Fig. 6: The short isoform of NHSL3 regulates migration persistence of single cells through its interaction with 14-3-3θ.
From: NHSL3 controls single and collective cell migration through two distinct mechanisms
a Prediction of direct partners of NHSL3 i3S using an AlphaFold2-based pipeline that decomposes tested proteins into evolutionary conserved domains. Partners bound to the unique C-terminal domain of i3S are shown. YWHAQ encoding 14-3-3θ displays the highest confidence score. Grey connections indicate protein-protein interaction reported in Biogrid. b 293 T cells are transiently co-transfected with plasmids expressing GFP-tagged NHSL3 i3S and PC-tagged 14-3-3 family proteins. Lysates and GFP immunoprecipitates are analysed by Western blots. c AlphaFold2-generated model of the interaction between NHSL3 i3S and 14-3-3θ. i3S residues that are critical for the interaction are displayed in full. d 293 T cells are transiently co-transfected with plasmids expressing the PC-tagged 14-3-3θ and the GFP-tagged i3S containing the indicated mutations. Lysates and GFP immunoprecipitates are analysed by Western blots. e Lysates from NHSL3 KO and NHSL3 KO stably expressing GFP or GFP-tagged NHSL3 i3S or the R135D derivative thereof are subjected to GFP immunoprecipitation and analysed by Western blots. f Migration persistence extracted from single cell trajectories of the same cell lines. Tracking 6.5 h, n = 60 cells. g Parental MCF10A, NHSL3 KO and KO cells stably expressing i3S or the R135D derivative are transfected with control or 14-3-3θ targeting siRNAs. Migration persistence is extracted from single cell trajectories and lysates from siRNA-transfected cells are analysed by Western blots. Tracking 6.5 h, n = 60 cells. Data are shown as mean ± SEM. Statistical significance is calculated with custom-made R programmes for single cell migration and p-values are indicated. Three biological repeats of each experiment gave similar results. Source data are provided as a Source Data file.
