Working strokes produced by curling protofilaments at disassembling microtubule tips can be biochemically tuned and vary with species

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    The authors measure the work output of shrinking mammalian microtubules, reporting results of fundamental importance that advance our mechanistic understanding of how shrinking microtubules exert forces on chromosomes during cell division. Carefully performed, technically advanced experiments and model-based quantitative data analysis provide compelling evidence for the authors' conclusions. This work will be of interest for cell biologists interested in cell division, biophysicists interested in force production by biopolymers, and structural biologists interested in microtubule dynamics.

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Abstract

The disassembly of microtubules can generate force and drive intracellular motility. During mitosis, for example, chromosomes remain persistently attached via kinetochores to the tips of disassembling microtubules, which pull the sister chromatids apart. According to the conformational wave hypothesis, such force generation requires that protofilaments curl outward from the disassembling tips to exert pulling force directly on kinetochores. Rigorously testing this idea will require modifying the mechanical and energetic properties of curling protofilaments, but no way to do so has yet been described. Here, by direct measurement of working strokes generated in vitro by curling protofilaments, we show that their mechanical energy output can be increased by adding magnesium, and that yeast microtubules generate larger and more energetic working strokes than bovine microtubules. Both the magnesium and species-dependent increases in work output can be explained by lengthening the protofilament curls, without any change in their bending stiffness or intrinsic curvature. These observations demonstrate how work output from curling protofilaments can be tuned and suggest evolutionary conservation of the amount of curvature strain energy stored in the microtubule lattice.

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  1. Author Response

    Reviewer #1 (Public Review):

    In a very interesting and technically advanced study, the authors measured the force production of curved protofilaments at depolymerizing mammalian microtubule ends using an optical trap assay that they developed previously for yeast microtubules. They found that the magnesium concentration affects this force production, which they argue based on a theoretical model is due to affecting the length of the protofilament curls, as observed previously by electron microscopy. Comparing with their previous force measurements, they conclude that mammalian microtubules produce smaller force pulses than yeast microtubules due to shorter protofilament curls. This work provides new mechanistic insight into how shrinking microtubules exert forces on cargoes such as for example kinetochores during cell …

  2. eLife assessment

    The authors measure the work output of shrinking mammalian microtubules, reporting results of fundamental importance that advance our mechanistic understanding of how shrinking microtubules exert forces on chromosomes during cell division. Carefully performed, technically advanced experiments and model-based quantitative data analysis provide compelling evidence for the authors' conclusions. This work will be of interest for cell biologists interested in cell division, biophysicists interested in force production by biopolymers, and structural biologists interested in microtubule dynamics.

  3. Reviewer #1 (Public Review):

    In a very interesting and technically advanced study, the authors measured the force production of curved protofilaments at depolymerizing mammalian microtubule ends using an optical trap assay that they developed previously for yeast microtubules. They found that the magnesium concentration affects this force production, which they argue based on a theoretical model is due to affecting the length of the protofilament curls, as observed previously by electron microscopy. Comparing with their previous force measurements, they conclude that mammalian microtubules produce smaller force pulses than yeast microtubules due to shorter protofilament curls. This work provides new mechanistic insight into how shrinking microtubules exert forces on cargoes such as for example kinetochores during cell division. The …

  4. Reviewer #2 (Public Review):

    Microtubules are regarded as dynamic tracks for kinesin and dynein motors that generate force for moving cargoes through cells, but microtubules also act as motors themselves by generating force from outward splaying protofilaments at depolymerizing ends. Force from depolymerization has been demonstrated in vitro and is thought to contribute to chromosome movement and other contexts in cells. Although this model has been in the field for many years, key questions have remained unanswered, including the mechanism of force generation, how force generated might be regulated in cells, and how this system might be tuned across cellular contexts or organisms. The barrier is that we lack an understanding of experimental conditions that can be used to control protofilament shape and energetics. This study by Murray …

  5. Reviewer #3 (Public Review):

    The authors used a previously established optical tweezers-based assay to measure the regulation of the working stroke of curled protofilaments of bovine microtubules by magnesium. To do so, the authors improved the assay by attaching bovine microtubules to trapping beads through an incorporated tagged yeast tubulin.

    The assay is state-of-the-art and provides a direct measurement of the stroke size of protofilaments and its dependence on magnesium.

    The authors have achieved all their goals and the manuscript is well written.

    The reported findings will be of high interest for the cell biology community.