Reversal of axonal growth defects in an extraocular fibrosis model by engineering the kinesin-microtubule interface

Nature Communications
Itsushi MinouraEtsuko Muto

Abstract

Mutations in human β3-tubulin (TUBB3) cause an ocular motility disorder termed congenital fibrosis of the extraocular muscles type 3 (CFEOM3). In CFEOM3, the oculomotor nervous system develops abnormally due to impaired axon guidance and maintenance; however, the underlying mechanism linking TUBB3 mutations to axonal growth defects remains unclear. Here, we investigate microtubule (MT)-based motility in vitro using MTs formed with recombinant TUBB3. We find that the disease-associated TUBB3 mutations R262H and R262A impair the motility and ATPase activity of the kinesin motor. Engineering a mutation in the L12 loop of kinesin surprisingly restores a normal level of motility and ATPase activity on MTs carrying the R262A mutation. Moreover, in a CFEOM3 mouse model expressing the same mutation, overexpressing the suppressor mutant kinesin restores axonal growth in vivo. Collectively, these findings establish the critical role of the TUBB3-R262 residue for mediating kinesin interaction, which in turn is required for normal axonal growth and brain development.

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Citations

May 10, 2017·Proceedings of the National Academy of Sciences of the United States of America·Brandon M BenselSusan P Gilbert
May 2, 2017·Human Molecular Genetics·Mary C Whitman, Elizabeth C Engle
May 31, 2018·The Journal of Biological Chemistry·Kellie A WollRoderic G Eckenhoff
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Feb 6, 2021·The Journal of Cell Biology·Rie AyukawaEtsuko Muto
Jun 4, 2021·Annual Review of Vision Science·Mary C Whitman

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Methods Mentioned

BETA
transfection
motility assay
dissection
gel filtration
fluorescence microscopy

Software Mentioned

Mark2
R Foundation for Statistical Computing
R statistical package ( R Foundation for Statistical Computing )
R package

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