Journal: bioRxiv

Article Title: CRB2 Depletion Induces YAP Signaling and Disrupts Mechanosensing in Podocytes

doi: 10.1101/2024.10.22.619513

Figure Lengend Snippet: CRB2 is expressed throughout the apical membrane of mature, immortalized human podocytes and demonstrates regional colocalization with the slit diaphragm protein TRPC6 (Inset). Scale bar, 25 µm.

Article Snippet: After washing the cells for 5 min twice, they were incubated with the following primary antibodies at 4°C overnight: nephrin rabbit polyclonal antibody (1:200, Thermo Fisher Scientific, PA5-20330), synaptopodin mouse monoclonal antibody (1:200, Progen Biotechnik, 61094), FAK1mouse monoclonal antibody (1:100, Millipore Sigma, 05-537), CRB2 rabbit polyclonal antibody (1:100, BIOSS, BS-14046R), TRPC6 mouse monoclonal antibody (1:100, Abcam, 105845), and at room temperature for one hour: paxillin rabbit monoclonal antibody (1:1200, Cell Signaling, 50195) in 1% BSA.

Techniques: Membrane

Journal: bioRxiv

Article Title: CRB2 Depletion Induces YAP Signaling and Disrupts Mechanosensing in Podocytes

doi: 10.1101/2024.10.22.619513

Figure Lengend Snippet: a) Phase contrast images and ERISM displacement maps of representative differentiated control (siScr) and CRB2 knockdown (si CRB2 ) podocytes on an ERISM substrate with an apparent stiffness of 53 kPa. Cell outlines are indicated by white-dashed lines. Groups were treated without (DMSO) or with 100 nM of K-975 for 24 hours. b – d) Mean indented volume (symbols), means (central lines) and ±SD (boxes) of single podocytes after 24 h of treatment with b) 100 nM of K-975, c) verteporfin with various of concentrations, and d) 100 nM of rapamycin for siScr and si CRB2 podocytes. All groups were treated with 0.2% of DMSO. N ≥ 10. Scale bar, 50 μm. Groups were compared using two-tailed t -tests with non-equal variance, n.s.: no significance, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001.

Article Snippet: After washing the cells for 5 min twice, they were incubated with the following primary antibodies at 4°C overnight: nephrin rabbit polyclonal antibody (1:200, Thermo Fisher Scientific, PA5-20330), synaptopodin mouse monoclonal antibody (1:200, Progen Biotechnik, 61094), FAK1mouse monoclonal antibody (1:100, Millipore Sigma, 05-537), CRB2 rabbit polyclonal antibody (1:100, BIOSS, BS-14046R), TRPC6 mouse monoclonal antibody (1:100, Abcam, 105845), and at room temperature for one hour: paxillin rabbit monoclonal antibody (1:1200, Cell Signaling, 50195) in 1% BSA.

Techniques: Control, Knockdown, Two Tailed Test

Journal: bioRxiv

Article Title: CRB2 Depletion Induces YAP Signaling and Disrupts Mechanosensing in Podocytes

doi: 10.1101/2024.10.22.619513

Figure Lengend Snippet: a) Family DUK40595 is a 2-generation Asian kindred from India. The proband (arrow head) has biopsy-proven FSGS and her younger male sibling has proteinuric nephropathy likely caused by the FSGS lesion. The cause of FSGS in both affected siblings was found to be a compound heterozygous mutation of CRB2 (FSGS-9). The pathogenic mutation is comprised of a previously described truncating frameshift mutation (p.Gly1036_Alafs*43) and a rare 9-bp deletion mutation (p.Leu1074_Asp1076del). b) Schematic of CRB2 protein functional domains. Family DUK40595 pathogenic mutations identified in the 3rd Laminin G domain (p.Gly1036Alafs*43, [* in orange]) and the 12th EGF-like domain (p.Leu1074_Asp1076del, [* in black]). c) The 9-bp, in-frame deletion results in the loss of three highly conserved amino acids (Asp-Leu-Phe) d) which significantly distorts the structure of the EGF-like domain.

