Dataset Information

Measuring the regulation of keratin filament network dynamics.

ABSTRACT: The organization of the keratin intermediate filament cytoskeleton is closely linked to epithelial function. To study keratin network plasticity and its regulation at different levels, tools are needed to localize and measure local network dynamics. In this paper, we present image analysis methods designed to determine the speed and direction of keratin filament motion and to identify locations of keratin filament polymerization and depolymerization at subcellular resolution. Using these methods, we have analyzed time-lapse fluorescence recordings of fluorescent keratin 13 in human vulva carcinoma-derived A431 cells. The fluorescent keratins integrated into the endogenous keratin cytoskeleton, and thereby served as reliable markers of keratin dynamics. We found that increased times after seeding correlated with down-regulation of inward-directed keratin filament movement. Bulk flow analyses further revealed that keratin filament polymerization in the cell periphery and keratin depolymerization in the more central cytoplasm were both reduced. Treating these cells and other human keratinocyte-derived cells with EGF reversed all these processes within a few minutes, coinciding with increased keratin phosphorylation. These results highlight the value of the newly developed tools for identifying modulators of keratin filament network dynamics and characterizing their mode of action, which, in turn, contributes to understanding the close link between keratin filament network plasticity and epithelial physiology.

SUBMITTER: Moch M

PROVIDER: S-EPMC3696749 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Measuring the regulation of keratin filament network dynamics.

Moch Marcin M Herberich Gerlind G Aach Til T Leube Rudolf E RE Windoffer Reinhard R

Proceedings of the National Academy of Sciences of the United States of America 20130611 26

The organization of the keratin intermediate filament cytoskeleton is closely linked to epithelial function. To study keratin network plasticity and its regulation at different levels, tools are needed to localize and measure local network dynamics. In this paper, we present image analysis methods designed to determine the speed and direction of keratin filament motion and to identify locations of keratin filament polymerization and depolymerization at subcellular resolution. Using these methods ...[more]

PMID: 23757496

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Project description:Keratin intermediate filaments are dynamic cytoskeletal components that are responsible for tuning the mechanical properties of epithelial tissues. Although it is known that keratin filaments (KFs) are able to sense and respond to changes in the physicochemical properties of the local niche, a direct correlation of the dynamic three-dimensional network structure at the single filament level with the microenvironment has not been possible. Using conventional approaches, we find that keratin flow rates are dependent on extracellular matrix (ECM) composition but are unable to resolve KF network organization at the single filament level in relation to force patterns. We therefore developed a novel method that combines a machine learning-based image restoration technique and traction force microscopy to decipher the fine details of KF network properties in living cells grown on defined ECM patterns. Our approach utilizes Content-Aware Image Restoration (CARE) to enhance the temporal resolution of confocal fluorescence microscopy by at least five fold while preserving the spatial resolution required for accurate extraction of KF network structure at the single KF/KF bundle level. The restored images are used to segment the KF network, allowing numerical analyses of its local properties. We show that these tools can be used to study the impact of ECM composition and local mechanical perturbations on KF network properties and corresponding traction force patterns in size-controlled keratinocyte assemblies. We were thus able to detect increased curvature but not length of KFs on laminin-322 versus fibronectin. Photoablation of single cells in microprinted circular quadruplets revealed surprisingly little but still significant changes in KF segment length and curvature that were paralleled by an overall reduction in traction forces without affecting global network orientation in the modified cell groups irrespective of the ECM coating. Single cell analyses furthermore revealed differential responses to the photoablation that were less pronounced on laminin-332 than on fibronectin. The obtained results illustrate the feasibility of combining multiple techniques for multimodal monitoring and thereby provide, for the first time, a direct comparison between the changes in KF network organization at the single filament level and local force distribution in defined paradigms.

Dataset Information

Measuring the regulation of keratin filament network dynamics.

Publications

Measuring the regulation of keratin filament network dynamics.

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