scholarly journals G1/S transcription factors assemble in discrete clusters that increase in number as cells grow

2019 ◽  
Author(s):  
Labe Black ◽  
Sylvain Tollis ◽  
Guo Fu ◽  
Jean-Bernard Fiche ◽  
Savanna Dorsey ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Super Resolution ◽  
Temporal Organization ◽  
Stochastic Simulations ◽  
Live Cells ◽  
Number Of Clusters ◽  
Localization Microscopy ◽  
Spatio Temporal ◽  
Cluster Behavior ◽  
Target Promoters

AbstractThe spatio-temporal organization of transcription factor (TF)-promoter interactions is critical for the coordination of transcriptional programs. In budding yeast, the main G1/S transcription factors, SBF and MBF, are limiting with respect to target promoters in small G1 phase cells and accumulate as cells grow, raising the question of how SBF/MBF are dynamically distributed across the G1/S regulon. Super-resolution Photo-Activatable Localization Microscopy (PALM) mapping of the static positions of SBF/MBF subunits revealed that 85% were organized into discrete clusters containing ∼8 copies regardless of cell size, while the number of clusters increased with growth. Stochastic simulations with a mathematical model based on co-localization of promoters in clusters recapitulated observed cluster behavior. A prediction of the model that SBF/MBF should exhibit both fast and slow dynamics was confirmed in PALM experiments on live cells. This spatio-temporal organization of the TFs that activate the G1/S regulon may help coordinate commitment to division.

scholarly journals G1/S transcription factors assemble in increasing numbers of discrete clusters through G1 phase

2020 ◽  
Vol 219 (9) ◽  
Author(s):  
Labe Black ◽  
Sylvain Tollis ◽  
Guo Fu ◽  
Jean-Bernard Fiche ◽  
Savanna Dorsey ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Super Resolution ◽  
Temporal Organization ◽  
G1 Phase ◽  
Biophysical Parameters ◽  
Large Program ◽  
Spatio Temporal ◽  
Target Promoters ◽  
Start Transition ◽  
Commitment To Cell Division

In budding yeast, the transcription factors SBF and MBF activate a large program of gene expression in late G1 phase that underlies commitment to cell division, termed Start. SBF/MBF are limiting with respect to target promoters in small G1 phase cells and accumulate as cells grow, raising the questions of how SBF/MBF are dynamically distributed across the G1/S regulon and how this impacts the Start transition. Super-resolution Photo-Activatable Localization Microscopy (PALM) mapping of the static positions of SBF/MBF subunits in fixed cells revealed each transcription factor was organized into discrete clusters containing approximately eight copies regardless of cell size and that the total number of clusters increased as cells grew through G1 phase. Stochastic modeling using reasonable biophysical parameters recapitulated growth-dependent SBF/MBF clustering and predicted TF dynamics that were confirmed in live cell PALM experiments. This spatio-temporal organization of SBF/MBF may help coordinate activation of G1/S regulon and the Start transition.

scholarly journals 3D Interferometric Lattice Light-Sheet Imaging

2020 ◽  
Author(s):  
Bin Cao ◽  
Guanshi Wang ◽  
Jieru Li ◽  
Alexandros Pertsinidis
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Light Sheet ◽  
Detection Sensitivity ◽  
Live Cells ◽  
Background Suppression ◽  
Axial Resolution ◽  
Volumetric Imaging ◽  
Spatio Temporal ◽  
And Function

