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...
Cell stability and motility depends on a complex dynamic cytoplasmic scaffolding called the cytoskeleton. It is composed of actin filaments, intermediate filaments and microtubules, and interacts with neighbouring cells and the extracellular matrix via specialized adhesion sites -multimolecular complexes responsible for the transmission of mechanical force and regulatory signals. The dynamic behaviour of these subcellular structures in living cells can be analysed by fluorescence microscopy yielding series of 2D or 3D images. Towards a quantitative analysis, we present methods for the segmentation and motion estimation of cytoskeletal filaments as well as for the tracking of adhesion sites, allowing the quantification of cytoskeletal dynamics under different conditions.
In this paper, we propose and compare different methods for the 3D segmentation of keratin intermediate filaments (KFs) in images acquired using confocal laser scanning microscopy (CLSM). KFs are elastic cables forming a complex scaffolding within epithelial cells. They are involved in many basic cell functions. To understand the mechanisms of filament formation and network organisation under physiological and pathological conditions, quantitative measurements of dynamic network alterations are essential. Segmenting KFs is a key component for analyzing their dynamic and biomechanical properties. KFs were labeled with fluorescent keratins to allow high resolution imaging of network dynamics in native cells. Our segmentation methods follow the principle of ridge enhancement filtering and subsequent centerline extraction. The evaluation of the methods is two-fold: (i) We develop synthetic data that exhibit the characteristics of real CLSM data to evaluate the precision of the different methods in terms of centerline localisation and (ii) we perform a connected component analysis on the segmentation results of real KF data to assess whether the connectivity of highly complex networks is being preserved by the segmentation. Our evaluation shows that in the presence of strong noise and despite the highly anisotropic spatial resolution of CLSM images the proposed method is able to accurately localize the centerlines of the KFs and to preserve the KF networks' connectivity. Taken together this is a strong indicator that also the network topology is being preserved.
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