An automated<i>in vitro</i>motility assay for high-throughput studies of molecular motors

Korten, Till; Tavkin, Elena; Scharrel, Lara; Kushwaha, Vikas; Diez, Stefan

doi:10.1039/c8lc00547h

Cited by 7 publications

(5 citation statements)

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“…Since the microtubule gliding velocity is highly sensitive to changes in temperature [ 44 ], flow chambers were fixed onto a Peltier element to minimize temperature changes during the experiment and between single technical replicates. We recorded 10 s time-lapse movies at an acquisition rate of one frame per second and determined the median frame-to-frame velocities of the gliding microtubules (data pooled from ten different fields of view) before and after the addition of recombinant proteins or cell lysates using an automated filament tracking algorithm [ 45 , 46 ] ( Figure 1 B,C,D). With this setup at hand, we were able to obtain highly reproducible velocity measurements ( Figure S1E ).…”

Section: Resultsmentioning

confidence: 99%

“…Microtubule gliding velocities were derived from the time-lapse movies using an automated MATLAB (The MathWorks, Inc., Natick, MA, USA) script based on a microtubule tip-tracking algorithm developed in-house [ 45 , 46 ]. In brief, for each frame, the position of a microtubule tip was localized with sub-pixel resolution and compared to its position in the subsequent frame, thereby determining the frame-to-frame velocity.…”

Section: Methodsmentioning

confidence: 99%

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The ALS-Associated FUS (P525L) Variant Does Not Directly Interfere with Microtubule-Dependent Kinesin-1 Motility

Seifert

Drechsler

Japtok

et al. 2021

IJMS

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Deficient intracellular transport is a common pathological hallmark of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Mutations in the fused-in-sarcoma (FUS) gene are one of the most common genetic causes for familial ALS. Motor neurons carrying a mutation in the nuclear localization sequence of FUS (P525L) show impaired axonal transport of several organelles, suggesting that mislocalized cytoplasmic FUS might directly interfere with the transport machinery. To test this hypothesis, we studied the effect of FUS on kinesin-1 motility in vitro. Using a modified microtubule gliding motility assay on surfaces coated with kinesin-1 motor proteins, we showed that neither recombinant wildtype and P525L FUS variants nor lysates from isogenic ALS-patient-specific iPSC-derived spinal motor neurons expressing those FUS variants significantly affected gliding velocities. We hence conclude that during ALS pathogenesis the initial negative effect of FUS (P525L) on axonal transport is an indirect nature and requires additional factors or mechanisms.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The ALS-Associated FUS (P525L) Variant Does Not Directly Interfere with Microtubule-Dependent Kinesin-1 Motility

Seifert

Drechsler

Japtok

et al. 2021

IJMS

Self Cite

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“…These approaches were previously discussed in several reviews. , Similarly, the motor-associated filaments can be engineered by covalent and noncovalent modifications . Such modifications are ideally explored in high-throughput fashion and analyzed with tracking software specifically adapted to the task of identifying and characterizing filaments, such as the popular FIESTA package …”

Section: Optimizing Molecular Motors For Nanodevices: From Biomolecul...mentioning

confidence: 99%

Synthetic Systems Powered by Biological Molecular Motors

2019

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Biological molecular motors (or biomolecular motors for short) are nature’s solution to the efficient conversion of chemical energy to mechanical movement. In biological systems, these fascinating molecules are responsible for movement of molecules, organelles, cells, and whole animals. In engineered systems, these motors can potentially be used to power actuators and engines, shuttle cargo to sensors, and enable new computing paradigms. Here, we review the progress in the past decade in the integration of biomolecular motors into hybrid nanosystems. After briefly introducing the motor proteins kinesin and myosin and their associated cytoskeletal filaments, we review recent work aiming for the integration of these biomolecular motors into actuators, sensors, and computing devices. In some systems, the creation of mechanical work and the processing of information become intertwined at the molecular scale, creating a fascinating type of “active matter”. We discuss efforts to optimize biomolecular motor performance, construct new motors combining artificial and biological components, and contrast biomolecular motors with current artificial molecular motors. A recurrent theme in the work of the past decade was the induction and utilization of collective behavior between motile systems powered by biomolecular motors, and we discuss these advances. The exertion of external control over the motile structures powered by biomolecular motors has remained a topic of many studies describing exciting progress. Finally, we review the current limitations and challenges for the construction of hybrid systems powered by biomolecular motors and try to ascertain if there are theoretical performance limits. Engineering with biomolecular motors has the potential to yield commercially viable devices, but it also sharpens our understanding of the design problems solved by evolution in nature. This increased understanding is valuable for synthetic biology and potentially also for medicine.

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“…41,[43][44][45] Important insights into the working of MPs have been gained from in vitro gliding assay experiments. [46][47][48][49][50][51][52] In them, the MP tails are attached irreversibly to a substrate. The head domains of MPs can attach to conjugate filaments and, while walking on them, drive the filaments in the opposite direction.…”

Section: Introductionmentioning

confidence: 99%

Cooperation and competition in the collective drive by motor proteins: mean active force, fluctuations, and self-load

Chaudhuri

Chitrak

2023

Soft Matter

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An automatedin vitromotility assay for high-throughput studies of molecular motors

Abstract: Molecular motors, essential to force-generation and cargo transport within cells, are invaluable tools for powering nanobiotechnological lab-on-a-chip devices.

Cited by 7 publications

References 64 publications

The ALS-Associated FUS (P525L) Variant Does Not Directly Interfere with Microtubule-Dependent Kinesin-1 Motility

The ALS-Associated FUS (P525L) Variant Does Not Directly Interfere with Microtubule-Dependent Kinesin-1 Motility

Synthetic Systems Powered by Biological Molecular Motors

Cooperation and competition in the collective drive by motor proteins: mean active force, fluctuations, and self-load

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