scholarly journals A quantitative map of human Condensins provides new insights into mitotic chromosome architecture

2018 ◽  
Author(s):  
Nike Walther ◽  
M. Julius Hossain ◽  
Antonio Z. Politi ◽  
Birgit Koch ◽  
Moritz Kueblbeck ◽  
...  
Keyword(s):  
Mitotic Chromosome ◽  
Super Resolution ◽  
Maximum Size ◽  
Correlation Spectroscopy ◽  
Loop Model ◽  
Chromosome Architecture ◽  
Fluorescence Correlation ◽  
Chromatid Segregation ◽  
Early Anaphase ◽  
Super Resolution Microscopy

AbstractThe two Condensin complexes in human cells are essential for mitotic chromosome structure. We used homozygous genome editing to fluorescently tag Condensin I and II subunits and mapped their absolute abundance, spacing and dynamic localization during mitosis by fluorescence correlation spectroscopy-calibrated live cell imaging and super-resolution microscopy. While ∼35,000 Condensin II complexes are stably bound to chromosomes throughout mitosis, ∼195,000 Condensin I complexes dynamically bind in two steps, in prometaphase and early anaphase. The two Condensins rarely co-localize at the chromatid axis, where Condensin II is centrally confined but Condensin I reaches ∼50% of the chromatid diameter from its center. Based on our comprehensive quantitative data, we propose a three-step hierarchical loop model of mitotic chromosome compaction: Condensin II initially fixes loops of a maximum size of ∼450 kb at the chromatid axis whose size is then reduced by Condensin I binding to ∼90 kb in prometaphase and ∼70 kb in anaphase, achieving maximum chromosome compaction upon sister chromatid segregation.

scholarly journals A quantitative map of human Condensins provides new insights into mitotic chromosome architecture

2018 ◽  
Vol 217 (7) ◽  
pp. 2309-2328 ◽  
Author(s):  
Nike Walther ◽  
M. Julius Hossain ◽  
Antonio Z. Politi ◽  
Birgit Koch ◽  
Moritz Kueblbeck ◽  
...  
Keyword(s):  
Mitotic Chromosome ◽  
Maximum Size ◽  
Correlation Spectroscopy ◽  
Loop Model ◽  
Absolute Abundance ◽  
Superresolution Microscopy ◽  
Chromosome Architecture ◽  
Fluorescence Correlation ◽  
Chromatid Segregation ◽  
Early Anaphase

The two Condensin complexes in human cells are essential for mitotic chromosome structure. We used homozygous genome editing to fluorescently tag Condensin I and II subunits and mapped their absolute abundance, spacing, and dynamic localization during mitosis by fluorescence correlation spectroscopy (FSC)–calibrated live-cell imaging and superresolution microscopy. Although ∼35,000 Condensin II complexes are stably bound to chromosomes throughout mitosis, ∼195,000 Condensin I complexes dynamically bind in two steps: prometaphase and early anaphase. The two Condensins rarely colocalize at the chromatid axis, where Condensin II is centrally confined, but Condensin I reaches ∼50% of the chromatid diameter from its center. Based on our comprehensive quantitative data, we propose a three-step hierarchical loop model of mitotic chromosome compaction: Condensin II initially fixes loops of a maximum size of ∼450 kb at the chromatid axis, whose size is then reduced by Condensin I binding to ∼90 kb in prometaphase and ∼70 kb in anaphase, achieving maximum chromosome compaction upon sister chromatid segregation.

scholarly journals Single mRNP analysis by super-resolution microscopy and fluorescence correlation spectroscopy reveals that small mRNP granules represent mRNA singletons

2019 ◽  
Author(s):  
Àngels Mateu-Regué ◽  
Jan Christiansen ◽  
Frederik Otzen Bagger ◽  
Christian Hellriegel ◽  
Finn Cilius Nielsen
Keyword(s):  
Translational Control ◽  
Fluorescence Correlation Spectroscopy ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Super Resolution ◽  
Correlation Spectroscopy ◽  
Live Cells ◽  
Nuclear Pore ◽  
Fluorescence Correlation ◽  
Super Resolution Microscopy

