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. 2017 Jul 15;28(15):2123-2134.
doi: 10.1091/mbc.E16-09-0643. Epub 2017 May 24.

NEK7 Is Required for G1 Progression and Procentriole Formation

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Free PMC article

NEK7 Is Required for G1 Progression and Procentriole Formation

Akshari Gupta et al. Mol Biol Cell. .
Free PMC article

Abstract

The decision to commit to the cell cycle is made during G1 through the concerted action of various cyclin-CDK complexes. Not only DNA replication, but also centriole duplication is initiated as cells enter the S-phase. The NIMA-related kinase NEK7 is one of many factors required for proper centriole duplication, as well as for timely cell cycle progression. However, its specific roles in these events are poorly understood. In this study, we find that depletion of NEK7 inhibits progression through the G1 phase in human U2OS cells via down-regulation of various cyclins and CDKs and also inhibits the earliest stages of procentriole formation. Depletion of NEK7 also induces formation of primary cilia in human RPE1 cells, suggesting that NEK7 acts at least before the restriction point during G1. G1-arrested cells in the absence of NEK7 exhibit abnormal accumulation of the APC/C cofactor Cdh1 at the vicinity of centrioles. Furthermore, the ubiquitin ligase APC/CCdh1 continuously degrades the centriolar protein STIL in these cells, thus inhibiting centriole assembly. Collectively our results demonstrate that NEK7 is involved in the timely regulation of G1 progression, S-phase entry, and procentriole formation.

