Cell Polarity Complexes Research Articles

Collagen prolyl 4-hydroxylase subunit α member-induced head and neck squamous cell carcinoma aggressiveness is antagonized by LLGL2 via reduced expression of occludin.

There are three isoforms of human collagen prolyl 4-hydroxylases (C-P4Hs), each of which has been reported to play an important role in regulating the progression of a variety of human cancers. By analyzing TGCA datasets on human head and neck squamous cell carcinoma (HNSC), we find that a higher expression of all three C-P4HAs (the α subunit of C-P4Hs) is a superior prognostic indicator than a higher expression of two or a single C-P4HA. Unexpectedly, some patients with higher levels of three C-P4HAs survive longer than patients whose tumors have lower expression of C-P4HAs. Therefore, there may be molecule(s) that can negate the deleterious effects of overexpressing C-P4HAs during cancer progression. By constructing a functional protein interaction network of C-P4HAs and analyzing molecules whose expressions are correlated significantly with that of C-P4HAs, we identify scribble cell polarity complex component 2 (LLGL2) as a factor that antagonizes the effects of overexpressed C-P4HAs on HNSC. Silencing of LLGL2 in the human oral squamous cell line Cal-27 upregulates the expression of occludin and increases cancer cell invasion and migration. In contrast, knocking down C-P4HA alone inhibits cell migration and invasion. Furthermore, simultaneously downregulating three C-P4HAs has more pronounced effects on inhibiting cell migration and invasion. Accordingly, high LLGL2 expression is also a marker indicating improved prognosis in patients with HNSC. These results suggest that the interplay between LLGL2 and C-P4HAs may be targeted to mitigate HNSC tumorigenesis and progression.

Mesenchymal Vangl1 and Vangl2 facilitate airway elongation and widening independently of the planar cell polarity complex.

Adult mammalian lungs exhibit a fractal pattern, as each successive generation of airways is a fraction of the size of the parental branch. Achieving this structure likely requires precise control of airway length and diameter, as the embryonic airways initially lack the fractal scaling observed in the adult. In monolayers and tubes, directional growth can be regulated by the planar cell polarity (PCP) complex. Here, we characterized the roles of PCP complex components in airway initiation, elongation and widening during branching morphogenesis of the lung. Using tissue-specific knockout mice, we surprisingly found that branching morphogenesis proceeds independently of PCP complex function in the lung epithelium. Instead, we found a previously unreported Celsr1-independent role for the PCP complex components Vangl1 and Vangl2 in the pulmonary mesenchyme, where they are required for branch initiation, elongation and widening. Our data thus reveal an explicit function for Vangl1 and Vangl2 that is independent of the core PCP complex, suggesting a functional diversification of PCP complex components in vertebrate development. These data also reveal an essential role for the embryonic mesenchyme in generating the fractal structure of airways in the mature lung.

Abstract 372: Cell polarity proteins as novel regulators of macropinocytosis in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is considered one of the deadliest cancers. This is likely because it is detected at a very late stage and the therapeutic approaches that have been used for its treatment have proven to be quite ineffective. Therefore, new therapeutic strategies are needed for a better prognosis in patients suffering of pancreatic cancer. PDAC cells often rewire their metabolism to survive and progress. This metabolic rewiring permits the acquisition of nutrients by the cells when these are exposed to a poor nutrient microenvironment. One of the most used processes in nutrient stress conditions is the “cell-drinking” pathway macropinocytosis, an endocytic pathway that cancer cells exploit to support cancer metabolism in such unfavorable conditions. Macropinocytosis inhibition has shown to suppress PDAC growth in mice and thus, it has become a promising therapeutic target for PDAC treatment. Here, we describe that atypical Protein Kinase C (aPKC) isoforms Protein Kinase C zeta (PKCζ) and Protein Kinase C iota (PKCι) regulate PDAC macropinocytosis in the context of glutamine stress. Genetic depletion and pharmacological inhibition of aPKC impair macropinocytosis promotion in PDAC cells exposed to a glutamine-deprived environment and in cells treated with DON, a glutamine analog that blocks glutamine metabolism. In epithelial cells, aPKCs are known to regulate the establishment of cell polarity in association with the scaffold proteins Par3 and Par6, constituting the cell polarity complex, which controls the function of different targets such as Par1 kinases. Loss of Par3, Par6 and Par-1a affects glutamine stress associated macropinocytosis in a similar extent than aPKC depletion, presenting a novel role of the cell polarity protein network in the modulation of macropinocytic uptake. Citation Format: Guillem Lambies Barjau, Szu-Wei Lee, Cosimo Commisso. Cell polarity proteins as novel regulators of macropinocytosis in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 372.