Article Snippet: After washing the cells for 5 min twice, they were incubated with the following primary antibodies at 4°C overnight: nephrin rabbit polyclonal antibody (1:200, Thermo Fisher Scientific, PA5-20330), synaptopodin mouse monoclonal antibody (1:200, Progen Biotechnik, 61094), FAK1mouse monoclonal antibody (1:100, Millipore Sigma, 05-537), CRB2 rabbit polyclonal antibody (1:100, BIOSS, BS-14046R), TRPC6 mouse monoclonal antibody (1:100, Abcam, 105845), and at room temperature for one hour: paxillin rabbit monoclonal antibody (1:1200, Cell Signaling, 50195) in 1% BSA.

Techniques: Mutagenesis, Functional Assay

Journal: bioRxiv

Article Title: CRB2 Depletion Induces YAP Signaling and Disrupts Mechanosensing in Podocytes

doi: 10.1101/2024.10.22.619513

Figure Lengend Snippet: Representative kidney biopsy images with CRB2 and Nephrin staining in a healthy human glomerulus (control), a patient with FSGS due to a pathogenic TRPC6 mutation and a patient with biopsy proven FSGS bearing the pathogenic truncating p.Gly1036Alafs*43 mutation. The uniform distribution of podocyte CRB2 staining in the control is largely preserved but mildly irregular in the patient with the TRPC6 mutation. Notably, in the patient with FSGS due to the truncating CRB2 mutation, CRB2 staining is obliterated, consistent with the predicted effect of the mutation to prevent the transmembrane insertion of the protein.

Article Snippet: After washing the cells for 5 min twice, they were incubated with the following primary antibodies at 4°C overnight: nephrin rabbit polyclonal antibody (1:200, Thermo Fisher Scientific, PA5-20330), synaptopodin mouse monoclonal antibody (1:200, Progen Biotechnik, 61094), FAK1mouse monoclonal antibody (1:100, Millipore Sigma, 05-537), CRB2 rabbit polyclonal antibody (1:100, BIOSS, BS-14046R), TRPC6 mouse monoclonal antibody (1:100, Abcam, 105845), and at room temperature for one hour: paxillin rabbit monoclonal antibody (1:1200, Cell Signaling, 50195) in 1% BSA.

Techniques: Staining, Control, Mutagenesis

Journal: bioRxiv

Article Title: CRB2 Depletion Induces YAP Signaling and Disrupts Mechanosensing in Podocytes

doi: 10.1101/2024.10.22.619513

Figure Lengend Snippet: a) Plot of relative quantification of CRB2 gene expression in scrambled siRNA (siScr) and CRB2 -knockdown (si CRB2 ) podocytes from three quantitative polymer chain reaction (qPCR) replicates. Mean values (boxes) and standard deviation (error bar). Groups were compared using a paired-sample t -test with non-equal variance, **: p ≤ 0.005. b) Phase contrast and immunofluorescence images of nephrin (red), synaptopodin (red) and nuclei (detected by DAPI, blue) for proliferating (at 33 °C) and 12-day-differentiaed (at 37 °C) siScr and si CRB2 podocytes. Cell outlines are indicated by white-dashed lines in the immunofluorescence images. Scale bars, 50 μm. c) Comparison of cell area between proliferating and 12-day-differentiated podocytes (dots), showing mean (lines) and ±1 SD (boxes) for the siScr and si CRB2 cells. Groups were compared using two-tailed t -tests with non-equal variance, n.s.: p > 0.05, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001.

Article Snippet: After washing the cells for 5 min twice, they were incubated with the following primary antibodies at 4°C overnight: nephrin rabbit polyclonal antibody (1:200, Thermo Fisher Scientific, PA5-20330), synaptopodin mouse monoclonal antibody (1:200, Progen Biotechnik, 61094), FAK1mouse monoclonal antibody (1:100, Millipore Sigma, 05-537), CRB2 rabbit polyclonal antibody (1:100, BIOSS, BS-14046R), TRPC6 mouse monoclonal antibody (1:100, Abcam, 105845), and at room temperature for one hour: paxillin rabbit monoclonal antibody (1:1200, Cell Signaling, 50195) in 1% BSA.