Understanding cellular structure and function requires live-cell imaging with high spatio-temporal resolution and high detection sensitivity. Direct visualization of molecular processes using single-molecule/super-resolution techniques has thus been transformative. However, extracting the highest-resolution 4D information possible from weak and dynamic fluorescence signals in live cells remains challenging. For example, some of the highest spatial resolution methods, e.g. interferometric (4Pi) approaches1-6 can be slow, require high peak excitation intensities that accelerate photobleaching or suffer from increased out-of-focus background. Selective-plane illumination (SPIM)7-12 reduces background, but most implementations typically feature modest spatial, especially axial, resolution. Here we develop 3D interferometric lattice light-sheet (3D-iLLS) imaging, a technique that overcomes many of these limitations. 3D-iLLS provides, by virtue of SPIM, low light levels and photobleaching, while providing increased background suppression and significantly improved volumetric imaging/sectioning capabilities through 4Pi interferometry. We demonstrate 3D-iLLS with axial resolution and single-particle localization precision down to <100nm (FWHM) and <10nm (1σ) respectively. 3D-iLLS paves the way for a fuller elucidation of sub-cellular phenomena by enhanced 4D resolution and SNR live imaging.

scholarly journals FIFO ATP synthase responds to glycolysis inhibition by localization into the inner boundary membrane

2018 ◽  
Author(s):  
K. Zalyevskiy ◽  
F. Hager ◽  
C. P. Richter ◽  
K. Psathaki ◽  
T. Appelhans ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Significant Proportion ◽  
Tca Cycle ◽  
Longitudinal Axis ◽  
Temporal Organization ◽  
Live Cells ◽  
Mitochondrial Atp Synthase ◽  
Key Enzyme ◽  
Spatio Temporal ◽  
Boundary Membrane

ABSTRACTMitochondrial F1F0ATP synthase is the key enzyme to fuel the cell with essential ATP. Strong indications exist that the respiratory chain and the ATP synthase are physically separated within cristae. How static this organization is, is largely unknown. Here, we investigated the effect of substrate restriction on mitochondrial respiration and the spatio-temporal organization of ATP synthase. By superresolution microscopy, the localization and mobility of single labelled mitochondrial ATP synthase was determined in live cells. We found, that the ATP synthase under oxidative respiration displayed a clear localization and confined mobility in cristae. Trajectories of individual ATP synthase proteins show a perpendicular course to the longitudinal axis of the respective mitochondrion, exactly following the ultrastructure of cristae. When substrate for TCA cycle and respiration was limited, a significant proportion of ATP synthase localized from cristae to the inner boundary membrane, and only less mobile ATP synthase remained in cristae. These observations showing the plasticity of the spatio-temporal organisation of ATP synthase can explain why ATP synthase show interactions with proteins in distinct mitochondrial subcompartments such as inner boundary membrane, cristae junctions and cristae.

scholarly journals Nanoscale spatio-temporal diffusion modes measured by simultaneous confocal and STED imaging

2018 ◽  
Author(s):  
Falk Schneider ◽  
Dominic Waithe ◽  
Silvia Galiani ◽  
Jorge Bernadino de la Serna ◽  
Erdinc Sezgin ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Molecular Diffusion ◽  
Correlation Spectroscopy ◽  
Stimulated Emission ◽  
Temporal Organization ◽  
Live Cells ◽  
Apparent Diffusion Coefficients ◽  
Diffusion Dynamics ◽  
Spatio Temporal

AbstractThe diffusion dynamics in the cellular plasma membrane provides crucial insights into the molecular interactions, organization and bioactivity. Fluorescence correlation spectroscopy combined with super-resolution stimulated emission depletion nanoscopy (STED-FCS) measures such dynamics with high spatial and temporal resolution and reveals nanoscale diffusion characteristics by measuring the molecular diffusion in conventional confocal mode and super-resolved STED mode sequentially. However, to directly link the spatial and the temporal information, a method that simultaneously measures the diffusion in confocal and STED modes is needed. Here, to overcome this problem, we establish an advanced STED-FCS measurement method; line interleaved excitation scanning STED-FCS (LIESS-FCS) which discloses the molecular diffusion modes at different spatial positions with a single measurement. It relies on fast beam-scanning along a line with alternating laser illumination that yields, for each pixel, the apparent diffusion coefficients for two different observation spot sizes (conventional confocal and super-resolved STED). We demonstrate the potential of the LIESS-FCS approach with simulations and experiments on lipid diffusion in model and live cell plasma membranes. We also apply LIESS-FCS to investigate the spatio-temporal organization of GPI-anchored proteins in the plasma membrane of live cells which interestingly show multiple diffusion modes at different spatial positions.

