scholarly journals Bridging the spatiotemporal scales of macromolecular transport in crowded biomimetic systems

Soft Matter ◽  
2019 ◽  
Vol 15 (6) ◽  
pp. 1200-1209 ◽  
Author(s):  
Kathryn Regan ◽  
Devynn Wulstein ◽  
Hannah Rasmussen ◽  
Ryan McGorty ◽  
Rae M. Robertson-Anderson
Keyword(s):  
Transport Properties ◽  
Single Molecule ◽  
Complex Dynamics ◽  
Single Molecules ◽  
Macromolecular Transport ◽  
Biomimetic Systems ◽  
Spatiotemporal Scales

We combine single-molecule conformational tracking with digital Fourier microscopy to couple crowding-induced conformations and trajectories of single molecules with ensemble-averaged transport properties across an unprecedented spatiotemporal range to elucidate the complex dynamics of large DNA crowded by custom-designed networks of actin and microtubules.

scholarly journals Bridging the spatiotemporal scales of macromolecular transport in crowded biomimetic systems

2018 ◽  
Author(s):  
Kathryn Regan ◽  
Devynn Wulstein ◽  
Hannah Rasmussen ◽  
Ryan McGorty ◽  
Rae M. Robertson-Anderson
Keyword(s):  
Single Molecule ◽  
Biological Molecules ◽  
Biological Macromolecules ◽  
Dna Dynamics ◽  
Dna Transport ◽  
Macromolecular Transport ◽  
Dna Compaction ◽  
Biomimetic Systems ◽  
Conformational Properties ◽  
Spatiotemporal Scales

AbstractCrowding plays a key role in the transport and conformations of biological macromolecules. Gene therapy, viral infection and transfection require DNA to traverse the crowded cytoplasm, including a heterogeneous cytoskeleton of filamentous proteins. Given the complexity of cellular crowding, the dynamics of biological molecules can be highly dependent on the spatiotemporal scale probed. We present a powerful platform that spans molecular and cellular scales by coupling single-molecule conformational tracking (SMCT) and selective-plane illumination differential dynamic microscopy (SPIDDM). We elucidate the transport and conformational properties of large DNA, crowded by custom-designed networks of actin and microtubules, to link single-molecule conformations with ensemble DNA transport and cytoskeleton structure. We show that actin crowding leads to DNA compaction and suppression of fluctuations, combined with anomalous subdiffusion and heterogeneous transport, whereas microtubules have much more subdued impact across all scales. Interestingly, in composite networks of both filaments, microtubules primarily govern single-molecule DNA dynamics whereas actin governs ensemble transport.

scholarly journals Three-dimensional total-internal reflection fluorescence nanoscopy with nanometric axial resolution by photometric localization of single molecules

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Alan M. Szalai ◽  
Bruno Siarry ◽  
Jerónimo Lukin ◽  
David J. Williamson ◽  
Nicolás Unsain ◽  
...  
Keyword(s):  
Single Molecule ◽  
Total Internal Reflection ◽  
Single Molecules ◽  
Fluorescence Microscope ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Axial Position ◽  
Localization Microscopy ◽  
Localization Precision ◽  
Single Molecule Localization Microscopy

AbstractSingle-molecule localization microscopy enables far-field imaging with lateral resolution in the range of 10 to 20 nanometres, exploiting the fact that the centre position of a single-molecule’s image can be determined with much higher accuracy than the size of that image itself. However, attaining the same level of resolution in the axial (third) dimension remains challenging. Here, we present Supercritical Illumination Microscopy Photometric z-Localization with Enhanced Resolution (SIMPLER), a photometric method to decode the axial position of single molecules in a total internal reflection fluorescence microscope. SIMPLER requires no hardware modification whatsoever to a conventional total internal reflection fluorescence microscope and complements any 2D single-molecule localization microscopy method to deliver 3D images with nearly isotropic nanometric resolution. Performance examples include SIMPLER-direct stochastic optical reconstruction microscopy images of the nuclear pore complex with sub-20 nm axial localization precision and visualization of microtubule cross-sections through SIMPLER-DNA points accumulation for imaging in nanoscale topography with sub-10 nm axial localization precision.

Interfacial charge transfer events of BODIPY molecules: single molecule spectroelectrochemistry and substrate effects

2014 ◽  
Vol 16 (42) ◽  
pp. 23150-23156 ◽  
Author(s):  
Jia Liu ◽  
Caleb M. Hill ◽  
Shanlin Pan ◽  
Haiying Liu
Keyword(s):  
Charge Transfer ◽  
Single Molecule ◽  
Single Molecules ◽  
Transfer Mechanism ◽  
Substrate Effects ◽  
Interfacial Charge Transfer ◽  
Charge Transfer Mechanism ◽  
Nanostructured Substrates ◽  
Interfacial Charge

BODIPY dye single molecules on nanostructured substrates are studied with a single molecule spectroelectrochemistry technique to reveal the heterogeneous charge transfer mechanism.

scholarly journals A method for imaging single molecules at the plasma membrane of live cells within tissue slices

