scholarly journals Dissection of molecular assembly dynamics by tracking orientation and position of single molecules in live cells

2016 ◽  
Vol 113 (42) ◽  
pp. E6352-E6361 ◽  
Author(s):  
Shalin B. Mehta ◽  
Molly McQuilken ◽  
Patrick J. La Riviere ◽  
Patricia Occhipinti ◽  
Amitabh Verma ◽  
...  
Keyword(s):  
Single Molecule ◽  
Leading Edge ◽  
Living Cells ◽  
Molecular Assembly ◽  
Higher Order ◽  
Live Cells ◽  
Actin Network ◽  
Position And Orientation ◽  
Orientation Of Molecules

Regulation of order, such as orientation and conformation, drives the function of most molecular assemblies in living cells but remains difficult to measure accurately through space and time. We built an instantaneous fluorescence polarization microscope, which simultaneously images position and orientation of fluorophores in living cells with single-molecule sensitivity and a time resolution of 100 ms. We developed image acquisition and analysis methods to track single particles that interact with higher-order assemblies of molecules. We tracked the fluctuations in position and orientation of molecules from the level of an ensemble of fluorophores down to single fluorophores. We tested our system in vitro using fluorescently labeled DNA and F-actin, in which the ensemble orientation of polarized fluorescence is known. We then tracked the orientation of sparsely labeled F-actin network at the leading edge of migrating human keratinocytes, revealing the anisotropic distribution of actin filaments relative to the local retrograde flow of the F-actin network. Additionally, we analyzed the position and orientation of septin-GFP molecules incorporated in septin bundles in growing hyphae of a filamentous fungus. Our data indicate that septin-GFP molecules undergo positional fluctuations within ∼350 nm of the binding site and angular fluctuations within ∼30° of the central orientation of the bundle. By reporting position and orientation of molecules while they form dynamic higher-order structures, our approach can provide insights into how micrometer-scale ordered assemblies emerge from nanoscale molecules in living cells.

scholarly journals Dissection of molecular assembly dynamics by tracking orientation and position of single molecules in live cells

2016 ◽  
Author(s):  
Shalin B. Mehta ◽  
Molly McQuilken ◽  
Patrick La Riviere ◽  
Patricia Occhipinti ◽  
Amitabh Verma ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescence Polarization ◽  
Single Molecules ◽  
Living Cells ◽  
Molecular Assembly ◽  
Higher Order ◽  
Actin Network ◽  
Molecular Assemblies ◽  
Position And Orientation ◽  
Orientation Of Molecules

AbstractRegulation of order, such as orientation and conformation, drives the function of most molecular assemblies in living cells, yet remains difficult to measure accurately through space and time. We built an instantaneous fluorescence polarization microscope, which simultaneously images position and orientation of fluorophores in living cells with single-molecule sensitivity and a time resolution of 100ms. We developed image acquisition and analysis methods to track single particles that interact with higher-order assemblies of molecules. We tracked the fluctuations in position and orientation of molecules from the level of an ensemble of fluorophores down to single fluorophores. We tested our system in vitro using fluorescently labeled DNA and F-actin in which the ensemble orientation of polarized fluorescence is known. We then tracked the orientation of sparsely labeled F-actin network at the leading edge of migrating human keratinocytes, revealing the anisotropic distribution of actin filaments relative to the local retrograde flow of the F-actin network. Additionally, we analyzed the position and orientation of septin-GFP molecules incorporated in septin bundles in growing hyphae of a filamentous fungus. Our data indicate that septin-GFP molecules undergo positional fluctuations within, ∼350nm of the binding site and angular fluctuations within ∼30° of the central orientation of the bundle. By reporting position and orientation of molecules while they form dynamic higher-order structures, our approach can provide new insights into how micron-scale ordered assemblies emerge from nanoscale molecules in living cells.Significance StatementIn living cells, the 3D architecture of molecular assemblies such as chromosomes, lipid bilayers, and the cytoskeleton is regulated through the interaction among their component molecules. Monitoring the position and orientation of constituent molecules is important for understanding the mechanisms that govern the structure and function of these assemblies. We have developed an instantaneous fluorescence polarization microscope to track the position and orientation of fluorescently labeled particles, including single molecules, which form micron-scale macromolecular assemblies in living cells. Our imaging approach is broadly applicable to the study of dynamic molecular interactions that underpin the function of micron-scale assemblies in living cells.

