Journal: Cell reports

Article Title: Ccdc85c-Par3 condensates couple cell polarity with Notch to control neural progenitor proliferation.

doi: 10.1016/j.celrep.2023.112677

Figure Lengend Snippet: Figure 2. Ccdc85c coiled-coil domain specifically binds to the 4N2 region of Par3 (A) Co-immunoprecipitation (coIP) confirms the interaction between Myc-Par3 and FLAG-Ccdc85c. (B) Schematic diagrams showing the domain organizations of Par3. A summary of the binding properties of various Par3 fragments to Ccdc85c (50–160), as shown in (E), is given at the right of the panel. (C) Schematic diagrams showing the domain organizations of Ccdc85c. A summary of the binding properties of various Ccdc85c fragments to Par3C (aa 686– 1340), as shown in (D), is given at the right of the panel. (D) GST pull-down assay of Trx-Par3C with MBP-His-GST-tagged hCcdc85c (1–180, 50–160) and GST-tagged hCcdc85c fragments (181–419). (E) GST pull-down assay of various Trx-tagged human Par3 fragments with MBP-His-Ccdc85c (50–160). (F) Analytical gel filtration analysis showing that Trx-Par3 (1070–1217) forms a stable complex with MBP-His-Ccdc85c (50–160). The elution volumes of the molecular size markers are indicated at the top of the panel. (G) ITC-based measurement of the binding between Trx-Par3 (1070–1217) and MBP-His-Ccdc85c (50–160).

Article Snippet: Mouse Numb and human Par3 plasmid were co-transfected into HEK293T cells (from ATCC) using PEI (Poly-sciences) as the transfection reagent.Cellswereharvested36hpost transfectionand lysed in lysisbuffer.

Techniques: Immunoprecipitation, Binding Assay, Pull Down Assay

Journal: Cell reports

Article Title: Ccdc85c-Par3 condensates couple cell polarity with Notch to control neural progenitor proliferation.

doi: 10.1016/j.celrep.2023.112677

Figure Lengend Snippet: Figure 3. Ccdc85c binds to Par3 via the electrostatic interaction (A) Multiple sequence alignment of Ccdc85c and Par3 in different species and color coded by sequence identity in blue or pink, respectively. The residues responsible for electrostatic interaction are indicated with red rectangles. (B) GST pull-down assay showing that NaCl significantly weakens the interaction between MBP-His-Ccdc85c (50–160) and Trx-Par3 (1070–1217) in a dose- dependent manner. (C) Mutagenesis of the positively charged clusters in Trx-Par3 (1070–1217) impairs the binding to MBP-His-Ccdc85c (50–160). K1115–1116R1118/A is referred to as K1115A, R1137Q1139R1140R1142Q1143/A as R1137A, and R1157–1159/A as R1157A.

Article Snippet: Mouse Numb and human Par3 plasmid were co-transfected into HEK293T cells (from ATCC) using PEI (Poly-sciences) as the transfection reagent.Cellswereharvested36hpost transfectionand lysed in lysisbuffer.

Techniques: Sequencing, Pull Down Assay, Mutagenesis, Binding Assay

Journal: Cell reports

Article Title: Ccdc85c-Par3 condensates couple cell polarity with Notch to control neural progenitor proliferation.

doi: 10.1016/j.celrep.2023.112677

Figure Lengend Snippet: Figure 4. The interaction between Ccdc85c and Par3 is required for the maintenance of RGC in the VZ (A) Representative images of brain sections electroporated with plasmids expressing BLBP-GFP together with either plasmid expressing scramble, ShCcdc85c, ShCcdc85c and hCCDC85C, or ShCcdc85c and hCCDC85C-D/A. Expression of hCCDC85C-D/A-carrying mutations disrupted the interaction between Ccdc85c and Par3, which could not rescue the delamination of RGC from the VZ, causing the downregulation of Ccdc85c. The identity of the RGC is verified by staining for Pax6. (B and C) Quantifications of the distribution of GFP+ cells in VZ (B) and IZ (C). (D) Quantifications of the percentage of GFP+ and Pax6+ cells. Scale bar: 50 mm. At least three brains were analyzed. Data are shown as mean ± SEM. One-way ANOVA test was used. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Mouse Numb and human Par3 plasmid were co-transfected into HEK293T cells (from ATCC) using PEI (Poly-sciences) as the transfection reagent.Cellswereharvested36hpost transfectionand lysed in lysisbuffer.

Techniques: Expressing, Plasmid Preparation, Staining

Journal: Cell reports

Article Title: Ccdc85c-Par3 condensates couple cell polarity with Notch to control neural progenitor proliferation.

