Three-color single-molecule imaging reveals conformational dynamics of dynein undergoing motility

Niekamp, Stefan; Stuurman, Nico; Zhang, Nan; Vale, Ronald D.

doi:10.1073/pnas.2101391118

Cited by 15 publications

(11 citation statements)

References 56 publications

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“…The different tags have also been combined to achieve multicolor and orthogonal labeling, going beyond the standard use of SNAP–CLIP and ACP–MCP or S6–A1 couples. Single dynein molecules were labeled simultaneously with Halo and YBBR tags [ 196 ] and with SNAP, Halo and YBBR tags for three-color imaging [ 197 ]; SNAP-β2-adrenergic receptor and HALO-transferrin receptor were orthogonally and simultaneously labeled [ 198 ].…”

Section: Fluorescent Probes For Single Molecule Microscopymentioning

confidence: 99%

Choosing the Probe for Single-Molecule Fluorescence Microscopy

Spagnolo

Luin

2022

IJMS

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Probe choice in single-molecule microscopy requires deeper evaluations than those adopted for less sensitive fluorescence microscopy studies. Indeed, fluorophore characteristics can alter or hide subtle phenomena observable at the single-molecule level, wasting the potential of the sophisticated instrumentation and algorithms developed for advanced single-molecule applications. There are different reasons for this, linked, e.g., to fluorophore aspecific interactions, brightness, photostability, blinking, and emission and excitation spectra. In particular, these spectra and the excitation source are interdependent, and the latter affects the autofluorescence of sample substrate, medium, and/or biological specimen. Here, we review these and other critical points for fluorophore selection in single-molecule microscopy. We also describe the possible kinds of fluorophores and the microscopy techniques based on single-molecule fluorescence. We explain the importance and impact of the various issues in fluorophore choice, and discuss how this can become more effective and decisive for increasingly demanding experiments in single- and multiple-color applications.

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Section: Fluorescent Probes For Single Molecule Microscopymentioning

confidence: 99%

Choosing the Probe for Single-Molecule Fluorescence Microscopy

Spagnolo

Luin

2022

IJMS

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“…FluoroCubes have enabled high precision particle tracking experiments for the long-term study of molecular motors, , and there are many additional potential application areas for FluoroCube technology. One useful feature in fluorescence is the ability to directly interrogate molecular interactions and conformations using Förster resonance energy transfer (FRET). − FRET is a process wherein energy transfers from an excited donor fluorophore to an acceptor fluorophore within a characteristic radius of typically ∼5 nm.…”

mentioning

confidence: 99%

Ultra-photostable DNA FluoroCubes: Mechanism of Photostability and Compatibility with FRET and Dark Quenching

Blanchard

Duran

et al. 2022

Nano Lett.

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DNA-based FluoroCubes were recently developed as a solution to photobleaching, which is a ubiquitous limitation of fluorescence microscopy (Niekamp; Stuurman; Vale Nature Methods, 2020). FluoroCubes, which are not as compact ∼4 × 4 × 5.4 nm 3 fourhelix bundles coupled to ≤6 fluorescent dyes, remain fluorescent up to ∼50× longer than single dyes and emit up to ∼40× as many photons. The current work answers two important questions about FluoroCubes. First, what is the mechanism by which photostability is enhanced? Second, are FluoroCubes compatible with Forster resonance energy transfer (FRET) and similar techniques? We use single particle photobleaching studies to show that photostability arises through interactions between the fluorophores and the four-helix DNA bundle. Supporting this, we discover that smaller ∼4 × 4 × 2.7 nm 3 FluoroCubes also confer ultraphotostability. However, we find that certain dye−dye interactions negatively impact FluoroCube performance. Accordingly, 4-dye FluoroCubes lacking these interactions perform better than 6-dye FluoroCubes. We also demonstrate that FluoroCubes are compatible with FRET and dark quenching applications.

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“…Consider the case where either experiments or high-resolution simulations have been performed and both the free energy and mechanical energy landscapes of the object in isolation are well known. For the case of dynein, the illustrative motor protein discussed in the introduction, both mechanical [34,35] and kinetic [12,36] information is available to parameterise the system. For proteins and unfolding in general, Hughes et al provide a detailed review [32].…”

Section: External Forcesmentioning

confidence: 99%

A generalised mechano-kinetic model for use in multiscale simulation protocols

Hanson

2020

Preprint

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Many biophysical systems and proteins undergo mesoscopic conformational changes in order to perform their biological function. However, these conformational changes often result from a cascade of atomistic interactions within a much larger overall object. For simulations of such systems, the computational cost of retaining high-resolution structural and dynamical information whilst at the same time observing large scale motions over long times is high. Furthermore, this cost is only going to increase as ever larger biological objects are observed experimentally at high resolution.With insight from the theory of Markov state models and transition state theory, we derive a generalised mechano-kinetic simulation framework which aims to compensate for these disparate time scales. The framework models continuous dynamical motion at coarse-grained length scales whilst simultaneously including fine-detail, chemical reactions or conformational changes implicitly using a kinetic model. The kinetic model is coupled to the dynamic model in a highly generalised manner, such that it can be applied to any defined continuous energy landscape, and indeed, any existing dynamic simulation framework. We present a series of analytical examples to validate the framework, and showcase its capabilities for studying more complex systems by designing a simulation of minimalist molecular motor.

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Three-color single-molecule imaging reveals conformational dynamics of dynein undergoing motility

Cited by 15 publications

References 56 publications

Choosing the Probe for Single-Molecule Fluorescence Microscopy

Choosing the Probe for Single-Molecule Fluorescence Microscopy

Ultra-photostable DNA FluoroCubes: Mechanism of Photostability and Compatibility with FRET and Dark Quenching

A generalised mechano-kinetic model for use in multiscale simulation protocols

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