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Keratin are among the most abundant proteins in epithelial cells. Functions of the keratin network in cells are shaped by their dynamical organization. Using a collection of experimentally-driven mathematical models, different hypotheses for the turnover and transport of the keratin material in epithelial cells are tested. The interplay between turnover and transport and their effects on the keratin organization in cells are hence investigated by combining mathematical modeling and experimental data. Amongst the collection of mathematical models considered, a best model strongly supported by experimental data is identified. Fundamental to this approach is the fact that optimal parameter values associated with the best fit for each model are established. The best candidate among the best fits is characterized by the disassembly of the assembled keratin material in the perinuclear region and an active transport of the assembled keratin. Our study shows that an active transport of the assembled keratin is required to explain the experimentally observed keratin organization.

The human uterine epithelium is characterised by remarkable plasticity with cyclic changes in differentiation that are controlled by ovarian steroid hormones to optimise conditions for embryo implantation. To understand whether and how cell-cell adhesion is affected, the localisation of junction proteins was studied throughout the menstrual cycle. Expression patterns were examined by immunofluorescence in 36 human endometrial specimens of different cycle stages. Antibodies against the desmosomal proteins desmoplakin 1/2 (Dp 1/2) and desmoglein 2 (Dsg 2), the adherens junction proteins E-cadherin and β-catenin and also the common junctional linker protein plakoglobin showed a strong subapical staining during the proliferative phase until the early luteal phase (day 20). In the mid- to late luteal phase, however, these junctional proteins redistributed over the entire lateral plasma membranes. In contrast, tight junction proteins (ZO-1, claudin 4) remained at their characteristic subapical position throughout the menstrual cycle. mRNA levels of Dp 1/2, E-cadherin and ZO-1 obtained by real time RT-PCR were not significantly changed during the menstrual cycle. The observed redistribution of desmosomes and adherens junctions coincides with the onset of the so called implantation window of human endometrium. We propose that this change is controlled by ovarian steroids and prepares the endometrium for successful trophoblast invasion.

Desmosomes are prominent cell adhesion structures that are major stabilizing elements, together with the attached cytoskeletal intermediate filament network, of the cytokeratin type in epithelial tissues. To examine desmosome dynamics in tightly coupled cells and in situations of decreased adhesion, fluorescent desmosomal cadherin desmocollin 2a (Dsc2a) chimeras were stably expressed in human hepatocellular carcinoma-derived PLC cells (clone PDc-13) and in Madin-Darby canine kidney cells (clone MDc-2) for the continuous monitoring of desmosomes in living cells. The hybrid polypeptides integrated specifically and without disturbance into normal-appearing desmosomes that occurred in association with typical cytokeratin filament bundles. Tracking of labeled adhesion sites throughout the cell cycle by time-lapse fluorescence microscopy revealed that they were immobile and that they maintained their structural integrity for long periods of time. Time-space diagrams further showed that desmosomal positioning was tightly controlled, even during pronounced cell shape changes, although the desmosomal arrays extended and contracted, suggesting that they were interconnected by a flexible system with intrinsic elasticity. Double-fluorescence microscopy detecting Dsc2a chimeras together with fluorescent cytokeratin 18 chimeras revealed the association and synchronous movement of labeled desmosomes and fluorescent cytokeratin filaments. Only a minor destabilization of desmosomes was observed during mitosis, demonstrated by increased diffuse plasma membrane fluorescence and the fusion of desmosomes into larger structures. Desmosomes did not disappear completely at any time in any cell, and residual cytokeratin filaments remained in association with adhesion sites throughout cell division. On the other hand, a rapid loss of desmosomes was observed upon calcium depletion, with irreversible uptake of some desmosomal particles. Simultaneously, diffusely distributed desmosomal cadherins were detected in the plasma membrane that retained the competence to nucleate the reformation of desmosomes after the cells were returned to a standard calcium-containing medium. To examine the molecular stability of desmosomes, exchange rates of fluorescent chimeras were determined by fluorescence recovery after photobleaching, thereby identifying considerable Dsc2a turnover with different rates of fluorescence recovery for PDc-13 cells (36+/-17% recovery after 30 minutes) and MDc-2 cells (60+/-20% recovery after 30 minutes). Taken together, our observations suggest that desmosomes are pliable structures capable of fine adjustment to functional demands despite their overall structural stability and relative immobility.

