Tracking Single Particles and Elongated Filaments with Nanometer Precision

Ruhnow, Felix; Zwicker, David; Diez, Stefan

doi:10.1016/j.bpj.2011.04.023

Cited by 304 publications

(287 citation statements)

References 37 publications

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“…1 B for the definition of these motility parameters), we used the open-source tracking software FIESTA (20). We found that all three quantities decreased significantly upon increasing the roadblock concentration (Fig.…”

Section: Resultsmentioning

confidence: 98%

“…S6). Two-dimensional trajectories of AuNP-loaded motors were then projected onto the MT centerline using FIESTA (20). Single-molecule conditions, i.e., conditions under which AuNP transportation was driven by individual motors, were ensured by optimizing the incubation ratios of AuNPs to motors such that the motility parameters of loaded and unloaded motors in the absence and presence of roadblocks were the same (Supporting Material D, Fig.…”

Section: Motors Circumvent Roadblocks By Unbiased Sideshifting To Thementioning

confidence: 99%

“…Fluorescence data MT snapshots and streams of GFP-labeled motors were tracked using FIESTA (20). Tracked motor trajectories (minimum duration: 4 frames) were double-checked by eye to avoid computer misinterpretations.…”

Section: Image Analysismentioning

confidence: 99%

See 2 more Smart Citations

Kinesin-1 Motors Can Circumvent Permanent Roadblocks by Side-Shifting to Neighboring Protofilaments

Schneider

Korten

Walter

et al. 2015

Biophysical Journal

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Obstacles on the surface of microtubules can lead to defective cargo transport, proposed to play a role in neurological diseases such as Alzheimer's. However, little is known about how motor proteins, which follow individual microtubule protofilaments (such as kinesin-1), deal with obstacles on the molecular level. Here, we used rigor-binding mutants of kinesin-1 as roadblocks to permanently obstruct individual microtubule binding sites and studied the movement of individual kinesin-1 motors by single-molecule fluorescence and dark-field scattering microscopy in vitro. In the presence of roadblocks, kinesin-1 often stopped for ∼ 0.4 s before either detaching or continuing to move, whereby the latter circumvention events occurred in >30% after a stopping event. Consequently, and in agreement with numerical simulations, the mean velocity, mean run length, and mean dwell time of the kinesin-1 motors decreased upon increasing the roadblock density. Tracking individual kinesin-1 motors labeled by 40 nm gold particles with 6 nm spatial and 1 ms temporal precision revealed that ∼ 70% of the circumvention events were associated with significant transverse shifts perpendicular to the axis of the microtubule. These side-shifts, which occurred with equal likelihood to the left and right, were accompanied by a range of longitudinal shifts suggesting that roadblock circumvention involves the unbinding and rebinding of the motors. Thus, processive motors, which commonly follow individual protofilaments in the absence of obstacles, appear to possess intrinsic circumvention mechanisms. These mechanisms were potentially optimized by evolution for the motor's specific intracellular tasks and environments.

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Section: Resultsmentioning

confidence: 98%

Section: Motors Circumvent Roadblocks By Unbiased Sideshifting To Thementioning

confidence: 99%

See 1 more Smart Citation

Kinesin-1 Motors Can Circumvent Permanent Roadblocks by Side-Shifting to Neighboring Protofilaments

Schneider

Korten

Walter

et al. 2015

Biophysical Journal

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“…Many particle tracking methods solve the correspondence problem in a spatially global manner (Chetverikov and Verestói, 1999;Genovesio et al, 2006;Ruhnow et al, 2011;Sbalzarini and Koumoutsakos, 2005;Thomann et al, 2002;Tvaruskó et al, 1999;Veenman et al, 2003). For example, some level of global optimization can be achieved using graph theory, as in the publicly available software ParticleTracker (Sbalzarini and Koumoutsakos, 2005).…”

Section: Background Informationmentioning

confidence: 99%

Quantitative comparison of multiframe data association techniques for particle tracking in time-lapse fluorescence microscopy

Smal

Meijering

2015

Medical Image Analysis

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“…Q-dots were tracked from sequential TIFF images using FIESTA software 52 . The obtained trajectories were further analysed with MatLab 2013b (MathWorks).…”

Section: Imaging and Data Processingmentioning

confidence: 99%

Dynamic formation of a microchannel array enabling kinesin-driven microtubule transport between separate compartments on a chip

et al. 2015

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Microtubules driven by kinesin motors have been utilised as "molecular shuttles" in microfluidic environments with potential applications in autonomous nanoscale manipulations such as capturing, separating, and/or concentrating biomolecules. However, the conventional flow cell-based assay has difficulty in separating bound target molecules from free ones even with buffer flushing because molecular manipulations by molecular shuttles take place on a glass surface and molecular binding occurs stochastically; this makes it difficult to determine whether molecules are carried by molecular shuttles or by diffusion. To address this issue, we developed a microtubule-based transport system between two compartments connected by a single-micrometre-scale channel array that forms dynamically via pneumatic actuation of a polydimethylsiloxane membrane. The device comprises three layers-a control channel layer (top), a microfluidic channel layer (middle), and a channel array layer (bottom)-that enable selective injection of assay solutions into a target compartment and dynamic formation of the microchannel array. The pneumatic channel also serves as a nitrogen supply path to the assay area, which reduces photobleaching of fluorescently labelled microtubules and deactivation of kinesin by oxygen radicals. The channel array suppresses cross-contamination of molecules caused by diffusion or pressure-driven flow between compartments, facilitating unidirectional transport of molecular shuttles from one compartment to another. The method demonstrates, for the first time, efficient and unidirectional microtubule transport by eliminating diffusion of target molecules on a chip and thus may constitute one of the key aspects of motor-driven nanosystems.

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Tracking Single Particles and Elongated Filaments with Nanometer Precision

Cited by 304 publications

References 37 publications

Kinesin-1 Motors Can Circumvent Permanent Roadblocks by Side-Shifting to Neighboring Protofilaments

Kinesin-1 Motors Can Circumvent Permanent Roadblocks by Side-Shifting to Neighboring Protofilaments

Quantitative comparison of multiframe data association techniques for particle tracking in time-lapse fluorescence microscopy

Dynamic formation of a microchannel array enabling kinesin-driven microtubule transport between separate compartments on a chip

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