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.
Highlights
Mutations in human b3-tubulin (TUBB3) cause an ocular motility disorder termed congenital fibrosis of the extraocular muscles type 3 (CFEOM3)
We used a baculovirus/insect cell expression system to express and purify the following three types of recombinant tubulin dimers composed of human a1-and b3-tubulin (TUBA1B and TUBB3, respectively): wild type (WT), R262C and R262H (Supplementary Fig. 2a)[19]
The WT, R262H and R262A tubulins were purified using His and FLAG tags and polymerized into MTs (Supplementary Fig. 2b), but the yield of R262C tubulin was too low for use in biochemistry experiments due to the low solubility of the protein
Summary
Mutations in human b3-tubulin (TUBB3) cause an ocular motility disorder termed congenital fibrosis of the extraocular muscles type 3 (CFEOM3). In a CFEOM3 mouse model expressing the same mutation, overexpressing the suppressor mutant kinesin restores axonal growth in vivo. 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. The unique properties of TUBB3 critically depends on its exact sequence, and mutations in TUBB3 in humans are associated with a spectrum of brain malformation and neurological disorders[9,10,11]. Patients show hypoplasia of the oculomotor nerves and dysgenesis of the corpus callosum, anterior commissure and corticospinal tracts[11] These defects point to an essential function for TUBB3 in the processes of axonal outgrowth and maintenance during development. Because the R262 mutation is the most common mutation in patients with CFEOM3, delineating its role in kinesin motility is essential for understanding the disease mechanism
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