Techniques: Expressing, Knockdown, Polymer, Standard Deviation, Immunofluorescence, Comparison, Two Tailed Test

Journal: bioRxiv

Article Title: CRB2 Depletion Induces YAP Signaling and Disrupts Mechanosensing in Podocytes

doi: 10.1101/2024.10.22.619513

Figure Lengend Snippet: a ) Luciferase reporter assay of average expression of transcriptional enhanced associate domain (TEAD) in siScr and si CRB2 podocytes. N = 6. b ) Western blots of non-phosphorylated YAP (np-YAP), Thrombospondin-1 (THBS-1) and total YAP expression in siScr and si CRB2 podocytes. c ) and d ) Relative expression level of np-YAP and THBS-1 compared to total YAP for both cell lines using data obtained from b). e ) Enzyme-linked immunoassay (ELISA) analysis of expression of YAP-target genes for both cell lines. TGF, transforming growth factor; CTGF, Connective tissue growth factor; Cyr61, Cysteine-rich angiogenic inducer 61; CCN1/2, Cellular communication network factor 1 or 2; EN1, Endothelin 1. N = 6. Mean values (boxes) and ±S.E.M. (error bars). Groups in panels a), c), d), and e) were compared using two-tailed t -tests with non-equal variance, n.s.: p > 0.05, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001.

Article Snippet: After washing the cells for 5 min twice, they were incubated with the following primary antibodies at 4°C overnight: nephrin rabbit polyclonal antibody (1:200, Thermo Fisher Scientific, PA5-20330), synaptopodin mouse monoclonal antibody (1:200, Progen Biotechnik, 61094), FAK1mouse monoclonal antibody (1:100, Millipore Sigma, 05-537), CRB2 rabbit polyclonal antibody (1:100, BIOSS, BS-14046R), TRPC6 mouse monoclonal antibody (1:100, Abcam, 105845), and at room temperature for one hour: paxillin rabbit monoclonal antibody (1:1200, Cell Signaling, 50195) in 1% BSA.

Techniques: Luciferase, Reporter Assay, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: bioRxiv

Article Title: CRB2 Depletion Induces YAP Signaling and Disrupts Mechanosensing in Podocytes

doi: 10.1101/2024.10.22.619513

Figure Lengend Snippet: a) Phase-contrast images and b) ERISM displacement maps of representative control (siScr) and CRB2 knock-down (si CRB2 ) podocytes on ERISM substrates with an apparent stiffness of 74 kPa on Day 1, 8 and 12 of the differentiation at 37 °C. Scale bars, 50 μm. c-f) Evolution of mean indented sensor volume (symbols) and ±S.E.M. (error bars) of siScr (green) and si CRB2 (orange) podocytes during differentiation, measured on ERISM substrates with an apparent stiffness of 11, 53, 74 and 142 kPa, respectively. N ≥ 20 for each group. g) Relative change of mean indented volume between Day 12 and Day 1 of differentiation (symbols) and ±SD (error bars) of siScr (green) and si CRB2 (orange) podocytes. Relative knockdown (KD) effect, i.e. the ratio of relative change, si CRB2 /siScr cell line, for ERISM substrate of different stiffness (grey symbols, right y-axis). Groups were compared using two-tailed t -tests with non-equal variance, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001.

Article Snippet: After washing the cells for 5 min twice, they were incubated with the following primary antibodies at 4°C overnight: nephrin rabbit polyclonal antibody (1:200, Thermo Fisher Scientific, PA5-20330), synaptopodin mouse monoclonal antibody (1:200, Progen Biotechnik, 61094), FAK1mouse monoclonal antibody (1:100, Millipore Sigma, 05-537), CRB2 rabbit polyclonal antibody (1:100, BIOSS, BS-14046R), TRPC6 mouse monoclonal antibody (1:100, Abcam, 105845), and at room temperature for one hour: paxillin rabbit monoclonal antibody (1:1200, Cell Signaling, 50195) in 1% BSA.

Techniques: Control, Knockdown, Two Tailed Test

Journal: bioRxiv

Article Title: CRB2 Depletion Induces YAP Signaling and Disrupts Mechanosensing in Podocytes

doi: 10.1101/2024.10.22.619513

Figure Lengend Snippet: a) Composite immunofluorescence images of the representative cells shown in Figure 5. F-actin (labelled by TRITC-phalloidin, green), paxillin (red) and nuclei (labelled by DAPI, blue) of a differentiated control podocyte (left), a zoom-in on the area marked by the white square (middle), and same staining for a differentiated si CRB2 podocyte (right). b) Corresponding ERISM displacement and c) Fourier-filtered ERISM maps, with black lines denoting the outlines of paxillin-rich areas. Scale bars, 50 μm. F-actin, filamentous actin.

