THESIS
2019
xviii, 151 pages : color illustrations ; 30 cm
Abstract
Brush border microvilli are actin-based protrusions lining the apical surface of
epithelial cells in intestines and proximal tubules of kidneys. Similar with the relatively
well-characterized stereocilia in cochlea, brush border microvilli also form bundle-like
protrusions and use cadherin-based tip-links to connect adjacent protrusions. For the
stereocilia, the cytoplasmic face of the tip-link is stabilized and regulated by a protein
complex composed of USH1C (aka Harmonin or AIE-75), USH1G and MYO7A.
However, the mechanistic basis of tip-link complex organization in microvilli is poorly
understood when compared with the stereocilia. In my thesis study, I systematically
characterize the following pairs of interactions: CDHR2 and USH1C, USH1C and
MYO7B, USH1C and ANKS4B, ANKS4B...[
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Brush border microvilli are actin-based protrusions lining the apical surface of
epithelial cells in intestines and proximal tubules of kidneys. Similar with the relatively
well-characterized stereocilia in cochlea, brush border microvilli also form bundle-like
protrusions and use cadherin-based tip-links to connect adjacent protrusions. For the
stereocilia, the cytoplasmic face of the tip-link is stabilized and regulated by a protein
complex composed of USH1C (aka Harmonin or AIE-75), USH1G and MYO7A.
However, the mechanistic basis of tip-link complex organization in microvilli is poorly
understood when compared with the stereocilia. In my thesis study, I systematically
characterize the following pairs of interactions: CDHR2 and USH1C, USH1C and
MYO7B, USH1C and ANKS4B, ANKS4B and MYO7B. We show that USH1C,
ANKS4B, and MYO7B form a stable tripartite complex via these identified multivalent
interactions for anchoring microvilli tip-link cadherins. Despite having only USH1C in
common, the microvilli and the stereocilia tip-link complexes are formed via strikingly
similar interaction modes. By using X-ray crystallography, we resolved 3 complex
structures: USH1C NPDZ1/ANKS4B SAM-PBM, ANKS4B CEN/MYO7B NMFS
and USH1C PDZ3/MYO7B CMF. The first two structures revealed similar binding
modes with their counterparts in stereocilia. The USH1C PDZ3/MYO7B CMF
complex structure reveal a novel binding mode both for MyTH4-FERM tandems and
for PDZ domains. I further revealed MYO7A CMF also interacts USH1C PDZ3 with
similar binding mode. Using purified entire MYO7B tail (M7BNC), full-length
ANKS4B and USH1C, I reconstitute the tripartite complex M7BNC/USH1C/ANKS4B,
and found that they can form highly concentrated condensates via multivalent
interaction mediated liquid-liquid phase separation (LLPS). In heterologous cells, both
MYO7A/USH1C/ANKS4B and MYO7B/USH1C/USH1G form condensed droplet via
LLPS. Usher syndrome disease mutations can affect the multivalent interactions and
reduce the LLPS capability of both MYO7B/USH1C/USH1G and
MYO7A/USH1C/ANKS4B. We propose that the upper tip-link densities of stereocilia
are formed by MYO7A/USH1C/ANKS4B through LLPS.
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