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Hear Res
2007 Feb 01;2241-2:15-26. doi: 10.1016/j.heares.2006.11.004.
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Myosin VI and VIIa distribution among inner ear epithelia in diverse fishes.
Coffin AB
,
Dabdoub A
,
Kelley MW
,
Popper AN
.
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Unconventional myosins are critical motor proteins in the vertebrate inner ear. Mutations in any one of at least six different myosins can lead to human hereditary deafness, but the precise functions of these proteins in the ear are unknown. This study uses a comparative approach to better understand the role of myosins VI and VIIa in vertebrate ears by examining protein distribution for these two myosins in the ears of evolutionarily diverse fishes and the aquatic clawed toad Xenopus laevis. Both myosins are expressed in the inner ears of all species examined in this study. Myo7a localizes to hair cells, particularly the actin-rich hair bundle, in all species studied. Myo6 also localizes to hair cells, but its distribution differs between species and end organs. Myo6 is found in hair bundles of most fish and frog epithelia examined here but not in anterior and posterior utricular hair bundles of American shad. These results show that myo7a distribution is highly conserved in diverse vertebrates and suggest functional conservation as well. The finding of myo6 in fish and Xenopus hair bundles, however, suggests a novel role for this protein in anamniotic hair cells. The lack of myo6 in specific American shad utricular hair bundles indicates a unique quality of these cells among fishes, perhaps relating to ultrasound detection capability that is found in this species.
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17204383
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Fig. 2.
Antibody characterization and controls. (A) Myo6 western blot. Myo6 antibody labels one
band of the expected molecular weight in mouse, Xenopus and zebrafish (~150 kDa). (B)
Myo7a western blot. Anti-myo7a labels a single band of ~200 kDa in zebrafish and American
shad. This band corresponds to the smaller of two labeled bands obtained from mouse cochlea.
(C–H) Epitope expression in cultured cells demonstrating antibody binding to zebrafish
myosins. GFP-labeled cells are positively transfected with the specific construct (myo6a,
myo6a, or myo7a, see below), while GFP-negative cells were not transfected and therefore
serve as negative controls for immunolabeling. (C–E) myo6a, (F) myo6b, (G) myo7a, (H)
control. (C) Myo6a-transfected NIH-3T3 cell, shown here expressing GFP. (D) The myo6a transfected
cell from panel C, immunolabeled for myo6. This demonstrates that the myo6
antibody binds to zebrafish myo6a. (E) Triple-labeled myo6a-transfected cell showing GFP
expression from panel C (green), myo6 immunocytochemistry from panel D (red) and DAPI
labeling (blue), which shows the successfully transfected cell surrounded by non-transfected
cells. (F) Triple-labeled (anti-GFP, anti-myo6, DAPI) NIH3T3 cell expressing myo6b. This
cell demonstrates that the myo6 antibody also binds zebrafish myo6b. (G) Triple-labeled (antiGFP,
anti-myo7a, DAPI) HEK293 cells transfected with myo7a, showing that the myo7a
antibody binds zebrafish myo7a. (H) NIH3T3 cell transfected with myo6b but not labeled with
anti-myo6, showing that GFP fluorescence does not bleed-through to red fluorescent channels.
(I–K) Immunocytochemistry controls in whole-mount inner ear epithelia. Each panel is a
merged image of phalloidin (green) and anti-myo6 (red) images. (I) Zebrafish utricle processed
for myo6 immunocytochemistry but with primary antibody omitted, demonstrating no bleedthrough
of the phalloidin fluorescence to the red channel. (J) American shad utricle labeled with anti-myo6 but with the secondary antibody omitted. The plane of section is at the cuticular
plate level, showing no myo6 staining in this region. (K) American shad utricle with clear
myosin labeling in the cuticular plate region. Phalloidin was omitted, showing that myo6
staining is genuine and not caused by bleed-through from the green channel. Scale bar in C is
25 μm and applies to panels C–H. Scale bars in I–K are 2 μm.
Fig. 5.
Myosin VI distribution in fish and Xenopus utricular hair cells. (A–C) Lake sturgeon, (D–F)
zebrafish, (G–I) oscar, (J–L) Xenopus. Arrows in C, F, I, and L indicate hair bundles that are
immunolabeled for myo6. In the oscar utricle (G–I), this staining is very faint and may be
artifact. All images are from whole-mount epithelia but cryosectioned tissue shows identical
labeling patterns (data not shown). All scale bars are 2 μm.
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