doi: 10.1016/j.celrep.2023.112677

Figure Lengend Snippet: Figure 5. Ccdc85c binding relieves Par3 from the auto-inhibited conformation to undergo phase separation and bind to Numb (A and B) Par3/Ccdc85c complex forms condensed puncta in living cells. (A) Representative images showing subcellular localization of Par3 and Ccdc85c, when expressed in COS7 cells. When expressed alone, mCherry-Ccdc85c (50– 160) and GFP-Par3 both showed diffused cytoplasmic localization. Co-expression of Ccdc85c and Par3 WT, but not the R1137A mutant, formed scattered bright puncta in cells. Nuclei were stained by DAPI. Scale bar: 20 mm. (B) Statistical data for (A). n represents the number of independent experimental cell culture batches, with >600 cells counted for each batch. Specimens’ statistics are presented as mean ± SEM; n.s., not significant, *p < 0.05, **p < 0.01, and ***p < 0.001 using one-way ANOVA with Tukey’s multiple comparison test. (C) Competition assay showing that MBP-His-Ccdc85c (50–160) could weaken the interaction between GST-Par3 PDZ1 and HA-Par3C in a dose-dependent manner. High-concentration salt (1 M NaCl) could also disrupt PDZ1/Par3C packing. HA-Par3 was detected by western blotting, and the other proteins were stained by Coomassie brilliant blue. (D) Quantification of FRET efficiency for CFP-YFP (n = 11), CFP-Par3-YFP (n = 11), CFP-Par3-YFP co-expressed with Ccdc85c (n = 11), and CFP-Par3D4N1/N2- YFP (n = 11). Error bars represent SEM. ****p < 0.0001. (E) coIP assay showing that MBP-His-Ccdc85c (50–160) can enhance the interaction between full-length FLAG-Numb and Myc-Par3 in a dose-dependent manner. MBP-His-Ccdc85c (50–160) was stained by Coomassie brilliant blue, and the other proteins were detected by western blotting.

Article Snippet: Mouse Numb and human Par3 plasmid were co-transfected into HEK293T cells (from ATCC) using PEI (Poly-sciences) as the transfection reagent.Cellswereharvested36hpost transfectionand lysed in lysisbuffer.

Techniques: Binding Assay, Expressing, Mutagenesis, Staining, Cell Culture, Comparison, Competitive Binding Assay, Concentration Assay, Western Blot, Co-Immunoprecipitation Assay

Journal: Cell reports

Article Title: Ccdc85c-Par3 condensates couple cell polarity with Notch to control neural progenitor proliferation.

doi: 10.1016/j.celrep.2023.112677

Figure Lengend Snippet: Figure 6. Ccdc85c promotes the recruitment and concentration of Numb to Par3 condensates (A) Co-expression of mCherry-Numb, GFP-Par3 WT, or R1137A mutant and FLAG-Ccdc85c in COS7 cells. Numb was enriched in Par3 puncta when co-ex- pressed with Ccdc85c, whereas the introduction of the Par3 R1137A mutation or the absence of Ccdc85c led to diffused localization of Numb and Par3 in the cytoplasm. Scale bar: 20 mm. (B) Statistical data for (A). n represents the number of independent experimental cell culture batches, with >600 cells counted for each batch. Specimens’ statistics are presented as mean ± SEM; n.s., not significant, *p < 0.05, **p < 0.01, and ***p < 0.001 using one-way ANOVA with Tukey’s multiple comparison test. (C) Representative time-lapse FRAP images showing that GFP-Numb, GFP-Par3, and GFP-Ccdc85c signals within the condensed puncta recovered within a few minutes after photobleaching. Scale bar: 10 mm. (D) Statistical data for (C). The green curve represents the averaged FRAP data of 20 puncta from 10 cells. The black curve represents the averaged FRAP data of 15 puncta from 8 cells. The red curve represents the averaged FRAP data of 20 puncta from 14 cells. Time 0 refers to the time point of the photobleaching pulse. All data are represented as mean ± SD.

Article Snippet: Mouse Numb and human Par3 plasmid were co-transfected into HEK293T cells (from ATCC) using PEI (Poly-sciences) as the transfection reagent.Cellswereharvested36hpost transfectionand lysed in lysisbuffer.

Techniques: Concentration Assay, Expressing, Mutagenesis, Cell Culture, Comparison

Journal: Cell reports

Article Title: Ccdc85c-Par3 condensates couple cell polarity with Notch to control neural progenitor proliferation.

doi: 10.1016/j.celrep.2023.112677

Figure Lengend Snippet: Figure 7. Ccdc85c acts through Notch signaling to regulate the maintenance of RGCs in the VZ (A) Representative images of brain sections showing that over-expression of plasmids expressing NICD (Notch intracellular domain) or shRNAs against Numb/ Numb-like (ShNb/Nbl) could rescue the deamination of RGCs from the VZ caused by the downregulation of Ccdc85c. (B) Quantification of the number of GFP+ cells located at the VZ surface. (C) Quantification of the percentage of proliferating cells (GFP and Ki67 double positive). Scale bar: 50 mm. At least three brains were analyzed. Data are shown as mean ± SEM. One-way ANOVA test was used. ***p < 0.001. (D) Working model. Ccdc85c interacts with the apical polarity protein Par3, which promotes the PSCP of Par3 and the recruitment of Numb, the Notch regulator. This, in turn, leads to the activation of Notch signaling and promotes RGC proliferation. In contrast, Par3 undergoes an auto-inhibited conformation that cannot recruit Numb. The released Numb antagonizes Notch activity, which leads to RGC differentiation.

Article Snippet: Mouse Numb and human Par3 plasmid were co-transfected into HEK293T cells (from ATCC) using PEI (Poly-sciences) as the transfection reagent.Cellswereharvested36hpost transfectionand lysed in lysisbuffer.

Techniques: Over Expression, Expressing, Activation Assay, Activity Assay