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

Korchagina, Julia Jurievna (2013) In vitro and in vivo analysis of the assembly of the non-collagenous tectorial membrane matrix. Doctoral thesis (PhD), University of Sussex.

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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. β-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.

Item Type: Thesis (Doctoral)
Schools and Departments: School of Life Sciences > Neuroscience
Subjects: Q Science > QH Natural history > QH0301 Biology
Q Science > QP Physiology > QP0351 Neurophysiology and neuropsychology > QP0431 Senses > QP0448 Special senses > QP0471 Equilibrium. Labyrinths of the ear. General works
Depositing User: Library Cataloguing
Date Deposited: 10 Dec 2013 15:00
Last Modified: 17 Sep 2015 14:56

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