XB-ART-56703Mol Biol Cell January 1, 2020; 31 (9): 930-943.
14-3-3 targets keratin intermediate filaments to mechanically sensitive cell-cell contacts.
Intermediate filament (IF) cytoskeletal networks simultaneously support mechanical integrity and influence signal transduction pathways. Marked remodeling of the keratin IF network accompanies collective cellular morphogenetic movements that occur during early embryonic development in the frog Xenopus laevis. While this reorganization of keratin is initiated 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 colocalizes with keratin filaments near cell-cell junctions in migrating mesendoderm. Coimmunoprecipitation, mass spectrometry, and bioinformatic analyses indicate 14-3-3 is associated with Keratin 19 (K19) 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 K19 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 IFs and is involved in their reorganization to sites of cell adhesion.
PubMed ID: 32074004
PMC ID: PMC7185971
Article link: Mol Biol Cell
Genes referenced: cdh3 krt19 krt8.2 pmch ywhab
Article Images: [+] show captions
|FIGURE 1:. 14-3-3 protein expression is ubiquitous across early embryonic stages and tissues. (A) Whole embryo lysates (1% Triton X-100) were immunoblotted for 14-3-3 using a pan antibody that detects multiple isoforms. Each lane represents approximately 50 μg. (B) Colored schematic of a bisected Xenopus embryo at gastrula depicting major tissue divisions. The tissues include the animal cap (AC), mesendoderm (ME), marginal zone (MZ), vegetal hemisphere (VG), and whole embryo lysate (WEL). (C) Embryos were dissected into separate tissues and corresponding lysates (1% Triton X-100) were immunoblotted using pan 14-3-3 antibody to examine expression across the gastrulating embryo. Each lane represents a portion of protein equivalent to approximately 1 embryo.|
|FIGURE 2:. Xenopus K19 associates with 14-3-3 proteins and C-cadherin. (A) Pan 14-3-3 immunoprecipitates (1% Tergitol type NP-40) from whole embryo lysates prior to band extraction and processing using LC/MS-MS. Prominent bands at 48, 30, and 28 kDa were processed. Heavy chain IgG from the antibody used for IP was not excised. (B) Table summary of relevant proteins detected in gel extracts processed using LC/MS-MS. Experiments were conducted using 14-3-3 immunoprecipitates from whole embryo lysates (WEL) as well as lysates from mesendoderm (ME) tissue only. Analysis was performed using Scaffold 4.7.3. (C) Summary schematic of K19 peptides (red) detected in the 48 kDa sample. Peptides are depicted within the context of the K19 primary structure and alongside described (green) and predicted (blue) possible 14-3-3 interaction sites. (D) 14-3-3 proteins were immunoprecipitated (1% Tergitol type NP-40) from whole embryo lysates and immunoblotted for C-cadherin, K19, and Vinculin. C-cadherin band is denoted by an arrow. The bottom band is yolk protein from sample.|
|FIGURE 3:. Filaments recruited to cell–cell adhesions associate with 14-3-3. (A–C) Single plane confocal images showing a sagittal perspective of a cryosectioned gastrulating embryo labeled immunocytochemically for 14-3-3 proteins (red) and K8 (green). Areas where filamentous colocalization was detected in B are illustrated by arrows in all three panels. (D) Cartoon schematic depicting orientation of cells in A–C. The blue arrow indicates direction of tissue migration. (E) Maximum intensity projection of confocal z-stack of cells shown in A–D to show more comprehensive filament distribution. (F–H) Leading edge of a mesendoderm explant demonstrating association between 14-3-3 and keratins (pan-keratin antibody labeling) at a cell–cell interface (arrowheads). Closer inspection of the keratin morphology at this area (F’–H’) reveals filamentous 14-3-3 labeling. (I) Explant schematic depicting the cell pair (F–H) relative to the rest of the tissue. Images are z-stacks (maximum intensity projection). Scale bars are 10 μm.|
|FIGURE 7:. 14-3-3 proteins target keratins to cell–cell adhesions. (A) Schematic of fusion peptides created by the insertion of R18 or R18M (R18/M) into FLAG/eGFP-K19. This construct was generated using full-length K19.L from X. laevis. (B) Protein lysates from stage 10.5 Xenopus embryos expressing C-cadherin-eGFP and either FLAG-R18-K19 or FLAG-R18M-K19 were prepared in 1% Triton X-100. FLAG constructs were immunoprecipitated and analyzed by immunoblot for associated proteins. (C) Protein lysates from stage 10.5 Xenopus embryos expressing human FLAG-14-3-3β and either eGFP-R18-K19 or eGFP-R18M-K19 were prepared in 1% Tergitol-type NP-40. FLAG constructs were immunoprecipitated and analyzed by immunoblot for associated proteins. (D, E) Explanted mesendoderm cells mosaically expressing eGFP-R18M-K19 (in D) or eGFP-R18-K19 (in E). (F, G) Explanted mesendoderm coexpressing mem-RFP and eGFP-R18M-K19 (in F) or eGFP-R18-K19 (in G). Arrows indicate areas where filament densities have localized. Images are z-stacks (maximum intensity projection). Scale bars are 10 μm.|
References [+] :
Acehan, Plakoglobin is required for effective intermediate filament anchorage to desmosomes. 2008, Pubmed