A role for the Golgi matrix protein giantin in ciliogenesis through control of the localization of dynein-2

Abstract

The correct formation of primary cilia is central to the development and function of nearly all cells and tissues. Cilia grow from the mother centriole by extension of a microtubule core, the axoneme, which is then surrounded with a specialized ciliary membrane that is continuous with the plasma membrane. Intraflagellar transport moves particles along the length of the axoneme to direct assembly of the cilium and is also required for proper cilia function. The microtubule motor, cytoplasmic dynein-2 mediates retrograde transport along the axoneme from the tip to the base; dynein-2 is also required for some aspects of cilia formation. In most cells, the Golgi lies adjacent to the centrioles and key components of the cilia machinery localize to this organelle. Golgi-localized proteins have also been implicated in ciliogenesis and in intraflagellar transport. Here, we show that the transmembrane Golgi matrix protein giantin (GOLGB1) is required for ciliogenesis. We show that giantin is not required for the Rab11-Rabin8-Rab8 pathway that has been implicated in the early stages of ciliary membrane formation. Instead we find that suppression of giantin results in mis-localization of WDR34, the intermediate chain of dynein-2. Highly effective depletion of giantin or WDR34 leads to an inability of cells to form primary cilia. Partial depletion of giantin or of WDR34 leads to an increase in cilia length consistent with the concept that giantin acts through dynein-2. Our data implicate giantin in ciliogenesis through control of dynein-2 localization.

Keywords: Cilia; Dynein; Golgi.

Fig. 1.

Giantin is required for ciliogenesis. (A,B) Cells were transfected with either control (A, GL2) or giantin (B, Giantin siRNA #1) siRNAs to suppress expression of giantin and serum starved for 24 hours. Cells were immunolabelled for giantin and acetylated tubulin and images acquired at the same settings for each slide. Boxed regions are enlarged in the lower panels; the lower box in the Gi #1 suppression panels shows a contrast enhanced version to highlight the remaining giantin. (C) Immunoblots showing effective suppression of giantin and lamin A/C as indicated. α-tubulin serves as a loading control. (D) Immunofluorescence showing effective suppression of giantin using siRNA #1 and #2. (E) Quantification of the percentage of ciliated cells following depletion of giantin or IFT20. (F) Immunoblotting with an anti-GFP antibody shows overexpression of IFT20-GFP and its effective suppression by siRNA. (G) Validation of suppression of giantin using siRNA transfection with additional independent siRNAs. (H) All four giantin siRNA duplexes result in statistically detectable reduction in ciliogenesis (n = 3 independent labelling experiments on the same population of labelled cells; *P<0.01 using ANOVA with Dunnett's post-hoc test). Note that results in panels C–E come from the same set of experiments. Results in E and F are from a distinct set of three independent experiments. Giantin siRNA #2 gives a more variable level of suppression yet on average still results in a decrease in ciliogenesis. Error bars represent s.d. Scale bars: 10 µm.

Fig. 2.

Expression of FLAG-giantin but not IFT20-GFP rescues the ability of giantin-depleted cells to form primary cilia. (A) The defect in ciliogenesis (acetylated tubulin in red) is rescued by expression of FLAG-giantin (green). (B) Enlargements from the boxed regions in A. Note the clearly defined rim-like labelling of the Golgi with FLAG-giantin and the presence of primary cilia in these cells. (C,D) IFT20-GFP expression does not rescue the ability of giantin-depleted cells to form primary cilia. 50 cells were examined that showed localization of IFT20-GFP to the Golgi and none contained a primary cilium. Arrow indicates Golgi-localized IFT20-GFP (left) and centrosomal acetylated tubulin (right). Scale bars: 10 µm.

Fig. 3.

Stable suppression of giantin in hTERT-RPE1 cells using short hairpin RNAs. (A) Immunofluorescence shows loss of giantin (green) whereas GM130 (red) remains localized to the enlarged and more dispersed Golgi. (B) Quantification of loss of giantin in stably depleted cells. All five shRNAs show a statistically detectable reduction in giantin labelling. (C) Stable suppression of giantin expression results in defects in ciliogenesis (examples are shown only from shRNAs #1 and #2). There is no statistically detectable difference between shRNA #4 and control. Error bars represent s.d. (D) Cells showing the most effective depletion of giantin fail to generate primary cilia in response to serum withdrawal. Scale bars: 10 µm.

Fig. 4.

Giantin is required during the early stages of ciliogenesis independently of the Rab11–Rabin8–Rab8 pathway. (A) Wild-type or giantin depleted cells were serum starved and the emergence of the cilium measured using acetylated tubulin. (B) Depletion of giantin, unlike that of Rab11A and Rab11B, does not disrupt the centrosomal accumulation of Rabin8. (C) Validation of inhibition of ciliogenesis in those cells evaluated in B. Error bars represent s.d.

Fig. 5.

Giantin is required to maintain cilia length but is not required for ciliogenesis in vivo. (A) Giantin depletion results in a partial reduction in the number of ciliated cells. Error bars represent s.d. **P<0.005. (B) The length of the remaining cilia from those experiments in A was measured. Note the statistically significant increase in cilia length with giantin siRNA #1. ***P<0.001. (C) Fibroblasts from wild-type rats or (D) ocd/ocd rats were serum starved for 24 hours and immunolabelled to detect acetylated tubulin. (E) Quantification of cilia length in wild-type versus ocd/ocd fibroblasts. No statistical difference was detected using a Mann–Whitney test but cilia >3.5 µm are only seen in ocd/ocd rat cells. Scale bar: 10 µm.

Fig. 6.

WDR34 is required for ciliogenesis. Cells were transfected with siRNA duplexes to suppress WDR34. (A–C) Silencing of WDR34 (red) causes a defect in ciliogenesis (acetylated tubulin labelling, green). Boxed regions show enlargements. Note that in C, the lower inset illustrates that a robust loss of WDR34 correlates with a failure to form cilia. The upper box shows that those cells that are less effectively suppressed still retain the ability to form primary cilia and that these remaining cilia are longer than controls (compare with A). (D) Immunoblotting of WDR34-depleted cells and lamin-A/C-depleted control, α-tubulin is used as a loading control. (E) Quantification of the ciliogenesis defect on WDR34 suppression. *P<0.01 using ANOVA with Dunnett's post-hoc test. Error bars represent s.d. (F) As in C, remaining cilia in WDR34-depleted cells were significantly longer than in control cells. (G) The typical pericentrosomal accumulation of WDR34 seen in control cells (arrows) is lost on suppression of giantin. (H) Quantification of loss of pericentrosomal WDR34 in giantin-depleted cells. *P<0.01 from ANOVA with Dunnett's post-hoc test comparing samples to the GL2 control. (I) Images show that the level of giantin remaining in cells correlates well with the pericentrosomal accumulation of WDR34. Insets to the lower panels illustrate how very low effective suppression of giantin results in a near complete loss of detectable WDR34 (arrowhead), whereas a less-effective suppression of giantin correlates with some faint but detectable pericentrosomal WDR34 labelling (arrow). (J) Quantification shows that the level of expression of giantin correlates well with the pericentrosomal accumulation of WDR34 (red dots indicate control; blue dots, giantin-depleted cells). The correlation (grey line) is significant (Spearman's correlation, P = 0.016; Pearson's correlation P = 0.036). Scale bars: 10 µm.