University of Sussex
Korchagina,_Julia_Jurievna.pdf (12.96 MB)

In vitro and in vivo analysis of the assembly of the non-collagenous tectorial membrane matrix

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posted on 2023-06-08, 16:31 authored by Julia Jurievna Korchagina
Alpha- and beta-tectorin (Tecta and Tectb) are major non-collagenous components of the tectorial membrane (TM). The presence of a zona pellucida (ZP) domain in both tectorins suggests that Tecta and Tectb can form hetero- or homopolymers. It is unclear, however, how these proteins assemble to form the TM matrix. The mechanisms of apical targeting, secretion and processing of the tectorins are also unexplored. I used fluorescently-tagged tectorin constructs for stable transfection into polarised epithelial MDCK cells or transient expression in mouse cochlear cultures to develop an in vitro model of TM matrix assembly. Significant amounts of matrix were not observed with stable tectorin expression in monolayer cultures of MDCK cells. In contrast, I observed substantial amounts of dense extracellular matrix on the apical surfaces of outgrowth zone cells when cochlear cultures were transiently transfected with either Tecta or Tectb. When ectopically expressed in hair cells, Tecta and Tectb locate to the distal tips of the hair bundle. To study the role of the inner-ear protein Ceacam16 in hearing, we generated a Ceacam16 functional null mouse model. The Ceacam16 gene was inactivated by targeted replacement of exons 2-5 with the bacterial lacZ gene. ß-gal staining I performed reveals that Ceacam16 is expressed in the epithelial cells of the spiral limbus and inner sulcus, and in both the pillar cells and Deiter’s cells. I first detected the presence of Ceacam16 in the TM at P12, four days before the defined striated-sheet matrix is observed. Transmission electron microscopy reveals a complete loss of striated-sheet matrix in Ceacam16 null mice in comparison to the wild-type. The results of this thesis suggest neonatal mouse cochlear cultures as a model for studying tectorin-based extracellular matrix production and also reveal that Ceacam16 is required for normal formation and/or maintenance of striated-sheet matrix.


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