FLUORESCENCE RECOVERY KINETICS UNCOVER TWO DISTINCT ACTIN POPULATION DURING STEREOCILIA DEVELOPMENT

Inner ear hair cells are essential for hearing, and this function is supported by specialized rod-like projections called stereocilia located on their apical surface. Each stereocilium contains long, unbranched actin filaments bundled together by actin crosslinking proteins, with their barbed ends oriented toward the tip and pointed ends toward the base. During development, stereocilia increase in both length and width, largely due to the elongation and addition of actin filaments. When actin is overexpressed in hair cells, EGFP-actin monomers first incorporate at the stereocilia tips and later along the shaft, supporting a tip-down model where actin added at the tip moves downward along the shaft.To examine actin dynamics, we used Fluorescence Recovery After Photobleaching (FRAP) on transgenic mice expressing EGFP-actin. Our results showed actin turnover occurring at both the tip and shaft in P2 and P4 mice, but by P6, turnover was restricted mainly to the tips. Quantitative analysis revealed that the size of the tip and shaft regions remains constant despite increases in fluorescence intensity, which challenges the simple tip-down model predicting expansion of the tip zone to fill the shaft. Additionally, the differing recovery rates between tip and shaft suggest distinct regulatory mechanisms for actin in these regions.Further investigations involved treating explants with the actin-capping drug Cytochalasin D (CytoD), followed by FRAP analysis after drug removal. EGFP-actin incorporation was uniform along the stereocilia, indicating reduced turnover at the tips and suggesting that actin addition along the shaft occurs independently of tip assembly. CytoD treatment caused a reduction in stereocilia width at P4 but not at P6, highlighting the increased stability of the shaft actin at later developmental stages. This stabilization depends on the actin crosslinker protein ESPN, as stereocilia in jerker mice lacking ESPN displayed highly dynamic actin that recovered rapidly after photobleaching.The two main conclusions from this study are that actin along the stereocilia shaft becomes progressively more stable during development due to ESPN-mediated crosslinking, and that actin incorporation along the shaft proceeds independently of assembly at the stereocilia tips.