Abstract
During development of the chick cochlea, actin crosslinkers and barbed-end cappers presumably influence growth and remodeling of the actin paracrystal of hair cell stereocilia. We used mass spectrometry to identify and quantify major actin-associated proteins of the cochlear sensory epithelium from E14 to E21, when stereocilia widen and lengthen. Tight actin crosslinkers (i.e. fascins, plastins, and espin) are expressed dynamically during cochlear epithelium development between E7 and E21, with FSCN2 replacing FSCN1 and plastins remaining low in abundance. Capping protein, a barbed-end actin capper, is located at stereocilia tips; it is abundant during growth phase II, when stereocilia have ceased elongating and are increasing in diameter. Capping protein levels then decline during growth phase III, when stereocilia reinitiate barbed-end elongation. Although actin crosslinkers are readily detected by electron microscopy in developing chick cochlea stereocilia, quantitative mass spectrometry of stereocilia isolated from E21 chick cochlea indicated that tight crosslinkers are present there in stoichiometric ratios relative to actin that are much lower than their ratios for vestibular stereocilia. These results demonstrate the value of quantitation of global protein expression in chick cochlea during stereocilia development.
Highlights
From the ‡Oregon Hearing Research Center and Vollum Institute, Oregon Health and Science University, Portland, Oregon 97239; §Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239; ¶National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda Maryland 20892; ʈDepartment of Public Health and Preventive Medicine, Oregon Health and Science University, Portland, Oregon 97239
Mass Spectrometry of Developing Cochlear Epithelia—To determine the expression profile of actin crosslinkers and other actin-cytoskeleton proteins during development of the chick cochlea, we examined organs from E14 and 16, which represent the beginning and end of the diameter-widening phase, and 18 and E21, during and at the end of the renewed elongation phase
To identify proteins at each developmental stage, we carried out liquid chromatography-tandem mass spectrometry (LC-MS/MS) shotgun proteomics
Summary
Two actin crosslinkers have been identified in chick cochlea hair bundles. The early observation that plastin 1 (PLS11; known as fimbrin and I-plastin) is present in mature and developing chick stereocilia led to the suggestion that it is the primary crosslinker in cochlear stereocilia [9]. In E21 chick vestibular hair bundles, fascin 2 (FSCN2) is the most abundant actin crosslinker [13, 14]; its paralog fascin 1 (FSCN1) was detected in bundles, albeit at much lower levels These experiments demonstrated that ESPN is present at low abundance in vestibular stereocilia; ESPN is unlikely to be the major crosslinker but instead exerts its effects by catalyzing filament elongation and widening of parallel actin bundles [15]. Low-abundance proteins are detected less reliably, the profusion of stereocilia actin filaments in the chick cochlea suggests that crosslinkers and cappers responsible for assembly of the stereocilium cytoskeleton should be relatively abundant in the whole cochlear epithelium. We examined the proteome of the peeled chick cochlear epithelia throughout important steps in stereocilia development These mass spectrometry experiments highlight the importance of FSCN2 and CAPZ, and were validated by additional quantitative RT-PCR, immunoblotting, and immunocytochemistry experiments. We show here that FSCN2 expression mirrors stereocilia lengthening; we found transient expression of CAPZ (and twinfilin) that could account for halted filament elongation seen during phase II and at the end of phase III
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