Abstract
Uneven distribution and local concentration of protein complexes on distinct membrane cortices is a fundamental property in numerous biological processes, including Drosophila neuroblast (NB) asymmetric cell divisions and cell polarity in general. In NBs, the cell fate determinant Numb forms a basal crescent together with Pon and is segregated into the basal daughter cell to initiate its differentiation. Here we discover that Numb PTB domain, using two distinct binding surfaces, recognizes repeating motifs within Pon in a previously unrecognized mode. The multivalent Numb-Pon interaction leads to high binding specificity and liquid-liquid phase separation of the complex. Perturbations of the Numb/Pon complex phase transition impair the basal localization of Numb and its subsequent suppression of Notch signaling during NB asymmetric divisions. Such phase-transition-mediated protein condensations on distinct membrane cortices may be a general mechanism for various cell polarity regulatory complexes.
Highlights
1, 100%), majority of pon mutant NB clones contained multiple Dpn-positive cells (12.1, n = 26, Fig. 8a–c, g)
Summary of the quantitative binding constants between various Numb PTB and Pon fragments derived from the ITC-based titration assays shown in Supplementary Fig. 2b–d. e In HEK293T cells, N139A,Y154EPon, N169A,F186EPon, 4MPon, or 6MPon mutation abolished or significantly impaired the interaction between Numb and Pon. f NumbC90W, NumbG192D, and NumbC90W,G192D could not coimmunoprecipitate with PonWT
We found that Numb PTB recognizes the AB motif repeats of its adaptor Pon in a previously unrecognized manner
Summary
The rescuing efficiency of endogenous Numb basal localization and NB over-proliferative phenotype in pon mutant NBs by various Flag-Pon mutants was nicely correlated with their abilities to induce LLPS through Numb binding in vitro and in heterologous cells Further supporting the above conclusion, larval brain treated with 1,6-hexanediol, which can disturb Numb/Pon LLPS assemblies in vitro (Fig. 5c, d), exhibited defective localizations of both endogenous Numb and Pon in a concentration-dependent manner (Fig. 9a, c). In these NBs, while Pon localized in cytoplasm, Numb was cortically localized with weak cytoplasm, reminiscent of its localization in pon mutant NB. Removal of 1,6-hexanediol restored the formation of Numb and Pon crescents in dividing NBs (Fig. 9b, c), demonstrating that the dynamic basal condensation of Numb/ Pon is mediated by LLP
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