a, Illustration of the work flow. Red fluorescent microtubule seeds (short red lines), were flowed into the microfluidic device and grafted on micropatterned lines (grey lines) (step I). Free green fluorescent tubulin dimers were then added onto the seeds to grow microtubules (green lines) (step II). The same mix was then flowed normal to the microtubules and used as a hydrodynamic constraint to bend microtubules (step III). Red fluorescent beads were added to the mix to measure fluid flow next to microtubules.
b, Time lapse sequence of microtubule (green) bending in response to fluid flow. Scale bar is 3 μm. Overlay of images during the bending steps conveniently shows the magnitude of microtubule deformation (right image).

a, Sequence of bending cycles. Images show the overlay of microtubule deformations during each successive bending cycle. Bending flows were applied during 10 seconds. A pause of 10 second was respected between bending steps. Deformations appeared larger cycle after cycle. Scale bar is 3 μm.
b, Overlay of microtubule maximal deformation during each bending cycle. Distinct colors have been attributed to each bending cycle. Scale bar is 3 μm.
c, Measurements of microtubule persistent length evolution over the successive bending cycles. Delay between bending cycles was 10 s. Microtubule persistent lengths were normalized to their initial value. Error bars correspond to the standard deviation calculated from 5 consecutive frames for each bending cycle. As a test of tendency, Spearman correlation tests for persistent length values over the successive cycles were performed. Green curves show microtubules for which the persistent length was not significantly affected over the bending cycles, whereas red curves show those that have been soften during the cyclic stress.
d, Comparison of initial microtubule persistent length for microtubules assembled in the presence of 14 μM or 26 μM of free tubulin dimers. Values were compared with an unpaired t test (two-tailed).
e, Overlay of microtubule maximal deformation during bending cycles for microtubules assembled in the presence of 14 μM or 26 μM of free tubulin dimers.
f, Measurements of microtubule persistent length evolution over the successive bending cycles for microtubules assembled in the presence of various concentration of free tubulin dimers (26 μM: n=9; 20 μM: n=15, 18 μM: n=15; 14 μM: n=13). Microtubule persistent lengths were normalized to their initial values. Values correspond to average persistent length of individual measurements shown in . Error bars show standard deviation between distinct microtubule bending experiments. All curves were considered significantly different using a two-way ANOVA test, except 14 and 18 μM. A T test on the average persistent length showed all these differences became significant at the fourth bending cycle.

a, Sequence of bending cycles. Images show the overlay of microtubule deformations during each successive bending cycle. Bending flows were applied during 10 seconds. A pause of 100 second was respected between bending steps. Deformations appeared similar for all cycles. Scale bar is 3 μm.
b, Overlay of microtubule maximal deformation during each bending cycle. Distinct colors have been attributed to each bending cycle. Scale bar is 3 μm.
c, Measurements of microtubule persistent length evolution over the successive bending cycles. Delay between bending cycles was 100 s. Error bars correspond to the standard deviation calculated from 5 consecutive frames for each bending cycle. As a test of tendency, Spearman correlation tests for persistent length values over the successive cycles were performed. Green curves show microtubules for which the final persistent length was not significantly different from their initial one, whereas red curves show those that have been soften during the cyclic stress.
d, Evolution of microtubule persistent length following five rapid bending cycles. Delay between the five first bending cycles was 10 s. To distinguish softening from non-softening microtubules, we used the distance between the minimum normalized Lp and the Lp during the first cycle (=1) as a parameter for a k-means clustering. The clustering algorithm found one group with a low distance, which corresponds to non-softening microtubules (shown on the left graph), and another group with a high distance, corresponding to softening microtubules (shown on the right graph). The recovery after a 100 s pause time was assessed by comparing Lp before and after the pause with a Wilcoxon matched-pairs test. Microtubules displaying significant recovery are shown in blue, the other in orange.

a, Image sequence showing a microtubule assembled with red tubulin (t=−160s), bathed in a solution of green tubulin (t=−45s), damaged (but not cut) with laser pulses (t=0) and let in the presence of green tubulin for 70s. Green tubulin was washed away and replaced by red tubulin. Removal of the green fluorescent background revealed incorporation of tubulin dimers at the damaged site (arrow). Scale bar is 5 μm.
b, Experimental procedure to grow microtubules with red tubulin and bend them in the presence of green tubulin.
c, Image sequence showing from left to right: a red microtubule, the overlay of 5 bending cycles in the presence of green tubulin, the 1-minute-long pause, followed by the green tubulin washout revealing incorporation of tubulin dimers along the microtubule length (arrow).
d, Averages of 30 images taken successively in the red and green channels at the end of the image sequence shown in c in order to improve signal to noise ratio. Stretches of green tubulin along the red lattice become more visible (arrows).
e, Green fluorescence intensity linescan along the microtubule length on the image shown in d. Arrows correspond to incorporation sites between the microtubule seed (right peak) and the growing end (left peak).
f, Measurement of external tubulin incorporation site positions along microtubule length. Distances were normalized with respect to microtubule length.
g, Experimental procedure to grow microtubules with red tubulin and flow green tubulin along their length without bending them.
h, Image sequence showing from left to right: a red microtubule, the overlay of pictures taken during green tubulin flow along microtubule length, the 1-minute-long pause, followed by the green tubulin washout revealing the absence of incorporation of tubulin dimers along the microtubule length.
i, Averages of 30 images taken successively in the red and green channels at the end of the image sequence shown in h in order to improve signal to noise ratio. No stretch of green tubulin could be detected along the red lattice.
j, Green fluorescence intensity linescan along the microtubule length on the image shown in i. Fluorescence linescans showed no fluorescence peak between the microtubule seed (right peak) and the growing end (left peak).

a, This graph represents the parameters used for the simulation. Microtubules had a length L, and a bending stiffness κ₀. Their stiffness was locally reduced to κ_p/κ₀ over a normalized length Δs/L positioned at the arc length s₀₀/L. In this example as well as in all simulations s₀/L = 0.33.
b, Examples of microtubule equilibrium shapes in response to distinct combinations local softenings parameters (κ_p/κ₀ and Δs/L) leading to the same apparent bending stiffness (<κ>/κ₀=0.5). Shown are also the fits against a model with the constant stiffness 0.5 (full lines).
c, Measurement of the distance ω/L between the shapes of experimental microtubules to shapes of microtubules with a constant rigidity as explained in Methods section. Two examples illustrate a high and low distance suggestive of local and global softening process respectively.
d, Comparison of distances to homogeneous microtubules between experimental observations and simulated microtubules. Colored lines show the theoretical dependence of the distance ω/L on the microtubule deflection Δx/L for different extent of the softened region Δs/L (different colors) and the same given apparent stiffness ⟨κ⟩/κ₀ = 0.5. Stars correspond to experimental measurements of the distance ω/L and the deflection Δx/L for different deflection for different microtubules. Shown are only values obtained from microtubules with an apparent stiffness comprised between 0.4 and 0.6 after five bending cycles.

Speculative interpretation of microtubule lattice defect contribution to microtubule deformation, softening and self-healing under mechanical stress. Illustration by Dr. Agnieszka Kawska at IlluScientia.com.