Prolonged overexpression of PLK4 leads to formation of centriole rosette clusters that are connected via canonical centrosome linker proteins

Abstract

Centrosome amplification is a hallmark of cancer and PLK4 is one of the responsible factors for cancer associated centrosome amplification. Increased PLK4 levels was also shown to contribute to generation of cells with centriole amplification in mammalian tissues as olfactory neuron progenitor cells. PLK4 overexpression generates centriole rosette (CR) structures which harbor more than two centrioles each. Long term PLK4 overexpression results with centrosome amplification, but the maturation of amplified centrioles in CRs and linking of PLK4 induced amplified centrosomes has not yet been investigated in detail. Here, we show evidence for generation of large clustered centrosomes which have more than 2 centriole rosettes and define these structures as centriole rosette clusters (CRCs) in cells that have high PLK4 levels for 2 consecutive cell cycles. In addition, we show that PLK4 induced CRs follow normal centrosomal maturation processes and generate CRC structures that are inter-connected with canonical centrosomal linker proteins as C-Nap1, Rootletin and Cep68 in the second cell cycle after PLK4 induction. Increased PLK4 levels in cells with C-Nap1 and Rootletin knock-out resulted with distanced CRs and CRCs in interphase, while Nek2 knock-out inhibited separation of CRCs in prometaphase, providing functional evidence for the binding of CRC structures with centrosomal linker proteins. Taken together, these results suggest a cell cycle dependent model for PLK4 induced centrosome amplification which occurs in 2 consecutive cell cycles: (i) CR state in the first cell cycle, and (ii) CRC state in the second cell cycle.

Keywords: Centrosome amplification; Centrosome linkers; PLK4.

Figure 1

Long term induction of PLK4 leads to the formation of CRCs. (A) Over-expression of PLK4 for 24 h generates cells with 2 centriole rosettes. U2OS cells were transfected with GFP-tagged PLK4 (green) and stained for Centrin-3 (red). The left panel shows a high-resolution confocal image, and the right panel shows a 3D reconstruction of the confocal image. (B) Induction of PLK4 for 48 h generates cells with multiple CRs. PLK4 expression was induced with dox (48 h), and cells were stained with DAPI (DNA, blue), $\mathrm{\gamma }$-tubulin (centrosome, green) and Centrin-3 (centriole, red). The left panel shows an interphase cell with a CRC, and the right panel shows multipolar metaphase formation with CRs in each pole. (C) The localization of CEP152 (green) and Centrin-3 (red) in PLK4-induced cells for 24 h (upper panel) and 48 hours (bottom panel). (D) The localization of CEP170 (green) and Centrin-3 (red) in PLK4-induced cells for 24 h (upper panel) and 48 hours (bottom panel). The right panels show normalized fluorescence intensities in C and D. (E) The quantification of mother centrioles and total centriole numbers in PLK4-induced cells for 24 h and 48 h. n:100, N:2 biological repeats. (F) The quantification of mature-mother centrioles and total centriole numbers in PLK4-induced cells for 24 h and 48 h. n:100, N:2 biological repeats. Raw counting data of 1E and 1F is available in Supplemental Table 1. (G) The localization of CEP164 (green) and Centrin-3 (red) in PLK4-induced cells for 24 h (upper panel) and 48 h (bottom panel). (H) Metaphase scoring of 24 h, 48 h and 72 h PLK4-induced U2OS cells. Illustrations in the upper panel represent different metaphase types (Blue: mitotic DNA, green: centrosome, red: centriole). Raw measurement data of 1E, 1F and 1H are available in Supplemental Table 1.

Figure 2

Centrosomal linker proteins connect CRs and CRCs. (A,B) Rootletin and CEP68 binds adjacent CRs in 24 hours PLK4 induced cells. (C,D) Rootletin and CEP68 binds CEP152 positive mature centrioles of CRCs in 48 hours PLK4 induced cells. (E,F) Mother centrioles in CRs and CRCs are positive for C-Nap1 (E) and Nek2 (F).

