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. 2009 Sep 23;4(9):e7097.
doi: 10.1371/journal.pone.0007097.

MyosinVIIa interacts with Twinfilin-2 at the tips of mechanosensory stereocilia in the inner ear

Agnieszka K Rzadzinska  1 , Elisa M Nevalainen, Haydn M Prosser, Pekka Lappalainen, Karen P Steel
Affiliations

MyosinVIIa interacts with Twinfilin-2 at the tips of mechanosensory stereocilia in the inner ear

Agnieszka K Rzadzinska et al. PLoS One. .

Abstract

In vertebrates hearing is dependent upon the microvilli-like mechanosensory stereocilia and their length gradation. The staircase-like organization of the stereocilia bundle is dynamically maintained by variable actin turnover rates. Two unconventional myosins were previously implicated in stereocilia length regulation but the mechanisms of their action remain unknown. MyosinXVa is expressed in stereocilia tips at levels proportional to stereocilia length and its absence produces staircase-like bundles of very short stereocilia. MyosinVIIa localizes to the tips of the shorter stereocilia within bundles, and when absent, the stereocilia are abnormally long. We show here that myosinVIIa interacts with twinfilin-2, an actin binding protein, which inhibits actin polymerization at the barbed end of the filament, and that twinfilin localization in stereocilia overlaps with myosinVIIa. Exogenous expression of myosinVIIa in fibroblasts results in a reduced number of filopodia and promotes accumulation of twinfilin-2 at the filopodia tips. We hypothesize that the newly described interaction between myosinVIIa and twinfilin-2 is responsible for the establishment and maintenance of slower rates of actin turnover in shorter stereocilia, and that interplay between complexes of myosinVIIa/twinfilin-2 and myosinXVa/whirlin is responsible for stereocilia length gradation within the bundle staircase.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Pan-twinfilin localizes to the tips of shorter stereocilia.
Confocal images showing the distribution of pan-twinfilin in stereocilia bundles. Actin filaments were counterstained with rhodamine/phalloidin (red). A–F and H–Pan-twinfilin (green) localizes to tips of shorter stereocilia of inner (A–C, E–F), outer (D) and vestibular (H) hair cells of wild type adult mice at P40. E–F–magnified image of single stereocilium (from B) from the second row showing two distinct spots of pan-twinfilin staining on the surface of the tip. The length of the pan-twinfilin fluorescent spot (F, 470±70 nm) corresponds with the length of the tip measured on SEM image (G, 440±50 nm). F and G show different bundles in similar orientation. Scale bars: A, D, H–10 µm; B–C–5 µm, E–G - 1 µm.
Figure 2
Figure 2. Myosin VIIa is necessary for pan-twinfilin localization at the stereocilia tips.
Confocal images showing the distribution of pan-twinfilin in mutant stereocilia bundles. Actin filaments were counterstained with rhodamine/phalloidin (red). Images on the right show green channel. A - In mosaic auditory sensory epithelia of Myo7a4626SB/4626SB Hprt(Myo7a)Brd/+ females at P5 myosinVIIa immunofluorescence (green) is restricted to complemented hair cells with normal morphological appearance of stereocilia bundles. B, C - Pan-twinfilin immunofluorescence (green) at stereocilia tips is restricted to complemented inner hair cells (showing normal hair bundle morphology) while there is no staining in shorter stereocilia of non-complemented inner hair cells (asterisks). D–E - Pan-twinfilin (green) localized to the tips of all whirlin- and myosinXVa-deficient stereocilia on the apical surface of inner hair cells of adult Whrnwi/wi (D) and Myo15ash2/sh2 mice (E). Scale bars: A–10 µm; B–D and H–5 µm, E–G–1 µm.
Figure 3
Figure 3. Wild type full-length DsRED-myosinVIIa (red) co-localizes with GFP- twinfilin-2 (green) in filopodia tips.
Confocal images showing distribution of GFP-twinfilin-2, DsRED-myosinVIIa, in BHK-21 cells. Cortical actin was stained with AlexaFluor633/phalloidin (blue). A GFP-twinfilin-2 alone localizes predominantly along the filopodium length. B DsRED-myosinVIIa alone localizes predominantly along the filopodium length. C–E Co-transfection of GFP-Twf-2 and DsRED-Myo7a reveals co-localization of co-expressed proteins at the filopodium tip (arrow heads) and adhesion plaques (arrows) in representative BHK-21 cells. Scale bars: A–H -25 µm.
Figure 4
Figure 4. Levels of exogenous expression of DsRED-Myo7a correlate with lower number of filopodia in BHK-21 fibroblasts.
A–B Higher levels of DsRED-Myo7a fluorescence in cell cytoplasm correlated negatively with number of filopodia in cells expressing GFP-Myo15a. In fibroblast showing low levels of DsRED-Myo7a fluorescence filopodia were numerous and GFP-Myo15a was clearly visible at their tips. C In cells co-transfected with GFP-Myo15a, DsRED-Myo7a and caerulean-Twf-2 all three proteins localized to filopodia tips. Scale bars: A–B -50 µm, C–10 µm.
Figure 5
Figure 5. Twinfilin-2 interacts with myosinVIIa in vivo.
The interaction between twinfilin-2 and myosinVIIa was demonstrated by immunoprecipitation from protein lysates of inner ear tissue with anti myosinVIIa antibody (myoVIIa), anti twinfilin-2 antibody (twf-2), protein A-Sepharose and anti twinfilin-1 (twf-1) antibody followed by the immunoblot with a anti-myosinVIIa antibody (A) and anti-twinfilin-2 antibody (B). Procedures for immunoprecipitations and Western blots are described under Materials and Methods (Supp. information). Twinfilin-2 has a molecular weight of 39 kD. MyosinVIIa has a molecular weight of about 250 kD; the band at 100 kD may be a result of protein degradation.
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