scholarly journals CTCF-mediated Chromatin Structures Dictate the Spatio-temporal Propagation of Replication Foci

2019 ◽  
Author(s):  
Qian Peter Su ◽  
Ziqing Winston Zhao ◽  
Luming Meng ◽  
Miao Ding ◽  
Weiwei Zhang ◽  
...  
Keyword(s):  
Super Resolution ◽  
S Phase ◽  
Temporal Organization ◽  
Replication Origins ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Replication Foci ◽  
Spatio Temporal ◽  
Optical Reconstruction ◽  
Epigenetic Signatures

ABSTRACTMammalian DNA replication is initiated at numerous replication origins, which are clustered into thousands of replication domains (RDs) across the genome. However, it remains unclear whether the replication origins within each RD are activated stochastically. To understand how replication is regulated at the sub-RD level, we directly visualized the spatio-temporal organization, morphology, and in situ epigenetic signatures of individual replication foci (RFi) across S-phase using super-resolution stochastic optical reconstruction microscopy (STORM). Importantly, we revealed a hierarchical radial pattern of RFi propagation that reverses its directionality from early to late S-phase, and is diminished upon caffeine treatment or CTCF knockdown. Together with simulation and bioinformatic analyses, our findings point to a ‘CTCF-organized REplication Propagation’ (CoREP) model. The CoREP model suggests a non-random selection mechanism for replication activation mediated by CTCF at the sub-RD level, as well as the critical involvement of local chromatin environment in regulating replication in space and time.

scholarly journals Imaging of native transcription factors and histone phosphorylation at high resolution in live cells

2018 ◽  
Vol 217 (4) ◽  
pp. 1537-1552 ◽  
Author(s):  
Sascha Conic ◽  
Dominique Desplancq ◽  
Alexia Ferrand ◽  
Veronique Fischer ◽  
Vincent Heyer ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase Ii ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Super Resolution ◽  
Cell Types ◽  
Fluorescent Labeling ◽  
Live Cell ◽  
Biological Information ◽  
Live Cells

Fluorescent labeling of endogenous proteins for live-cell imaging without exogenous expression of tagged proteins or genetic manipulations has not been routinely possible. We describe a simple versatile antibody-based imaging approach (VANIMA) for the precise localization and tracking of endogenous nuclear factors. Our protocol can be implemented in every laboratory allowing the efficient and nonharmful delivery of organic dye-conjugated antibodies, or antibody fragments, into different metazoan cell types. Live-cell imaging permits following the labeled probes bound to their endogenous targets. By using conventional and super-resolution imaging we show dynamic changes in the distribution of several nuclear transcription factors (i.e., RNA polymerase II or TAF10), and specific phosphorylated histones (γH2AX), upon distinct biological stimuli at the nanometer scale. Hence, considering the large panel of available antibodies and the simplicity of their implementation, VANIMA can be used to uncover novel biological information based on the dynamic behavior of transcription factors or posttranslational modifications in the nucleus of single live cells.

scholarly journals Single-molecule Photoswitching and Localization

2011 ◽  
Vol 64 (5) ◽  
pp. 503 ◽  
Author(s):  
Sebastian van de Linde ◽  
Steve Wolter ◽  
Markus Sauer
Keyword(s):  
Single Molecule ◽  
Optical Resolution ◽  
Super Resolution ◽  
Image Plane ◽  
Localization Microscopy ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Resolution Imaging ◽  
Spatio Temporal ◽  
Optical Reconstruction ◽  
20 Nm