Figure 3

CRCs inter-connect with planar or circular types of linking. (A) Representative confocal images of planar oriented and circular oriented Rootletin linker in cells with CRCs. (B) Percentage of CRC arrangement type in cells with CRCs. Rootletin and CEP68 staining were independently quantified; dots represent biological repeats, and lines display the mean of repeats. (C) Diameter of Rootletin ring in cells that CRCs are bound with circular oriented linker. Dots represent measurement of diameter in individual cells. (n: 102 circular CRC from 2 independent experiment, line represent mean value.) (D) Left panel: Representative images of circular linked CRCs with different number of mother centrioles. Scale bars: 1 $\mathrm{\mu }$m. Right panel: Diameter of Rootletin ring in circular oriented CRCs are increased in cells with >6 mother centrioles (n: 112 circular linked CRC from 2 independent experiment). Lines represent median and interquartile range in G and H. Raw measurement data of 3B, 3C and 3D are available in Supplemental Table 2.

Figure 4

The regulation of CRC binding and separation involves centrosomal linkers. (A) Western blot showing PLK4 induction in C-Nap1, Rootletin and Nek2 individual knock-out U2OS cell clones. (B) CR (top panel) and CRC (bottom panel) formations in C-Nap1 KO (left panel), Rootletin KO (middle panel) and Nek2 KO (right panel) U2OS cells. (C) CR distances (top panel) and CRC diameters (bottom panel) in cells synchronized with aphidicolin and released for 4 hours. n: 100 for each group, pooled from 4 independent experiment. (D) CR distances (top panel) and CRC diameters (bottom panel) in cells synchronized with DTB and released for 6 hours. n: 100 for each group, pooled from 4 independent experiment. (E) CR distances (top panel) and CRC diameters (bottom panel) in prometaphase cells synchronized with aphidicolin and STLC. n: 100 for each group, combined from 4 independent experiment. Median and interquartile range is shown on plots. Frequency distributions are calculated with non-linear gaussian regression and raw measurement data of 4C, 4D and 4E are available in Supplemental Table 3.

Figure 5

Nek2 regulates CRC separation. (A) Western blot showing Nek2 over-expression in U2OS-WT and U2OS-KO cell groups. (B) CRC diameters of Nek2 over-expressing WT, C-Nap1 KO and Rootletin KO U2OS cells in interphase. Left side p values indicate comparisons with control groups in Fig. 4C bottom panel. n: 100 for each group, pooled from 3 independent experiment. (C) CRC diameters in Nek2 over-expressing WT, C-Nap1 KO and Rootletin KO cells in prometaphase. Left side p values indicate comparisons with control groups in Fig. 4E bottom panel. n: 100 for each group, combined from 3 independent experiment. (B,C): Median and interquartile range is shown on plots. Frequency distributions are calculated with non-linear gaussian regression and raw measurement data of 5B and 5C are available in Supplemental Table 3.

Figure 6

Centriole rosette clusters develop during the second cell cycle following PLK4 induction. (A) Cell cycle synchronization using double thymidine block (DTB). (B) Representative images of CRs and CRCs in cells at various cell cycle phases. (C) Quantification of mother centrioles and total centrioles in cells with CRs and CRCs across different cell cycle stages. Raw measurement data is available in Supplemental Table 4. (D) A representative image of a cell containing two distinct CRCs in 36h PLK4 induced cells.

Figure 7

A model for the generation of CRCs. A newly divided G1 cell possesses two centrioles, one mother and one daughter. As the cell progresses into the S phase, a daughter centriole transforms into a young mother centriole, and increased PLK4 levels generate more than one procentriole on the wall of both mother centrioles, leading to the formation of centriole rosettes (CRs). When a cell with two CRs divides into two daughter cells, each daughter cell inherits one CR. Daughter centrioles within a CR disengage after division and become linked to each other with centrosome linker proteins. As the cell proceeds into the next S phase, daughter centrioles mature into young mother centrioles, and centrosome rosette clusters (CRCs) are produced by the generation of procentrioles on all mother centrioles through continued PLK4 overexpression .