Article Snippet: After washing the cells for 5 min twice, they were incubated with the following primary antibodies at 4°C overnight: nephrin rabbit polyclonal antibody (1:200, Thermo Fisher Scientific, PA5-20330), synaptopodin mouse monoclonal antibody (1:200, Progen Biotechnik, 61094), FAK1mouse monoclonal antibody (1:100, Millipore Sigma, 05-537), CRB2 rabbit polyclonal antibody (1:100, BIOSS, BS-14046R), TRPC6 mouse monoclonal antibody (1:100, Abcam, 105845), and at room temperature for one hour: paxillin rabbit monoclonal antibody (1:1200, Cell Signaling, 50195) in 1% BSA.

Techniques: Immunofluorescence, Control, Staining

Journal: bioRxiv

Article Title: CRB2 Depletion Induces YAP Signaling and Disrupts Mechanosensing in Podocytes

doi: 10.1101/2024.10.22.619513

Figure Lengend Snippet: a) Representative immunofluorescence images of F-actin (labelled by TRITC-phalloidin, green), paxillin (red) and nuclei (labelled by DAPI, blue) for 12-day-differentiated control (siScr) and CRB2 knockdown (si CRB2 ) podocytes. Scale bars, 50 μm. F-actin, filamentous actin; YAP, yes-associated protein. b) Western blot of phosphorylated paxillin, focal adhesion kinase (p-paxillin, p-FAK) and total FAK in siScr and si CRB2 podocytes. Since the experiment was not repeated, we did not perform quantitative analysis. Mean fluorescence intensity of c) F-actin and e) paxillin per cell for both cell lines. d) Actin cytoskeleton order parameter (nematic order, S ) for both cell lines. S = 0 for isotropic, non-polarized and S = 1 for perfectly linearly polarized actin cytoskeleton. f) Numbers of focal adhesion (FA) clusters per cell (quantified via the counts of paxillin dots in immunofluorescence images), g) mean size of individual FAs and h) ratio of total FA area across a cell to its size for differentiated siScr and si CRB2 podocytes. Shown are the values for individual cells (symbols), the mean (central lines) and ±SD (boxes). At least 6 cells were measured per group. Groups were compared using two-tailed student t -tests with non-equal variance, n.s.: no significance, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001.

Article Snippet: After washing the cells for 5 min twice, they were incubated with the following primary antibodies at 4°C overnight: nephrin rabbit polyclonal antibody (1:200, Thermo Fisher Scientific, PA5-20330), synaptopodin mouse monoclonal antibody (1:200, Progen Biotechnik, 61094), FAK1mouse monoclonal antibody (1:100, Millipore Sigma, 05-537), CRB2 rabbit polyclonal antibody (1:100, BIOSS, BS-14046R), TRPC6 mouse monoclonal antibody (1:100, Abcam, 105845), and at room temperature for one hour: paxillin rabbit monoclonal antibody (1:1200, Cell Signaling, 50195) in 1% BSA.

Techniques: Immunofluorescence, Control, Knockdown, Western Blot, Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: CRB2 Depletion Induces YAP Signaling and Disrupts Mechanosensing in Podocytes

doi: 10.1101/2024.10.22.619513

Figure Lengend Snippet: Focal Adhesion Kinase (FAK) staining is used to identify focal adhesions by indirect immunofluorescence imaging in siScr and si CRB2 podocytes (Insets). Although the number of focal adhesions per cell is similar in both lines, the focal adhesion density is greater in si CRB2 podocytes due to their significantly reduced cell area as shown in of the main text. Scale bar, 25 µm.

Article Snippet: After washing the cells for 5 min twice, they were incubated with the following primary antibodies at 4°C overnight: nephrin rabbit polyclonal antibody (1:200, Thermo Fisher Scientific, PA5-20330), synaptopodin mouse monoclonal antibody (1:200, Progen Biotechnik, 61094), FAK1mouse monoclonal antibody (1:100, Millipore Sigma, 05-537), CRB2 rabbit polyclonal antibody (1:100, BIOSS, BS-14046R), TRPC6 mouse monoclonal antibody (1:100, Abcam, 105845), and at room temperature for one hour: paxillin rabbit monoclonal antibody (1:1200, Cell Signaling, 50195) in 1% BSA.

