Regulator of calcineurin-2 is a centriolar protein with a role in cilia length control

Abstract

Almost every cell in the human body extends a primary cilium. Defective cilia function leads to a set of disorders known as ciliopathies, which are characterised by debilitating developmental defects that affect many tissues. Here, we report a new role for regulator of calcineurin 2 (RCAN2) in primary cilia function. It localises to centrioles and the basal body and is required to maintain normal cilia length. RCAN2 was identified as the most strongly upregulated gene from a comparative RNAseq analysis of cells in which expression of the Golgi matrix protein giantin had been abolished by gene editing. In contrast to previous work where we showed that depletion of giantin by RNAi results in defects in ciliogenesis and in cilia length control, giantin knockout cells generate normal cilia after serum withdrawal. Furthermore, giantin knockout zebrafish show increased expression of RCAN2. Importantly, suppression of RCAN2 expression in giantin knockout cells results in the same defects in the control of cilia length that are seen upon RNAi of giantin itself. Together, these data define RCAN2 as a regulator of cilia function that can compensate for the loss of giantin function.

Keywords: Calcineurin; Cilia; Giantin; Golgi; RCAN2.

Fig. 1.

Giantin knockout RPE1 cells have no obvious defects in ciliogenesis. (A) Representative images of RPE1 WT and giantin KO cells fixed after 24 h of serum starvation and labelled for Arl13B (red) and acetylated tubulin (green); nuclei are stained with DAPI (blue). Scale bars: 10 μm and 5 μm (inset). (B) Quantification of the experiment represented in A (n=3). The mean percentage of cells producing cilia does not change upon KO of giantin. Error bars represent s.d., Mann-Whitney U-test. (C) TEM images of (i) serial and (ii) single plane 70 nm sections through representative WT and giantin KO cilia at two different magnifications.

Fig. 2.

RCAN2 localises to centrioles. (A) qPCR of two giantin zebrafish mutant lines (Q2948X and X3078) compared with wild-type sibling controls shows that rcan2 is strongly upregulated relative to the housekeeping gene actb1. In contrast, hprt1 and gapdh are not upregulated. (B) Endogenous RCAN2 (red) localises to centrioles labelled with γ-tubulin (green) in hTERT-RPE1 cells. Scale bar: 10 μm. (C) RCAN2 is frequently (25±11%, n=3 independent experiments) found concentrated at the mother centriole (from which the cilium extends) as shown by acetylated tubulin labelling of the ciliary axoneme (arrowhead). Where only one puncta of RCAN2 labelling is found (asterisk; 7±3% of cells, n=3 independent experiments), a ciliary axoneme is always found extending from this centriole. (D) The distal appendage protein CEP170 is associated with the brighter of the two RCAN2-positive centrioles (arrowhead) as well as with single centrioles positive for CEP170 (asterisk). Boxes are 5×5 µm; >50 cells were analysed in each of the independent experiments in B-D.

Fig. 3.

RCAN2 compensates for loss of function of giantin in cilia length control. (A) Depletion of RCAN2 using two different siRNA duplexes (1 and 2) is effective as judged by reduced centriolar labelling compared with cells transfected with control siRNA (GL2). Two examples are shown in each case. Scale bars: 1 µm. (B) Quantification of experiment shown in A. Depletion of RCAN2 results in shorter cilia in WT RPE-1 cells but longer cilia in giantin KO cells compared with GL2-transfected controls. (C) Depletion of RCAN2 does not affect the ability of cells to produce cilia. (D) Quantification of cilia length in RCAN2-depleted WT and giantin KO RPE-1 cells. Depletion of RCAN2 in wild-type RPE-1 cells results in a decrease in cilium length. In contrast, depletion of RCAN2 in giantin KO RPE-1 cells increases cilia length, even when compared with WT cells. Statistical significance was tested using one-way, non-parametric ANOVA (Kruskal-Wallis test) with Dunn's multiple comparisons test. N≥35 cilia were measured in each case from 3 independent biological replicates; ns, not significant.

Fig. 4.

Giantin KO cells show higher levels of NFAT1 activation. (A) Expression of myc-RCAN2 (red in merge) prevents cilia formation as shown by acetylated tubulin or Arl13b (both pseudocoloured green in merge; images were obtained from cells triple labelled with Alexa Fluor 488, 568 and 647). 80% of myc-RCAN2-expressing cells failed to extend a cilium (n=100). (B) In 5% of these myc-RCAN2-expressing cells, we observed ‘decapitated’ cilia, positive for Arl13b but not for acetylated tubulin. (C) Representative images showing WT and giantin KO and knockdown (KD) cells grown in serum or serum deprived for 24 h and then immunolabelled for NFAT1. Scale bars: 10 μm. (D) Quantification of the ratio of nuclear to cytoplasmic NFAT1 staining intensity in experiments represented in A [n=3, bars represent median and interquartile range, P-values calculated using non-parametric ANOVA (Kruskal-Wallis test with Dunn's multiple comparisons test)].