Figures

FIGURE 1:
FIGURE 1:
Depletion of NEK7 causes a G1-phase arrest. (A) Asynchronous U2OS cells were transfected with control or NEK7 siRNAs for 48 h, and the cells were analyzed for their DNA content profiles. (B) Schematic of the experimental conditions in C–E. U2OS cells were transfected with control or NEK7 siRNAs for 8 h and then synchronized at early mitosis with 100 ng/ml nocodazole (Noc) for 16 h. The cells were then released into fresh medium and collected at the indicated time points. (C) The DNA content profiles for individual time points. (D, E) Total cell lysates were analyzed by immunoblotting using antibodies against the indicated proteins at least twice. All samples treated with control and NEK7 siRNAs were blotted on the same membrane; they have been separated for clarity. The cells used in E are the same as those in C. Red arrowheads indicate band-shifted proteins. (F, G) U2OS cells were treated as in A, and total cell lysates were analyzed by immunoblotting against the indicated antibodies.
FIGURE 2:
FIGURE 2:
NEK7-depleted cells show cell cycle progression defects other than a G1-phase arrest. (A) Schematic of the experimental conditions in B–D. U2OS cells were transfected with control or NEK7 siRNAs for 8 h and then synchronized at S-phase with 2 mM thymidine (Thy) for 16 h. The cells were then released into fresh medium and collected at the indicated time points. (B) The DNA content profiles for individual time points. (C, D) Total cell lysates were analyzed by immunoblotting using antibodies against the indicated proteins at least twice. All samples treated with control and NEK7 siRNAs were blotted on the same membrane; they have been separated for clarity. The cells used in D are the same as those in B.
FIGURE 3:
FIGURE 3:
NEK7 is required for procentriole formation. (A) Asynchronous U2OS cells were transfected with control or NEK7 siRNAs for 48 h, and the cells were fixed and immunostained using the indicated antibodies. DNA is shown in blue. Insets are magnified views of the centrosomes. Scale bar, 5 μm. (B) Fluorescence intensities of the indicated proteins at the centrioles quantified on an arbitrary scale. (C) Percentage of interphase cells with the indicated centriolar markers. More than 50 cells were counted in each experimental group. (D) U2OS cells were treated as in A, and total cell lysates were analyzed by immunoblotting against the indicated proteins. (E) U2OS cells were transfected with control or NEK7 siRNAs. After 24 h, they were transfected with PLK4-ΔPEST-Flag for 24 h and then fixed. (F) GFP-STIL U2OS cells were transfected with control or NEK7 siRNAs, simultaneously induced for overexpression of GFP-STIL, and fixed after 48 h. Cells in E and F were immunostained with antibodies specific to STIL or GFP (red) and Flag or Centrin (green). Scale bar, 500 nm. (G) Population distributions of interphase cells containing centriolar STIL foci. More than 50 cells expressing high and comparable fluorescence intensities of PLK4-ΔPEST-Flag/GFP-STIL were counted in each experimental group. All histogram values are mean percentages ± SD from three independent experiments. **p < 0.01; one-tailed t test.
FIGURE 4:
FIGURE 4:
Depletion of NEK7 induces ciliogenesis. RPE1 cells were transfected with control and NEK7 siRNAs for a total of 48 h, and additional samples were simultaneously treated with a control siRNA and released into serum-free medium for a total of 48 h. (A) The cells were then fixed and stained with the indicated antibodies. DNA is shown in blue. Scale bar, 5 μm. (B) Percentage of interphase cells that were ciliated. (C) Percentage of ciliated cells that exhibited STIL foci at the basal bodies. More than 50 cells were counted in each experimental group. All histogram values are mean percentages ± SD from three independent experiments. *p < 0.05; n.s., not significant (one-tailed t test). (D) Magnified views of centriolar proteins at the base of cilia in the indicated cells. Cells were prepared as in A. Scale bar, 1 μm. (E) Total cell lysates in each condition were analyzed by immunoblotting against the indicated antibodies.
FIGURE 5:
FIGURE 5:
The centriolar protein STIL is targeted for proteasomal degradation by the APC/CCdh1 in NEK7-depleted cells. (A, C) U2OS cells were transfected with control or NEK7 siRNAs for 24 h and then transfected with the indicated vectors for another 24 h. Total cell lysates were analyzed by immunoblotting using STIL, SAS-6, or tubulin antibodies. Right, approximate protein sizes in kilodaltons. Red arrowheads indicate expected bands for different constructs. (B) Schematic of the HA-STIL constructs used in A and E. WT, wild type; ΔM, Δ(601-1017); ΔC, Δ(1018-1287); N, Δ(601-1287). (D) U2OS cells were transfected with control or NEK7 siRNAs for 48 h, and 10 μM MG132 was added for the last 6 h of the siRNA treatment. (E) U2OS cells were treated as in A, and 10 μM MG132 was added to the last 6 h of the experiment.
FIGURE 6:
FIGURE 6:
Localization patterns of the APC/C cofactor Cdh1 in control and NEK7-depleted cells. U2OS cells were transfected with control (A) or NEK7 (C) siRNAs for 48 h, and the cells were fixed and immunostained with antibodies specific to Cdh1 (green) and centrin (red). DNA is shown in blue. Insets are magnified views of the centrosomes. Scale bar, 5 μm. (B) The fluorescence intensities of Cdh1 at the centrosomes were quantified on an arbitrary scale at different cell cycle phases and are indicated as box plots. *p < 0.05; **p < 0.01 (one-tailed t test). (D) U2OS cells were imaged by 3D-SIM to address the localization of Cdh1 around the centrosomes. The fluorescence intensities of centrosomal Cdh1 are not comparable between images in D. Scale bar, 500 nm.
FIGURE 7:
FIGURE 7:
Centrosomal accumulation of Cdh1 in NEK7-depleted cells is PCM independent. U2OS cells were transfected with control (A, C), CEP192 (D), or NEK7 (A, E) siRNAs for 48 h, and the cells were fixed and immunostained with the indicated antibodies. DNA is shown in blue. Insets are magnified views of the centrosomes. Scale bars, 500 nm (A), 5 μm (C). (B) Approximate outer diameters of the indicated proteins at mitotic centrosomes. (F, G) Fluorescence intensities of Cdh1 and CEP192 at the centrosomes were quantified on an arbitrary scale at different cell cycle phases and are indicated as box plots. **p < 0.01; n.s., not significant (one-tailed t test).
FIGURE 8:
FIGURE 8:
Depletion of NEK7 inhibits G1 progression and centriole duplication. (Top) Schematic of how progression through G1 and the G1/S transition is regulated in control cells. During early G1, increases in cyclin D–CDK4/6 activity are necessary to pass the restriction point, and these complexes are subsequently inactivated in late G1. The G1/S transition largely depends on cyclin E/CDK2 activity, and inactivation of the APC/CCdh1 is essential for S-phase entry. At the same time, various procentriolar proteins are expressed that are required for the initiation of centriole duplication. Toward S-phase, cyclin A replaces cyclin E in the complex with CDK2 to complete DNA replication, and procentriole assembly is completed as well. (Bottom) In the absence of NEK7, the expression of various cyclins is inhibited; in particular, expression of the early G1 cyclin D is blocked. Low levels of cyclin D could lead to an arrest at the restriction point, which induces ciliogenesis. In the absence of cyclin D–CDK4/6 activity, expression of late G1 cyclins and CDKs is also reduced. In addition, inactivation of the APC/CCdh1 is also inhibited, and these aberrant conditions cause a G1 arrest in NEK7-depleted cells. Cdh1 also accumulates at the centriole walls in these cells, which may inhibit procentriole formation.

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