The keratin–desmosome scaffold: pivotal role of desmosomes for keratin network morphogenesis

Desmosome-anchored keratin intermediate filaments (KFs) are essential for epithelial coherence. Yet, desmosomal KF attachment and network organization are still unexplored in vivo. We, therefore, monitored KF network morphogenesis in fluorescent keratin 8 knock-in murine embryos revealing keratin enrichment at newly formed desmosomes followed by KF formation, KF elongation and KF fusion. To examine details of this process and its coupling to desmosome formation, we studied fluorescent keratin and desmosomal protein reporter dynamics in the periphery of expanding HaCaT keratinocyte colonies. Less than 3 min after the start of desmosomal proteins clustering non-filamentous keratin enriched at these sites followed by KF formation and elongation. Subsequently, desmosome-anchored KFs merged into stable bundles generating a rim-and-spokes system consisting of subcortical KFs connecting desmosomes to each other and radial KFs connecting desmosomes to the cytoplasmic KF network. We conclude that desmosomes are organizing centers for the KF cytoskeleton with a hitherto unknown nucleation capacity. Electronic supplementary material The online version of this article (10.1007/s00018-019-03198-y) contains supplementary material, which is available to authorized users.


Supplementary Figures
The keratin-desmosome scaffold becomes increasingly complex over time in HaCaT keratinocytes. Double immunofluorescence microscopy was performed in HaCaT cells 3, 5 and 7 days after seeding detecting desmosomes by anti-DspI and keratins by anti-keratin staining. The images show maximum intensity projections of multiple planes at low magnification in the top 3 image rows and of the boxed areas at higher magnification in the fourth row. X-z orthogonal views along the broken lines marked in the third row are presented at the bottom. Note the predominance of radial desmosome-attached KFs at left and the increasing number of interdesmosomal KFs in the middle and at right, which are difficult to delineate at increasing cell density because cells start to grow on top of each other forming an incomplete double layer.

Fig. S2
Plakophilin isoforms are differentially distributed in desmosomes of HaCaT keratinocytes. The double immunofluorescence micrographs show the different Pkp isoforms in relation to DspI in expanding HaCaT colonies 3 days after seeding. Note that Pkp3 labels the most peripheral DspI-positive desmosomes and is replaced by Pkp2 further inward, while Pkp1 cannot be detected at this early time point after seeding. Pkp1 fluorescence is only noted at later time points in suprabasal HaCaT cells (bottom right; Pkp1 control at day 6).

Fig. S3
Suprabasal cells are connected to cells below and to neighboring cells by a highly-ordered keratindesmosome-scaffold. a-b The fluorescence images show HK5-EYFP/Dsc2-mCerulean and K14-mCerulean/Dsg2-mCherry distribution patterns in confluent, formaldehyde-fixed HaCaT keratinocytes at day 6 after seeding. Note that the transfected cells are completely surrounded by non-transfected cells (nuclei stained with DAPI). The images depict thick keratin filament bundles connecting highly-ordered rows of desmosomes.

Fig. S4
Desmosomal-cadherins show higher turnover of the rapidly exchangeable pool than desmoplakin. The data correspond to those shown in Fig. 6b and show FRAP at 3 min intervals (n=30 cells except for FRAP measurements of DspI-EGFP (n=31)). Curves were fitted to data in SigmaPlot (equation: exponential rise to maximum with 2 parameters). Data points: mean. Error bars: standard error.

Movie Legends
Movie 1 Keratin filaments nucleate close to the cortical interdesmosomal network in murine blastocysts. The overview at left shows an animated maximum intensity projection of the fluorescence recorded in the trophectoderm of a late murine Krt8-YFP knock-in blastocyst (see also corresponding Fig. 1c). The time-lapse fluorescence images at right were recorded at the cell border of two trophectoderm cells of the same blastocyst every 62.5 s (see also corresponding Fig. 1d). The top reveals the appearance of growing keratin particles from the interdesmosomal subcortical keratin system. These motile and highly flexible particles fuse and enlarge before merging with the cytoplasmic KF network (arrowheads delineate a few selected examples; contrast was enhanced per frame to compensate for bleaching). The lower plane shows the same region at enhanced contrast settings to unravel further details in the selected focal planes.

Movie 2
The 3D animation of a z-stack recording depicts the fluorescence of keratin and desmosome reporters in a confluent HaCaT cell culture. The images (maximum intensity projection) were obtained from mixed HaCaT cell cultures containing cells producing HK5-EYFP (clone B10) delineating KFs and Dsc2-mCerulean delineating desmosomes (for details see legend to corresponding Fig. 2b). The animation first shows rotating views of the keratin-desmosome fluorescence, then rotating views of the desmosome fluorescence by itself, and then top-to-bottom views of single focal planes of double keratin-desmosome fluorescence.
Movie 3 Scroll through confocal sections reveals organizational details of the keratin-desmosome scaffold in a confluent HaCaT cell culture. The images were obtained from HaCaT cells producing HK5-EYFP delineating KFs and Dsc2-mCerulean delineating desmosomes (for details see legend to corresponding Fig. 2b). Note the presence of desmosome-anchored interdesmosomal and radial KFs in different focal planes.
Movie 4 Time-lapse image series depicts the coordinated dynamics of HK5-YFP and Dsc2-mCerulean in a confluent HaCaT culture. The images (maximum intensity projections) were recorded every 55 s in HaCaT cells producing HK5-EYFP delineating KFs and Dsc2-mCerulean delineating desmosomes (for details see legend to corresponding Fig. 2b). Note the coordinated movement of desmosomes with attached subcortical and radial KFs.

Movie 5
The time-lapse image series depicts the coordinated dynamics of keratin 14-mCerulean and DspI-mApple in a confluent HaCaT cell culture. The images (maximum intensity projections; 45 s recording intervals) were obtained from HaCaT cells producing Keratin 14-mCerulean and DspI-mApple delineating KFs and desmosomes (for details see legend to corresponding Fig. 2c). Note the coordinated movement of desmosomes with attached subcortical and radial KFs. Movie 9 Subcortical, interdesmosomal keratin filaments merge into bundles. The time-lapse series (maximum intensity projections; 60 s recording intervals) shows Keratin 14-mCerulean and Dsg2-mCherry fluorescence at the cell border in a HaCaT cell colony 3 days after seeding (see also corresponding Fig. 5a). Note the fusion of color-coded interdesmosomal KFs into KF bundles with tightly-spaced Dsg2-mCherry-positive desmosomes producing a pearls-on-a-string pattern.
Movie 10 Interdesmosomal and radial keratin filaments merge into bundles. The time-lapse series (maximum intensity projections; 26 s recording intervals) shows HK5-EYFP and Dsc2-mCerulean fluorescence at the cell border in a HaCaT cell colony 3 days after seeding (see also corresponding Fig. 5b). Note the fusion of color-coded interdesmosomal and radial KFs into KF bundles with tightlyspaced and enlarged Dsc2-mCerulean-positive desmosomes.