Shifts in keratin isoform expression activate motility signals during wound healing

Abstract

Keratin intermediate filaments confer structural stability to epithelial tissues, but the reason this simple mechanical function requires a protein family with 54 isoforms is not understood. During skin wound healing, a shift in keratin isoform expression alters the composition of keratin filaments. If and how this change modulates cellular functions that support epidermal remodeling remains unclear. We report an unexpected effect of keratin isoform variation on kinase signal transduction. Increased expression of wound-associated keratin 6A, but not of steady-state keratin 5, potentiated keratinocyte migration and wound closure without compromising mechanical stability by activating myosin motors to increase contractile force generation. These results substantially expand the functional repertoire of intermediate filaments from their canonical role as mechanical scaffolds to include roles as isoform-tuned signaling scaffolds that organize signal transduction cascades in space and time to influence epithelial cell state.

Keywords: intermediate filaments; keratin; myosin; signal transduction; wound healing.

Figure 1.. Epidermal remodeling triggers a keratin isoform switch.

A. Main keratin isoforms expressed in steady-state and actively remodeling epidermis. B. Top: A skin excision specimen includes healing edges from the prior biopsy wound. Middle: Hematoxylin and eosin (H&E)-staining. Dotted outlines, epidermal migration fronts. Epidermal layers: B, basal; S, spinous (suprabasal); C, cornified. Bottom: Immunofluorescence labeling of wound-associated K6 and K17, and steady-state basal epidermal K5. C. Intermediate filament expression in acute (Top, GSE97615) and chronic (Bottom, GSE80178) human skin wounds and intact skin controls. D-E. Immunofluorescence of steady-state K5 and wound-associated K6 (D) or K17 (E) at keratinocyte monolayer wound edges. F. Keratin transcript levels measured by qRT-PCR from intact or wounded keratinocyte monolayers (see Methods). n = 3 monolayers per group. G-H. Stratified epidermal cultures. G, H&E staining after different growth periods. H, Immunofluorescence labeling of basal epidermal keratin K5 and differentiated epidermal keratin K10. I. Top: Schematic of the epidermal culture wound model (see Methods). Middle: H&E staining after 72 hours of migration. Bottom: Immunofluorescence of wound-associated K6 and K17, and steady-state basal K5. Dashed vertical lines, image re-alignment correcting for folds in the tissue section. See also Figure S1B.

Figure 2.. Wound-associated K6A supports epidermal migration without compromising mechanical stability.

A. Diagram of the gain-of-function keratin expression model. Cultured keratinocytes were transduced with lentivirus to express tagged keratin, then divided by fluorescence activated cell sorting based on keratin expression levels. B. Western blot analysis of endogenous (K5, K6) and exogenously expressed (K5-G, K6-R) keratins in parental (wt), K5^high, and K6A^high cell lines (see also Figure S1E). Intermediate bands likely represent degradation products of the tagged constructs. Right: Ratio of K5-G or K6-R to K5 or K6. n = 4 samples per group. C. Proliferation of K5^high and K6A^high cells in culture measured by uptake of 5-ethynyl-2’-deoxyuridine (EdU). n = 6 samples per group. D. Thin-section transmission electron microscopy images of typical desmosomes with keratin intermediate filament insertions. E. Mechanical stability of keratinocyte monolayers measured using a fragmentation assay (see Methods). n = 12 monolayers per group. F. Paired epidermal cultures were grown from K5^high or K6A^high cells. Following barrier removal and 12-hours of migration, the remaining gap was measured in H&E-stained sections. n = 8–9 epidermal culture pairs per group. G-J. Paired epidermal cultures grown from K5^high and K6A^high cells on opposite sides of each barrier. Cultures were imaged live following barrier removal (G; see also Video S1). Red and green lines, farthest migration extent of the K6A^high and K5^high halves at each time point. H, Farthest distance migrated by the K6A^high half minus farthest distance migrated by the K5^high half for each culture pair. I, Average migration speed for each culture half over 20-hours. J, Time needed for the K5^high and K6A^high halves of each culture pair to reach distance checkpoints. Dots below the diagonal represent culture pairs where the K6A^high half reached the checkpoint first; dots above the diagonal represent culture pairs where the K5^high half reached the checkpoint first. n = 16 epidermal culture pairs.

