Alternative splicing in class V myosins determines association with Rab10

Abstract

Rab proteins influence vesicle trafficking pathways through the assembly of regulatory protein complexes. Previous investigations have documented that Rab11a and Rab8a can interact with the tail region of myosin Vb and regulate distinct trafficking pathways. We have now determined that a related Rab protein, Rab10, can interact with myosin Va, myosin Vb, and myosin Vc. Rab10 localized to a system of tubules and vesicles that have partially overlapping localization with Rab8a. Both Rab8a and Rab10 were mislocalized by the expression of dominant-negative myosin V tails. Interaction with Rab10 was dependent on the presence of the alternatively spliced exon D in myosin Va and myosin Vb and the homologous region in myosin Vc. Yeast two-hybrid assays and fluorescence resonance energy transfer studies confirmed that Rab10 binding to myosin V tails in vivo required the alternatively spliced exon D. In contrast to our previous work, we found that Rab11a can interact with both myosin Va and myosin Vb tails independent of their splice isoform. These results indicate that Rab GTPases regulate diverse endocytic trafficking pathways through recruitment of multiple myosin V isoforms.

FIGURE 1.

Tissue distribution of human myosin Va and myosin Vb splice isoforms. A, schematic of the alternative exon organization in the tails of myosin Va and myosin Vb. It is known that exons B, D, and F are subject to alternative splicing in myosin Va, whereas there is only evidence that exon D is alternatively spliced in myosin Vb, which does not contain exon F. B, alignment of exon D sequences from mouse and human myosin V's. myosin Va and myosin Vb both contain exon D (amino acids 1320-1346 of myosin Va and 1315-1340 of myosin Vb), whereas myosin Vc contains an exon D-like region (amino acids 1124-1147 of human myosin Vc) that is not known to be alternatively spliced. Alignment of the exon D regions from all three motors reveals a high degree of homology, especially in the center of the exon. Asterisks indicate amino acid identities. C, PCR-based analysis of human tissue panels reveals the alternative splicing pattern of exon D in myosin Va and myosin Vb. Primers flanking the region encoding exon D for both motors were used to amplify cDNA from human MTC™ panels (Clontech). cDNA amplified from HeLa cell RNA as well as myosin Va and myosin Vb tail constructs were used as positive controls. Variants expressing exon D (upper bands) and lacking exon D (lower bands) were visible. Per., peripheral; Pos., positive.

FIGURE 2.

Endogenous Rab10 localizes to a system of tubules and vesicles and partially co-localizes with Rab8a- and myc-EHD3-labeled tubules. A-C, HeLa cells stained with anti-Rab8a, anti-Rab10, or anti-Rab11a polyclonal primary and Cy3-labeled secondary antibodies were imaged by confocal microscopy. In a typical HeLa culture ∼17% (±1.2 S.E.) of cells showed a tubular Rab8a or Rab10 pattern, whereas the rest showed a vesicular distribution. D and E, HeLa cells transfected with mCherry-Rab10 (left) and stained with anti-Rab8a polyclonal primary and Alexa488-labled secondary antibodies (center). The merged image (right) shows that Rab8a localized with Rab10 on the peripheral ends of some but not all tubules. F and G, HeLa cells transfected with Myc-tagged EHD3 and stained with Alexa-488-labeled secondary antibodies (center) were co-stained with anti-Rab10 or anti-Rab8a polyclonal primary antibodies and Alexa-568-labeled secondary antibodies (left). Merged images (right) demonstrate that Rab10 only partially co-localizes with EHD3 (see inset). Scale bars represent 10 μmin panels A-D, F, and G and 5 μm in panel E.

FIGURE 3.

