Strong cytoskeleton activity on millisecond timescales upon particle binding revealed by ROCS microscopy

Felix Jünger, Alexander Rohrbach

Cells change their shape within seconds, cellular protrusions even on subsecond timescales enabling various responses to stimuli of approaching bacteria, viruses or pharmaceutical drugs. Typical response patterns are governed by a complex reorganization of the actin cortex, where single filaments and molecules act on even faster timescales. These dynamics have remained mostly invisible due to a superposition of slow and fast motions, but also due to a lack of adequate imaging technology. Whereas fluorescence techniques require too long integration times, novel coherent techniques such as ROCS microscopy can achieve sufficiently high spatiotemporal resolution. ROCS uses rotating back‐scattered laser light from cellular structures and generates a consistently high image contrast at 150nm resolution and frame rates of 100 Hz ‐ without fluorescence or bleaching. Here, we present an extension of ROCS microscopy that exploits the principles of dynamic light scattering for precise localization, visualization and quantification of the cytoskeleton activity of mouse macrophages. The locally observed structural reorganization processes, encoded by dynamic speckle patterns, occur upon distinct mechanical stimuli, such as soft contacts with optically trapped beads. We find that a substantial amount of the near‐membrane cytoskeleton activity takes place on millisecond timescales, which is much faster than reported ever before.

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Native kinesin-1 does not bind preferentially to GTP-tubulin-rich microtubules in vitro

Qiaochu Li, Stephen J. King, Jing X

Molecular motors such as kinesin-1 work in small teams to actively shuttle cargos in cells, for example in polarized transport in axons. Here, we examined the potential regulatory role of the nucleotide state of tubulin on the run length of cargos carried by multiple kinesin motors, using an optical trapping-based in vitro assay. Based on a previous report that kinesin binds preferentially to GTP-tubulin-rich microtubules, we anticipated that multiple-kinesin cargos would run substantially greater distances along GMPCPP microtubules than along GDP microtubules. Surprisingly, we did not uncover any significant differences in run length between microtubule types. A combination of single-molecule experiments, comparison with previous theory, and classic microtubule affinity pulldown assays revealed that native kinesin-1 does not bind preferentially to GTP-tubulin-rich microtubules. The apparent discrepancy between our observations and the previous report likely reflects differences in post-translational modifications between the native motors used here and the recombinant motors examined previously. Future investigations will help shed light on the interplay between the motor's post-translational modification and the microtubule's nucleotide-binding state for transport regulation in vivo.

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Dynein arms are strain-dependent direction-switching force generators

Chikako Shingyoji, Izumi Nakano, Yuichi Inoue and Hideo Higuchi

Dynein is a minus-end-directed motor that can generate (forward) force to move along the microtubule toward its minus end. In addition, axonemal dyneins were reported to oscillate in the generation of forward force, and cytoplasmic dynein is observed to generate bidirectional forces in response to defined chemical states. Both dyneins can also respond to mechanically applied force. To test whether axonemal dynein can switch direction of force generation, we measured force using an optical trap and UV-photolysis of caged ATP. We observed that isolated dynein could repeatedly generate force in both directions along the microtubule. Bidirectional force was also observed for dynein arms that are still attached on the doublet microtubules. Axonemal dynein generated force to move backward (∼4 pN) as well as forward (5-6 pN) along microtubules. Furthermore, backward force could be stimulated by plus-end directed external force applied to axonemal dynein prior to ATP application. The results show that axonemal dynein is unique exhibiting multiple modes of force generation including backward and forward force, oscillatory force and slow, repetitive bidirectional force. The results also demonstrate that mechanical strain is important for switching the directionality of force generation in axonemal dyneins. This article is protected by copyright. All rights reserved.

DOI