We have previously described vulva carcinoma-derived A-431 subclone AK13-1, which stably expresses fluorescently labeled cytokeratin filaments (CKFs). Time-lapse fluorescence microscopy of these cells permits the continuous monitoring of the dynamics of the CKF cytoskeleton in vivo. To study mechanisms and principles of CKF disassembly as it occurs, e.g., during mitosis and liver disease, we have treated cells with the phosphatase inhibitor okadaic acid (OA), which induces complete CKF network breakdown within 3-5 h without significantly affecting the organization of the actin- and tubulin-based cytofilaments. In time-lapse movies, we find that the network breakdown starts at the cell periphery and proceeds toward the cell center, where residual filaments become compacted into a prominent perinuclear ring. The progressing disassembly is paralleled by an increase of diffuse fluorescence throughout the cytoplasm and the appearance of non-filamentous spheroidal aggregates. They are formed in the filament-free cell periphery from non-filamentous precursors and can sometimes be detected in the proximity of desmosomes. Other aggregates are either found in close apposition to CKFs or are generated directly from the compacted perinuclear material. Primary granules later fuse, thereby producing structures of considerable size. We show that CKF network breakdown and granule formation rely on metabolic energy and that the continued presence of OA is needed for its completion. We conclude that phosphorylation/dephosphorylation is an important mechanism regulating CKF network dynamics in vivo with far-reaching implications for the understanding of epithelial plasticity and pathology.

In order to study the dynamics of gap junctions in living cells, a cDNA was expressed in hepatocellular carcinoma-derived PLC cells coding for chimerical polypeptide Cx.EGFP-1, which consists of rat connexin32 and enhanced green fluorescent protein (EGFP). Cx.EGFP-1 was integrated into gap junctions, and the emitted epifluorescence reliably reported the distribution of the chimera. Therefore, stably transfected PLC clone PCx-9 was used to examine the dynamic behavior of gap junctions by time-lapse fluorescence microscopy. The pleomorphic fluorescent junctional plaques were highly motile within the plasma membrane. They often fused with each other or segregated into smaller patches, and fluctuation of fluorescence was detected within individual gap junctions. Furthermore, the uptake of junctional fragments into the cytoplasm of live cells was documented as originating from dynamic invaginations that form long tubulovesicular structures that pinch off. Endocytosis and subsequent lysosomal degradation, however, appeared to contribute only a little to the rapid gap junction turnover (determined half-life of 3.3 h for Cx.EGFP-1), since most cytoplasmic Cx.EGFP-1 fluorescence did not colocalize with the endocytosed fluid phase marker horseradish peroxidase or the receptor-specific endocytotic ligand transferrin and since it was distinct from lysosomes. Disassembly of gap junctions was monitored in the presence of the translation-inhibitor cycloheximide and showed increased endocytosis and continuous reduction of junctional plaques. Highly motile cytoplasmic microvesicles, which were detectable as multiple, weakly fluorescent puncta in all movies, are proposed to contribute significantly to gap junction morphogenesis by the transport of small subunits between biosynthetic, degradative, and recycling compartments.

Keratins are wide used markers for malignant epithelial cells, but their primary function is stabilization of epithelial tissues. Nevertheless, during metastasis, epithelia cells change from sessile cells into migrating cells. Consequently the stable keratin filament network connected to desmosomes has to undergo significant alterations which are required in a moving cell. Here we describe the involvement of keratins in the migration processes, which is typical for malignant cells. We want to answer the question how an altered keratin network of a migrating cell is built up, where this process takes place and how it is regulated. Using time lapse video microscopy of cells expressing fluorescently labelled keratins, we could show the formation of the new keratin network in the emerging lamellipodia of migrating cells. Close to the leading edge of lamellipodia, in direct proximity to focal contacts, the birth of keratin filament precursors (KFPs) could be observed as newly appearing fluorescent granules that subsequently elongate. During this elongation period, the KFPs moved retrograde along actin fibers and merged with the existing keratin network which was located further backwards in the lamellipodium. To further examine the origin of keratin protein forming KFPs, we fused photoactivable GFP (paGFP) to keratin. By examination of living cells expressing this protein, we could follow the dissociation of soluble keratin-paGFP from existing perinuclear filaments and the subsequent incorporation of keratin-paGFP in newly forming GFPs. We propose that these processes are part of a continuous turnover cycle of the keratin network, which enables the fast organization of the keratin network in the lamellipodia of migrating epithelial cells. [ABSTRACT FROM AUTHOR]