—figure supplement 2
Rescue of RNAi phenotypes by ectopic expression of SKA1-Cerulean or CENP-X-Cerulean.
Live imaging of P. patens protonemal apical cells expressing SKA1-Cerulean (A) or CENP-X-Cerulean (C) in the SKA1 5’UTR RNAi or CENP-X 5’UTR RNAi lines, respectively. RNAi was induced by addition of …
Figure 3
Cytokinesis failure in somatic cells can generate plants with whole-genome duplication.
(A) Quantification of the nuclear DNA content. Anaphase/telophase cells were used as a standard for 1N nuclei (light blue). Interphase cells randomly selected in the control line mostly had double …
Videos
Video 1
Localization of the centromere and CCAN proteins during cell division.
Live-cell imaging was conducted in P. patens protonemal cells expressing mCherry-tubulin (magenta) and one of the following tagged proteins(green): Citrine-CENP-A, KNL2-Citrine, Citrine-CENP-C, …
Video 2
Transient disappearance of CENP-C from the kinetochore after cell division.
Live-cell imaging was conducted in P. patens protonemal cells expressing mCherry-tubulin (magenta) and one of the following tagged proteins (green): Citrine-CENP-A, Citrine-CENP-C and KNL2-Citrine. …
Video 3
Localization of the C-termini tagged CENP-C and CENP-O.
Live-cell imaging was conducted in P. patens protonemal cells expressing mCherry-tubulin (magenta) and one of the following tagged proteins(green):CENP-C-Citrine and CENP-O-Citrine. Images are …
Video 4
Localization of the Mis12, KNL1, Nuf2 and SKA1 during cell division.
Live-cell imaging was conducted in P. patens protonemal cells expressing mCherry-tubulin or GFP-tubulin (magenta) and one of the following tagged proteins: Mis12-mCherry, KNL1-Citrine, Nuf2-Citrine …
Video 5
Mitotic defects observed in RNAi lines targeting CENP-C, Nnf1, Nuf2 and KNL1.
Representative images of mitotic progression and defects caused by depletion of four kinetochore proteins. White boxes indicate normal cell division in the control line (GH). White arrowheads show …
Video 6
Chromosome missegregation after RNAi.
Representative images of mitotic progression and chromosome missegregation caused by depletion of CENP-A or CENP-X or SKA1. RNAi was induced by addition of β-estradiol to the growth medium at final …
Video 7
Cytokinesis defect associated with lagging chromosomes in anaphase.
Representative images of correlation between lagging chromosomes and cytokinesis defect in CENP-A exon RNAi and SKA1 5’UTR RNAi lines. Note that minor lagging chromosomes observed in the midzone for …
Video 8
Visualization of the cell plate formation using FM4-64 dye.
Representative images of cytokinesis in the control GH line (upper row), SKA1 5’UTR RNAi line with minor lagging chromosomes (middle row), and with persistent lagging chromosomes (bottom row). Cell …
Video 9
Mitotic entry of the multi-nucleated cell in P. patens.
SKA1 5’UTR RNAi was induced by addition of β-estradiol to the growth medium at final concentration of 5 µM, 5–6 days prior to observation. Multi-nucleated cells resulting from cytokinesis failure …
Additional files
-
Supplementary file 1
Physcomitrella patens transgenic lines generated in this study.
- https://doi.org/10.7554/eLife.43652.025
-
Supplementary file 2
Plasmids and primers used in this study.
- https://doi.org/10.7554/eLife.43652.026
-
Supplementary file 3
Protein alignments used for the phylogeny analysis.
- https://doi.org/10.7554/eLife.43652.027
-
Transparent reporting form
- https://doi.org/10.7554/eLife.43652.028