Figure 3.. Wound-associated K6A supports keratinocyte migration in monolayers and single cells.

A. Diagram of a mosaic monolayer containing K5^high (green), K6A^high (red), K5^high/K6A^high (K5^hi6^hi, yellow), and K5^low/K6A^low (K5^lo6^lo, grey) cells during migration. B. Top: Keratin expression regions. Bottom: Local migration speeds calculated using a computer vision pipeline (see Methods). White lines, 200-μm band tracking the wound edge. See also Video S2. C. Average local migration speed 10-μm to 200-μm from the wound edge aligned relative to the global peak in migration speed. n = 36 scratch wounds. D. Relative local migration speed within each keratin expression region (compared to average across all regions) at different time points relative to the peak in migration speed. n = 36 scratch wounds. E-F. Pairwise comparison of local migration speed between keratin expression regions at the peak in migration speed (E) and 2-hours later (F). Each point represents one scratch wound. n = 36 scratch wounds. G-I. Movement of sparsely seeded, individual K5^high and K6A^high keratinocytes. G, Aligned cell trajectories. Rings, quantiles of maximum distance from the origin. See also Video S3. H-I, Mean track velocity (H) and mean squared displacement rate (I) in K5^high and K6A^high cells. n = 7–10 movies with 144–232 tracks per group.

Figure 4.. Wound-associated K6A increases cellular force generation by activating myosin.

A. Keratin filaments in K5^high and K6A^high cells. Right, pseudo-color overlay of filament images captured 3 minutes apart. B. Filament networks delineated by a computer vision pipeline (see Methods). As in (A), overlay of networks 3 minutes apart highlights filament movement. See also Video S4. C. Schematic of filament dynamics score calculation (see Methods). D. Smoothed maps of local filament dynamics scores over different time intervals. E. Comparison of filament dynamics scores between K5^high and K6A^high cells. Each data point represents spatially averaged filament dynamics scores for a cell. n = 110–139 cells in 24–25 movies per group. F. Comparison of filament dynamics scores between keratinocytes expressing a chimeric keratin containing only the head domain of K6A joined to the remainder of K5 (K6A^h5^rt) with K5^high cells. n = 44–47 cells in 28 movies per group. See also Figure S4D. G. Traction force microscopy (TFM) of K5^high and K6A^high cells. Left, reconstructed traction vectors (arrows) on a heatmap of force magnitude and a watermark of keratin filament TIRF images. Rings, visible borders and expanded boundary for strain energy calculation (see also Figure S4F). Right, strain energy density. Outlined dots, connected between groups, mean values of experimental replicates in separate batches of TFM substrates. n = 51–58 cells per group. H. TFM of cells expressing a chimeric keratin containing the head domain of K6A joined to the remainder of K5 (K6A^h5^rt) or full-length K5. See also Figure S4H. n = 40 cells per group. I. Diagram of the proximity ligation assay (PLA). J. PLA detecting activated myosin by colocalization of phosphorylated myosin regulatory light chain (pRLC) and non-muscle myosin heavy chain (MYH9). Left, annotated images of K5^high (green outline) and K6A^high (red outline) cells. White diamonds, PLA detections (see Methods). Heatmap, detection density. Right, PLA detection density in K5^high and K6A^high cells. Outlined dots, connected between groups, mean values of experimental replicates. n = 66–80 cells per group in 37 total images. K-L. PLA detecting activated myosin near keratin filaments by colocalization of pRLC and K5 (K) or K17 (L). K, n = 94–115 cells per group in 20 images. L, 99–108 cells per group in 23 images. M-N. PLA detecting total myosin near keratin filaments by colocalization of total myosin regulatory light chain (RLC) and K5 (M) or K17 (N). M, n = 117–119 cells per group in 19 images. N, 121–129 cells per group in 21 images. O. Schematic diagram of the proposed model. K6A-containing filaments preferentially recruit regulatory kinases, reducing the dimensionality of kinase–RLC interaction to increase myosin activation. Question marks indicate that the identity of the kinase and the mechanism of kinase–intermediate filament association remain to be determined.