Ki-67 is a PP1-interacting protein that organises the mitotic chromosome periphery
- University of Edinburgh, United Kingdom
- Riken Advanced Science Institute, Japan
- MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, United Kingdom
- Brunel University, United Kingdom
Figures
Figure 1 with 2 supplements
Ki-67 is evolutionary related to Repo-Man but shows distinct behaviour during mitosis.
(A1) Schematic representations of evolutionarily conserved regions in human Repo-Man and Ki-67 proteins (shown approximately to scale). (A2) E-values corresponding to global profile-to-sequence …
Figure 1—figure supplement 1
Characterisation of Ki-67 RNAi.
Two siRNAs against Ki-67 deplete the protein efficiently in HeLa cells. Top and middle panels show two different exposures of the same blot.
Figure 1—figure supplement 2
Distribution of PP1gamma in mitosis after the Ki-67 siRNA.
In cells depleted of Ki-67, PP1 is still recruited to the nucleolus in interphase (panels 4–4′) and to kinetochores in metaphase (panels 5–5′) but its levels are lower on anaphase chromosomes …
Figure 2 with 3 supplements
Ki-67 is required for targeting of nucleolar proteins to the chromosome periphery in mitosis.
(A) Localisation of endogenous nucleolin is aberrant in mitotic cells after Ki-67 depletion (panels 2, 3, 5). HeLa cells were transfected with Ki-67 RNAi oligo 5 (panels 2, 3, 5) or control oligos …
Figure 2—figure supplement 1
Distribution of nucleolin in mitosis following exposure of cells to different Ki-67 siRNA oligonucleotides with and without rescue by Ki-67 cDNA.
RNAi rescue experiment. Representative microscopy images for quantifications shown in Figure 2B. HeLa cells depleted of Ki-67 were transfected with either mCherry:Ki-67wt (panels 2, 5) or …
Figure 2—figure supplement 2
Distribution of nucleolin in mitosis following exposure of cells to different Ki-67 siRNA oligonucleotides.
HeLa cells were transfected with Ki-67 RNAi oligo 1, 2 or 5 or control oligos and stained for nucleolin. Nucleolin localisation was classified as for Figure 2B (diffuse, aberrant, and big foci) and …
Figure 2—figure supplement 3
Distribution of NIFK in mitosis following Ki-67 depletion.
NIFK T234 phosphorylation is regulated normally in the presence and absence of Ki-67. Hela cells were transfected with Ki-67 RNAi oligo 5 (panels 3, 4) or control oligos (panels 1, 2) and stained …
Figure 3 with 1 supplement
Ki-67 organizes the mitotic chromosome periphery.
(A) CLEM of HeLa cells transfected with GFP-PerP-C and control oligos (top panels) or Ki-67 oligos (bottom panels). Mitotic cells from control or Ki-67 RNAi with visible GFP aggregates (arrow) were …
Figure 3—figure supplement 1
Depletion of Ki-67 does not leave electron-dense nucleolar remnants in the cytoplasm.
CLEM of HeLa cells transfected with PERP-C and control oligos (left panels) or Ki-67 oligonucleotides (right panels). Mitotic cells from control or Ki-67 RNAi with visible GFP aggregates (green …
Figure 4 with 1 supplement
Ki-67 localisation is not dependant on other c-PERPS tested.
(A) Ki-67 localisation was analysed following the RNAi depletion of several novel c-PERPs (PERP B, D, and E). Following a 48 hr knock-down period, cells were fixed and labelled with anti-Ki67 …
Figure 4—figure supplement 1
Depletion of cPERPs by RNAi.
As no antibodies are available yet targeting these novel cPERPs, HeLa cells were transfected with GFP:PERP cDNA with or without the co-transfection of siRNA. Cells were harvested after 48 hr and …
Figure 5
Ki-67 is does not function to protect chromosomes from DNA damage or provide structural maintenance.
(A) Representative overview images of HeLa cells transfected with control or Ki-67 specific siRNA oligos probed with anti-53bp antibodies to assess levels of DNA damage. Scale bar 10 μm. (B) A bar …
Figure 6 with 1 supplement
Segregation of nucleolin.
(A) In cells depleted of Ki-67 the nucleolus never disassembles completely during mitosis and B23 never accumulates around the mitotic chromosomes. Time-lapse imaging of GFP:B23 in HeLa cells after …
Figure 6—figure supplement 1
Electron micrographs of interphase nuclei from Ki-67 RNAi cells.
Three major components of the nucleolus are recognised: Fibrillar centres (FC), dense fibrillar component (DFC), and granular component (GC). Scale bar 2 μm.
Figure 7 with 3 supplements
The nucleoli of Ki-67 depleted cells are fewer, smaller, and functionally repressed.
Normal cell, nucleus, and nucleolus size is compromised following depletion of Ki-67. (A) Representative images showing a reduced cell size and nucleolar size phenotype in Ki-67 depleted cells, …
Figure 7—figure supplement 1
Ki-67 depletion influences cell size, nuclei size, and nucleoli size.
A 2D scatter plot showing combined nuclei area on the Y axis, vs cell area on the X axis, for control (green) and Ki-67 depleted (red) cells. Each individual translucent dot represents one cell. …
Figure 7—figure supplement 2
Ki-67 depletion has only minor effects on the cell cycle detected by FACS.
(top) HeLa cells transfected with control or Ki-67 siRNA oligos were incubated for 48, 72, or 96 hr, before cell-cycle analysis using FACS. Gated cells were manually categorised into cell-cycle …
Figure 7—figure supplement 3
Ki-67 depletion influences nucleolar size.
A bar graph showing the mean cross-sectional area measurements of nucleoli from control and Ki-67 depleted cells. Nucleoli measurements are on a per cell basis and placed in ascending order, from …
Figure 8
Ki-67 depletion affects nuclear architecture.
(A) The position of a chromosome 13p (marked by a LacO array:LaciGFP) in HT1080 cells (1) was assessed in control and Ki67 RNAi experiments. 150 nuclei from three independent experiments were imaged …
