De novo formation of cytokeratin filament networks originates from the cell cortex in A-431 cells
Windoffer R, Leube RE, 2001
Of the three major cytoskeletal filament systems, the intermediate filaments are the least understood. Since they differ fundamentally from the actin- and microtubule-based networks by their lack of polarity, it has remained a mystery how and where these principally endless filaments are formed. Using a recently established epithelial cell system in which fluorescently labeled intermediate filaments of the cytokeratin type can be monitored in living cells, we address these issues.
By multidimensional time-lapse fluorescence microscopy, we examine de novo intermediate filament network formation from non-filamentous material at the end of mitosis and show that it mirrors disassembly. It is demonstrated that filament formation is initiated from the cell cortex without focal preference after cytokinesis. Furthermore, it is shown that this process is dependent on energy, on the integrity of the actin filament network and the microtubule system, and that it can be inhibited by the tyrosine phosphatase inhibitor pervanadate. Based on these observations, a two-step working model is proposed involving (1) interactions within the planar cortical layer acting as an organizing center forming a two-dimensional network and (2) subsequent radial dynamics facilitating the formation of a mature three-dimensional network.
Animated 3-D reconstruction from a stack of fluorescence images of human CK13-EGFP chimera HK13-1 in stably transfected AK13-1 cells during interphase. Note the typical CKF organization with thick perinuclear cables extending toward the cell periphery to desmosomal contact sites and the relative scarcity of filament bundles in cortical domains of the free cell edge.
Animated 3-D reconstruction from a stack of fluorescence images of human CK13-EGFP chimera HK13-1 in stably transfected AK13-1 cells during metaphase. No filaments are seen but instead strong, diffuse fluorescence is detectable throughout the cytoplasm, and aggregated material is seen in the form of variably shaped granules and short rods predominantly in the cortical region.
Fluorescence microscopy showing stages of CKF formation at the end of mitosis in A-431 clone AK13-1 stably expressing human CK13-EGFP chimera HK13-1. At time point 0 min the cell has entered cytokinesis and formation of the cleavage furrow is in progress. Granules and rodlets are seen in the cytoplasm and are enriched in the cell cortex at that time. Some cytoplasmic rodlets further elongate (2.5 min) but disappear later (22.5 min). Rebuilding of a typical CKF network originates from the cell periphery and proceeds toward the cell center. Note that diffuse cytoplasmic fluorescence decreases during filament formation. Simultaneously, cells flatten and re-establish extensive contacts with neighboring cells.
Using confocal scanning laser microscopy images were obtained from human vulvar carcinoma-derived A-431 subclone AK13-1 stably expressing fluorescent CKFs containing human CK13-EGFP chimera HK13-1. The movie shows five focal planes from bottom to top. At the beginning of the recording (early prophase) partial filament breakdown has already taken place in central cytoplasmic regions and increased diffuse cytoplasmic staining can be seen. At that time point the nucleus is still intact. Filament disassembly continues from the center of the cell towards the periphery, where the originally fine network concentrates in more solid structures and disintegrates into granules. Note, that at the end of the sequence CKFs are completely absent. Instead, significant diffuse cytoplasmic staining and granules, most of which are located in the cell periphery, are detected in the rounded cells. At some intermediate time points rodlets arise in the cytoplasm (e.g., level 2, 7.5 min) but disappear later.
Time-lapse fluorescence microscopy of dividing AK13-1 cells depicting the distribution of human CK13-EGFP chimera HK13-1 in the presence of 0.05% [w/v] sodium azide and 50 mM deoxyglucose. The movie shows only one of several confocal image planes. Note the differences to the control movie 5.
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Time-lapse fluorescence microscopy of dividing AK13-1 cells depicting the distribution of human CK13-EGFP chimera HK13-1 in the presence of nocodazole (1 µM) to disrupt microtubules.The movie shows only one of several confocal image planes.
Download: .mp4 / .wmv