MPDZ is associated with immune infiltration and regulates migration and invasion by switching YAP1 phosphorylation in colorectal cancer

BackgroundMultiple PDZ Domain Crumbs Cell Polarity Complex Component (MPDZ) is involved in a few human cancers. However, the features and potential mechanisms of MPDZ in progression of colorectal cancer (CRC) remains unknown. MethodsThe prognostic role of MPDZ in CRC was determined by Kaplan-Meier and univariate regression analysis. Enrichment analysis was performed to characterize crucial pathways of MPDZ. Immune infiltration and immunotherapeutic outcome were further evaluated. CCK8, EDU, transwell, and wound healing assay were used to assess the influence of MPDZ on pernicious performance of CRC cells. CD8+ T cells and CRC cells were co-cultured to explore the effect of MPDZ on the tumor microenvironment. qRT–PCR, western blot, immunoprecipitation (IP), and methylated RNA immunoprecipitation (me-RIP) were implemented in seeking for the potential mechanisms of MPDZ in CRC. ResultsCRC patients with elevated MPDZ expression suffered from significantly worse prognosis. Enrichment analysis revealed that MPDZ involved in pathways related to metastasis and cell cycle in CRC. In addition, MPDZ expression were related to several immunoinhibitors and had the ability to predict immunotherapy response. Finally, in vitro assays demonstrated that MPDZ knockdown inhibited migration, invasion and immune evasion of CRC cells. Mechanistically, MPDZ knockdown enhanced YAP1 phosphorylation by increased LATS1 expression. Moreover, m6A-MPDZ mRNA may be recognized and degraded by m6A recognition protein YTHDF2. ConclusionsMPDZ was critical for CRC development and could be a promising candidate for the treatment of CRC patients.

Helicobacter pylori and Epstein-Barr virus infection in cell polarity alterations.

The asymmetrical distribution of the cellular organelles inside the cell is maintained by a group of cell polarity proteins. The maintenance of polarity is one of the vital host defense mechanisms against pathogens, and the loss of it contributes to infection facilitation and cancer progression. Studies have suggested that infection of viruses and bacteria alters cell polarity. Helicobacter pylori and Epstein-Barr virus are group I carcinogens involved in the progression of multiple clinical conditions besides gastric cancer (GC) and Burkitt's lymphoma, respectively. Moreover, the coinfection of both these pathogens contributes to a highly aggressive form of GC. H. pylori and EBV target the host cell polarity complexes for their pathogenesis. H. pylori-associated proteins like CagA, VacA OipA, and urease were shown to imbalance the cellular homeostasis by altering the cell polarity. Similarly, EBV-associated genes LMP1, LMP2A, LMP2B, EBNA3C, and EBNA1 also contribute to altered cell asymmetry. This review summarized all the possible mechanisms involved in cell polarity deformation in H. pylori and EBV-infected epithelial cells. We have also discussed deregulated molecular pathways like NF-κB, TGF-β/SMAD, and β-catenin in H. pylori, EBV, and their coinfection that further modulate PAR, SCRIB, or CRB polarity complexes in epithelial cells.

Multiple PDZ domain protein regulates sperm motility through CatSper channel

Multiple PDZ domain protein (MPDZ) is a component of the crumbs cell polarity complex and is ubiquitously expressed in mammalian tissues. In mouse sperm, MPDZ was identified as a regulator of the sperm acrosome reaction in the past two decades. Here, a relationship between MPDZ and sperm motility was discovered. During breeding, we found that MPDZ-null mice had smaller litter size. The Ca2+ signal in mouse sperm decreased. Computer-assisted semen analysis revealed that MPDZ-null males had reduced mouse sperm motility. In humans, MPDZ expression is positively associated with sperm motility. Considering the regulatory role of the CatSper channel in sperm motility and fertility, we identified the expression profile of CatSper subunits (CatSper1–4) and found a reduction at both the transcriptional and translational levels in MPDZ-null mouse spermatozoa. However, in vitro, only CatSper1/2 expression was upregulated in MPDZ-overexpressing GC2 cells. Meanwhile, we found an upregulated cell Ca2+ signal when MPDZ was overexpressed in GC2 cells. Mechanical analysis revealed that MPDZ bound to the signal transducer and activator of transcription 3 (Stat3) and promoted its phosphorylation at tyrosine 705 (Y705) to upregulate CatSper1/2 expression. Inhibition of Stat3 (Y705) phosphorylation or Stat3 expression attenuated the effect of MPDZ on CatSper1/2 expression. These results suggested that MPDZ was responsible for the transcriptional regulation of the CatSper channel, at least in part, to regulate Ca2+ signal and sperm motility. To the best of our knowledge, this is the first study to highlight the importance of MPDZ acting as a regulator of sperm motility.

