LED illumination for video‐enhanced DIC imaging of single microtubules

Bormuth, Volker; Howard, Jonathon; Schäffer, Erik

doi:10.1111/j.1365-2818.2007.01756.x

Cited by 54 publications

(51 citation statements)

References 20 publications

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“…To ensure the functionality of the motors, we measured the speed of Kip3-coated microspheres by video tracking (30) with the optical trap turned off. We determined the speed by linear fits to the tracked x position.…”

Section: Applying Sideward Loads With Optical Tweezersmentioning

confidence: 99%

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The Kinesin-8 Kip3 Switches Protofilaments in a Sideward Random Walk Asymmetrically Biased by Force

Bugiel

Böhl

Schäffer

2015

Biophysical Journal

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Molecular motors translocate along cytoskeletal filaments, as in the case of kinesin motors on microtubules. Although conventional kinesin-1 tracks a single microtubule protofilament, other kinesins, akin to dyneins, switch protofilaments. However, the molecular trajectory-whether protofilament switching occurs in a directed or stochastic manner-is unclear. Here, we used high-resolution optical tweezers to track the path of single budding yeast kinesin-8, Kip3, motor proteins. Under applied sideward loads, we found that individual motors stepped sideward in both directions, with and against loads, with a broad distribution in measured step sizes. Interestingly, the force response depended on the direction. Based on a statistical analysis and simulations accounting for the geometry, we propose a diffusive sideward stepping motion of Kip3 on the microtubule lattice, asymmetrically biased by force. This finding is consistent with previous multimotor gliding assays and sheds light on the molecular switching mechanism. For kinesin-8, the diffusive switching mechanism may enable the motor to bypass obstacles and reach the microtubule end for length regulation. For other motors, such a mechanism may have implications for torque generation around the filament axis.

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Section: Applying Sideward Loads With Optical Tweezersmentioning

confidence: 99%

“…Unless noted otherwise, all chemicals are from Sigma-Aldrich (St. Louis, MO). Microtubules were visualized with differential interference contrast employing a light-emitting diode (30).…”

Section: Introductionmentioning

confidence: 99%

The Kinesin-8 Kip3 Switches Protofilaments in a Sideward Random Walk Asymmetrically Biased by Force

Bugiel

Böhl

Schäffer

2015

Biophysical Journal

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“…Images were collected either every 10 s or every 100 ms (in streaming mode). In differential interference contrast (DIC) experiments, we used the setup described previously (20). Data were analyzed by making kymographs from acquired images using Fiji software, which were then used to measure the parameters of microtubule dynamics.…”

Section: Methodsmentioning

confidence: 99%

Stu2, the Budding Yeast XMAP215/Dis1 Homolog, Promotes Assembly of Yeast Microtubules by Increasing Growth Rate and Decreasing Catastrophe Frequency

Podolski

Mahamdeh

Howard

2014

Journal of Biological Chemistry

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Background:The reported inhibition of microtubule growth by Stu2 is difficult to reconcile with its cellular phenotypes. Results: Using microscopy assays, we found that Stu2 increases the growth rate and decreases the catastrophe frequency of yeast microtubules. Conclusion: Stu2 promotes microtubule growth, with considerably higher activity on budding yeast microtubules. Significance: The biochemical properties of Stu2 reported here account for the mitotic phenotypes observed in cells.

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“…To quantify the movement of the beads due to microtubule growth and shrinkage, we recorded DIC movies at 30 Hz, averaged over 10 frames, resulting in 3 frames per second. The implementation of video-enhanced DIC using a light-emitting diode was described previously (33). Bead positions were tracked in recorded movies by using a customwritten MATLAB script.…”

Section: Methodsmentioning

confidence: 99%

“…The dynamics were characterized by alternating periods of growth and shrinkage. Beads were trapped using optical tweezers (31)(32)(33)(34), brought into the vicinity of the growing end of a microtubule, and then positioned on it to allow binding to occur. When the optical trap was switched off, the bound XMAP215-coated bead remained attached to the microtubule tip.…”

Section: Xmap215-coated Beads Remain Attached To Growing and Shrinkingmentioning

confidence: 99%

The growth speed of microtubules with XMAP215-coated beads coupled to their ends is increased by tensile force

Trushko

Schäffer

Howard

2013

Proc. Natl. Acad. Sci. U.S.A.

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The generation of pulling and pushing forces is one of the important functions of microtubules, which are dynamic and polarized structures. The ends of dynamic microtubules are able to form relatively stable links to cellular structures, so that when a microtubule grows it can exert a pushing force and when it shrinks it can exert a pulling force. Microtubule growth and shrinkage are tightly regulated by microtubule-associated proteins (MAPs) that bind to microtubule ends. Given their localization, MAPs may be exposed to compressive and tensile forces. The effect of such forces on MAP function, however, is poorly understood. Here we show that beads coated with the microtubule polymerizing protein XMAP215, the Xenopus homolog of Dis1 and chTOG, are able to link stably to the plus ends of microtubules, even when the ends are growing or shrinking; at growing ends, the beads increase the polymerization rate. Using optical tweezers, we found that tensile force further increased the microtubule polymerization rate. These results show that physical forces can regulate the activity of MAPs. Furthermore, our results show that XMAP215 can be used as a handle to sense and mechanically manipulate the dynamics of the microtubule tip.microtubule polymerase | microtubule dynamics | optical trap X MAP215 is a microtubule plus-end binding protein that alters microtubule dynamics (1-4) by promoting microtubule growth. It was identified as a factor increasing microtubule polymerization rates in Xenopus egg extracts (5). In vivo studies have shown that disruption of XMAP215 function leads to short interphase microtubules, reduced microtubule growth rate, and increased frequency of microtubule catastrophe and pause events (6-11). Depletion of XMAP215 also results in short, abnormally formed mitotic spindles and short astral microtubules (7,12,13). In vitro studies have demonstrated that XMAP215 binds preferentially to the microtubule plus end and follows the growth and shrinkage of the microtubule tip, catalyzing microtubule growth (14-16). In addition to localization at the microtubule plus end and centrosomes (17-19), proteins of the XMAP215/Dis1 family are found at the kinetochores (9, 20, 21), which are sites of high force during cell division. Microtubules themselves can exert pulling and pushing forces (22)(23)(24)(25)(26)(27)(28)(29)(30). Therefore, the localization of XMAP215 at microtubule ends suggests that XMAP215 can function under load. However, the effect of forces on XMAP215 activity has not been directly analyzed. In this study, we investigated the influence of forces-directed toward the microtubule plus and minus end-on XMAP215 activity in vitro. Microtubule growth and shrinkage were monitored with high spatial and temporal resolution by visualizing the position of XMAP215-coated polystyrene microspheres bound to the tips of dynamic microtubules. The microspheres, so-called "beads," served as handles to apply forces on XMAP215 molecules using optical tweezers. Results XMAP215-Coated Beads Remain Attached to Growing ...

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LED illumination for video‐enhanced DIC imaging of single microtubules

Cited by 54 publications

References 20 publications

The Kinesin-8 Kip3 Switches Protofilaments in a Sideward Random Walk Asymmetrically Biased by Force

The Kinesin-8 Kip3 Switches Protofilaments in a Sideward Random Walk Asymmetrically Biased by Force

Stu2, the Budding Yeast XMAP215/Dis1 Homolog, Promotes Assembly of Yeast Microtubules by Increasing Growth Rate and Decreasing Catastrophe Frequency

The growth speed of microtubules with XMAP215-coated beads coupled to their ends is increased by tensile force

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