Force-dependent vinculin binding to talin in live cells: a crucial step in anchoring the actin cytoskeleton to focal adhesions

2014 ◽  
Vol 306 (6) ◽  
pp. C607-C620 ◽  
Author(s):  
Hiroaki Hirata ◽  
Hitoshi Tatsumi ◽  
Chwee Teck Lim ◽  
Masahiro Sokabe
Keyword(s):  
Actin Cytoskeleton ◽  
Focal Adhesions ◽  
Living Cells ◽  
Live Cells ◽  
Mechanical Forces ◽  
Actin Network ◽  
Crucial Step

Mechanical forces play a pivotal role in the regulation of focal adhesions (FAs) where the actin cytoskeleton is anchored to the extracellular matrix through integrin and a variety of linker proteins including talin and vinculin. The localization of vinculin at FAs depends on mechanical forces. While in vitro studies have demonstrated the force-induced increase in vinculin binding to talin, it remains unclear whether such a mechanism exists at FAs in vivo. In this study, using fibroblasts cultured on elastic silicone substrata, we have examined the role of forces in modulating talin-vinculin binding at FAs. Stretching the substrata caused vinculin accumulation at talin-containing FAs, and this accumulation was abrogated by expressing the talin-binding domain of vinculin (domain D1, which inhibits endogenous vinculin from binding to talin). These results indicate that mechanical forces loaded to FAs facilitate vinculin binding to talin at FAs. In cell-protruding regions, the actin network moved backward over talin-containing FAs in domain D1-expressing cells while it was anchored to FAs in control cells, suggesting that the force-dependent vinculin binding to talin is crucial for anchoring the actin cytoskeleton to FAs in living cells.

scholarly journals SIMILARITY OF THE KINETICS OF INVERTASE ACTION IN VIVO AND IN VITRO. II

1932 ◽  
Vol 16 (2) ◽  
pp. 233-242 ◽  
Author(s):  
B. G. Wilkes ◽  
Elizabeth T. Palmer
Keyword(s):  
Physiological Activity ◽  
Outer Region ◽  
Living Cells ◽  
Yeast Cells ◽  
Live Cells ◽  
Intracellular Enzyme ◽  
Relationship Of ◽  
Kinetics Of

1. The pH-activity relationship of invertase has been studied in vivo and in vitro under identical external environmental conditions. 2. The effect of changing (H+) upon the sucroclastic activity of living cells of S. cerevisiae and of invertase solutions obtained therefrom has been found, within experimental error, to be identical. 3. The region of living yeast cells in which invertase exerts its physiological activity changes its pH freely and to the same extent as that of the suspending medium. It is suggested that this may indicate that this intracellular enzyme may perform its work somewhere in the outer region of the cell. 4. In using live cells containing maltase, no evidence of increased sucroclastic activity around pH 6.9, due to the action of Weidenhagen's α-glucosidase (maltase), was found.

scholarly journals Single-Molecule Kinetics in Living Cells

2019 ◽  
Vol 88 (1) ◽  
pp. 635-659 ◽  
Author(s):  
Johan Elf ◽  
Irmeli Barkefors
Keyword(s):  
Single Molecule ◽  
Living Cells ◽  
The Past

In the past decades, advances in microscopy have made it possible to study the dynamics of individual biomolecules in vitro and resolve intramolecular kinetics that would otherwise be hidden in ensemble averages. More recently, single-molecule methods have been used to image, localize, and track individually labeled macromolecules in the cytoplasm of living cells, allowing investigations of intermolecular kinetics under physiologically relevant conditions. In this review, we illuminate the particular advantages of single-molecule techniques when studying kinetics in living cells and discuss solutions to specific challenges associated with these methods.