SummarySmall cytoplasmic mRNP granules are implicated in mRNA transport, translational control and decay. Employing Super-resolution Microscopy and Fluorescence Correlation Spectroscopy, we analyzed the molecular composition and dynamics of single cytoplasmic YBX1_IMP1 mRNP granules in live cells. Granules appeared elongated and branched with patches of IMP1 and YBX1 distributed along mRNA, reflecting the attachment of the two RNA-binding proteins in cis. Particles form at the nuclear pore and are spatially segregated from translating ribosomes, so the mRNP is a repository for mRNAs awaiting translation. In agreement with the average number of mRNA-binding sites derived from CLIP analyses, individual mRNPs contain 5 to 15 molecules of YBX1 and IMP1 and a single poly(A) tail identified by PABPC1. Taken together, we conclude that small cytoplasmic mRNP granules are mRNA singletons, thus depicting the cellular transcriptome. Consequently, expression of functionally related mRNAs in RNA regulons is unlikely to result from coordinated assembly.

scholarly journals Characterization of Porous Materials by Fluorescence Correlation Spectroscopy Super-resolution Optical Fluctuation Imaging

ACS Nano ◽  
2015 ◽  
Vol 9 (9) ◽  
pp. 9158-9166 ◽  
Author(s):  
Lydia Kisley ◽  
Rachel Brunetti ◽  
Lawrence J. Tauzin ◽  
Bo Shuang ◽  
Xiyu Yi ◽  
...  
Keyword(s):  
Porous Materials ◽  
Fluorescence Correlation Spectroscopy ◽  
Super Resolution ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation

scholarly journals Diffusion and interaction dynamics of the cytosolic peroxisomal import receptor PEX5

2021 ◽  
Author(s):  
Silvia Galiani ◽  
Katharina Reglinski ◽  
Pablo Carravilla ◽  
Aurelien Barbotin ◽  
Iztok Urbančič ◽  
...  
Keyword(s):  
Molecular Interactions ◽  
Super Resolution ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation ◽  
Interaction Dynamics ◽  
Diffusion Dynamics ◽  
Interaction Partners ◽  
Target Binding ◽  
Import Receptor

Measuring diffusion dynamics in living cells is essential for the understanding of molecular interactions. While various techniques have been used to explore such characteristics in the plasma membrane, this is less developed for measurements inside the cytosol. An example of cytosolic action is the import of proteins into peroxisomes, via the peroxisomal import receptor PEX5. Here, we combined advanced microscopy and spectroscopy techniques such as fluorescence correlation spectroscopy (FCS) and super-resolution STED microscopy to present a detailed characterization of the diffusion and interaction dynamics of PEX5. Among other features, we disclose a slow diffusion of PEX5, independent of aggregation or target binding, but associated with cytosolic interaction partners via its N-terminal domain. This sheds new light on the functionality of the receptor in the cytosol. Besides specific insights, our study highlights the potential of using complementary microscopy tools to decipher molecular interactions in the cytosol via studying their diffusion dynamics.

scholarly journals Computationally-efficient spatiotemporal correlation analysis super-resolves anomalous diffusion

2020 ◽  
Author(s):  
Shawn Yoshida ◽  
William Schmid ◽  
Nam Vo ◽  
William Calabrase ◽  
Lydia Kisley
Keyword(s):  
Correlation Analysis ◽  
Anomalous Diffusion ◽  
Super Resolution ◽  
Correlation Spectroscopy ◽  
Brownian Diffusion ◽  
Computationally Efficient ◽  
Fluorescence Correlation ◽  
Diffusion Dynamics ◽  
Nanoscale Structure ◽  
User Friendly

AbstractAnomalous diffusion dynamics in confined nanoenvironments govern the macroscale properties and interactions of many biophysical and material systems. Currently, it is difficult to quantitatively link the nanoscale structure of porous media to anomalous diffusion within them. Fluorescence correlation spectroscopy super-resolution optical fluctuation imaging (fcsSOFI) has been shown to extract nanoscale structure and Brownian diffusion dynamics within gels, liquid crystals, and polymers, but has limitations which hinder its wider application to more diverse, biophysically-relevant datasets. Here, we parallelize the least-squares curve fitting step on a GPU improving computation times by up to a factor of 40, implement anomalous diffusion and two-component Brownian diffusion models, and make fcsSOFI more accessible by packaging it in a user-friendly GUI. We apply fcsSOFI to simulations of the protein fibrinogen diffusing in polyacrylamide of varying matrix densities and super-resolve locations where slower, anomalous diffusion occurs within smaller, confined pores. The improvements to fcsSOFI in speed, scope, and usability will allow for the wider adoption of super-resolution correlation analysis to diverse research topics.

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