Journal: bioRxiv

Article Title: Domain-specific mechanisms of YAP1 variants in ocular coloboma revealed by in-vitro and organoid studies

doi: 10.1101/2025.10.28.685193

Figure Lengend Snippet: YAP1 expression during early human eye development. (A) Expression of phospho-YAP1 (P-YAP1(Ser127)) and YAP1 in human embryonic ocular tissue (CS15-16)[Scale: 50µm]. (B) Expression of YAP1 co-localised with VSX2+ retinal progenitors in day 35 retinal organoids [Scale: 40µm]. (C) Levels of 4/8 YAP1 isoforms in iPSCs, day 35 (D35) and day 60 (D60) retinal organoids. POM: periocular mesenchyme. pR: presumptive retina. pRPE: presumptive retinal pigmented epithelium. OF: optic fissure. iPSC: induced pluripotent stem cell.

Article Snippet: To visualise the impact of the studied genetic variants, we used YAP1 structure models 3KYS (YAP1-TEAD complex) and 5YDY (YAP1-LATS complex), both extracted from the Protein Databank (PDB)[ , ].

Techniques: Expressing

Journal: bioRxiv

Article Title: Domain-specific mechanisms of YAP1 variants in ocular coloboma revealed by in-vitro and organoid studies

doi: 10.1101/2025.10.28.685193

Figure Lengend Snippet: Modelling effects of YAP1 variants on protein structure. (A) Pedigree for YAP1 p.Glu356Asp. (B) Location of YAP1 variants from this study in different protein domains. (C-G) Introduction of YAP1 variants in solved protein structures. (C-E) YAP1-TEAD complex: p.Val55Leu, p.Met86Thr, p.Phe95Ser. (F-G) YAP1-LATS: p.Lys254Arg, p.Ser257Phe. Green and blue dots represent favourable van der Waals interactions, yellow and pink spikes represent unfavourable atomic overlaps. (H) Western blot showing non-degraded YAP1 variant proteins.

Article Snippet: To visualise the impact of the studied genetic variants, we used YAP1 structure models 3KYS (YAP1-TEAD complex) and 5YDY (YAP1-LATS complex), both extracted from the Protein Databank (PDB)[ , ].

Techniques: Western Blot, Variant Assay

Journal: bioRxiv

Article Title: Domain-specific mechanisms of YAP1 variants in ocular coloboma revealed by in-vitro and organoid studies

doi: 10.1101/2025.10.28.685193

Figure Lengend Snippet: YAP1 variants affect protein localisation and transcriptional activity. (A) Sub-cellular localisation of EYFP-YAP1 wild-type (WT), p.Met86Thr and p.Ser257Phe variants. [Scale: 50µm] (B) Quantification of relative nuclear vs. cytoplasmic localisation of YAP1 variants with respect to WT EYFP-YAP1, with experimental control p.Ser127Ala. >100 cells analysed from 3 biological replicates (N=3, n≥100). Data presented as box and whiskers plot representing 25 th to 75 th percentile with a line at the median (C) Fold change in TEAD-dependent luciferase assay of EYFP-YAP1 WT, variants and control p.Ser94Ala. N=3. (D) Fold-change in expression of endogenous CYR61 and CTGF by EYFP-YAP1 WT, variants and control p.Ser94Ala. N=3. Bar charts represent mean ± SD. Statistical significance was defined as: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****); ns = not significant.

Article Snippet: To visualise the impact of the studied genetic variants, we used YAP1 structure models 3KYS (YAP1-TEAD complex) and 5YDY (YAP1-LATS complex), both extracted from the Protein Databank (PDB)[ , ].

Techniques: Activity Assay, Control, Luciferase, Expressing

Journal: bioRxiv

Article Title: Domain-specific mechanisms of YAP1 variants in ocular coloboma revealed by in-vitro and organoid studies

doi: 10.1101/2025.10.28.685193

Figure Lengend Snippet: Quantification of YAP1-TEAD1 binding by fluorescence cross correlation spectroscopy (FCCS) (A) Schematic of FCCS principle to measure diffusion and protein-protein interaction strength of fluorescent tagged YAP1 and TEAD1 in the nucleus. (B) Representative image of YAP1 and TEAD1 expression in HEK293 LTV cells. (C) Representative (i) auto-and (ii) cross-correlation data showing raw data and fit lines for total EYFP-YAP1 and TEAD1-mScarlet and complexed fluorescent proteins (YAP1-TEAD1). (D) Dissociation constant (Kd) of YAP1-TEAD1 binding affinity of EYFP-YAP1 WT, variants and control p.Ser94Ala. N≥3. Bars show mean ± SD. (E) Diffusion rate for EYFP-YAP1 WT, variants and control p.Ser94Ala measured from auto-correlation data. All data points plotted with bars at mean. (F) Luciferase activity vs YAP1-TEAD1 dissociation constant (inverse of binding affinity) for EYFP-YAP1 WT and variants. Error bars are ± SD. Statistical significance was defined as: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****); ns = not significant.

Article Snippet: To visualise the impact of the studied genetic variants, we used YAP1 structure models 3KYS (YAP1-TEAD complex) and 5YDY (YAP1-LATS complex), both extracted from the Protein Databank (PDB)[ , ].

Techniques: Binding Assay, Fluorescence, Spectroscopy, Diffusion-based Assay, Expressing, Control, Luciferase, Activity Assay

Journal: bioRxiv

Article Title: Domain-specific mechanisms of YAP1 variants in ocular coloboma revealed by in-vitro and organoid studies

doi: 10.1101/2025.10.28.685193

Figure Lengend Snippet: Functional role of YAP1 in day 40 retinal organoids. (A-B) YAP1 expression after 7 day selinexor (nuclear protein export inhibitor) treatment in organoids and quantification of YAP1 nucleus vs cytoplasmic localisation compared to DMSO control group. [Scale: 10µm]. (C) Cleaved caspase3 and Phosphohistone H3 (PHH3) expression in retinal organoids after 7 day o.25 uM Verteporfin (VPF; YAP1-TEAD inhibitor) treatment of organoids and control DMSO group [Scale: 50µm]. (D-E) Comparison of proliferative (PHH3+) and apoptotic (Caspase3+) cells after VPF treatment compared to DMSO control group. Quantification of PHH3+ cells per 10 6 DAPI intensity units showed a significant reduction in proliferative activity in VPF-treated organoids compared to control. Apoptosis was calculated as the percentage of cleaved Caspase3 signal relative to total DAPI intensity per region of interest (ROI). Data is presented as mean ± SD. N=3. Statistical significance was defined as: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****); ns = not significant.

Article Snippet: To visualise the impact of the studied genetic variants, we used YAP1 structure models 3KYS (YAP1-TEAD complex) and 5YDY (YAP1-LATS complex), both extracted from the Protein Databank (PDB)[ , ].

Techniques: Functional Assay, Expressing, Control, Comparison, Activity Assay