14-3-3 serves as a major regulator of keratin intermediate filament dynamics in vivo. Migratory mesendoderm tissue of the Xenopus embryo is used to show that the dynamic reorganization of keratin filaments, a consequence of force on cell-cell adhesions, is mediated by a novel association between 14-3-3 and Keratin 19.

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.

The significance of cytoplasmic intermediate filament proteins has previously been examined largely through various genetic approaches, including knockdown, knockout and transgenic overexpression. Few studies to date have attempted to examine the role of specifically the filamentous intermediate filament network in orchestrating various cell functions. To directly assess the role of the filamentous keratin intermediate filament network in regulation of cellular behavior, we created a PhotoActivatable disruptor of keratin Intermediate Filaments (PA-dIF). This genetically encoded construct consists of a peptide derived from the 2B2 region of Keratin 8 fused to the photosensitive LOV2 domain from Avena sativa phototropin-1. Upon 458 nm photoirradiation, PA-dIF disrupts keratin intermediate filaments in multiple species and cell types. Marked remodeling of the keratin intermediate filament network accompanies collective cellular morphogenetic movements that occur during gastrulation and neurulation in the Xenopus laevis frog embryo. Light-based activation of PA-dIF was able to disrupt keratin intermediate filaments in Xenopus cells and lead to tissue-specific disruption of morphogenetic processes. Altogether our data show a fundamental requirement for keratin intermediate filaments in orchestrating morphogenetic movements during early embryonic development that have yet to be revealed in other model systems. Moreover, our data validate the utility of a new genetically encoded photoactivatable tool for the disruption and examination of intermediate filaments. Döring and Stick, 1990; Peter and Stick, 2015), assemble into a filamentous network in the nucleus. All other intermediate filament proteins assemble as filaments in the cytoplasm. Cytoplasmic intermediate filaments form five subfamilies that collectively include keratins, vimentin, desmin, neurofilaments, among many others. In addition to their conserved amino acid sequences, motifs and structure, a key defining feature of intermediate filaments is that they convey mechanical strength to cells and cellular structures. Through linkage to cell adhesions, association with the nuclear membrane, and interactions with other cytoskeletal networks, intermediate filaments have important roles in regulating cell shape, nuclear morphology, and consequently cellular function (Sanghvi-Shah and Weber, 2017). The diversity of proteins, and the cytoplasmic filaments that they make, creates small but significant differences in assembly mechanics and the micromechanical properties of filaments (Block et al., 2015).