Mariani RA , Paranjpe S , Dobrowolski R , Weber GF .

R01 AG062475 NIA NIH HHS , P30 NS046593 NINDS NIH HHS , R15 HD084254 NICHD NIH HHS

	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 4:. 14-3-3 proteins are distributed proximally to cell–cell adhesions. (A) Representative linescan analysis of fluorescence across cellular compartments indicated in B. Lines extended from the approximate middle of the cell to the area just prior to C-cadherin signal and to the onset of C-cadherin signal. Measurements were taken from the cell center (purple rectangle), proximal to the adhesion (teal rectangle), and at the adhesion (yellow rectangle). Each rectangle represents 0.5 μm in length. (B) Immunofluorescence image of a leading edge mesendoderm cell labeled for 14-3-3 and expressing C-cadherin (X. laevis origin, eGFP label). Colored arrows indicate regions represented by rectangles in A. (C, D) Comparison of the mean increase in 14-3-3 signal and C-cadherin signal from the cell center to the area proximal to the cell–cell adhesion (in C) and at the cell–cell adhesion (in D). Analysis was performed using paired sample t tests, ***p < 0.001. Error bars are ± SEM. (E) Representative linescan analysis of fluorescence across cellular compartments indicated in F. Lines extended from the approximate middle of the cell to the onset of C-cadherin signal. Measurements were taken from the cell center (purple rectangle) and at the adhesion (yellow rectangle). Each rectangle represents 0.5 μm in length. (F) Immunofluorescence image of mesendoderm cells expressing C-cadherin (eGFP) and mCherry-K19 (X. laevis origin). Colored arrows indicate regions represented by rectangles in E. Scale bars are 10 μm. (G) Scatterplot of the mean relative fluorescence intensities of C-cadherin and 14-3-3 in 20 cells near and at the cell–cell contact zone. (H) Scatterplot of the mean relative fluorescence intensities of C-cadherin and K19 in 20 cells imaged by confocal in the basal plane and a higher junctional plane. Percentages indicate the proportion of corresponding mean intensities in each quadrant.
	FIGURE 5:. Mesendoderm keratin filament dynamic exchange is decreased by 14-3-3 inhibition. (A) Immunoblot analyses of protein extracts (1% Triton X-100) of stage 10.5 Xenopus embryos expressing mCh-R18 or mCh-R18M. (B) Coimmunoprecipitation (1% Tergitol type NP-40) performed using stage 10.5 lysates expressing human FLAG-14-3-3 β with either mCherry-R18 or mCherry-R18M. (C, D) Still images from photobleach and recovery time lapse movies (Supplemental Movie S1). Mesendoderm explants expressing either 14-3-3 inhibitor peptide mCherry-R18 or control peptide mCherry-R18M (red) with eGFP-K19 (green) were exposed to GFP photobleaching and fluorescence recovery at the site was measured. (C’–D’’) Enlarged view of the region of filament bleaching (white boxes) during recovery measurements. The time annotations in seconds refer to start of capture (t = 0 s) and end of capture (t = 330 s). (E) Representative analysis plotting fluorescence recovery against time of image capture. (F) Comparison of mean eGFP-K19 recovery rate in explants expressing either mCherry-R18 or mCherry-R18M. Analysis was performed using a one-tailed t test, *p < 0.05. Error bars are ± SEM. Scale bars are 10 μm.
	FIGURE 6:. 14-3-3 is necessary for targeting of keratin to cell–cell contacts. (A–C) Mesendoderm cell pair establishing de novo cell–cell contact after collision. Cells are expressing mCherry-R18M and eGFP-K19. The arrowhead depicts the cell–cell adhesion where keratin densities (asterisk) have localized. Image stack is 35 slices (10.54 μm). (D–F) Postcollision mesendoderm cell pair expressing mCherry-R18 and eGFP-K19. The bracket depicts the cell–cell adhesion that demonstrates a gap where keratin filaments have failed to reorganize. Image stack is 40 slices (10.92 μm). (G) Comparison of the number of keratin gaps at cell–cell adhesions in postcollision cell pairs expressing either mCherry-R18M or mCherry-R18. Analysis was performed using a z-test for proportions from two samples, ***p < 0.001. Error bars represent the 99.9% CI for each sample proportion. Fluorescent images are z-stacks (maximum intensity projection). Brightfield images are single planes. Scale bars are 20 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.

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