Fig. 4 |. The association of BSL1 with the polarity complex promotes BIN2 partitioning to the nucleus.

a, Left: representative confocal images showing changes in BIN2 membrane/nucleus (M/N) partitioning in consecutive stomatal lineage cells co-expressing BIN2p::BIN2–YFP (yellow) and 35Sp::mCherry– TUA5 (cyan). Arrows indicate preferential membrane localization and polarization of BIN2 in the MMC. White arrowhead indicates preferential nuclear localization of BIN2 in the SLGC. Cyan arrowheads indicate the position of the PPB. The cartoon depicts the corresponding protein localization of BIN2 (orange) at each stage shown above. Scale bar, 5 μm. Right: quantification of the membrane/nuclear (M/N) partition of BIN2–YFP in pre-divisional cells (PrC or MMC) versus that in post-divisional SLGCs. Box plots show the first and third quartiles, median (line) and mean (cross). Two-tailed Student’s t-test. n, number of cells. ***P < 0.0001. b, Left, representative confocal images showing the expression patterns of BIN2p::BIN2–YFP (yellow) in indicated genetic backgrounds. bsl-quad, quadruple loss-of-function mutant; BSL1⁺⁺ or BSL1^D584N++, plants overexpressing BSL1 or phosphatase-dead BSL1^D584N (both driven by the stomata lineage TMM promoter; elevated transcript levels shown in Extended Data Fig. 4a). Inset, enlarged views show the subcellular partitioning of BIN2. Arrowheads indicate the preferential nuclear localization of BIN2. Scale bar, 10 μm. Right, quantification of M/N partitioning of BIN2– YFP. Absolute fluorescence intensity values from the membrane or nuclear regions of a cell were taken. All images were captured with same settings and z-stacked for measurement. Box plots show the first and third quartiles, median (line) and mean (cross). One-way ANOVA with Tukey’s post-hoc tests were performed to compare with the WT. n, number of cells. ***P < 0.0001. c, BSL1 but not BSL1^D584N interferes with the interaction of BIN2 with BASL. Top, schematic of the co-IP assay used to monitor the ability of BSL1/BSL1^D584N–FLAG to affect the interaction between BIN2–YFP and Myc–BASL. Bottom, results were detected by western blotting. Specifically, the designated protein fusions were overexpressed and purified from N. benthamiana leaf cells. BIN2–YFP and Myc–BASL were co-expressed in the presence or absence of BSL1/BSL1^D584N–FLAG. The interactions of BIN2–BASL were assayed by the amount of Myc–BASL being immunoprecipitated by BIN2–YFP bound to GFP-Trap agarose beads. Quantification of the amount of BASL interacting with BIN2 is shown as the mean ± s.d., n = three biological replicates.