Fig. 6.

MT detyrosination amplifies kinesin-1-based transport of APC via a positive feedback loop. (A) Representative point-scanning confocal maximum-intensity projected images of RPE-1 cells treated with indicated siRNAs on linear micropatterns immunostained with antibodies against detyrosinated (Detyr. Tubulin) and total α-tubulin with DAPI as a DNA counterstain. (Scale bar, 20 µm.) (B) Scatter plot of normalized detyrosinated tubulin intensity from immunostained RPE-1 cells on linear micropatterns following indicated treatments. (N, n): siCTRL (74,5), siAPC (24,2), and siKIF5B (42,3). N, number of cells; n, number of independent experiments. The scatter plots include mean ± SD. ***P ≤ 0.001 (C) Positive feedback loop between microtubule detyrosination and kinesin-mediated transport of APC during cell migration. (D) Symmetry breaking of APC cytoplasmic concentration profiles in the model. Green curve, a unique stable symmetric state in the model without feedback, $\chi =1$ ; red curve, an asymmetric polarized state with strong feedback, $\chi =11$ . (E) The model predicts a pitchfork bifurcation as the positive feedback strength exceeds a critical value ${\chi }_{\mathit{cr}}$ . Solid lines indicate stable states; dashed line, unstable. The two asymmetric states ${\sigma }^{+}$ and ${\sigma }^{-}$ differ only in the direction of cellular polarization. (F) MT detyrosination reduces polarization threshold ${\chi }_{\mathit{cr}}$ and increases cell asymmetry magnitude $\sigma$.$\sigma$ is plotted vs. the strength of feedback $\chi$ at the indicated on the figure values of detyrosination effect strength $\alpha$ . Respective values of the critical feedback strength ${\chi }_{\mathit{cr}}(\alpha )$ are shown by empty circles. (G) Dynamics of cellular polarization in the model projected on the plane of APC cytoplasmic concentrations at the right and left cell ends (see text for details). Red dots are stable asymmetric states; green dot, unstable symmetric saddle state $S$ that separates two asymmetric states. (H) Polarization efficiency $E_{\mathit{pol}}$ with (Right) and without (Left) the contribution of MT detyrosination shown as a surface over a log-scaled domain of normalized APC diffusion coefficient $d$ and APC MT association constant $K_a$ . Note that the maximal value of $E_{\mathit{pol}}$ on the Right panel is ~15 fold higher than on the Left. Star indicates an example of a parameter set with high polarization efficiency when the detyrosination is included and no polarization without it.