Targeting of CRMP-2 to the primary cilium is modulated by GSK-3β

Abstract

CRMP-2 plays a pivotal role in promoting axon formation, neurite outgrowth and elongation in neuronal cells. CRMP-2's role in other cells is unknown. Our preliminary results showed CRMP-2 expression in cilia of fibroblasts. To localize CRMP-2, define its role and study the regulation of CRMP-2's expression in cilia we carried out the following experiments. We find that in fibroblasts CRMP-2 localizes to the centrosome and is associated with the basal body and -at a low level- is present in primary cilia. Phosphorylated pCRMP-2 can only be detected at the basal body. RNAi knockdown of CRMP-2 interfered with primary cilium assembly demonstrating a critical requirement for CRMP-2. Deletion analysis of CRMP-2 identified a 51 amino acid sequence in the C-terminus that is required for targeting to the basal body and primary cilium. This domain contains GSK-3β phosphorylation sites as well as two repeats of the VxPx motif, previously identified as a cilium targeting signal in other primary cilium proteins. To our surprise, mutation of the CRMP-2 VxPx motifs did not eliminate primary cilium targeting. Instead, mutation of the GSK-3β phosphorylation sites abolished CRMP-2 targeting to the primary cilium without affecting basal body localization. Treatment of cells with lithium, a potent GSK-3β inhibitor, or with two specific GSK-3β inhibitors (the L803-mts peptide inhibitor and CHIR99021) resulted in cilium elongation and decreased basal body levels of pCRMP-2 as well as increased levels of total CRMP-2 at the primary cilium. In summary, we identified CRMP-2 as a protein critically involved in primary cilia formation. To our knowledge this is the first demonstration of modulation of primary cilium targeting by GSK-3β.

Figure 1. CRMP-2 localizes to the centrosome/basal body.

Human foreskin fibroblasts were stained with anti-CRMP-2 antibody (green signal), and co-stained with anti-β-tubulin antibody (A and B; red signal) to illustrate microtubule organizing center and spindle poles, respectively, anti-γ-tubulin antibody (red signal) to reveal the centrosomes (C) or anti-acetylated tubulin antibody (red signal) to visualize the primary cilia (D). A) CRMP-2 is at the centrosome in interphase cells (arrowheads). B) CRMP-2 shows diffuse staining in mitotic cells. C) CRMP-2 and γ-tubulin colocalize (arrows). D) Small amounts of CRMP-2 are at the basal body (arrow) and primary cilia (arrowhead): the inset is an enlarged image of the indicated region. The last column in panels A–D shows merged images. Bars indicate 10 μm. Magnification: 100X. E) Immuno electron microscopy shows CRMP-2 at the basal body (bb: arrows) and the primary cilium (pc). Arrowheads indicate CRMP-2 at microtubules emanating from the basal body, and asterisks indicate CRMP-2 occasionally observed at the shaft of the primary cilium. Magnification: 50,000X.

Figure 2. CRMP-2-GFP expression pattern in fibroblasts.

Human foreskin fibroblasts were transfected with CRMP-2-GFP (green signal), fixed and stained with anti-acetylated tubulin antibodies (red signal). The last column shows merged images. A) GFP signal was observed at the basal body (arrow) and –at a low level- at the primary cilium (arrowhead). The inset shows an offset of an enlarged part of the image to compare staining patterns for acetylated tubulin and CRMP-2-GFP. B) and C) show examples of transfected cells with CRMP-2-GFP in concentrations in the primary cilium (arrows). D) Shows an example of the unequal distribution of CRMP-2-GFP occasionally observed: the inset shows an offset for comparison of the signals. Bars indicate 10 μm. Magnification: 100X.

Figure 3. CRMP-2 is required for ciliogenesis.

CRMP-2 expression was knocked down using siRNA. Efficacy was measured in human foreskin fibroblasts by western blotting (A) and by immunofluorescence of transfected cells (B). A) Human foreskin fibroblasts were transfected with iLenti-Scramble-siRNA-GFP (lane 1) or iLenti-CRMP-2-siRNA-GFP (lane 2) and endogenous CRMP-2 protein expression was analyzed by western blotting using anti-CRMP-2 antibodies. Both constructs express GFP independent of the siRNA, as a marker for cells positively transfected. Blots were reprobed using anti-actin antibodies. B) Human foreskin fibroblasts were transfected with the constructs listed in A) and analyzed for CRMP-2 protein expression in GFP positive cells. Cells expressing CRMP-2 siRNA (green signal) have a significantly reduced level of CRMP-2 (red signal); GFP-negative cells show normal CRMP-2 (arrows). C) Primary cilia in human foreskin fibroblasts transfected with CRMP-2 siRNA (green signal) were visualized using anti-acetylated tubulin antibodies (red signal). A majority of CRMP-2 siRNA expressing cells lacked a primary cilium. Surrounding untransfected cells were not affected. D) A scrambled siRNA (green signal) had no effect on cilium formation using anti-acetylated tubulin antibodies (red signal). In B0, C) and D) bars indicate 10 μm. Magnification: 100X. E) Quantitation of CRMP-2 siRNA transfection experiments in RPE cells and human foreskin fibroblasts (HFSK) shows a significant difference (P<0.05) between the percentage of cells lacking a primary cilium after CRMP-2 siRNA expression (siRNA) compared to cells expressing scrambled siRNA (control). Multiple experiments were done and >200 GFP-positive cells were measured in each condition.

