Hearing and vestibular function depend on precise variations in stereocilium dimensions according to position in the hair bundle and hair cell type and location in the cochlea or vestibular system. At the core of the stereocilium is a specialized cytoskeletal element, the parallel actin bundle. It displays the hallmarks of a supramolecular scaffold that determines the dimensions, placement and physical properties of stereocilia. Parallel actin bundles are held together by actin-bundling proteins, which cross-link neighboring actin filaments. We discovered and are characterizing a family of novel actin-bundling proteins called the espins. We showed that espin proteins are present throughout the body of stereocilia and the target of the jerker mutation, which causes deafness and vestibular dysfunction in mice. Our scanning electron microscopic analysis of jerker homozygotes, which lack espin proteins, showed that espins are required for stereocilia to undergo a 2-fold increase in diameter during morphogenesis. This led us to hypothesize that espins are required to assemble additional layers of actin filaments at periphery of the pre-existing actin bundle. Espins are encoded by a single gene, but are produced in different sized isoforms (28-91 kDa) with different amino-terminal extensions. We showed that the 116-residue carboxy-terminal actin-bundling module of espins is necessary and sufficient for actin bundling, but shows no obvious resemblance to other actin-bundling proteins. We also characterized the xAB, an additional actin filament-binding site present only in the extended amino-termini of the large espin isoforms, espin 1 and espin 2. We determined that the xAB increases the size of actin bundles formed by espins in vitro and inhibits actin fluorescence recovery (treadmilling) in espin-containing microvilli. We also found that the xAB in the espin 1 isoform is autoinhibited, but can be activated by a peptide in myosin III, a motor that is responsible for compartmentalizing espin 1 to the tip of stereocilia This was an indication that espin isoforms have different activities and localizations in stereocilia. To test our hypothesis that the different espin isoforms play distinct roles in stereocilia, we will compare the activities and localizations of the isoforms we have found in stereocilia. We will use isoform-specific polyclonal antibodies to localize the isoforms during the morphogenesis of stereocilia. In addition, we will use confocal microscopy, negative staining and total interference reflection fluorescence microscopy to examine their effects on actin bundling and polymerization in vitro. To test our hypothesis that espin level determines stereocilium actin filament number and diameter, we will use scanning and transmission electron microscopy to characterize stereocilium morphogenesis and actin bundle organization in our CBA/CaJ congenic mice that lack espins (jerker homozygotes) or contain ~1.5-times normal levels of espins owing to stable expression of an espin-BAC transgene. To deduce the molecular mechanism of actin filament cross-linking by the espin actin-bundling module, we will use scanning alanine mutagenesis to map its actin-binding sites and X-ray crystallography to determine its 3-dimensional structure.

Public Health Relevance

Hearing and balance depend on hair cells in the inner ear, in particular their small fingerlike extensions called stereocilia. We have discovered and are characterizing a family of novel stereocilia proteins called the espins, which are the target of mutations that cause deafness and vestibular problems in mice and humans and are required to form and maintain stereocilia. We have obtained evidence that different espin family members play distinct roles in stereocilia, and we are figuring out how they function.

Agency
National Institute of Health (NIH)
Institute
National Institute on Deafness and Other Communication Disorders (NIDCD)
Type
Research Project (R01)
Project #
5R01DC004314-18
Application #
9593501
Study Section
Auditory System Study Section (AUD)
Program Officer
Watson, Bracie
Project Start
2000-01-01
Project End
2019-11-30
Budget Start
2018-12-01
Budget End
2019-11-30
Support Year
18
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Northwestern University at Chicago
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
005436803
City
Chicago
State
IL
Country
United States
Zip Code
60611
Ahmed, Zubair M; Jaworek, Thomas J; Sarangdhar, Gowri N et al. (2018) Inframe deletion of human ESPN is associated with deafness, vestibulopathy and vision impairment. J Med Genet 55:479-488
Winkelman, Jonathan D; Suarez, Cristian; Hocky, Glen M et al. (2016) Fascin- and ?-Actinin-Bundled Networks Contain Intrinsic Structural Features that Drive Protein Sorting. Curr Biol 26:2697-2706
Zheng, Lili; Beeler, Dina M; Bartles, James R (2015) Characterization and regulation of an additional actin-filament-binding site in large isoforms of the stereocilia actin-bundling protein espin. J Cell Sci 128:2208
Zheng, Lili; Beeler, Dina M; Bartles, James R (2014) Characterization and regulation of an additional actin-filament-binding site in large isoforms of the stereocilia actin-bundling protein espin. J Cell Sci 127:1306-17
Sekerkov√°, Gabriella; Richter, Claus-Peter; Bartles, James R (2011) Roles of the espin actin-bundling proteins in the morphogenesis and stabilization of hair cell stereocilia revealed in CBA/CaJ congenic jerker mice. PLoS Genet 7:e1002032
Zheng, Lili; Zheng, Jing; Whitlon, Donna S et al. (2010) Targeting of the hair cell proteins cadherin 23, harmonin, myosin XVa, espin, and prestin in an epithelial cell model. J Neurosci 30:7187-201
Odeh, Hana; Hunker, Kristina L; Belyantseva, Inna A et al. (2010) Mutations in Grxcr1 are the basis for inner ear dysfunction in the pirouette mouse. Am J Hum Genet 86:148-60
Shin, Homin; Purdy Drew, Kirstin R; Bartles, James R et al. (2009) Cooperativity and frustration in protein-mediated parallel actin bundles. Phys Rev Lett 103:238102
Lieleg, Oliver; Schmoller, Kurt M; Purdy Drew, Kirstin R et al. (2009) Structural and viscoelastic properties of actin networks formed by espin or pathologically relevant espin mutants. Chemphyschem 10:2813-7
Sekerkova, Gabriella; Zheng, Lili; Mugnaini, Enrico et al. (2008) Espin actin-cytoskeletal proteins are in rat type I spiral ganglion neurons and include splice-isoforms with a functional nuclear localization signal. J Comp Neurol 509:661-76

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