Abstract P2-11-35: A tight junctional scaffold protein MUPP1/MPDZ and its docking proteins, angiomotins in breast cancer

Abstract A tight junctional scaffold protein MUPP1/MPDZ and its docking proteins, angiomotins in breast cancer Han Gao1, Fiona Ruge1, Lin Ye1, Jane Lane1, Eleri Davies2, Wen G. Jiang1, Tracey A. Martin1 1Cardiff University School of Medicine, Heath Park, Cardiff CF14 4XN, UK 2Wales Breast Centre, University Llandough Hospital, Cardiff CF64 2XX, UK Introduction. MUPP1 (Multiple PDZ Domain Crumbs Cell Polarity Complex Component) or MPDZ is a scaffold protein with multiple PDZ domains, critical for multiple protein docking and protein-protein interactions in cells. MUPP1 is particularly interesting as it offers docking sites for a number of tight junction (TJ) proteins and proteins known to regulate cell junction and motility, including angiomotin (AMOT) and angiomotin-like proteins, the latter being important in regulating cell migration of endothelial cells and an important member in TJ protein complexes. The MUPP1 protein complex at cell junctions also includes CAR/CXADR (coxsackie adenovirus receptor) and members of the ERM (Moesin-Ezrin-Radixin) protein family. It was proposed that the junctional-related MUPP1 docking protein complex may collectively have a role in the development and progression of cancer including breast cancer. The present study examined the profile and integrated expression of these MUPP1 docking molecules in relationship with the outcome, clinical and pathological factors of breast cancer. Methods. Using the datasets of an existing breast cancer cohort at Cardiff, we visited the expression profile of the MUPP1/MPDZ docking molecules including MUPP1, four members of the ERM family, including ezrin, moesin, radixin and EHM2 (also known as NF2) and CAR/CXADR (coxsackie adenovirus receptor). The profile and integrated expression of the profile were analysed against clinical, pathological and outcome factors and also the hormone receptor status of breast cancers. Results. MUPP1 is a weakly linked to the disease progression, so as angiomotin, angiomotin-like1 and angiomotin-like2. Alone, only EHM2 showed significance in linking with the survival of the patient’s OS (p=0.38), neither of the remaining MUPP1 complex molecules, including MUPP1, AMOT, AMOTL1, AMOTL2, ezrin, moesin and radixin was able to offer a significant predictive value for the survival of the patients. However, it was found that MUPP1 has a significant correlation with AMOTs, EHM2 and ezrin. When these significantly correlated molecules were integrated in the analysis, the integrated expression pattern showed a highly significant value in evaluating the overall survival (p=0.003) and disease-free survival (p=0.007). Both of these predictive powers are independent in a multivariate analyses including the clinical and hormone receptors for OS (p=0.022, Hazard Ratio (HR)=1.352 95%CI (1.044-1.752)) and RFS (p=0.023, HR=1.241 (95% CI. 1.031-1.498)). It was interesting to note that the link between the expression pattern and OS was more prominent in ER negative (p=0.018) than in ER positive tumours (p=0.177). The same was observed with disease free survival for ER negative (p=0.011) compared with ER positive (p=0.462) tumours. Non-triple negative breast cancer had shown a similar sensitivity compared with triple negative breast cancer. Discussion. MUPP1, a PDZ domain containing docking protein closed linked with TJ structure, forms an interesting expression profile with its docking molecules including angiomotins and EHM2 that allows evaluation of disease progress and clinical outcome of the patients with breast cancer. Citation Format: Han Gao, Fiona Ruge, Lin Ye, Jane Lane, Eleri Davies, Wen G. Jiang, Tracey A. Martin. A tight junctional scaffold protein MUPP1/MPDZ and its docking proteins, angiomotins in breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-11-35.