Figure 4. CRMP-2 mutation analysis delineates the primary cilium targeting sequence.

A) Domain structure of wild type CRMP-2 (wt) and corresponding regions present in the mutants (mut1 - mut8) are indicated. DHC, dynein heavy chain interacting domain; Nb, Numb interacting domain; Mt, microtubule-binding domain; KLC, kinesin light chain interacting domain. CTS, cilium targeting capability, based on observations of mutant protein expression patterns in transfected fibroblasts. B) Immunofluorescence analysis of human foreskin fibroblasts expressing mut7-GFP or mut8-GFP as indicated. Human foreskin fibroblasts were transfected with mut7-GFP or mut8-GFP (green signal) and analyzed using anti-acetylated tubulin antibodies (red signal). Mut7-GFP localizes to the basal body (top panels: arrow) in cells lacking a primary cilium, and to the cilium in cells that express the organelle (middle panels). Mut8-GFP does not localize to basal body or cilium. Bars indicate 10 μm. Magnification: 100X. C) Nucleotide sequence and deduced amino acid sequence of the CRMP-2 51 residue C-terminal region present in mut7. Val and Pro residues in the VxPx sequences are indicated in blue and Thr and Ser residues in known GSK-3β phosphorylation sites are indicated in red (T509, T514 and S518). D) Point mutations were introduced to mutate the VxPx sites to AxAx (mut9: purple lettering) and GSK-3β phosphorylation sites to Ala (mut-10: red lettering). E) Analysis of the localization of mut9-GFP (top panels) and mut10-GFP (bottom panels) (green signals) was done by transfection of human foreskin fibroblasts and staining for acetylated tubulin (red signals). Although the intensity of mut9-GFP was lower than wild type, mut9-GFP was observed to localize to both the basal body (arrow) and the cilium. The bottom panels show that mut10-GFP only localizes to the basal body (arrow), not the cilium. Bars indicate 10 μm. Magnification: 100X.

Figure 5. Inhibition of GSK-3β enhances CRMP-2 levels in elongated primary cilia.

A) The effect of inhibition of GSK-3β on amount and localization of endogenous phosphorylated pCRMP-2 was analyzed by indirect immunofluorescence using anti-pCRMP-2(Thr514) antibodies (green signals) in untreated RPE cells and in RPE cells treated with the indicated GSK-3β inhibitors: lithium, CHIR99021 and the L803-mts peptide inhibitor. Primary cilia were visualized using anti-acetylated tubulin antibodies (red signals). Arrows point to pCRMP-2 at the basal body. No phosphorylated pCRMP-2 was seen in primary cilia using this method. The right column shows merged images. Bars indicate 10 μm. Magnification: 100X. B) To analyze the effect of inhibition of GSK-3β on total CRMP-2 protein, we attempted to analyze endogenous CRMP-2 in RPE cells before and after treatment with the GSK-3β inhibitor CHIR99021 (top panels). However, signal was too low to provide for reliable quantitative data and we therefore resorted next to using RPE cells transfected with CRMP-2-GFP. CRMP-2-GFP in transfected RPE cells was analyzed in untreated cells (“untreated”, middle panels) or after treatment with CHIR99021 (“CHIR99021”, bottom panels). Primary cilia were visualized using anti-acetylated tubulin antibodies (“acetylated tub”, red signals). Note the presence of CRMP-2-GFP in untreated cells predominantly at the basal body (middle panels, arrowhead). GSK-3β inhibition caused a readily detectable increase in CRMP-2-GFP in primary cilia (bottom panels, arrow). Bars indicate 10 μm.

Figure 6. Model for regulation of CRMP-2 localization to primary cilia.

The model schematically presents that the majority of CRMP-2 in normal serum-starved cells is phosphorylated by GSK-3β and resides at the basal body. After GSK-3β inhibition or mutation of CRMP-2 GSK-3β phosphorylation sites, CRMP-2′s presence in the primary cilium is enhanced.