Fig 1: Myo5a schematic. Schematic of Myo5a mRNA/cDNA, depicting regions encoding the major structural domains of the Myo5a protein monomer: the ATP hydrolyzing/actin binding motor domain, the light-chain binding IQ domain, the central dimerization domain, the alternative exon domain, and the globular tail/cargo binding domain. Three coiled-coil regions (CC1, CC2, and CC3) have been described and are shown in the second line; CC1 and CC2 are within the dimerization domain, while CC3 is intact only in variants that do not contain exon F (such as the predominant brain form of Myo5a, ABCE), since it includes part of exon E sequence and part of globular tail domain sequence. CC3 is bisected by exon F, if present. Alternative exons are shown in the expansion; sequence encoding the constitutive exons A, C and E and the optional exons B, D and F are indicated by solid and speckled patterns, respectively. The red arrow indicates the position of the molecular defect in the Myo5a gene in the DBA mouse, which impairs mRNA splicing. Below, the products of detrusor nested PCR reactions are shown, as are the locations of restriction enzyme cleavage sites. The absence of exon B (and its diagnostic Sty I restriction site) in detrusor nested PCR products is indicated by the dashed line. The red lines indicate the positions of the RT-PCR assays employed.
Fig 2: Subcellular localisation of EGFP–MyRIP mutants in HUVECs – mutation of the actin-binding region does not perturb endogenous MyoVa recruitment to WPBs. (A) Top, schematic structure of human MyRIP domains and position of the mutated amino acids. Below, confocal immunofluorescence image of a single HUVEC expressing EGFP–MyRIP WT and labelled with phalloidin (red) and specific antibodies against EGFP (green) and VWF (blue). Regions indicated by white boxes are shown as greyscale inserts. (B–D) As for A but for cells expressing EGFP–MyRIP A751P (B), EGFP–MyRIP 4A (C) and EGFP–MyRIP A751P 4A (D). (E,F) Confocal immunofluorescence images of single HUVECs expressing EGFP–MyRIP WT (E) or EGFP–MyRIP 4A (F), and labelled with specific antibodies to EGFP (green) and MyoVa (red). Greyscale images below show, on the same scale, endogenous MyoVa immunoreactivity from regions indicated in the MyRIP-expressing (i) and non-expressing (ii) cells. Scale bars: 10 µm.
Fig 3: Expression of Myo5a in bladder tissue. (A–C) Laser scanning confocal microscopy of sections from WT detrusor detected Myo5a immunoreactivity (red staining) on nerve fibers that were immunoreactive for the pan-neuronal marker synaptophysin (Syp, green staining). The merged image on the right shows co-localization of these markers in yellow. (D–F) Immunoreactivity for Myo5a (red staining) and vesicular acetylcholine transporter (VAChT, green staining) in WT bladder muscularis. Co-localization of these two proteins in the merged panel (yellow) indicates expression of Myo5a in cholinergic fibers of the urinary bladder. Arrows indicate serosa. (Scale bar = 50 µm; Mag ×40).
Fig 4: Relative expression of variant exons in WT and DBA tissues. cDNAs made from total RNA of brain, skin and detrusor of WT animals (black circles, N = 4) and DBA animals (gray squares, N = 3) were assayed in triplicate for Myo5a exon B or for exon F with their specific TaqMan assays, and for 18S rRNA as an internal control. Expression was determined by the ∆∆Ct method and graphed relative to WT within each tissue. Horizontal line indicates average relative expression ±SEM. (A) Expression of exon B in brain was not different in DBA compared to WT (p = 0.67). In detrusor and skin, exon B was not detected (nd) and therefore differences between strains could not be determined. (B) Expression of exon F was lower in DBA brain, skin and detrusor (*p < 0.05) than in corresponding WT tissues.
Fig 5: Myo5a exons D and F in bladders from WT and DBA mice. (A) Nested PCR fragments from detrusors of WT (N = 6) and DBA (N = 5) animals were prepared with primer pair 3 and electrophoresed. A representative agarose gel with bands corresponding to fragments with and without exon D from three WT and three DBA detrusors is shown, with individual WT and DBA brain samples as controls. Exposure of the 50 bp standard lane, with weighted 350 bp band, was adjusted separately. (B) The intensity of +D bands is graphed as a proportion of total intensity in each lane (sum of +D and −D) and plotted for WT (black circles), and DBA (gray squares). Horizontal line indicates average intensity in % ± SEM for the +D band in all replicates. The comparison between WT and DBA detrusor was significant (*p = 0.015). (C) Nested PCR fragments from detrusor of three WT and three DBA animals were prepared with primer pair 4, digested with Ban II to cleave the +D band, and electrophoresed. The relative positions of detrusor exon F-containing digestion fragments including or lacking exon D are indicated, and a flanking lane with 100 bp standards is marked. Contrast for the standard lane was adjusted separately. (D) Data are graphically represented; the horizontal line indicates average intensities in % ± SEM for PCR product containing exon D (black circles, WT; gray squares, DBA) are graphed. Coincidence of exons D and F in the same cDNA fragment was significantly reduced in DBA detrusor (p = 0.03).
Supplier Page from MilliporeSigma for Anti-Myosin Va (LF-18) antibody produced in rabbit