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Detection of cytokeratin dynamics by time-lapse fluorescence microscopy in living cells

Windoffer R, Leube RE, 1999

To monitor the desmosome-anchored cytokeratin network in living cells fusion protein HK13-EGFP consisting of human cytokeratin 13 and the enhanced green fluorescent protein was stably expressed in vulvar carcinoma-derived A-431 cells. It is shown for A-431 subclone AK13-1 that HK13-EGFP emits strong fluorescence in fixed and living cells, being part of an extended cytoplasmic intermediate filament network that is indistinguishable from that of parent A-431 cells.


Biochemical, immunological and ultrastructural analyses demonstrate that HK13-EGFP behaves identically to the endogenous cytokeratin 13 and is therefore a reliable in vivo tag for this polypeptide and the structures formed by it. Time-lapse fluorescence microscopy reveals that the cytokeratin 13-containing network is in constant motion, resulting in continuous restructuring occurring in single and migratory cells, as well as in desmosome-anchored cells. Two major types of movement are distinguished: (i) oscillations of mostly long filaments, and (ii) an inward-directed flow of fluorescence originating as diffuse material at the cell periphery and moving in the form of dots and thin filaments toward the deeper cytoplasm where it coalesces with other filaments and filament bundles.


Both movements are energy dependent and can be inhibited by nocodazole, but not by cytochalasin D. Finally, disassembly and reformation of cytokeratin filament networks are documented in dividing cells revealing distinct and rapidly occurring stages of cytokeratin organisation and distribution.

Caption of the corresponding Figure 5:


Three representative fluorescence micrographs taken from a time-lapse recording showing alterations in distribution of HK13-EGFP in a living AK13-1 cell. The movie covers 46 minutes and is composed of pictures taken every 60 seconds showing that the HK13-EGFP- containing filaments are highly dynamic structures with only few present during the entire observation period (arrowheads) but most disappearing (small arrows denoting filament gone by 23 minutes; large arrows delineating filament gone after 46 minutes). Two types of movement are discernible in the movie but can not be visualised in static micrographs: (i) slow filament undulations predominantly occurring at the central parts of the cell, and (ii) inward-directed flow of fluorescence from the cell periphery toward the inner cytoplasm.


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Caption of the corresponding Figure 6:


Images taken from a series of fluorescence recordings of a single AK13-1 cell illustrating inward movement of HK13- EGFP from the cell periphery toward inner circularly-arranged filament bundles. Images are shown inversely to optimize views. To measure the inward-directed peripheral movement, fluorescent ‘dots’ were traced through several frames. Circles mark four such dots whose position was followed. The path taken is shown in the first enlarged and duplicated micrograph by black lines with measuring points indicated by white dots. The complexity and dynamics of the movements can only be appreciated from the movie that was made of pictures taken at 120 second intervals and shows a 240 minutes observation period encompassing also the frames depicted in this Fig. 6.


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Caption of the corresponding Figure 7:


Photomicrographs taken from movie 3 depicting HK13-EGFP fluorescence in several AK13-1 cells before and during treatment with 100 nM nocodazole. The movie consists of pictures taken every 120 seconds over a time period of 115 minutes (55 minutes before and 60 minutes after addition of the drug). The wavy movement of CKFs and the inward-directed fluctuations of fluorescence are apparent before treatment (compare e.g. regions marked by arrows in A and B). Upon addition of the drug, cells briefly flatten and almost all movement stops abruptly (compare e.g. arrows in C and D). Note again that only the movie allows full appreciation of this effect.


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Caption of corresponding Figure 8:


HK13-EGFP fluorescence of two AK13-1 cells before and during cytochalasin D incubation (0.1 m M). Images are from movie 4 (pictures taken every 60 seconds for 115 minutes) which is available at: The cells show an extended cytoplasmic filament network at the beginning of the recording period ( - 55 minutes). Cells were first observed for 55 minutes in medium without cytochalasin D showing the characteristic inward flow of fluorescence from the cell periphery and the wavy movement of inner filaments (see movie). After addition of cytochalasin D to the culture medium cells contract and CKFs condense around the nucleus (arrowheads in image taken at 41 minutes). Despite this condensation the wavy movement and inward-directed movement are still maintained. The latter is illustrated in images taken at 41 minutes and 51 minutes (see also insets at two times higher magnification) by arrow that marks a fluorescent dot that moved appr. 1.6 µm toward the cell centre during this time (bracket), i.e. appr. 266 nm/minute roughly corresponding to the mean speed of inward-movement determined from untreated cells. Note that the complexity of dynamic movements can only be seen in the movie in which presentation was optimised to follow the comparatively weak fluorescence in the cell periphery.


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Caption of the corresponding Figure 10:


HK13-EGFP fluorescence microscopy during mitotic stages of a single AK13-1 cell. The whole sequence of pictures taken at 5 minute intervals (recording time 280 minutes) is available as movie 5. Time point 0 min shows a cell that has already rounded up and whose CKFs have mostly aggregated around the nucleus. Late at prophase (50 min) a similar pattern is still prevalent, but aggregates and remaining filaments are disassembled abruptly within less than 10 minutes (60 min). Note that at this time point only diffuse staining and speckles/granular spheroidal aggregates are seen. This fluorescence pattern is maintained during the next hour with some enrichment at opposite cell poles (120 min). After cytokinesis (130 min) a submembraneous enrichment of diffuse staining is visible which further accumulates symmetrically to the plane of division at 140 min. Filaments emanating from the strongly fluorescent material are seen 10 minutes later (150 min). A CK network with perinuclearly located CKFs are seen 125 minutes later (275 min).


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Caption of the corresponding Figure 11:


Fluorescence microscopy depicting HK13-EGFP in AK13-1 cells during mitosis. Cells were synchronized by two successive thymidine blocks and time-lapse fluorescence microscopy was performed 10 hours after release of the second block. The four micrographs are taken from movie 6 that was recorded at 1 minute intervals (recording time 72 min). Note the changes in fluorescence patterns during successive stages of mitosis and compare them to Fig. 10. Toward the end of the observation period the focus was adjusted to a plane where the filamentous network in neighbouring cells and of the flattening mitotic cells could be resolved.


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