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Structural, Biochemical and In Silico Studies of Atypical Cadherins Involved in Inner Ear Hair Cell Organization and Mechanotransduction

Tamilselvan, Elakkiya
http://orcid.org/0000-0003-2347-6805
http://rave.ohiolink.edu/etdc/view?acc_num=osu17013871841299

Abstract Details

2023, Doctor of Philosophy, Ohio State University, Biophysics.
Cadherins are a family of large transmembrane glycoproteins instrumental in facilitating organ formation during morphogenesis in vertebrates and invertebrates. At the cellular level, they are involved in adhesion, signaling, recognition, mechanotransduction, and motility. In the modern classification of the cadherin superfamily, classical cadherins with five extracellular cadherin (EC) repeats as well as clustered and non-clustered -protocadherins with six or seven EC repeats have been well-studied and their homophilic/heterophilic interactions with molecules on the same (cis) cell or opposite (trans) cells have been characterized. Complexity arises when the number of EC repeats increases with diverse Ca2+ coordination at linker regions between two consecutive EC repeats. In larger cadherins, such as cadherin-23 (CDH23), protocadherin-15 (PCDH15) and cadherin epithelial growth factor (EGF) Laminin-G (LAG or LamG) seven pass G-type receptor-1 (CELSR1), the structural flexibility afforded by different Ca2+ coordination plays determinant roles in their adhesion capacity during inner-ear mechanotransduction and planar cell polarity (PCP). CDH23 and PCDH15, each with 27 and 11 EC repeats, connect two adjacent hair- like protrusions known as stereocilia together atop of a hair cell, the primary mechanosensory cell in the inner ear. Through heterophilic interactions between their first two N-terminal EC repeats, CDH23 and PCDH15 form a filament known as the tip link. In response to sound, stereocilia undergo displacement and the tip link experiences tension, which opens the ion-conducting mechanotransduction channels on the tip-link’s lower end to send signals to the brain. The heterophilic trans tetrameric complex formed by CDH23 and PCDH15, and the cis interactions along the length of PCDH15 have been well-studied in the past but full-length ectodomain structures and high-resolution structural models of complete CDH23 and PCHD15 ectodomain have not been resolved. Here, we present the structure of a PCDH15 segment and protocols for expression and purification of full-length ectodomains that allowed us to obtain several 2-D classes and a cryogenic-electron microscope (cryo-EM) envelope of PCDH15 along with cryo-EM images of CDH23. An in silico constructed model of PCDH15 fitted well to the ab inito envelope and 2D classes. We similarly use structures and AlphaFold2 (AF2) predictions to build a CDH23 ectodomain model that is representative of some of the conformations observed using cryo-EM. Unlike the PCDH15 dimeric ectodomain, the conformations of CDH23 are numerous and lack stable cis interactions. While this posed a difficulty, our high-resolution structural models of CDH23 and PCDH15 provide the opportunity to study these proteins in atomic-level detail for the first time in response to force as expected in the inner ear. Another cadherin that is also found in the inner ear but functions in a different capacity is CELSR1. Forming a transmembrane protein complex with two other core PCP proteins in the apical region of cells, CELSR1 is involved in the organization of hair cells in the cochlea to maximize efficiency of signal transduction. CELSR1, an adhesion G-protein coupled receptor (GPCR) family member, is one of the most highly conserved cadherins since multicellularity arose in evolution. With nine EC repeats in the ectodomain, CELSR1 is involved in homophilic interactions that help propagate PCP, a property in which cells undergo coordinated alignment along the tissue plane. We have used a myriad of biophysical and biochemical techniques along with crystal structures of EC1-4 and EC4-7 to show that CELSR1 is weakly adhesive and that there is a flexible linker region in its ectodomain. We also propose the existence of a cis mediated interaction at the C-terminal end of the CELSR1 cadherin ectodomain similar to what is observed for PCDH15. In addition, we have also built two hybrid models for CELSR1 that resemble in vitro states of its paralog, CELSR2, observed using high-speed atomic force microscopy. Our results support a signaling function for CELSR1. Overall, our high-resolution structural models of CDH23, PCDH15, and CELSR1 based on cryo-EM images and constructed using structures of smaller fragments solved by X-ray crystallography and AF2 predictions provide a framework for studying other elongated cadherins in the superfamily.
Marcos Sotomayor (Advisor)
247 p.
Biochemistry; Biophysics
cadherins; inner ear; hearing; morphogenesis; CDH23; PCDH15; Planar cell polarity; cell orientation; CELSR1; adhesion GPCR; X-ray crystallography; molecular dynamics simulations

Recommended Citations

Citations

  • Tamilselvan, E. (2023). Structural, Biochemical and In Silico Studies of Atypical Cadherins Involved in Inner Ear Hair Cell Organization and Mechanotransduction [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu17013871841299

    APA Style (7th edition)

  • Tamilselvan, Elakkiya. Structural, Biochemical and In Silico Studies of Atypical Cadherins Involved in Inner Ear Hair Cell Organization and Mechanotransduction. 2023. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu17013871841299.

    MLA Style (8th edition)

  • Tamilselvan, Elakkiya. "Structural, Biochemical and In Silico Studies of Atypical Cadherins Involved in Inner Ear Hair Cell Organization and Mechanotransduction." Doctoral dissertation, Ohio State University, 2023. http://rave.ohiolink.edu/etdc/view?acc_num=osu17013871841299

    Chicago Manual of Style (17th edition)

osu17013871841299
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This open access ETD is published by The Ohio State University and OhioLINK.