Fluorescence microscopic imaging and image analysis of the cytoskeleton

Herberich, Gerlind; Würflinger, Thomas; Sechi, Antonio; Windoffer, Reinhard; Leube, Rudolf E.; Aach, Til

doi:10.1109/acssc.2010.5757755

Cited by 8 publications

(6 citation statements)

References 19 publications

(20 reference statements)

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“…First, a basic Gaussian filter was used for noise reduction. An optimally oriented flux filter was then applied [ 32 ]. It enhances curvilinear structures, notably filaments, in the image thereby further suppressing noise.…”

Section: Methodsmentioning

confidence: 99%

Effects of Plectin Depletion on Keratin Network Dynamics and Organization

et al. 2016

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The keratin intermediate filament cytoskeleton protects epithelial cells against various types of stress and is involved in fundamental cellular processes such as signaling, differentiation and organelle trafficking. These functions rely on the cell type-specific arrangement and plasticity of the keratin system. It has been suggested that these properties are regulated by a complex cycle of assembly and disassembly. The exact mechanisms responsible for the underlying molecular processes, however, have not been clarified. Accumulating evidence implicates the cytolinker plectin in various aspects of the keratin cycle, i.e., by acting as a stabilizing anchor at hemidesmosomal adhesion sites and the nucleus, by affecting keratin bundling and branching and by linkage of keratins to actin filament and microtubule dynamics. In the present study we tested these hypotheses. To this end, plectin was downregulated by shRNA in vulvar carcinoma-derived A431 cells. As expected, integrin β4- and BPAG-1-positive hemidesmosomal structures were strongly reduced and cytosolic actin stress fibers were increased. In addition, integrins α3 and β1 were reduced. The experiments furthermore showed that loss of plectin led to a reduction in keratin filament branch length but did not alter overall mechanical properties as assessed by indentation analyses using atomic force microscopy and by displacement analyses of cytoplasmic superparamagnetic beads using magnetic tweezers. An increase in keratin movement was observed in plectin-depleted cells as was the case in control cells lacking hemidesmosome-like structures. Yet, keratin turnover was not significantly affected. We conclude that plectin alone is not needed for keratin assembly and disassembly and that other mechanisms exist to guarantee proper keratin cycling under steady state conditions in cultured single cells.

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Section: Methodsmentioning

confidence: 99%

Effects of Plectin Depletion on Keratin Network Dynamics and Organization

et al. 2016

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“…Image corruption by noise leads to a granular, discontinuous appearance of the curvilinear KFs, preventing reliable measurement of KF motion. To reduce noise without blurring filaments, curvelet transform-based denoising was used (15,38). Curvelets are ridge-like basis functions localized in space, frequency, and orientation.…”

Section: Methodsmentioning

confidence: 99%

“…Motion analysis. Local KF motion was measured using a deformable registration algorithm (15,40) based on free-form deformations. A deformation grid was superimposed on the image.…”

Section: Methodsmentioning

confidence: 99%

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Measuring the regulation of keratin filament network dynamics

Moch¹,

Herberich

Aach

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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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.keratin filament turnover | keratin filament assembly | keratin filament disassembly | growth factor | live cell imaging M echanical properties of cells and tissues are determined by the cytoskeleton, which consists of complex filament networks. Among these networks, the intermediate filament-based network of epithelial cells is the most diverse. More than 50 keratin intermediate filament (KF) polypeptides are expressed in specific combinations in different epithelial cell types. Their importance for epithelial homeostasis is underscored by functions that go beyond cell mechanics as a basis of migration and tissue formation, and contribute to organelle trafficking, protein translation, signaling, immune response, and cell survival (1-4).The continuous inward-directed movement of KFs and the repetitive cycles of KF assembly in the cell periphery and KF disassembly in the more central cytoplasm are striking features of the keratin network in cultured cells (2, 3, 5). These features were determined from time-lapse recordings of fluorescent proteinlabeled keratins, fluorescence recovery after photobleaching (FRAP) experiments, and photoactivation analyses (6, 7). It has been suggested that keratin cycling is needed to establish cell type-specific network architecture and that keratin cycling supports network remodeling while maintaining network integrity, for example, during wound healing and differentiation (2, 5). The regulatory factors affecting keratin cycling and the parts of the cycle subject to regulation ar...

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“…Various automated and semi-automated computational tools have been developed to recognize filamentous objects in medical imaging [ 12 , 13 , 14 , 15 , 16 ]. Locating ridges in a 2D image is a well-studied problem in mathematics and computer vision [ 17 ]; however, the 3D equivalent is a newer area of research [ 18 ].…”

Section: Introductionmentioning

confidence: 99%

Tracing Actin Filament Bundles in Three-Dimensional Electron Tomography Density Maps of Hair Cell Stereocilia

et al. 2018

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Cryo-electron tomography (cryo-ET) is a powerful method of visualizing the three-dimensional organization of supramolecular complexes, such as the cytoskeleton, in their native cell and tissue contexts. Due to its minimal electron dose and reconstruction artifacts arising from the missing wedge during data collection, cryo-ET typically results in noisy density maps that display anisotropic XY versus Z resolution. Molecular crowding further exacerbates the challenge of automatically detecting supramolecular complexes, such as the actin bundle in hair cell stereocilia. Stereocilia are pivotal to the mechanoelectrical transduction process in inner ear sensory epithelial hair cells. Given the complexity and dense arrangement of actin bundles, traditional approaches to filament detection and tracing have failed in these cases. In this study, we introduce BundleTrac, an effective method to trace hundreds of filaments in a bundle. A comparison between BundleTrac and manually tracing the actin filaments in a stereocilium showed that BundleTrac accurately built 326 of 330 filaments (98.8%), with an overall cross-distance of 1.3 voxels for the 330 filaments. BundleTrac is an effective semi-automatic modeling approach in which a seed point is provided for each filament and the rest of the filament is computationally identified. We also demonstrate the potential of a denoising method that uses a polynomial regression to address the resolution and high-noise anisotropic environment of the density map.

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Fluorescence microscopic imaging and image analysis of the cytoskeleton

Cited by 8 publications

References 19 publications

Effects of Plectin Depletion on Keratin Network Dynamics and Organization

Effects of Plectin Depletion on Keratin Network Dynamics and Organization

Measuring the regulation of keratin filament network dynamics

Tracing Actin Filament Bundles in Three-Dimensional Electron Tomography Density Maps of Hair Cell Stereocilia

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