scholarly journals tdLanYFP, a yellow, bright, photostable and pH insensitive fluorescent protein for live cell imaging and FRET-based sensing strategies

2021 ◽  
Author(s):  
Y. Bousmah ◽  
H. Valenta ◽  
G. Bertolin ◽  
U. Singh ◽  
V. Nicolas ◽  
...  
Keyword(s):  
Single Molecule ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy ◽  
Live Cell ◽  
Live Cells

AbstractYellow fluorescent proteins (YFP) are widely used as optical reporters in Förster Resonance Energy Transfer (FRET) based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pHs. In fact, today, there is no yellow variant derived from the EYFP with a pK1/2 below ∼5.5. Here, we characterize a new yellow fluorescent protein, tdLanYFP, derived from the tetrameric protein from the cephalochordate B. lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133 000 mol−1.L.cm−1, it is, to our knowledge, the brightest dimeric fluorescent protein available, and brighter than most of the monomeric YFPs. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and preserves this property in live cells. As a consequence, tdLanYFP allows the imaging of cellular structures with sub-diffraction resolution with STED nanoscopy. We also demonstrate that the combination of high brightness and strong photostability is compatible with the use of spectro-microscopies in single molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pHs. Finally, we show that tdLanYFP can be a FRET partner either as donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFPa very attractive yellow fluorescent protein for long-term or single-molecule live-cell imaging that is also suitable for FRET experiment including at acidic pH.

scholarly journals Raman2RNA: Live-cell label-free prediction of single-cell RNA expression profiles by Raman microscopy

2021 ◽  
Author(s):  
Koseki J. Kobayashi-Kirschvink ◽  
Shreya Gaddam ◽  
Taylor James-Sorenson ◽  
Emanuelle Grody ◽  
Johain R. Ounadjela ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Molecule ◽  
Vibrational Energy ◽  
Temporal Dynamics ◽  
Expression Profiles ◽  
Raman Microscopy ◽  
Live Cells ◽  
Label Free ◽  
Imaging Data

Single cell RNA-Seq (scRNA-seq) and other profiling assays have opened new windows into understanding the properties, regulation, dynamics, and function of cells at unprecedented resolution and scale. However, these assays are inherently destructive, precluding us from tracking the temporal dynamics of live cells, in cell culture or whole organisms. Raman microscopy offers a unique opportunity to comprehensively report on the vibrational energy levels of molecules in a label-free and non-destructive manner at a subcellular spatial resolution, but it lacks in genetic and molecular interpretability. Here, we developed Raman2RNA (R2R), an experimental and computational framework to infer single-cell expression profiles in live cells through label-free hyperspectral Raman microscopy images and multi-modal data integration and domain translation. We used spatially resolved single-molecule RNA-FISH (smFISH) data as anchors to link scRNA-seq profiles to the paired spatial hyperspectral Raman images, and trained machine learning models to infer expression profiles from Raman spectra at the single-cell level. In reprogramming of mouse fibroblasts into induced pluripotent stem cells (iPSCs), R2R accurately (r>0.96) inferred from Raman images the expression profiles of various cell states and fates, including iPSCs, mesenchymal-epithelial transition (MET) cells, stromal cells, epithelial cells, and fibroblasts. R2R outperformed inference from brightfield images, showing the importance of spectroscopic content afforded by Raman microscopy. Raman2RNA lays a foundation for future investigations into exploring single-cell genome-wide molecular dynamics through imaging data, in vitro and in vivo.

scholarly journals High throughput mechanobiology: Force modulation of ensemble biochemical and cell-based assays

2020 ◽  
Author(s):  
Ália dos Santos ◽  
Natalia Fili ◽  
David S. Pearson ◽  
Yukti Hari-Gupta ◽  
Christopher P. Toseland
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Receptor Activation ◽  
Magnetic Tweezers ◽  
Live Cells ◽  
Mechanical Stimuli ◽  
Cellular Assays ◽  
Novel Approach ◽  
Microplate Reader

