The Journal of Neuroscience, October 4, 2006, ():
A New Compartment at Stereocilia Tips Defined by Spatial and Temporal Patterns of Myosin IIIa Expression
J. Neurosci. Schneider et al.
26: 10243
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplemental Figure 1
In Situ hybridization confirms gene expression of myosin IIIa in adult hair cells
In situ hybridization of cryosections of the inner ear of P8 (a,b) and P50 (c,d) mice show myosin IIIa expression in inner and outer hair cells of the basal turn of the cochlea (a,c, arrowheads) as well as in hair cell of the ampullae (b,d, arrowheads). Bar = 20μm.
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Supplemental Figure 2
Electron micrographs of thin section perpendicular to rat stereocilia (a-c) and a cross-section grazing the tip of a chick stereocilium (d) immunogold-labeled for myosin IIIa show labeling at the tips of stereocilia. With the post-embedding labeling procedure we used only the antigen sites close to the surface of the section are available for interaction with the antibody. Therefore each section shows a limited view of the distribution of myosin IIIa. However, the images shown above, which are representative of more than 40 images collected, are consistent with the thimble-like pattern of distribution of myosin IIIa seen in the immunofluorescence images.”. Bar = 100 nm.
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Supplemental Figure 3
Myosin IIIa ∆K mutations induce length changes in actin protrusions.
Confocal images of cultured organ of Corti (a,b) and vestibular tissue transfected with GFP-myosin ΔK and ΔK-34 (green) and then counterstained for actin with phalloidin (red) after 18 to 24 hours of transgene expression. Transfections with myosin IIIa ΔK (a) and ΔK-34 (c) often result in disruption of the staircase organization of the stereocilia bundle. Note that some stereocilia of the bundle are longer than those of the untransfected control-cell (arrows in a and c). Transfected supporting cells transfected with either GFP-myosin IIIa ΔK (b) or ΔK-34 (c) show elongated microvilli expressing at their tips (arrowheads). Compare the elongated microvilli in the transfected cells in b and c to the short and barely visible microvilli in untransfected supporting cells or to the supporting cell transfected with WT GFP-myosin IIIa in the inset in b. Bar = 2 μm
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Supplemental Figure 4
Diagram illustrating the proposed distribution of myosins Ic, IIIa, and XVa at and near the stereocilia tip.
Myosin XVa is restricted to the stereocilia cap or tip density at the ends of the actin paracrystal, and myosin IIIa is localized to a thimble-like region at and around the tips of stereocilia (denoted between brackets). Myosin Ic overlaps with the myosin IIIa compartment but extends down the shafts of stereocilia.
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Supplemental Movie 1 and 2
Hypermorphic myosin IIIa ∆K localizes to stereocilia tips and exhibits intrafilopodial motility.
A time-lapse movie (movie 1) of a COS-7 cell transfected with myosin IIIa ΔK. A series of 20 confocal sections were collected sequentially for each time point for a total of 20 minutes and viewed as the extended focus projection. Elapsed time is shown in the upper right corner. The width of the entire image is 30 µm. The majority of filopodia have intense fluorescence at their tips. A close up view (movie 2) of a selected region of the same cell show intrafilopodial motility.
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Movie 2
