‘White wave’ analysis of epithelial scratch wound healing reveals how cells mobilise back from the leading edge in a myosin-II-dependent fashion

Abstract

Collective cell migration is absolutely essential for a wide variety of physiological episodes including the re-epithelialisation component of tissue repair. However, the investigation of such processes has been frustrated by difficulties in quantitatively analysing the behaviours of a large body of cells within a migrating epithelial sheet, which previously required manually tracking a large number of individual cells, or using advanced computational techniques. Here, we describe a novel and simpler image subtraction method with which we can visualise and quantify collective cell mobilisation as a ‘white wave’ that propagates back from the leading edge of a scratch-wounded monolayer of cultured epithelial cells. Using this technique, we show that actomyosin constriction negatively regulates cell mobilisation and that the advancement of cell sheets and the mobilisation of rows of cells behind their leading edges are independently regulated. We also show that there is a finite limit to the number of rows of cells mobilised after wounding. Moreover, our data suggest that enhancing cell mobilisation, by release from myosin II contractility, accelerates the healing of large wounds in the long term, thus raising the possibility that the cell mobilisation ‘wave’ we reveal here might be a therapeutic target for improving wound healing.

Re-epithelialization of cutaneous wounds in adult zebrafish combines mechanisms of wound closure in embryonic and adult mammals

“…Basal epidermal cells behind the LE also formed cryptic lamellipodia (Fig. 2G, lower panel), as has been suggested previously from in vitro experiments (Matsubayashi et al, 2011). Only in rare cases did individual LE keratinocytes form lamellipodia that projected beyond the otherwise homogeneous lamellipodial front (Fig.…”

“…In cultured epithelial cells, closure of scratched wounds is not only achieved by activities at the front row, but also involves several rows of following epithelial cells that migrate as coherent cell sheets (Farooqui and Fenteany, 2005;Fenteany et al, 2000;Matsubayashi et al, 2011). We observed a similar phenomenon in keratinocytes following the LE in zebrafish wounds in vivo, with (cryptic) lamellipodia also formed by second and third row keratinocytes (Fig.…”

“…By contrast, in partial-thickness wounds, both mechanisms are at play, explaining their higher closure rates (500 µm/h) compared with full-thickness wounds (250 µm/h). The even lower closure rates of cultured keratinocytes in scratch assays (50-70 µm/ h) (Matsubayashi et al, 2011) might be due to the absence of such epithelial rearrangement further away from the LE, because here, cells are mono-layered and closure is solely dependent on active cell migration. Of note, this 'pushing' from the back, including the elongation/stretching of cells, seems to be largely independent of the 'pulling' at the front.…”

Myosin II is not required for Drosophila tracheal branch elongation and cell intercalation

“…Therefore, TC migration does not rely on actomyosin contractibility, posing the question: which molecular players might be involved in TC force generation? Interestingly, collective movement of cells depends on actin-based filopodia and lamellipodia (Mayor and Etienne-Manneville, 2016), and was shown in several cases to be independent of MyoII activity (Matsubayashi et al, 2011;SerraPicamal et al, 2012). Furthermore, several studies showed that a downregulation of MyoII is required for effective collective cell migration (Hidalgo-Carcedo et al, 2011;Omelchenko and Hall, 2012;Yamada and Nelson, 2007).…”

Advances in Wound-Healing Assays for Probing Collective Cell Migration

“…Mechanical scraping can be performed using a pipette tip, needle, bladder, razor, rubber policeman, cotton bud, or Teflon spatula for creating a single scratch or a plastic hair comb or wounder for creating multiple scratches simultaneously. [7][8][9][10][11][12][13] To standardize the process and increase the throughput, the scratching assay can be implemented using a robotic system. 14 To improve the reproducibility and minimize the surface damage, a drill press assay has been developed.…”