Ciliary transition zone activation of phosphorylated Tctex-1 controls ciliary resorption, S-phase entry and fate of neural progenitors

Abstract

Primary cilia are displayed during the G(0)/G(1) phase of many cell types. Cilia are resorbed as cells prepare to re-enter the cell cycle, but the causal and molecular link between these two cellular events remains unclear. We show that Tctex-1 phosphorylated at Thr 94 is recruited to ciliary transition zones before S-phase entry and has a pivotal role in both ciliary disassembly and cell cycle progression. However, the role of Tctex-1 in S-phase entry is dispensable in non-ciliated cells. Exogenously adding a phospho-mimic Tctex-1(T94E) mutant accelerates cilium disassembly and S-phase entry. These results support a model in which the cilia act as a brake to prevent cell cycle progression. Mechanistic studies show the involvement of actin dynamics in Tctex-1-regulated cilium resorption. Tctex-1 phosphorylated at Thr 94 is also selectively enriched at the ciliary transition zones of cortical neural progenitors, and has a key role in controlling G(1) length, cell cycle entry and fate determination of these cells during corticogenesis.

Figure 1

Tctex-1 is involved in cilium-dependent cell-cycle re-entry. (a) Diagrams depicting the timeline of transfection experiments and the plasmids used for transfection. (b) Representative immunoblots containing lysates of control or Tctex-1-sh transfected RPE-1 cells were probed with indicated Abs. The time points at which cells were harvested after serum re-addition are shown in hrs. (c) Quantification results of (b); the signal levels were normalized with the dynein intermediate chain (DIC) signal (mean ± s.e.m.; n=3 experiments, ** p< 0.01, t-test). (d–h) BrdU incorporation indices of synchronized RPE-1 or IFT20KD-RPE-1 cells (d), HeLa or COS-7 (e), 3T3 cells (f, h), and WT or Ift88 mutant MEF cells (g). BrdU incorporation of the control cells was taken to be 100%. All data shown are mean ± s.e.m.; n= 5, 4, 7, 3, and 7 experiments for figures (d), (e), (f), (g), and (h), respectively; an average of 400 cells were counted in each experiment; ** p< 0.01, *** p< 0.001; t-test for (d)–(g); *p<0.05, one-way ANOVA test for (h). (i) Immunoblotting assays demonstrated the reduction of endogenous Tctex-1 levels in various cell types transfected with Tctex-1-sh plasmid.

Figure 2

Temporal activation of phospho(T94)Tctex-1 at the transition zone and its function in ciliary disassembly. (a) Immunofluorescence of phospho(T94)Tctex-1 (green), acetylated α-tubulin (red), and γ-tubulin (cyan) in quiescent RPE-1 cells or cells treated with serum for 2 hr or 24 hr. DAPI: nuclei (blue). Bar= 5 µm. (b) Representative immunoblots show the levels of (pan)Tctex-1 and phospho-Tctex-1 in cells harvested after serum addition. To improve the detection of phospho-Tctex-1, immunoprecipitation was first carried out using a saturated amount of anti-(pan)Tctex-1 Ab, and the immunoprecipitates were electrophoresed and immunoblotted with phospho(T94)Tctex-1 Ab. Anti-HA Ab was used as an immunoprecipitation Ab control (Ctrl). The relative expression level of phospho(T94)Tctex-1 level was normalized with the total amount of immunoprecipitated Tctex-1. (c) Relative phospho(T94)Tctex-1 immunofluorescence intensity in RPE-1 cells post serum re-addition. The ratios of intensity of phospho(T94)Tctex-1:γ-tubulin were quantified by MetaMorph software (means±s.e.m.; n=3 experiments). (d) Diagram depicting the timeline of cilium disassembly experiments. (e) The fractions of transfected RPE-1 cells containing a cilium harvested at various time points after serum addition are shown. An average of 500 cells were counted for each experiment (means ± s.e.m.; n=3 experiments). (f) Quantification of the lengths of cilia in transfected 3T3 cells harvested after serum addition (means ± s.e.m.; n=3 experiments). (g) The fractions of transfected cells containing a cilium harvested at various time points after serum addition are scored and statically analyzed as described above (mean ± s.e.m.; n=5 experiments; *p<0.05, p**<0.01; one-way ANOVA).

