Direct measurement of conformational strain energy in protofilaments curling outward from disassembling microtubule tips
- University of Washington, United States
- UT Southwestern Medical Center, United States
Figures
Figure 1
Measuring the tubulin conformational wave with a feedback-controlled laser trap.
(a) A bead is tethered to the side of a microtubule via a single antibody bound to the C-terminal tail of β-tubulin and placed under tension using the laser trap. The trap is feedback-controlled to …
Figure 2 with 1 supplement
Tubulin waves generate large forces.
(a, b) Mean pulse risetime versus force (a) and distributions of risetime at indicated forces (b) for wild-type microtubules. The mean risetime across all forces is depicted by the dashed line in (a)…
Figure 2—figure supplement 1
Properties of wild-type tubulin waves measured using different bead sizes.
(a – f) Mean amplitudes versus force (a, c, e) and distributions of amplitude at indicated forces (b, d, f) for pulses generated by wild-type yeast microtubules, measured with 320 nm beads (a, b), …
Figure 3 with 1 supplement
Proposed mechanisms underlying conformational wave-driven bead movement in the assay.
(a) Initially, when a bead is placed under tension it rests against the microtubule wall at a secondary contact point. (b) In the lateral push scenario, the curling protofilaments push laterally …
Figure 3—figure supplement 1
A rare example record in which the initial pulse, from a stable baseline, was followed by bead relaxation toward the trap center and then by a second pulse (double arrow).
Such secondary pulses were seen in only 2% of all recorded events (18 of 760). These rare secondary pulses might be generated by axial pulling. However, the lack of any relaxation before the primary …
Figure 4 with 1 supplement
Stall forces and pulse amplitudes vary with bead size, but pulse energy is invariant.
(a) With increasing bead size, the leverage increases and therefore the trapping force required to completely suppress the pulses (i.e., the ‘stall force’) decreases. (b) Unloaded pulse amplitudes …
Figure 4—figure supplement 1
Estimation of strain energy per tubulin.
(a) Given the 23° curvature and 8 nm length of a tubulin dimer, a curl height of h = 20 nm implies that the curled segments are ~4 dimers in length. (b) A maximum of ~4 curls could push …
Figure 5 with 1 supplement
Hyperstable mutant microtubules produce slower pulses.
(a) Superposition of polymerized (’straight’, green) and unpolymerized (’curved’, blue) conformations of β-tubulin. Residue T238 is inaccessible to solvent and located on a helix (H7) that undergoes …
Figure 5—figure supplement 1
Hyperstable mutant T238V tubulin disassembles more slowly than wild-type.
(a) Selected images from a movie of an individual yeast microtubule dissembling in vitro, recorded by video-enhanced differential interference contrast (VE-DIC) microscopy. The white arrow marks the …
Figure 6
Hyperstable mutant microtubules produce pulses with identical energy.
(a) Mean pulse risetime versus force for mutant T238V microtubules. Wild-type data (from Figure 2a) is shown for comparison. The mean risetimes across all forces for T238V and wild-type microtubules …
Figure 7 with 1 supplement
Free energy landscape for a curling αβ-tubulin.
(a) The model considers a single αβ-tubulin (highlighted) as it bends outward from a microtubule. For simplicity, only two protofilaments are depicted. The curling subunit is shown (arbitrarily) at …
Figure 7—figure supplement 1
Free energy landscape for a single curling αβ-tubulin subunit, calculated by adding independent contributions from mechanical strain and lateral bonding.
(a) Mechanical strain energy, U, is calculated as a function of bend angle, φ, by assuming the αβ-tubulin subunit behaves like a slender elastic rod with a naturally bent shape, a bend angle of φ = 2…
Figure 8
More force is required to pluck hyperstable mutant tubulin subunits from the microtubule end.
(a) A bead is tethered to the end of a growing microtubule via a single antibody bound to the β-tubulin C-terminus and then tested with a 0.25 pN·s−1 force ramp. (b) Usually, detaching the bead by …
Author response image 1
Data recopied from Figure 4B.
Curves show predictions assuming a tether of 36 nm and a curl height, h, as indicated.
Videos
Video 1
Example of wave assay.
A bead tethered to the side of a coverslip-anchored microtubule is initially held under laser trap tension (here, ~1 pN). The distal plus end of the microtubule is severed by laser scissors (at 0 …
Video 2
Second example of wave assay.
A bead tethered to the side of a coverslip-anchored microtubule is initially held in the laser trap, at low tension (<1 pN). Feedback control is initiated (at −5.6 s) to apply higher tension (4 pN), …
Video 3
Example of plucking force assay.
A bead linked to the assembling plus end of a coverslip-anchored microtubule is subjected to increasing tension until the bead detaches. After bead detachment, the microtubule plus end disassembles, …
Additional files
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Supplementary file 1
All individual pulse measurements.
Individual amplitudes and risetimes for all recorded pulses, as well as the means and standard errors for each measurement condition, are given in the accompanying Excel spreadsheet. The spreadsheet also includes all the individual plucking force values.
- https://doi.org/10.7554/eLife.28433.019
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Source code 1
Custom software for controlling the laser trap.
- https://doi.org/10.7554/eLife.28433.020