Techniques: Staining, Immunofluorescence, Imaging

Journal: bioRxiv

Article Title: CRB2 Depletion Induces YAP Signaling and Disrupts Mechanosensing in Podocytes

doi: 10.1101/2024.10.22.619513

Figure Lengend Snippet: Mean indented volume (symbols), means (central lines) and ±SD (boxes) of individual 12-day differentiated podocytes after 24 h of verteporfin treatment at two different concentrations in dark and in DMSO control, for CRB2 knockdown (si CRB2 ) podocytes. Excessive cell death was observed for si CRB2 podocytes treated with 5 µM of verteporfin. All groups were treated with 0.2% of DMSO and N ≥ 6. Groups were compared using two-tailed t -tests with non-equal variance, n.s.: no significance, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001.

Article Snippet: After washing the cells for 5 min twice, they were incubated with the following primary antibodies at 4°C overnight: nephrin rabbit polyclonal antibody (1:200, Thermo Fisher Scientific, PA5-20330), synaptopodin mouse monoclonal antibody (1:200, Progen Biotechnik, 61094), FAK1mouse monoclonal antibody (1:100, Millipore Sigma, 05-537), CRB2 rabbit polyclonal antibody (1:100, BIOSS, BS-14046R), TRPC6 mouse monoclonal antibody (1:100, Abcam, 105845), and at room temperature for one hour: paxillin rabbit monoclonal antibody (1:1200, Cell Signaling, 50195) in 1% BSA.

Techniques: Control, Knockdown, Two Tailed Test

Journal: bioRxiv

Article Title: CRB2 Depletion Induces YAP Signaling and Disrupts Mechanosensing in Podocytes

doi: 10.1101/2024.10.22.619513

Figure Lengend Snippet: Schematic diagram of the possible signaling pathways involved upon CRB2 knockdown. a) In healthy podocytes, the Crumbs complex associates with the PAR complex, thereby facilitating normal cell spreading and contractility. Either the interaction with crumbs complex or the activation of MST1/2 and LATS1/2 signaling of the Hippo pathway may promote YAP phosphorylation (deactivation), leading to retention or degradation of cytoplasmic YAP. b) In CRB2 mutant-related nephropathy, the activity of Rac1 might be suppressed by the disassociation of the PAR complex from the Crumbs complex. Together with the activated TGF-β signaling, this may lead to a higher activity of RhoA/ROCK/Myosin II signaling, thereby resulting in a smaller cell spreading area and an enhanced cell contractility. In the absence of CRB2, upregulated RhoA-and FAK-mediated LATS1/2 suppression may also result in nuclear translocation of YAP and upregulation of YAP target genes such as THBS-1 , CTGF , CYR61 , EN1 and TGF-β , etc. FAK signaling is activated by THBS-1, leading to the upregulation of paxillin. Question marks, assumptions. Solid lines, normal signaling. Dashed lines, reduced signaling. AMOT, angiomotin. CRB2, crumbs 2. CTGF, connective tissue growth factor. CYR61, cysteine-rich angiogenic inducer 61. FAK, focal adhesion kinase. GTP, guanine triphosphate. LATS1/2, large tumor suppressor kinase 1/2. MST1/2, mammalian sterile 20-like kinase 1/2. P, phosphate. PAR, protease activated receptor complex. Rac1, Ras-related C3 botulinum toxin substrate 1. RhoA, Ras homolog family member A. ROCK, Rho-associated protein kinase. TEAD, transcriptional enhanced associate domain. TGF-β, transforming growth factor beta. THBS-1, thrombospondin 1. YAP, yes-associated protein.

Article Snippet: After washing the cells for 5 min twice, they were incubated with the following primary antibodies at 4°C overnight: nephrin rabbit polyclonal antibody (1:200, Thermo Fisher Scientific, PA5-20330), synaptopodin mouse monoclonal antibody (1:200, Progen Biotechnik, 61094), FAK1mouse monoclonal antibody (1:100, Millipore Sigma, 05-537), CRB2 rabbit polyclonal antibody (1:100, BIOSS, BS-14046R), TRPC6 mouse monoclonal antibody (1:100, Abcam, 105845), and at room temperature for one hour: paxillin rabbit monoclonal antibody (1:1200, Cell Signaling, 50195) in 1% BSA.

Techniques: Knockdown, Activation Assay, Mutagenesis, Activity Assay, Translocation Assay, Sterility