The molecular motor Myosin Va interacts with the cilia-centrosomal protein RPGRIP1L

Abstract

Myosin Va (MyoVa) is an actin-based molecular motor abundantly found at the centrosome. However, the role of MyoVa at this organelle has been elusive due to the lack of evidence on interacting partners or functional data. Herein, we combined yeast two-hybrid screen, biochemical studies and cellular assays to demonstrate that MyoVa interacts with RPGRIP1L, a cilia-centrosomal protein that controls ciliary signaling and positioning. MyoVa binds to the C2 domains of RPGRIP1L via residues located near or in the Rab11a-binding site, a conserved site in the globular tail domain (GTD) from class V myosins. According to proximity ligation assays, MyoVa and RPGRIP1L can interact near the cilium base in ciliated RPE cells. Furthermore, we showed that RPE cells expressing dominant-negative constructs of MyoVa are mostly unciliated, providing the first experimental evidence about a possible link between this molecular motor and cilia-related processes.

Figure 1. MyoVa interacts with the isoform c of RPGRIP1L.

(a) Schematic representation of the human MyoVa domain architecture highlighting the Motor domain (MD, yellow), the IQ motifs (blue), the alternatively spliced exons at the medial tail (A–E, exon F was omitted), and the GTD (pink) as well as the phosphorylation site at S1652 (top). (b) Comparison between the longest isoform of RPGRIP1L (isoform a) with that identified in this work (full-length isoform c). The isoform a is encoded by all the 27 exons of RPGRIP1L gene whereas isoform c lacks 46 residues (yellow) encoded by the exon 23. Both isoforms contain three C2 domains (green). (c) Activation of the LacZ and HIS3 reporter genes in YTH assays indicates that MyoVa-GTD interacts with the isoform c of RPGRIP1L. Yeast cells expressing only Gal4 Activation domain and/or LexA DNA-binding domain were used as negative controls.

Figure 2. MyoVa-GTD binds to the C2 domains of RPGRIP1L.

(a) Pull-down assays showing that 6xHis-MyoVa-GTD construct interacts with the three C2 domains of RPGRIP1L fused to GST. Bacteria expressing only 6xHis-MyoVa-GTD and GST were used as negative control. (b) Amino acid sequence identity and similarity between the C2 domains of human RPGRIP1L, according to structural alignment of the homology models of C2_MED and C2_CTERM (predicted using the HHPred server54) and the RMN structure of C2_NTERM (PDB ID: 2YRB) using the service PDBeFold. (c) MST assays showing that MyoVa-GTD binds to C2_NTERM (left), C2_MED (center) and C2_CTERM (right).

Figure 3. RPGRIP1L binds to a conserved binding site of class V myosins.

(a) YTH assays showing that mutations on the face C of MyoVa-GTD (W1713A, Y1721A, Q1755A and F1792A) disrupt the interaction between MyoVa-GTD and RPGRIP1L. (b) Surface representation of MyoVa-GTD (PDB ID: 4J5L) highlighting the residues identified as being involved in RPGRIP1L binding (pink), and other residues whose Ala mutants displayed auto-activation (K1757A, K1759A; grey) or a result similar to the wild-type MyoVa-GTD (yellow) in the YTH assay (panel A). The Rab11a-binding site, inferred from the crystal structure of MyoVb∙Rab11a complex (PDB ID: 4LX0), as well as the N- and C-termini of MyoVa-GTD (N-t and C-t) are indicated. (c) YTH assays showing that the isoform c of RPGRIP1L also interacts with MyoVb-GTD. Yeast cells expressing only Gal4 Activation domain and/or LexA DNA-binding domain were used as negative controls. (d) Phospho-mimetic (Ser to Glu) and non-phospho-mimetic (Ser to Ala) mutants of MyoVa-GTD interacted with RPGRIP1L in YTH assays, similarly to the wild-type protein. Yeast cells expressing only Gal4 Activation domain and/or LexA DNA-binding domain were used as negative controls.

Figure 4. MyoVa interacts with RPGRIP1L at the centrosome.

(a) PLA indicate the presence of endogenous complexes between MyoVa and RPGRIP1L (red dots) in a radius of 2 μm from the center of primary cilium base in 16% of hTERT RPE-1 ciliated cells (n = 522). Primary cilium axoneme is marked with acetylated-α-tubulin antibody (green). (b) The mean PLA dot count per cell was 2.12 ± 0.17 (mean ± SEM) in the assay and 0.26 ± 0.08 (mean ± SEM) in the control without primary antibodies (P < 0.005, two-tailed Student’s t-test).

Figure 5. Overexpression of dominant-negative constructs of MyoVa suppressed ciliogenesis.

Overexpression of EGFP-mGTD (a) or EGFP-GTD (b) in transfected RPE cells (n = 31 or 83) severely decreased the proportion of ciliated cells compared to their non-transfected neighbors (n = 170 or 373). Means ± SD represent the average data of 4 (a) or 17 (b) analyzed fields. (c) Overexpression of EGFP alone (negative control) did not affect the proportion of ciliated cells (n = 37) compared to the non-transfected condition (n = 238). Means ± SD represent the average data of eight analyzed fields. ns = nonsignificant; ****P ≤ 0.0001 (two-tailed Student’s t-test).