Centriole reduplication during prolonged interphase requires procentriole maturation governed by Plk1

Abstract

Supernumerary centrioles lead to abnormal mitosis, which in turn promotes tumorigenesis. Thus, centriole duplication must be coordinated with the cell cycle to ensure that the number of centrioles in the cell doubles precisely during each cell cycle. However, in some transformed cells, centrioles undergo multiple rounds of duplication (reduplication) during prolonged interphase. Mechanisms responsible for centriole reduplication are poorly understood. Here, we report that centrioles reduplicate consistently in cancerous and nontransformed human cells during G2 arrests and that this reduplication requires the activity of Polo-like kinase 1 (Plk1). We also find that a cell's ability to reduplicate centrioles during S arrests depends on the presence of activated (Thr210-phosphorylated) Plk1 at the centrosome. In the absence of activated Plk1, nascent procentrioles remain associated with mother centrioles, which prevents centriole reduplication. In contrast, if Plk1(pT210) appears at the centrosome, procentrioles mature, disengage from mother centrioles, and ultimately duplicate. Plk1 activity is not required for the assembly of procentrioles, however. Thus, the role of Plk1 is to coordinate the centriole duplication cycle with the cell cycle. Activation of Plk1 during late S/G2 induces procentriole maturation, and after this point, the centriole cycle can be completed autonomously, even in the absence of cell-cycle progression.

Figure 1

Centrioles reduplicate in G2-arrested cells. (A) Examples of typical centrosome configurations in RO-treated HeLa cells at indicated time points (30 hr corresponds to the beginning of G2 arrest, see Supplementary Experimental Procedures). Similar configurations were also observed in U2-OS cells (not shown). (30 hr) Two diplosomes in one cell; (40 hr) four individual centrioles in one cell; (48 hr) four diplosomes in one cell. Green, centrin-GFP; red, γ-tubulin (B) Centrin-GFP-positive structures also contain polyglutamylated tubulin, as evident from staining with GT335 antibody. (C) Percentages of cells with different number of centrioles as determined via centrin-GFP signal. (Also see Figure S2).

Figure 2

Procentrioles form, but do not mature, in Emi1-depleted cells. (A) Percentage of Emi1-depleted HeLa and U2-OS cells with two centrosomes at various time points after transfection. (B) Centrosome configuration in Emi1-depleted U2-OS cells 46 h after transfection. See text for details. (C) Serial-section EM analyses confirm that Emi1-depleted cells with grossly enlarged nuclei (yellow arrowheads) contain just two diplosomes with 150-200-nm short procentrioles (red arrows). (See also Figure S2 and Video S1).

Figure 3

pThr210 Plk1 is present at centrosomes under conditions that allow procentriole maturation and centriole reduplication. Cells either treated with HU, RO-3306, or depleted of Emi1 were fixed at indicated time points and analyzed by fluorescence microscopy. The pThr210 signal was prominent at the centrosome in RO-treated HeLa as well as in RO- and HU-treated U2-OS cells. In contrast, pThr210 was not detectable at the centrosome in HU-treated HeLa or in Emi1-depleted HeLa or U2-OS cells.

Figure 4

Activated Plk1 is necessary for centriole reduplication. (A-C) Expression of phosphomimicking Plk1 mutant (T210D) induces centriole reduplication in Emi1-depleted U2TR cells. Mitotic cells (collected by shake-off) were plated on coverslips and 1.5 hr later transected with Emi1 siRNA. Doxycycline (1 μM) was added 24-26h after shake off. (A) Typical centrosome configurations in non-induced (−dox) vs. induced (+dox) Emi1-depleted U2TR cells. Red, γ-tubulin (PCM); green, polyglutamylated tubulin (centrioles). (B) Percentage of cells with various numbers of centrosomes under two treatment conditions. (C) Procentrioles mature and disengage from mother centrioles upon expression of Plk1(T210D). Notice that in non-induced cells all centrioles are duplicated (green arrows in 38 hr −dox). However, procentrioles are small and do not contain hPOC-5 which is normally recruited to the distal end of growing procentrioles during G2. In induced cells, procentrioles become more prominent (green arrows in 38 hr +dox) and eventually do recruit hPOC-5 (blue arrows in 38 hr +dox). At a later time, induced cells contain both individual centrioles and diplosomes. hPOC5 is found in all individual centrioles. However, some procentrioles lack hPOC-5 or contain minimal amounts of this protein (red arrow, 48 hr +dox). (See also Figure S4). (D-E) Inhibition of Plk1 prevents centriole reduplication but not initiation of procentriole assembly. Mitotic HeLa cells (collected by shake-off) were plated on coverslips and 1.5 hr later treated with RO. 100 nM Plk1 inhibitor BI2536 was added 29 hr after shake-off. Alternatively, 1.5 hr after shakeof cell were transfected with siRNA against Plk1. (D) Percentage of cells with various centriole and centrosome numbers under different conditions. (E) Procentriole assembly in G2-arrested HeLa cells after selective ablation of the original procentriole. A procentriole (red arrow in 00:00) was ablated within the diplosome (compare 00:00 and 00:02) at 31h after shake-off. This operation resulted in the formation of a new procentriole (arrow in 15:00) on the mother centriole, as evidenced by centrin-GFP signal. (See also Figure S5).