Figure 6.
Pkp4 promotes formation of α-catenin–independent clusters associated with actin filaments. (a) Schematic representation of full-length α-catenin (αCat) and the truncated mutant αCatCH-Δ259, which contains only the β-catenin–binding N-terminal region (residues 1–259). The extended deletion removes all regions required for direct binding to F-actin (αABD) and for interactions with actin-associated proteins, including vinculin (Vin), afadin (Afad), ZO-1, and EPLIN, located within the middle subdomains (M1–M3). The mCherry tag (mCH) is indicated by a red circle; unstructured regions are shown as solid lines. Domain boundaries are labeled by residue numbers. (b) Western blot of WT A431 (A431), αCat-KO cells (αCat-KO), and αCat-KO cells expressing αCatCH-Δ259 (αCat-259) probed for E-cadherin (Ecad), α-catenin N terminus (αCat, N-term), p120, pkp4, and β-tubulin (tbl, loading control). Molecular weight markers (kDa) are shown at left. The positions of intact α-catenin (αCat) and the mutant (α259) are indicated. (c) Maximum-intensity x–y projections of αCatCH-Δ259 cells stained for mCH (red) together with E-cadherin (Ecad) or pkp4 (Pkp4), or together with p120 and pkp4 (pkp4 shown only in the zoomed region, blue). Bar, 20 μm. Enlarged regions (dashed boxes) are shown on right. Bar, 6 μm. Arrows indicate representative clusters. (d) Line-scan intensity profiles taken along the dashed line in c. (e) Ratios of fluorescence intensities in cluster versus inter-cluster regions for mCH, E-cadherin, pkp4, and p120 (n = 11 from two independent images). Statistical analysis: two-tailed Student’s t test: ns, nonsignificant; ****, P <0.0001. Bars indicate mean ± SD. (f) Maximum-intensity projections of αCatCH-Δ259 cells extracted with 1% Triton X-100 and stained for mCH (red) and F-actin or pkp4 (green). Bar, 20 μm. Enlarged regions (dashed boxes) are shown at the bottom rows. Bar, 10 μm. (g) PREM of αCatCH-Δ259 clusters. The left panel shows an overview of two overlapping cells; boxed region is enlarged at top middle. Additional panels show representative clusters from other cells in the same culture. Immunogold particles marking mCH (10 nm) in the overview image are pseudocolored red. Source data are available for this figure: SourceData F6.

Pkp4 promotes formation of α-catenin–independent clusters associated with actin filaments. (a) Schematic representation of full-length α-catenin (αCat) and the truncated mutant αCatCH-Δ259, which contains only the β-catenin–binding N-terminal region (residues 1–259). The extended deletion removes all regions required for direct binding to F-actin (αABD) and for interactions with actin-associated proteins, including vinculin (Vin), afadin (Afad), ZO-1, and EPLIN, located within the middle subdomains (M1–M3). The mCherry tag (mCH) is indicated by a red circle; unstructured regions are shown as solid lines. Domain boundaries are labeled by residue numbers. (b) Western blot of WT A431 (A431), αCat-KO cells (αCat-KO), and αCat-KO cells expressing αCatCH-Δ259 (αCat-259) probed for E-cadherin (Ecad), α-catenin N terminus (αCat, N-term), p120, pkp4, and β-tubulin (tbl, loading control). Molecular weight markers (kDa) are shown at left. The positions of intact α-catenin (αCat) and the mutant (α259) are indicated. (c) Maximum-intensity x–y projections of αCatCH-Δ259 cells stained for mCH (red) together with E-cadherin (Ecad) or pkp4 (Pkp4), or together with p120 and pkp4 (pkp4 shown only in the zoomed region, blue). Bar, 20 μm. Enlarged regions (dashed boxes) are shown on right. Bar, 6 μm. Arrows indicate representative clusters. (d) Line-scan intensity profiles taken along the dashed line in c. (e) Ratios of fluorescence intensities in cluster versus inter-cluster regions for mCH, E-cadherin, pkp4, and p120 (n = 11 from two independent images). Statistical analysis: two-tailed Student’s t test: ns, nonsignificant; ****, P <0.0001. Bars indicate mean ± SD. (f) Maximum-intensity projections of αCatCH-Δ259 cells extracted with 1% Triton X-100 and stained for mCH (red) and F-actin or pkp4 (green). Bar, 20 μm. Enlarged regions (dashed boxes) are shown at the bottom rows. Bar, 10 μm. (g) PREM of αCatCH-Δ259 clusters. The left panel shows an overview of two overlapping cells; boxed region is enlarged at top middle. Additional panels show representative clusters from other cells in the same culture. Immunogold particles marking mCH (10 nm) in the overview image are pseudocolored red. Source data are available for this figure: SourceData F6.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal