Future Challenges in Single-Molecule Fluorescence and Laser Trap Approaches to Studies of Molecular Motors

Elting, Mary Williard; Spudich, James A.

doi:10.1016/j.devcel.2012.10.002

Cited by 27 publications

(24 citation statements)

References 95 publications

(115 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As the technical details of both methods have been described and discussed in detail elsewhere, here we only describe the remaining steps needed to successfully set up single-molecule fluorescence and optical trapping experiments on dynein using the MT-filled slide chambers prepared in Sections 3.6.1–3.6.3. For more details on these assays, please refer to references [86–88,54]. …”

Section: Methodsmentioning

confidence: 99%

Combining Structure–Function and Single-Molecule Studies on Cytoplasmic Dynein

Rao

Hülsemann

Gennerich

2017

Single Molecule Analysis

View full text Add to dashboard Cite

Cytoplasmic dynein is the largest and most intricate cytoskeletal motor protein. It is responsible for a vast array of biological functions, ranging from the transport of organelles and mRNAs to the movement of nuclei during neuronal migration and the formation and positioning of the mitotic spindle during cell division. Despite its megadalton size and its complex design, recent success with the recombinant expression of the dynein heavy chain has advanced our understanding of dynein’s molecular mechanism through the combination of structure-function and single-molecule studies. Single-molecule fluorescence assays have provided detailed insights into how dynein advances along its microtubule track in the absence of load, while optical tweezers have yielded insights into the force generation and stalling behavior of dynein. Here, using the S. cerevisiae expression system, we provide improved protocols for the generation of dynein mutants and for the expression and purification of the mutated and/or tagged proteins. To facilitate single-molecule fluorescence and optical trapping assays, we further describe updated, easy-to-use protocols for attaching microtubules to coverslip surfaces. The presented protocols together with the recently solved crystal structures of the dynein motor domain will further simplify and accelerate hypothesis-driven mutagenesis and structure-function studies on dynein.

show abstract

Section: Methodsmentioning

confidence: 99%

Combining Structure–Function and Single-Molecule Studies on Cytoplasmic Dynein

Rao

Hülsemann

Gennerich

2017

Single Molecule Analysis

View full text Add to dashboard Cite

show abstract

“…Force generation depends on molecular motors, which often act cooperatively to generate forces exceeding the single-motor forces 6,7 . How cooperative motors are coordinated is an area of active research.…”

mentioning

confidence: 99%

Bacterial twitching motility is coordinated by a two-dimensional tug-of-war with directional memory

et al. 2014

View full text Add to dashboard Cite

Type IV pili are ubiquitous bacterial motors that power surface motility. In peritrichously piliated species, it is unclear how multiple pili are coordinated to generate movement with directional persistence. Here we use a combined theoretical and experimental approach to test the hypothesis that multiple pili of Neisseria gonorrhoeae are coordinated through a tug-of-war. Based on force-dependent unbinding rates and pilus retraction speeds measured at the level of single pili, we build a tug-of-war model. Whereas the one-dimensional model robustly predicts persistent movement, the two-dimensional model requires a mechanism of directional memory provided by re-elongation of fully retracted pili and pilus bundling. Experimentally, we confirm memory in the form of bursts of pilus retractions. Bursts are seen even with bundling suppressed, indicating re-elongation from stable core complexes as the key mechanism of directional memory. Directional memory increases the surface range explored by motile bacteria and likely facilitates surface colonization.

show abstract

“…These findings were almost always based on the development of new techniques to study muscle, but without the advantage of today’s accumulated knowledge and technology, such as fluorescence microscopy and basic molecular techniques. 5 For example, the sliding filament and cross-bridge theories depended on the development and application of electron and dual interference contrast microscopies. Similarly, the length-tension relationship, which provided a molecular basis for the Frank-Starling Law, depended on developing tools for accurate and simultaneous measurement of muscle, or sarcomere, length and force generation.…”

Section: Advanced Technologies Are Required To Study the Myofilament mentioning

confidence: 99%

The Myofilament Field Revisited in the Age of Cellular and Molecular Biology

Sadayappan

2017

Circulation Research

View full text Add to dashboard Cite

In the not too distant past, myofilament research attracted considerable attention after the discovery that mutations in myofilament genes cause cardiomyopathies. However, basic research has not been completely translated into therapies. This viewpoint discusses the need to develop innovative and integrative technologies and generate translatable models to uncover the complex biology of myofilament proteins to advance the field.

show abstract

Future Challenges in Single-Molecule Fluorescence and Laser Trap Approaches to Studies of Molecular Motors

Cited by 27 publications

References 95 publications

Combining Structure–Function and Single-Molecule Studies on Cytoplasmic Dynein

Combining Structure–Function and Single-Molecule Studies on Cytoplasmic Dynein

Bacterial twitching motility is coordinated by a two-dimensional tug-of-war with directional memory

The Myofilament Field Revisited in the Age of Cellular and Molecular Biology

Contact Info

Product

Resources

About