Espin 1 immunofluorescence distribution in stereocilia is similar to myosin IIIa Immunofluorescence confocal images show myosin IIIa (green) localized at the tips of stereocilia in rat cochlear hair cells at PD6 (a) matching the localization observed for espin 1 (green, b). In contrast, pan-espin labeling (green) appears along the entire stereocilia (c). Scale bar, 5 µm. Immunogold labeling shows espin 1 localized at the tip of stereocilia around the actin core (d, e) in contrast to labeling with a pan-espin antibody which shows localization throughout the entire stereocilia actin cores (f)in directly frozen (d) or plunge-frozen (e) adult rat cochlear (d) and vestibular (e, f) hair cells. Scale bars, 200 nm. (g) Immunofluorescence of espin 1 (green) localization in guinea pig vestibular stereocilia reveals a tip-to-base gradient that is more extended in longer stereocilia (inset T) than medium (inset M) and short (inset S) stereocilia. (h) Measurements of the green (espin 1) and red (actin) relative pixel intensity (rpi) of fluorescence along the stereocilia for each rectangular inset in g confirm this tip-to-base concentration gradient of espin 1 immunofluorescence. Longitudinal (i) as well as cross section (j) images reveal a thimble-like distribution of espin 1 (green) at tips of stereocilia in hair cells of P8 rat cochlea. (k-m) Immunofluorescence of rat cochlear hair cells in different developmental time points shows that espin 1 targets stereocilia tips as early as P0 (k) and undergoes progressive compartmentalization at the tips during P2 (l) and P4 (m), reaching a peak of intensity at ∼PD6 as shown in b. Scale bars, 5 µm. Antibodies: Espin 1 (PB539), myosin IIIa (PB638), and pan-espin (PB127). In all immunofluorescence images, the immunolabeling is visualized using Alexa-488-conjugated secondary antibody and F-actin (red) is visualized using Alexa 568-phalloidin.

Espin 1 alone or when co-overexpressed with myosin IIIa elongates stereocilia. (a) GFP-espin 1 localizes to stereocilia tips in a tip-to-base gradient distribution in transfected cultured organ of Corti hair cells. (b) High magnification close-up view and measurement of the relative pixel intensity (rpi) of GFP-espin 1 (green) and actin (Alexa 568-phalloidin, red) fluorescence along the distal portion of the stereocilia shown in the rectangular inset in (a) matches the tip-to-base concentration gradient observed for endogenous espin 1 (Fig. 1h). Organ of Corti (c) and vestibular (d) hair cells transfected with GFP-myosin IIIa show tip localization similar to espin 1. Co-transfection of vestibular hair cells with GFP-myoIIIa (arrow, green) and untagged espin 1 together (e and f) produce longer stereocilia than hair cells transfected with GFP-espin 1 alone (a) or with GFP-myosin IIIa alone (c and d). The average ratios of stereocilia length between transfected (H_T) and neighboring non-transfected (H_NT) cells, , are plotted in the bar graph in (g). GFP-espin 1 alone = 1.5 ± 0.67, n=19 (∼50% increase); GFP-myoIIIa alone = 1.1 ± 0.14, n=16 (∼10% increase); and GFP-myosin IIIa and espin 1 = 2.3 ± 0.69, n=14, (∼130% increase). Note that a value of ℓ=1 (indicated by the dotted line in the graph) corresponds to a zero percent increase in length. The error bars represent the standard deviation of the mean. ANOVA statistical analysis shows that the mean value for the hair cells co-transfected with espin1 and myosin IIIa is significantly higher than the cells transfected with espin 1 alone, with a p-value equal to 0.002. In contrast, the mean bundle heights for hair cells transfected with myosin IIIa alone were not significantly higher than the controls, with a p-value of .149. Scale bars, 5 µm.