2020 ◽  
Vol 153 (1) ◽  
Author(s):  
Gregory I. Mashanov ◽  
Tatiana A. Nenasheva ◽  
Tatiana Mashanova ◽  
Catherine Maclachlan ◽  
Nigel J.M. Birdsall ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Lines ◽  
Single Molecule ◽  
Cardiac Tissue ◽  
Single Molecules ◽  
Live Cells ◽  
Biological Macromolecules ◽  
Tissue Slices ◽  
Tissue Samples ◽  
Mammalian Cell Lines

Recent advances in light microscopy allow individual biological macromolecules to be visualized in the plasma membrane and cytosol of live cells with nanometer precision and ∼10-ms time resolution. This allows new discoveries to be made because the location and kinetics of molecular interactions can be directly observed in situ without the inherent averaging of bulk measurements. To date, the majority of single-molecule imaging studies have been performed in either unicellular organisms or cultured, and often chemically fixed, mammalian cell lines. However, primary cell cultures and cell lines derived from multi-cellular organisms might exhibit different properties from cells in their native tissue environment, in particular regarding the structure and organization of the plasma membrane. Here, we describe a simple approach to image, localize, and track single fluorescently tagged membrane proteins in freshly prepared live tissue slices and demonstrate how this method can give information about the movement and localization of a G protein–coupled receptor in cardiac tissue slices. In principle, this experimental approach can be used to image the dynamics of single molecules at the plasma membrane of many different soft tissue samples and may be combined with other experimental techniques.

Tunneling magnetoresistance and light modulation in Fe4N(La2/3Sr1/3MnO3)/C60/Fe4N single molecule magnetic tunnel junctions

2020 ◽  
Vol 8 (9) ◽  
pp. 3137-3146 ◽  
Author(s):  
Xuefei Han ◽  
Wenbo Mi ◽  
Dunhui Wang
Keyword(s):  
Transport Properties ◽  
Single Molecule ◽  
Tunnel Junctions ◽  
Magnetic Tunnel Junctions ◽  
Tunneling Magnetoresistance ◽  
Light Modulation ◽  
Spin Dependent Transport ◽  
Dependent Transport

Spin-dependent transport properties and light modulation of Fe4N/C60/Fe4N and LSMO/C60/Fe4N single molecule magnetic tunnel junctions.

scholarly journals Topology-dependent anomalous dynamics of ring and linear DNA are sensitive to cytoskeleton crosslinking

2019 ◽  
Vol 5 (12) ◽  
pp. eaay5912 ◽  
Author(s):  
Devynn M. Wulstein ◽  
Kathryn E. Regan ◽  
Jonathan Garamella ◽  
Ryan J. McGorty ◽  
Rae M. Robertson-Anderson
Keyword(s):  
Transport Properties ◽  
Single Molecule ◽  
Intracellular Transport ◽  
Conformational Stability ◽  
Conformational Dynamics ◽  
Single Mode ◽  
Dna Molecules ◽  
Linear Dna ◽  
Linear Chains ◽  
Anomalous Dynamics

Cytoskeletal crowding plays a key role in the diffusion of DNA molecules through the cell, acting as a barrier to effective intracellular transport and conformational stability required for processes such as transfection, viral infection, and gene therapy. Here, we elucidate the transport properties and conformational dynamics of linear and ring DNA molecules diffusing through entangled and crosslinked composite networks of actin and microtubules. We couple single-molecule conformational tracking with differential dynamic microscopy to reveal that ring and linear DNA exhibit unexpectedly distinct transport properties that are influenced differently by cytoskeleton crosslinking. Ring DNA coils are swollen and undergo heterogeneous and biphasic subdiffusion that is hindered by crosslinking. Conversely, crosslinking actually facilitates the single-mode subdiffusion that compacted linear chains exhibit. Our collective results demonstrate that transient threading by cytoskeleton filaments plays a key role in the dynamics of ring DNA, whereas the mobility of the cytoskeleton dictates transport of linear DNA.

scholarly journals Dynamics of DNA replication in a eukaryotic cell

2019 ◽  
Vol 116 (11) ◽  
pp. 4973-4982 ◽  
Author(s):  
Thomas Kelly ◽  
A. John Callegari
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Origin Recognition Complex ◽  
Complex Dynamics ◽  
Replication Timing ◽  
Eukaryotic Cell ◽  
Replication Initiation ◽  
Integrative Model ◽  
Genomic Locus ◽  
Two Factors

Each genomic locus in a eukaryotic cell has a distinct average time of replication during S phase that depends on the spatial and temporal pattern of replication initiation events. Replication timing can affect genomic integrity because late replication is associated with an increased mutation rate. For most eukaryotes, the features of the genome that specify the location and timing of initiation events are unknown. To investigate these features for the fission yeast, Schizosaccharomyces pombe, we developed an integrative model to analyze large single-molecule and global genomic datasets. The model provides an accurate description of the complex dynamics of S. pombe DNA replication at high resolution. We present evidence that there are many more potential initiation sites in the S. pombe genome than previously identified and that the distribution of these sites is primarily determined by two factors: the sequence preferences of the origin recognition complex (ORC), and the interference of transcription with the assembly or stability of prereplication complexes (pre-RCs). We suggest that in addition to directly interfering with initiation, transcription has driven the evolution of the binding properties of ORC in S. pombe and other eukaryotic species to target pre-RC assembly to regions of the genome that are less likely to be transcribed.

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