scholarly journals Mitochondrial adaptor TRAK2 activates and functionally links opposing kinesin and dynein motors

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Adam R. Fenton ◽  
Thomas A. Jongens ◽  
Erika L. F. Holzbaur
Keyword(s):  
Single Molecule ◽  
Binding Protein ◽  
Integrated Control ◽  
Coiled Coil ◽  
Single Molecule Imaging ◽  
Cell Lysates ◽  
Motor Complex

AbstractMitochondria are transported along microtubules by opposing kinesin and dynein motors. Kinesin-1 and dynein-dynactin are linked to mitochondria by TRAK proteins, but it is unclear how TRAKs coordinate these motors. We used single-molecule imaging of cell lysates to show that TRAK2 robustly activates kinesin-1 for transport toward the microtubule plus-end. TRAK2 is also a novel dynein activating adaptor that utilizes a conserved coiled-coil motif to interact with dynein to promote motility toward the microtubule minus-end. However, dynein-mediated TRAK2 transport is minimal unless the dynein-binding protein LIS1 is present at a sufficient level. Using co-immunoprecipitation and co-localization experiments, we demonstrate that TRAK2 forms a complex containing both kinesin-1 and dynein-dynactin. These motors are functionally linked by TRAK2 as knockdown of either kinesin-1 or dynein-dynactin reduces the initiation of TRAK2 transport toward either microtubule end. We propose that TRAK2 coordinates kinesin-1 and dynein-dynactin as an interdependent motor complex, providing integrated control of opposing motors for the proper transport of mitochondria.

scholarly journals The condensin complex is a mechanochemical motor that translocates along DNA

2017 ◽  
Author(s):  
Tsuyoshi Terekawa ◽  
Shveta Bisht ◽  
Jorine M. Eeftens ◽  
Cees Dekker ◽  
Christian H. Haering ◽  
...  
Keyword(s):  
Single Molecule ◽  
Atp Hydrolysis ◽  
Molecular Motor ◽  
Average Distance ◽  
Coiled Coil ◽  
Chromosome Organization ◽  
Single Molecule Imaging ◽  
Base Pairs ◽  
Translocation Mechanism ◽  
Mechanistic Basis

One Sentence SummarySingle-molecule imaging reveals that eukaryotic condensin is a highly processive DNA-translocating motor complex.AbstractCondensin plays crucial roles in chromosome organization and compaction, but the mechanistic basis for its functions remains obscure. Here, we use single-molecule imaging to demonstrate that Saccharomyces cerevisiae condensin is a molecular motor capable of ATP hydrolysis-dependent translocation along double-stranded DNA. Condensin’s translocation activity is rapid and highly processive, with individual complexes traveling an average distance of ≥10 kilobases at a velocity of ∼60 base pairs per second. Our results suggest that condensin may take steps comparable in length to its ∼50-nanometer coiled-coil subunits, suggestive of a translocation mechanism that is distinct from any reported DNA motor protein. The finding that condensin is a mechanochemical motor has important implications for understanding the mechanisms of chromosome organization and condensation.

Label-free, mass-sensitive single-molecule imaging using interferometric scattering microscopy

2020 ◽  
Author(s):  
Nikolas Hundt
Keyword(s):  
Single Molecule ◽  
Cellular Systems ◽  
Single Molecule Imaging ◽  
Label Free ◽  
Measurement Sensitivity ◽  
Mass Distributions ◽  
Quantitative Measurements ◽  
Contrast Mechanism ◽  
Cellular Components ◽  
Scattering Signal

Abstract Single-molecule imaging has mostly been restricted to the use of fluorescence labelling as a contrast mechanism due to its superior ability to visualise molecules of interest on top of an overwhelming background of other molecules. Recently, interferometric scattering (iSCAT) microscopy has demonstrated the detection and imaging of single biomolecules based on light scattering without the need for fluorescent labels. Significant improvements in measurement sensitivity combined with a dependence of scattering signal on object size have led to the development of mass photometry, a technique that measures the mass of individual molecules and thereby determines mass distributions of biomolecule samples in solution. The experimental simplicity of mass photometry makes it a powerful tool to analyse biomolecular equilibria quantitatively with low sample consumption within minutes. When used for label-free imaging of reconstituted or cellular systems, the strict size-dependence of the iSCAT signal enables quantitative measurements of processes at size scales reaching from single-molecule observations during complex assembly up to mesoscopic dynamics of cellular components and extracellular protrusions. In this review, I would like to introduce the principles of this emerging imaging technology and discuss examples that show how mass-sensitive iSCAT can be used as a strong complement to other routine techniques in biochemistry.

A Molecular Logic Gate Enables Super-Resolved Imaging of Intracellular Lipid Droplets

2019 ◽  
Author(s):  
Adam Eördögh ◽  
Carolina Paganini ◽  
Dorothea Pinotsi ◽  
Paolo Arosio ◽  
Pablo Rivera-Fuentes
Keyword(s):  
Single Molecule ◽  
Lipid Droplets ◽  
Neutral Lipids ◽  
Logic Gate ◽  
Living Cells ◽  
Three Dimensions ◽  
Single Molecule Imaging ◽  
Molecular Logic Gate ◽  
Molecular Logic ◽  
Cellular Compartments

<div>Photoactivatable dyes enable single-molecule imaging in biology. Despite progress in the development of new fluorophores and labeling strategies, many cellular compartments remain difficult to image beyond the limit of diffraction in living cells. For example, lipid droplets, which are organelles that contain mostly neutral lipids, have eluded single-molecule imaging. To visualize these challenging subcellular targets, it is necessary to develop new fluorescent molecular devices beyond simple on/off switches. Here, we report a fluorogenic molecular logic gate that can be used to image single molecules associated with lipid droplets with excellent specificity. This probe requires the subsequent action of light, a lipophilic environment and a competent nucleophile to produce a fluorescent product. The combination of these requirements results in a probe that can be used to image the boundary of lipid droplets in three dimensions with resolutions beyond the limit of diffraction. Moreover, this probe enables single-molecule tracking of lipids within and between droplets in living cells.</div>

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