PCARE requires coiled coil, RP62 kinase-binding and EVH1 domain-binding motifs for ciliary expansion

Abstract

Retinitis pigmentosa (RP) is a genetically heterogeneous form of inherited retinal disease that leads to progressive visual impairment. One genetic subtype of RP, RP54, has been linked to mutations in PCARE (photoreceptor cilium actin regulator). We have recently shown that PCARE recruits WASF3 to the tip of a primary cilium, and thereby activates an Arp2/3 complex which results in the remodeling of actin filaments that drives the expansion of the ciliary tip membrane. On the basis of these findings, and the lack of proper photoreceptor development in mice lacking Pcare, we postulated that PCARE plays an important role in photoreceptor outer segment disk formation. In this study, we aimed to decipher the relationship between predicted structural and function amino acid motifs within PCARE and its function. Our results show that PCARE contains a predicted helical coiled coil domain together with evolutionary conserved binding sites for photoreceptor kinase MAK (type RP62), as well as EVH1 domain-binding linear motifs. Upon deletion of the helical domain, PCARE failed to localize to the cilia. Furthermore, upon deletion of the EVH1 domain-binding motifs separately or together, co-expression of mutant protein with WASF3 resulted in smaller ciliary tip membrane expansions. Finally, inactivation of the lipid modification on the cysteine residue at amino acid position 3 also caused a moderate decrease in the sizes of ciliary tip expansions. Taken together, our data illustrate the importance of amino acid motifs and domains within PCARE in fulfilling its physiological function.

Figure 1

Bioinformatic analysis shows structural and functional amino acid motifs in PCARE protein. PCARE encodes a protein of 1289 amino acids. At the N-terminus of PCARE, the glycine residue at amino acid position 2 (p.Gly2), and the cysteine residue at amino acid position 3 (p.Cys3) exhibit tentative palmitoylation and myristoylation lipid modifications, respectively. As predicted with DISOPRED server, a helical structural domain is located between amino acids 171 and 338. Using Jalview, the following conserved SLiMs were predicted: two RP62 kinase-binding motifs, and three EVH1 domain-binding motifs. To generate PCARE mutant proteins, the p.Gly2 and p.Cys3 residues were substituted by the inactive amino acid alanine residue, whereas predicted protein domains that are annotated in this schematic were deleted via site-directed mutagenesis, including one mutant in which all three EVH1 domain-binding motifs were deleted.

Figure 2

PCARE mutants are expressed and stable. Wild-type and mutant PCARE constructs were transfected in HEK293T cells. Western blot analysis revealed stable expression of all PCARE constructs. Wild-type PCARE protein has a molecular weight of 140 kDa, the molecular weight of mutant PCARE proteins is approximately the same, except for the mutant carrying the helix deletion which is smaller. Tubulin was loaded for control of the total amount of protein.

Figure 3

Mutant PCARE isoforms, except for PCAREΔhelix, localize to the cilia. Wild-type and mutant HA-tagged PCARE constructs were overexpressed in hTERT RPE-1 cells. Upon visualization with an anti-HA antibody, wild-type HA-tagged PCARE and PCARE mutant constructs except for PCAREΔhelix are co-localized with ciliary axoneme staining (anti-HA in red, ARL13B in green, DAPI in blue). All scale bars are 20 μm.

Figure 4

PCARE mutants differentially form ciliary expansions upon co-transfection with WASF3 in hTERT RPE-1. (A) When counted in the IHC slide and quantified, with wild-type PCARE normalized to hundred percent, all PCARE mutants generated less ciliary tip expansions than the wild-type (type 1 in yellow, type 2 in green, type 3 in red). (B) hTERT RPE-1 cells were transfected with 3×HA-PCARE (wild-type or mutant) and 3×FLAG-WASF3 constructs in equal amounts. Three distinct types of the ciliary tip expansions can be distinguished: type 1, where PCARE and WASF3 co-localize into the cilia and form expansions of the ciliary tip, type 2, where the proteins co-localize to the cilia but formed no expansions and, finally, type 3, where the proteins are retained in the cytosol (3×HA-PCARE in green, WASF3 in red, ARL13B in magenta, DAPI in blue). Untransfected cells (Utr) are shown as a negative control. Scale bars 5 μm. (C) The sizes of the ciliary tip expansions of type 1 were counted and quantified using an automated Fiji script. The triple-deletion PCAREΔEVH1a–c and single-deletions of PCAREΔEVH1a and PCAREΔEVH1b showed a significant decrease in the size of the expansion tip (^***P ≤ 0.001, ^**P ≤ 0.01, ^*P ≤ 0.05, red line shows mean and SD).

Figure 5

PCARE mutants form ciliary expansions when co-expressed with WASF3 in IMCD-3 stable cell line. (A) When counted in the IHC slide and quantified, with wild-type PCARE normalized to hundred percent, all PCARE mutants generated less ciliary tip expansions than the wild-type (type 1 in yellow, type 2 in green, type 3 in red). (B) IMCD-3 cells stably expressing FLAG-tagged WASF3 were transfected with 3×HA-PCARE (wild-type or mutant) constructs. Three distinct types of the ciliary tip expansions can be distinguished: type 1, where PCARE and WASF3 co-localize into the cilia and form expansions of the ciliary tip, type 2, where the proteins co-localize to the cilia but formed no expansions and, finally, type 3, where the proteins co-localize into the cytosol (3×HA-PCARE in green, WASF3 in red, ARL13B in magenta, DAPI in blue). Untransfected cells (Utr) are shown as a negative control. Scale bars 5 μm. (C) The sizes of the ciliary tip expansions of type 1 were counted and quantified using an automated Fiji script. The removal of lipid modification from the p.Cys3, PCAREΔEVH1a and PCAREΔEVH1a–c led to a significant decrease in the ciliary tip expansion sizes. (^***P ≤ 0.001, ^**P ≤ 0.01, ^*P ≤ 0.05, red line shows mean and SD).