Mechanical and signaling roles for keratin intermediate filaments in the assembly and morphogenesis of mesendoderm tissue at gastrulation

Abstract

The coordination of individual cell behaviors is a crucial step in the assembly and morphogenesis of tissues. mesendoderm cells migrate collectively along a fibronectin (FN) substrate at gastrulation, but how the adhesive and mechanical forces required for these movements are generated and transmitted is unclear. Traction force microscopy (TFM) was used to establish that traction stresses are limited primarily to leading edge cells in mesendoderm explants, and that these forces are balanced by intercellular stresses in follower rows. This is further reflected in the morphology of these cells, with broad lamellipodial protrusions, mature focal adhesions and a gradient of activated Rac1 evident at the leading edge, while small protrusions, rapid turnover of immature focal adhesions and lack of a Rac1 activity gradient characterize cells in following rows. Depletion of keratin (krt8) with antisense morpholinos results in high traction stresses in follower row cells, misdirected protrusions and the formation of actin stress fibers anchored in streak-like focal adhesions. We propose that maintenance of mechanical integrity in the mesendoderm by keratin intermediate filaments is required to balance stresses within the tissue to regulate collective cell movements.

14-3-3 recruits keratin intermediate filaments to mechanically sensitive cell-cell contacts

“…Fluid flow shear stress of alveolar cells results in phosphorylation of known 14-3-3 sites in keratin 18 along with increases in dynamic exchange that are 14-3-3 dependent (Sivaramakrishnan et al, 2009). Similarly, we see decreased FRAP recovery when 14-3-3 is inhibited in eGFP-K19 expressing mesendoderm tissue, which is known to have intrinsic mechanical stresses through the cell-cell contacts (Weber et al, 2012;Sonavane et al, 2017). Expression of R18 peptide results in inhibition of 14-3-3 in multiple contexts throughout the cell rather than perturbation of the subset of 14-3-3 molecules that associate with the keratin-cadherin complex.…”

“…The association and function of 14-3-3 in relation to keratin intermediate filaments may provide an interface between cytoskeletal networks and a host of scaffolding proteins and signal transduction pathways (Margolis et al, 2006;Loschke et al, 2016;Sonavane et al, 2017). In turn, the local recruitment of keratin at sites of tension due to local signaling/phosphorylation events could promote a positive feedback loop for keratin filament assembly dynamics (Ridge et al, 2005;Woll et al, 2007;Ju et al, 2015;Sonavane et al, 2017). The enriched presence of 14-3-3 near both the cell-cell adhesion and in the lamellipodia suggest either non-intermediate filament related functions in the latter region or a possible function in intermediate filament formation (Windoffer et al, 2004(Windoffer et al, , 2011.…”

Intermediate filaments in developing neurons: Beyond structure

“…These phenotypes highlight the importance of IF proteins in imparting structural integrity to multicellular assemblies. It is of note that in Xenopus loss of keratin expression leads to early developmental abnormalities at the gastrulation stage more pronounced and earlier than those observed in mice (Baribault, Price, Miyai, & Oshima, 1993;Sonavane et al, 2017), raising interesting questions about redundancy among IF classes in mammals.…”

Desmoplakin is required for epidermal integrity and morphogenesis in theXenopus laevisembryo

“…For example, loss of keratin in the embryo results in misdirected lamellapodial protrusions and disorganized actin networks that translate into defects in tissue level collective migration (Sonavane et al, 2017). Thus, it is likely that keratins integrate the cell mechanics necessary for morphogenetic movements (Klymkowsky et al, 1992).…”