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

Abstract

Abbreviations: FRAP fluorescence recovery after photobleaching IFs intermediate filaments LC/MS-MS liquid chromatography, tandem mass spectrometry Abstract Intermediate filament cytoskeletal networks simultaneously support mechanical integrity and influence signal transduction pathways. Marked remodeling of the keratin intermediate filament network accompanies collective cellular morphogenetic movements that occur during early embryonic development in the frog Xenopus laevis. While this reorganization of keratin isinitiated by force transduction on cell-cell contacts mediated by C-cadherin, the mechanism by which keratin filament reorganization occurs remains poorly understood. In this work we demonstrate that 14-3-3 proteins regulate keratin reorganization dynamics in embryonic mesendoderm cells from Xenopus gastrula. 14-3-3 co-localizes with keratin filaments near cellcell junctions in migrating mesendoderm. Co-immunoprecipitation, mass spectrometry and bioinformatic analyses indicate Keratin 19 is a target of 14-3-3 in the whole embryo and, more specifically, mesendoderm tissue. Inhibition of 14-3-3 results in both the decreased exchange of keratin subunits into filaments and blocks keratin filament recruitment toward cell-cell contacts.Synthetically coupling 14-3-3 to Keratin 19 through a unique fusion construct conversely induces the localization of this keratin population to the region of cell-cell contacts. Taken together, these findings indicate that 14-3-3 acts on keratin intermediate filaments and is involved in their reorganization to sites of cell adhesion.

A novel photoactivatable tool for intermediate filament disruption indicates a role for keratin filaments in early embryogenesis

“…Nonetheless, Krt18 appeared to be the primary target for our dIF constructs in the early Xenopus embryo. Interestingly, we did not find Krt19 particularly enriched in CA-dIF expressing samples even though recent work from our laboratory indicates an important role for 14-3-3 binding to Krt19 for intermediate filament recruitment to cell-cell adhesions (Mariani et al, 2018). Conversely, in that study, we did not find Krt18 associated with 14-3-3.…”

“…Xenopus keratin 8 plasmid C2-eGFP-Krt8 construct was a kind gift from Dr. Victoria Allan and was subcloned into pCS2 + previously (Weber et al, 2012). Keratin 19 plasmid pCS2 + -eGFP-Krt19 was made by Richard Mariani (Mariani et al, 2018). PhotoActivatable disruptor of Intermediate Filaments (PA-dIF) and Constitutively Active disruptor of Intermediate Filaments (CA-dIF) were generated by cloning commercially synthesized Flag-tagged LOV-J-2B2 (Flag-PA-dIF) and LOV-J(I379E)-2B2 (Flag-CA-dIF) (Genewiz, NJ) into pCS2 + vector using EcoRI and XhoI restriction sites.…”

“…In addition, these findings provide evidence that suggests PA-dIF is effectively caged when maintained in the dark. (Hatzfeld and Weber, 1992;Steinert et al, 1993a;Strelkov et al, 2002;Helfand et al, 2011) (Weber et al, 2012;Mariani et al, 2018). To investigate the ability of dIF constructs to disrupt keratin networks in an in vivo model system, we co-injected mRNA encoding PA-dIF or CA-dIF with either eGFP-Krt8 or eGFP-Krt19 into the animal cap of (Fig.…”

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

“…14-3-3 proteins may be such factors. They have been shown to be involved in mechanosensitive keratin recruitment toward cell-cell contacts in migrating mesendoderm of the Xenopus laevis gastrula [57]. Given the evolutionary diversity and cell type-specificity of desmosome and KF composition these or other factors may drive this process in the different environments.…”

Keratin intermediate filaments: intermediaries of epithelial cell migration

“…Some of the most compelling evidence for a function of the interaction between keratins and cell junctions in cell movement comes from observations in the Xenopus laevis embryo [105,106]. In this system, KFs are linked to C cadherin-containing cell junctions.…”