Within only a few years super-resolution fluorescence imaging based on single-molecule localization and image reconstruction has attracted considerable interest because it offers a comparatively simple way to achieve a substantially improved optical resolution down to ∼20 nm in the image plane. Since super-resolution imaging methods such as photoactivated localization microscopy, fluorescence photoactivation localization microscopy, stochastic optical reconstruction microscopy, and direct stochastic optical reconstruction microscopy rely critically on exact fitting of the centre of mass and the shape of the point-spread-function of isolated emitters unaffected by neighbouring fluorophores, controlled photoswitching or photoactivation of fluorophores is the key parameter for resolution improvement. This review will explain the principles and requirements of single-molecule based localization microscopy, and compare different super-resolution imaging concepts and highlight their strengths and limitations with respect to applications in fixed and living cells with high spatio-temporal resolution.

scholarly journals Enlightening G-protein-coupled receptors on the plasma membrane using super-resolution photoactivated localization microscopy

2013 ◽  
Vol 41 (1) ◽  
pp. 191-196 ◽  
Author(s):  
Marco Scarselli ◽  
Paolo Annibale ◽  
Claudio Gerace ◽  
Aleksandra Radenovic
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Super Resolution ◽  
Molecular Size ◽  
G Protein Coupled Receptors ◽  
Temporal Organization ◽  
Localization Microscopy ◽  
Photoactivated Localization Microscopy ◽  
Super Resolution Microscopy ◽  
G Protein Coupled

The possibility to visualize and image the arrangement of proteins within the cell at the molecular level has always been an attraction for scientists in biological research. In particular, for signalling molecules such as GPCRs (G-protein-coupled receptors), the existence of protein aggregates such as oligomers or clusters has been the topic of extensive debate. One of the reasons for this lively argument is that the molecular size is below the diffraction-limited resolution of the conventional microscopy, precluding the direct visualization of protein super-structures. On the other hand, new super-resolution microscopy techniques, such as the PALM (photoactivated localization microscopy), allow the limit of the resolution power of conventional optics to be broken and the localization of single molecules to be determined with a precision of 10–20 nm, close to their molecular size. The application of super-resolution microscopy to study the spatial and temporal organization of GPCRs has brought new insights into receptor arrangement on the plasma membrane. Furthermore, the use of this powerful microscopy technique as a quantitative tool opens up the possibility for investigating and quantifying the number of molecules in biological assemblies and determining the protein stoichiometry in signalling complexes.

scholarly journals Coupling chromatin structure and dynamics by live super-resolution imaging

2019 ◽  
Author(s):  
R. Barth ◽  
K. Bystricky ◽  
H. A. Shaban
Keyword(s):  
Chromatin Structure ◽  
Cell Function ◽  
Super Resolution ◽  
Temporal Correlation ◽  
Temporal Organization ◽  
Chromatin Dynamics ◽  
Structure And Dynamics ◽  
Resolution Imaging ◽  
Spatio Temporal ◽  
Chromatin Structure And Dynamics

AbstractChromatin conformation regulates gene expression and thus constant remodeling of chromatin structure is essential to guarantee proper cell function. To gain insight into the spatio-temporal organization of the genome, we employ high-density photo-activated localization microscopy and deep learning to obtain temporally resolved super-resolution images of chromatin in living cells. In combination with high-resolution dense motion reconstruction, we confirm the existence of elongated ~ 45 to 90 nm wide chromatin ‘blobs’. A computational chromatin model suggests that these blobs are dynamically associating chromatin fragments in close physical and genomic proximity and adopt TAD-like interactions in the time-average limit. Experimentally, we found that chromatin exhibits a spatio-temporal correlation over ~ 4 μm in space and tens of seconds in time, while chromatin dynamics are correlated over ~ 6 μm and last 40 s. Notably, chromatin structure and dynamics are closely related, which may constitute a mechanism to grant access to regions with high local chromatin concentration.

Spatio-Temporal LED Driving for Subjective Super-Resolution of Grayscale Images

2019 ◽  
pp. 1243
Author(s):  
Kojiro Matsushita ◽  
Toyotaro Tokimoto ◽  
Kengo Fujii ◽  
Hirotsugu Yamamoto
Keyword(s):  
Super Resolution ◽  
Spatio Temporal ◽  
Grayscale Images
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