Espin 1 interacts with myosin IIIa through its ankyrin repeats domain (ARD) in transfected COS-7 cells. (a) mCherry-tagged espin 1 ARD shows filopodia tip localization when cotransfected with GFP-myoIIIa ΔK but fails to localize at the filopodia tips when co-transfected with GFP-myosin X or GFP-myosin XVa. Scale bar, 5 µm. (b) Selected video images from a time lapse video (Supplementary Information, Video S1) show co-transport of GFP-myoIIIa ΔK and mCherry-ARD during the formation and extension of filopodia in COS-7 cells. Scale bar, 2.5 µm. (c) Single representative frame from a time lapse video of a single filopodium (Supplementary Information, Video S2) graph with the normalized relative pixel intensity of the fluorescence along the filopodium show co-distribution (cross-correlation of the green and red intensity values = 0.990 for this frame; the average cross correlation for six randomly selected frames from the same video was 0.99) of GFP-myoIIIa ΔK and mCherry-ARD forming a tip to base gradient and co-localization of the green and red fluorescence puncta (arrowheads) corresponding to the co-transport of GFP-myoIIIa ΔK and mCherry-ARD trafficking into and out of the tip of the filopodia. Scale bar, 0.5 µm. (d) Schematic representation of the constructs analyzed in this figure. Legend: GST, glutathione S-transferase; GFP, green fluorescence protein; ARD, ankyrin repeats domain; myoIIIa ΔK, myosin IIIa with a deletion of it kinase domain; 3THDI and 3THDII, myosin IIIa tail homology domains 1 and 2, respectively. (e) Western blots of GST pull-downs show that purified GST-ARD co-precipitates with GFP-myoIIIa ΔK, but not with GST alone. Precipitates were detected using anti-GST (α-GST) and anti-GFP (α-GFP).

Myosin IIIa interacts with espin 1 through its 3THDI domain. (a) Schematic representation of the espin 1 and myosin IIIa constructs analyzed in this figure. Legend: ABM, actin binding module; WH2, Wiskott-Aldrich homology domain 2; GFP-myoIIIa Δ32, myosin IIIa lacking exon 32 which causes a frame shift rendering the protein without the 3THDI and 3THDII domains. (b) Co-expression of untagged espin 1 shows that GFP-myoIIIa, GFP-tailΔ3THDII, and GFP-3THDI (green) colocalize with espin 1 (red) along actin filament bundles. In contrast, GFP-myoIIIa Δ32, GFP-pre-, and post3THDI are dispersed in the cytoplasm, despite the presence of espin 1 bundles. Scale bar, 5 µm. (c) Western blots of GST pull-downs confirm that the 3THDI region of myosin IIIa is necessary and sufficient for binding to espin 1 ARD, as pre3THDI and post3THDI show no binding to GST-ARD. Precipitates were detected using α-GST and α-GFP.

Myosin IIIa and espin 1 synergistically elongate filopodia in COS-7 cells via espin 1 WH2 activity. Overexpression of either GFP-myoIIIa ΔK (a) or GFP-espin 1 (b) results in formation of short filopodia (mean lengths = 1.7 ± 0.83 µm and 1.3 ± 0.28 µm, respectively). In contrast, the co-expression of GFP-myoIIIa ΔK (green) and espin 1 (c) has a synergistic effect that generates extremely long filopodia (14.3 ± 9.1 µm). F-actin (red) is visualized using Alexa 568-phalloidin. (inset, c) Graph of the relative pixel intensity (rpi) of the GFP-myoIIIa ΔK distribution in the single filopodium indicated by the rectangle in c shows the characteristic tip-to-base decaying gradient. (d) Co-expression of full length GFP-myoIIIa and espin 1 produces a more limited tip localization of these proteins and elongation of filopodia (3.7 ± 3.2 µm) when compared to co-expression of GFP-myoIIIa ΔK and espin 1. (e) The enhanced elongated phenotype is restored to a limited extent (5.93 ± 3.10 µm) when COS-7 cells were co-transfected instead with GFP-myoIIIa K50R and espin 1. (f) The enhanced elongation phenotype is similar to c when the cell was co-transfected with GFP-myoIIIa ΔK,33,34 and espin 1 (10.02 ± 4.7 µm). (g) Co-expression of GFP-myoIIIa ΔK and espin 1 lacking ARD (espin 1 ΔARD, labeled with the pan espin antibody, red) but fails to elongate filopodia (2.05 ± 1.8 µm) and to show tip localization of espin 1 ΔARD (inset, g). (h) Co-expression of GFP-myoIIIa ΔK and espin 1 with a mutated WH2 domain (espin 1 mWH2, labeled with espin 1 antibody, red) fails to elongate filopodia (2.65 ± 1.5 µm) despite the fact that espin 1 mWH2 localizes to the tip and forms the tip to base gradient matching the distribution of the GFP-myoIIIa ΔK (inset, h). Scale bars, 2.5 µm. Measurements of filopodia lengths for each of the combinations shown in the panels above are presented as box-plots, with upper and lower whiskers representing the range, the top and bottom of the box representing the upper and lower 25th percentile, and the filled squares represent the mean values.