Only myosin V tails expressing exon D alter endogenous Rab10 distribution. A, HeLa cells transfected with EGFP-myosin Va tail splice isoform containing exon D (EGFP-MVa-tail +D) and stained for Rab10. EFGP-myosin Va tail +D caused endogenous Rab10 to mislocalize to EGFP-labeled puncta. B, EGFP-myosin Va tail-brain splice isoform, which lacks exon D (EGFP-MVa-tail -D), also localized to disperse puncta but was unable to recruit endogenous Rab10. C and D, HeLa cells transfected with EGFP-myosin Vb tail expressing exon D (EGFP-MVb-tail +D) or lacking exon D (EGFP-MVb-tail -D) and stained for Rab10. Both splice variants of EGFP-myosin Vb tail localized to a perinuclear cisternum, but only myosin Vb tail expressing exon D caused Rab10 to mislocalize to the same cisternum. E and F, HeLa cells transfected with wild-type EGFP-myosin Vc tail, which contains an exon D-like domain (EGFP-MVc-tail) or a synthetic construct lacking exon D (EGFP-MVc-tail ΔD) and stained for Rab10. Similar to myosin Va and myosin Vb, myosin Vc tail required exon D to recruit Rab10. Scale bars in all panels represent 10 μm. Percent co-localization (±S.E.) are listed in the merged images on the right of each panel (n ≥ 10). ^*, statistically significant difference comparing +D and -D constructs (p < 0.001).

FIGURE 4.

Myosin Va, Vb, and Vc tails alter endogenous Rab8a distribution. A and B, HeLa cells transfected with EGFP-myosin Va tail +D or EGFP-myosin Va tail -D and stained for Rab8a. As with Rab10, EGFP-myosin Va tail +D caused endogenous Rab8a to mislocalize to EGFP-labeled puncta, whereas EGFP-myosin Va tail -D did not co-localize with Rab8a. C and D, expression of either splice variant of EGFP-myosin Vb tail (+Dor -D) caused Rab8a to redistribute to the EGFP-labeled perinuclear cisternum. E and F, only wild-type EGFP-myosin Vc tail, which contains an exon D-like domain, was able to recruit Rab8a, whereas MVc-tail-ΔD did not. Scale bars in all panels represent 10 μm. Percent co-localization (±S.E.) are listed in the merged images on the right of each panel (n ≥ 10). ^*, statistically significant difference comparing +D and -D constructs (p < 0.001).

FIGURE 5.

Myosin Va and myosin Vb tails alter endogenous Rab11a distribution in an exon-independent manner. A and B, HeLa cells transfected with EGFP-myosin Va tail +D or EGFP-myosin Va tail -D and stained for endogenous Rab11a. Unlike Rab8a and Rab10, Rab11a co-localized with both splice isoforms of myosin Va tail in scattered puncta. C and D, similarly, expression of either splice isoform of EGFP-myosin Vb tail (+Dor -D) caused endogenous Rab11a to localize to the EGFP-labeled perinuclear cisterna. Scale bars in all panels represent 10 μm. Percent co-localization (±S.E.) are listed in the merged images on the right of each panel (n ≥ 10).

FIGURE 6.

Myosin Va and myosin Vb tails alter endogenous transferrin receptor (CD71) distribution. A and B, HeLa cells transfected with EGFP-myosin Va tail +D or EGFP-myosin Va tail -D were stained for endogenous transferrin receptor (CD71). Similar to Rab11a, CD71 co-localized with both splice isoforms of myosin Va tail in scattered puncta. C and D, likewise, expression of EGFP-myosin Vb tail +D or EGFP-myosin Vb tail -D caused endogenous CD71 to be recruited to the EGFP-labeled perinuclear cisterna. Scale bars in all panels represent 10 μm. Percent co-localization (±S.E.) are listed in the merged images on the right of each panel (n ≥ 10).

FIGURE 7.

Myosin V tails FRET with Rab8a, Rab10, and Rab11a in vivo. FRET microscopy was performed on HeLa cells grown on coverslips and cotransfected with mCerulean-tagged myosin Va tail with or without exon D (myosin Va +Dor -D), myosin Vb tail with or without exon D (myosin Vb +D or -D), and mVenus-tagged Rab8a, Rab10, or Rab11a. The fluorescence intensity of a photobleached region of interest was measured before (pre-) and after (post-) photobleaching, and the average of pre-bleach images was compared with the average of the post-bleach images after background subtraction. As a control, FRET was measured on cells expressing the mCerulean-tagged myosin V tails alone. Error bars indicate the S.E. Rab11a produced a positive FRET signal with all four myosin V tail constructs regardless of splicing, whereas Rab10 produced a positive FRET signal only with myosin Va and myosin Vb tails expressing exon D. In contrast, Rab8a produced a positive FRET signal only with myosin Va tail +D but with both myosin Vb tail +Das well as -D. No FRET was observed between myosin Va tail -D and either Rab8a or Rab10 or between myosin Vb tail -D and Rab10. ^*, statistically significant difference comparing +D and -D constructs (p < 0.001).