ABSTRACTMechanobiology is focused on how the physical forces and the mechanical properties of proteins, cells and tissues contribute to physiology and disease. While the response of proteins and cells to mechanical stimuli is critical for function, the tools to probe these activities are typically restricted to single molecule manipulations. Here, we have developed a novel microplate reader assay to encompass mechanical measurements with ensemble biochemical and cellular assays, using a microplate lid modified with magnets. This configuration enables multiple static magnetic tweezers to function simultaneously across the microplate, thereby greatly increasing throughput. The broad applicability and versatility of our approach has been demonstrated through in vitro force-induced enzymatic activity and conformation changes, along with force-induced receptor activation and their downstream signalling pathways in live cells. Overall, our methodology allows for the first-time ensemble biochemical and cell-based assays to be performed under force, in high throughput format. This novel approach would substantially add to the mechano-biological toolbox and increase the availability of mechanobiology measurements.

scholarly journals Synergy between Cyclase-associated protein and Cofilin accelerates actin filament depolymerization by two orders of magnitude

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Shashank Shekhar ◽  
Johnson Chung ◽  
Jane Kondev ◽  
Jeff Gelles ◽  
Bruce L. Goode
Keyword(s):  
Actin Filament ◽  
Single Molecule ◽  
Actin Filaments ◽  
Binding Protein ◽  
Actin Dynamics ◽  
Actin Network ◽  
Cellular Mechanisms ◽  
Actin Networks ◽  
Binding Event

AbstractCellular actin networks can be rapidly disassembled and remodeled in a few seconds, yet in vitro actin filaments depolymerize slowly over minutes. The cellular mechanisms enabling actin to depolymerize this fast have so far remained obscure. Using microfluidics-assisted TIRF, we show that Cyclase-associated protein (CAP) and Cofilin synergize to processively depolymerize actin filament pointed ends at a rate 330-fold faster than spontaneous depolymerization. Single molecule imaging further reveals that hexameric CAP molecules interact with the pointed ends of Cofilin-decorated filaments for several seconds at a time, removing approximately 100 actin subunits per binding event. These findings establish a paradigm, in which a filament end-binding protein and a side-binding protein work in concert to control actin dynamics, and help explain how rapid actin network depolymerization is achieved in cells.

scholarly journals Actin turnover–dependent fast dissociation of capping protein in the dendritic nucleation actin network: evidence of frequent filament severing

2006 ◽  
Vol 175 (6) ◽  
pp. 947-955 ◽  
Author(s):  
Takushi Miyoshi ◽  
Takahiro Tsuji ◽  
Chiharu Higashida ◽  
Maud Hertzog ◽  
Akiko Fujita ◽  
...  
Keyword(s):  
Single Molecule ◽  
Actin Filaments ◽  
Dissociation Rate ◽  
Actin Network ◽  
Lim Kinase ◽  
Capping Protein ◽  
Filament Dynamics ◽  
Actin Turnover ◽  
Kinetics Of

Actin forms the dendritic nucleation network and undergoes rapid polymerization-depolymerization cycles in lamellipodia. To elucidate the mechanism of actin disassembly, we characterized molecular kinetics of the major filament end-binding proteins Arp2/3 complex and capping protein (CP) using single-molecule speckle microscopy. We have determined the dissociation rates of Arp2/3 and CP as 0.048 and 0.58 s−1, respectively, in lamellipodia of live XTC fibroblasts. This CP dissociation rate is three orders of magnitude faster than in vitro. CP dissociates slower from actin stress fibers than from the lamellipodial actin network, suggesting that CP dissociation correlates with actin filament dynamics. We found that jasplakinolide, an actin depolymerization inhibitor, rapidly blocked the fast CP dissociation in cells. Consistently, the coexpression of LIM kinase prolonged CP speckle lifetime in lamellipodia. These results suggest that cofilin-mediated actin disassembly triggers CP dissociation from actin filaments. We predict that filament severing and end-to-end annealing might take place fairly frequently in the dendritic nucleation actin arrays.

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