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. 2015 Nov 17;13(7):1287-1294.
doi: 10.1016/j.celrep.2015.10.003. Epub 2015 Nov 5.

The Stereociliary Paracrystal Is a Dynamic Cytoskeletal Scaffold In Vivo

Philsang Hwang  1 , Shih-Wei Chou  1 , Zongwei Chen  1 , Brian M McDermott Jr  2
Affiliations

The Stereociliary Paracrystal Is a Dynamic Cytoskeletal Scaffold In Vivo

Philsang Hwang et al. Cell Rep. .

Abstract

Permanency of mechanosensory stereocilia may be the consequence of low protein turnover or rapid protein renewal. Here, we devise a system, using optical techniques in live zebrafish, to distinguish between these mechanisms. We demonstrate that the stereocilium's abundant actin cross-linker fascin 2b exchanges, without bias or a phosphointermediate, orders of magnitude faster (t1/2 of 76.3 s) than any other known hair bundle protein. To establish the logic of fascin 2b's exchange, we examine whether filamentous actin is dynamic and detect substantial β-actin exchange within the stereocilium's paracrystal (t1/2 of 4.08 hr). We propose that fascin 2b's behavior may enable cross-linking at fast timescales of stereocilia vibration while noninstructively facilitating the slower process of actin exchange. Furthermore, tip protein myosin XVa fully exchanges in hours (t1/2 of 11.6 hr), indicating that delivery of myosin-associated cargo occurs in mature stereocilia. These findings suggest that stereocilia permanency is underpinned by vibrant protein exchange.

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Figures

Figure 1
Figure 1. Stereociliary fascin 2b exchanges rapidly and without bias
(A) Hair bundle schematic (B) (top) Confocal image of a single stereocilium (arrowhead) containing GFP-fascin 2b (green) demonstrates even labeling of the protrusion’s core. Below is a schematic of the fusion protein. (C) A lateral crista hair bundle before (Pre) and after a photobleach, which targeted the mid-region (yellow bracket). This FRAP series is qualitative. (D) FRAP profiles from a quantitative series of images displayed in Figure S1A. Within 315 s, the mean intensity in the post-bleached region nearly matches the mean intensity along the length of the stereocilia (dashed line), indicating significant GFP-fascin 2b exchange. (E) Recovery plot from quantitative FRAP series in Figure S1A fit to a one-phase exponential function. Color-coded scale of recovery, after upper half (F) or lower half (G) of bundles were bleached (dashed line), demonstrates that GFP-fascin 2b migrates towards the stereociliary tips or bases, respectively. Blue-to-red color scale represents low-to-high GFP signal, respectively. (H) Bleaching entire bundle reveals migration of fusion protein from soma to stereocilia. (I) Models of fascin 2b movement in stereocilia. (J) Mean FRAP recovery halftimes from photobleach events of tops, middles, or bases of hair bundles containing GFP-fascin 2b from 7- or 4-dpf zebrafish or from middles of filopodia containing GFP-fascin 1 from COS-7 cells (Aratyn et al., 2007). *P< 0.005 (Student’s t test) indicates statistically distinct. Scale bars are 1 μm. See also Figures S1, S2, and S4, Tables S1 and S2, and Movie S1.
Figure 2
Figure 2. Rapid stereociliary fascin 2b exchange transpires without a phosphointermediate
(A) Phosphomutant GFP-S38A fascin 2b schematic. (B) Qualitative FRAP series of a hair bundle containing GFP-S38A fascin 2b before (Pre) and after photobleaching (yellow bracket). (C) Recovery plot from a hair bundle containing GFP-S38A fascin 2b from a quantitative FRAP series in Figure S1F, t1/2 = 73.6 s. (D) Phosphomimetic GFP-S38E fascin 2b schematic. (E) Bundle containing GFP-S38E fascin 2b before (Pre) and after photobleaching. (F) Recovery plot of E demonstrates exceedingly fast recovery, t1/2 = 4.68 s, of the phosphomimetic relative to wild-type fascin 2b. (G) Summary of recovery halftimes for GFP-fascin 2b, GFP-S38A fascin 2b (S38A), GFP-S38E fascin 2b (S38E), and GFP. Since GFP-S38A fascin 2b has a similar t1/2 value to GFP-fascin 2b, wild-type fascin 2b does not transition through a detectable phosphointermediate in stereocilia. *P< 0.0001 (Student’s t test) indicates statistically significant. Fish used were 7 dpf. Scale bars are 1 μm. (H) Model: The fascin 2b-F-actin binding cycle is independent of phosphorylation. In model, horizontally oriented fascin 2b protein indicates protein bound to F-actin. Vertically oriented fascin 2b protein is not bound to F-actin. Alternative model is shown in Figure S3B. See also Figure S3 and Tables S1 and S3.
Figure 3
Figure 3. β-actin exchanges on an hourly timescale in live zebrafish hair cells
Schematics of a hair bundle (A) and β-actin-mCherry (B). (C) Hair bundle (arrowhead) and cuticular plate (asterisk) labeled with β-actin-mCherry in transgenic zebrafish. (D) Confocal image of three stereocilia (arrowheads) from a splayed hair bundle reveals even labeling across each stereocilium. Splayed bundles are rare in this transgenic. Note: limited portions of each stereocilium are in the plane of focus. (E) Quantitative fluorescence recovery of a bundle containing β-actin-mCherry after a midsection bleach (yellow bracket). (F) Recovery plot reveals a t1/2 of 4.11 h for the hair bundle observed in E. The data points are fit to a one-phase exponential equation. Zebrafish examined at 7 dpf. Scale bar is 1 μm. See also Figure S4 and Table S1.
Figure 4
Figure 4. Robust myosin XVa exchange at the tips of mature stereocilia
(A) Schematic of bundle containing β-actin-mCherry (red) and GFP-myosin XVa (green). (B) Image of a bundle expressing GFP-myosin XVa (green) reveals fusion protein at stereociliary tips (Pre, left and right). Bundle is counterlabeled with β-actin-mCherry (Pre, left, red) in a doubly transgenic hair cell. After photobleaching the top half of bundle (only GFP signal is shown), the GFP-myosin XVa signal recovers completely by 24 h. Scale bar is 1 μm. (C) Time course of myosin XVa recovery. Zebrafish examined at 7 dpf. (D) Summary of recovery halftimes of fusion proteins in stereocilia and filopodia. Recovery halftime of myosin XVa in filopodia is estimated (Belyantseva et al., 2005). See also Table S1.
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