Fig. 5: Intracellular reconstitution studies of Nck1 assemblies.

a Strategy for construction of Y15-based Nck assemblies in living cells. b CLSM images of engineered assemblies and polymerized actin in living COS-7 cells. COS-7 cells were co-transfected with the Y15-AG-HA-encoded plasmid (200 ng), Y15-mCherry-Nck(1–258)-encoded plasmid (200 ng) and mTagBFP2-Lifeact-7-encoded plasmid (100 ng). The cells were cultured for 2 days and CLSM observations were performed. The lower panels show enlarged images of ROIs given in the upper panels. Scale bar, 10 µm (upper) and 5 µm (lower). c Effect of Nck domains on actin polymerization (left panel, series of Y15-tagged Nck used in this study; right panel, fluorescence intensities of Phallodin-iFluor 633 in cytosolic Y15-based assemblies). Transfected cells were stained with Phallodin-iFluor 633 after fixation and permeabilization. The actin intensities in cytosolic granules were quantified (n = 20, 40, 40, and 40 cells examined over three biologically independent experiments). d Density-dependency of Nck(1–258) in assemblies on actin polymerization. COS-7 cells were transfected with mixtures of Y15-AG-HA-encoded plasmid (250 ng) and Y15-mCherry-Nck(1–258)-encoded plasmid (0, 62.5, 125, 187.5, or 250 ng) (n = 50 cells examined over three biologically independent experiments). The Nck densities were calculated as mCherry intensity normalized against AG intensity in Y15-based assemblies. The boxplots are presented with the elements: center line, median; box limits, Q1 and Q3; whiskers, 1.5× interquartile range; points, outliers. p-values; two-tailed paired t-tests. Source data are provided as a Source data file.