GJA1-20k rescues gap junction permeability in C33A cells with GJA1 knockdown. (A) Images of cells scraped loaded with Lucifer yellow dilithium salt and rhodamine dextran. Each image is the mean of 30–34 individual images from each corresponding group. Scale bar = 100 μm. (B) Lucifer yellow spreading from the scraping lines from images in A. (C) Normalized areas of dye-coupled cells. Data were calculated by subtracting dextran-positive area from Lucifer yellow–positive area, the result of which was then divided by cut length. n = 30–34 images per group from three independent experimental repeats. Data are presented as box and whisker plots, with boxes showing median, 25th, and 75th percentile, with whiskers spanning the 10th to 90th percentile. ns = P > 0.05, **P < 0.01 by one-way ANOVA with Bonferroni’s post hoc test and multiple comparisons between each group with GJA1 siRNA. (D) Schematic flowchart summarizing the conclusion from Fig. 6 and Fig. 7. (E) Cx43 vesicles are trafficked on microtubules to the cell border to form Cx43 gap junctions. GJA1-20k expression directly reorganizes intracellular actin to produce an increased amount of actin puncta and stabilized F-actin fibers. Actin puncta, where Cx43 vesicles dock, serve as microtubule rest stops and F-actin fibers orient microtubules toward the cell border. Together, these populations of actin, directly produced by GJA1-20k’s functioning as an actin-capping protein, pattern microtubules to increase Cx43 trafficking, and the subsequent amount of Cx43 gap junctions at the cell border.