Figure 3

Phospho(T94)Tctex-1 and actin dynamics participate in ciliary resorption. (a) Fractions of cells displaying cilia in serum-starved RPE-1 cells, either untreated (none) or treated with GFP-9R, T94E-9R, or T94A-9R, harvested at indicated time points after the treatments. All data shown are mean ± s.e.m.; n=4 experiments, except untreated control, where n=3 experiments. (b) Cells in serum-free medium were treated with various peptides followed by the incubation with BrdU for additional 16 hr. Fractions of BrdU-labeled cells are shown (mean ± s.e.m.; n=7 experiments; **p< 0.01; one-way ANOVA). (c) Fraction of ciliated cells in control (DMSO) and CytoD (0.5 µM) treated cells post serum re-addition (mean ± s.e.m.; n=3 experiments). (d) Representative image of cilia displayed in control cells vs. CytoD treated cells 24-hr after serum re-addition. (e) Fractions of cells that had cilia after the addition of GFP-9R or T94E-9R for indicated time in the presence of DMSO (n=3 experiments) or CytoD (n= 5 experiments). All data shown are mean ± s.e.m.

Figure 4

Phospho(T94)Tctex-1 is specifically expressed at the transition zone of RG in developing neocortex. (a) Drawing depicting the radially elongated RG with their endfeet contacting both the ventricular and pial surfaces. The primary cilium, anchored on a basal body (BB), extends into ventricular spaces. The large majority of mitosis (M phase) occurs at the ventricular surfaces. Cells leave the cell cycle, migrate away from the ventricular zone (VZ), form multipolar post-mitotic neurons, and pause their migration in the intermediate zone (IZ) prior to reaching their cortical location. CP: cortical plate. (b) Anti-Tctex-1 mouse Ab recognized a single band of ~12-kDa on an immunoblot containing embryonic mouse brain lysates. Confocal images of E13 mouse cortical slice co-labeled for primary cilium marker Arl13b (green), Tctex-1 (red), and cell-cell junction marker ZO-1 (cy5). Bars= 20 µm (top panel); 2 µm (bottom panel). (c) Confocal images of the ventricle surfaces of E11-E17 neocortical slices that were triple-labeled for Arl13b (green), phospho(T94)Tctex-1 (red), and γ-tubulin (cyan). In these experiments, cortical slices were first incubated with phospho(T94)Tctex-1 Ab, followed by excess biotinylated goat-anti-rabbit Ab. The sections were then PFA fixed, incubated with anti-Arl13b and γ-tubulin Abs, followed by detection by using Alexa488-conjugated anti-rabbit Ab, Cy5-conjugated anti-mouse Ab, and Alexa568-conjugated streptavidin. (d) Representative enlarged view of (c) demonstrating that phospho(T94)Tctex-1 (red) was specifically located in the transition zone between Arl13b-labeled primary cilia (green) and γ-tubulin-labeld basal body (cyan) of the RG. (e, f) Confocal images of the ventricular zone double labeled with γ-tubulin Ab and phospho(T94)Tctex-1 Ab preabsorbed with the phosphopeptides corresponding to the antigen (e) or control peptides (f). Arrows in (e) point to the γ-tubulin labeled basal bodies and centrosomes, which lacked phospho(T94)Tctex-1 signal. Bar=5 µm. (g) Co-labeling of phospho(T94)Tctex-1 and Arl13b in 24-hr transfected cortical slices. Note that cells transfected with GFP control plasmid (left panel, green arrowhead) and neighboring non-transfected cells (arrows) displayed similar levels of phospho(T94)Tctex-1 at the endfeet. However, cells transfected with Tctex-1-sh had reduced phopsho(T94)Tctex-1 immunolabeling. Bar= 2 µm. (h) Confocal image of the post-mitotic neurons located in the intermediate zone region displayed no detectable phospho(T94)Tctex-1 (red) between the Arl13-labeled primary cilia (green) and γ-tubulin-labeled basal bodies (cyan). Bar= 5 µm.

Figure 5

Suppression of Tctex-1 in RG induced premature neuronal differentiation. (a) Cortical slices harvested 40 hr after electroporation with the indicated plasmids. GFP was detected by direct green immunofluoresence; DAPI: blue. The dashed lines depict the borders of the cortexes. The intermediate zone (IZ) is defined by the presence of tangentially oriented cells. (b, c) Representative images showing the labeling of Tuj1 (b) and brain lipid binding protein (BLBP) (c) of vector- and Tctex-1-sh transfected brain slices. Arrowheads point to the sites where the cell processes project from the cell bodies. (d) Percentage of total transfected GFP⁺ cells that were Tuj1⁺, Nestin⁺, or BLBP⁺. All data shown are mean ± s.e.m.; n=4 experiments, except BLBP staining, where n=5 experiments; an average of 600 cells were counted in each experiment; ***p<0.001; one-way ANOVA. (e) Representative images depicting the cortical distribution of GFP cells in the E18.5 brains (i.e., 5-days post electroporation). The bins of marginal zone (MZ), superficial (sCP), mid-, and deeper cortical plate (dCP) are defined, as described . (f) The numbers of cell bodies located in each bin were scored, and quantification of the cortical location of transfected cells (mean ± s.e.m.; n=3 experiments; ***P<0.001, t-test).

Figure 6

Phospho-Tctex-1 is required for the cell cycling of RG. (a) Representative ventricular zone images of transfected brains immunolabeled with P-H3. Arrowheads indicate cells that were double positive for both GFP and P-H3. (b) Representative confocal images of transfected cortical slices subjected to cell cycle exit analysis: immunofluoresence of GFP (green), 24-hr BrdU (red), Ki67 (cyan) are shown. Arrows point to the cells that were double positive for GFP and BrdU but negative for Ki67. Arrowheads point to the cells that were triple positive for GFP, BrdU, and Ki67. (c) Fractions of GFP, P-H3-double positive cells out of total GFP⁺ cells (or mitotic index) are shown as means ±s.e.m.; n= 3, 6, 3, 5, 6 experiments for vector, Tctex-sh, Tctex-1-sh/WT, Tctex-1-sh/T94E and Tctex-1-sh/T94A, respectively; ***p<0.001; one-way ANOVA. (d) Fractions of GFP⁺, BrdU⁺, Ki67⁻ cells out of total GFP⁺, BrdU⁺ cells (or cell cycle exit index) are listed as means ± s.e.m.; n=3 experiments; total 100 cells were scored; **p<0.01; one-way ANOVA.

Figure 7

Phenotypic characterization of loss-of-function of AurA and HDAC6, and gain-of-function of Tctex-1 in developing neocortex. (a) Cortical slices transfected with AurA-sh or HDAC6-sh plasmid (top panels) and their immunolabeling of P-H3 (red; middle panel). The schematic diagrams of these plasmids were shown in the bottom panel. (b) Cortical slices overexpressing the indicated Tctex-1 variants. Arrowheads point to P-H3-labeled, mitotic GFP⁺ cells at the ventricular zone. Bars=100 µm (top panel); 20 µm (bottom panel). (c) Mitotic indices of GFP transfected cells are shown as means ± s.e.m.; n=3 experiments; total 1,200 cells were scored; ** p < 0.01, one-way ANOVA. (d) The fractions of 2-hr BrdU incorporated, GFP⁺ cells out of total GFP⁺ cells in transfected brains, 24 hr and 40 hr post-electroporation (mean ± s.e.m.; n=4 experiments; total 700 cells were scored; *p < 0.05, t-Test). (e) Cumulative BrdU labeling curves of non-transfected, vector alone and T94E/GFP transfected cells (means ± s.e.m.; n=3 experiments; *p < 0.05, *** p < 0.001, one-way ANOVA). OE: overexpression.