Bi-allelic variations in CRB2, encoding the crumbs cell polarity complex component 2, lead to non-communicating hydrocephalus due to atresia of the aqueduct of sylvius and central canal of the medulla

Congenital hydrocephalus is a common condition caused by the accumulation of cerebrospinal fluid in the ventricular system. Four major genes are currently known to be causally involved in hydrocephalus, either isolated or as a common clinical feature: L1CAM, AP1S2, MPDZ and CCDC88C. Here, we report 3 cases from 2 families with congenital hydrocephalus due to bi-allelic variations in CRB2, a gene previously reported to cause nephrotic syndrome, variably associated with hydrocephalus. While 2 cases presented with renal cysts, one case presented with isolated hydrocephalus. Neurohistopathological analysis allowed us to demonstrate that, contrary to what was previously proposed, the pathological mechanisms underlying hydrocephalus secondary to CRB2 variations are not due to stenosis but to atresia of both Sylvius Aqueduct and central medullar canal. While CRB2 has been largely shown crucial for apico-basal polarity, immunolabelling experiments in our fetal cases showed normal localization and level of PAR complex components (PKCι and PKCζ) as well as of tight (ZO-1) and adherens (β-catenin and N-Cadherin) junction molecules indicating a priori normal apicobasal polarity and cell–cell adhesion of the ventricular epithelium suggesting another pathological mechanism. Interestingly, atresia but not stenosis of Sylvius aqueduct was also described in cases with variations in MPDZ and CCDC88C encoding proteins previously linked functionally to the Crumbs (CRB) polarity complex, and all 3 being more recently involved in apical constriction, a process crucial for the formation of the central medullar canal. Overall, our findings argue for a common mechanism of CRB2, MPDZ and CCDC88C variations that might lead to abnormal apical constriction of the ventricular cells of the neural tube that will form the ependymal cells lining the definitive central canal of the medulla. Our study thus highlights that hydrocephalus related to CRB2, MPDZ and CCDC88C constitutes a separate pathogenic group of congenital non-communicating hydrocephalus with atresia of both Sylvius aqueduct and central canal of the medulla.

Loss of the crumbs cell polarity complex disrupts epigenetic transcriptional control and cell cycle progression in the developing retina.

The crumbs cell polarity complex plays a crucial role in apical-basal epithelial polarity, cellular adhesion, and morphogenesis. Homozygous variants in human CRB1 result in autosomal recessive Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP), with no established genotype-phenotype correlation. The associated protein complexes have key functions in developmental pathways; however, the underlying disease mechanism remains unclear. Using the oko meduzym289/m289 (crb2a-/- ) zebrafish, we performed integrative transcriptomic (RNA-seq data) and methylomic [reduced representation bisulphite sequencing (RRBS)] analysis of whole retina to identify dysregulated genes and pathways. Delayed retinal cell specification was identified in both the crb2a-/- zebrafish and CRB1 patient-derived retinal organoids, highlighting the dysfunction of cell cycle modulation and epigenetic transcriptional control. Differential DNA methylation analysis revealed novel hypermethylated pathways involving biological adhesion, Hippo, and transforming growth factor β (TGFβ) signalling. By integrating gene expression with DNA methylation using functional epigenetic modules (FEM), we identified six key modules involving cell cycle control and disturbance of TGFβ, bone morphogenetic protein (BMP), Hippo, and SMAD protein signal transduction pathways, revealing significant interactome hotspots relevant to crb2a function and confirming the epigenetic control of gene regulation in early retinal development, which points to a novel mechanism underlying CRB1-retinopathies. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Molecular mechanism of core planar cell polarity complex function elucidated with single-molecule methods.

The planar cell polarity (PCP) signaling pathway polarizes epithelial cells along an axis parallel to the epithelial sheet in order to generate front-back asymmetry at the tissue level. Failures in PCP signaling underlie developmental defects and diseases such as neural tube and heart defects, deafness and contribute to cancer. Six core PCP proteins assemble into large, asymmetric complexes at cell-cell junctions, with Vang and Prickle recruited to one side of the cell-cell junction while Frizzled and Dishevelled are recruited to the other. How this asymmetric assembly process occurs is not known. Genetic evidence suggests that the process of symmetry breaking at PCP clusters is coupled to cluster formation. To test how this occurs, we developed a method to count the number of each of the six core proteins in individual PCP complexes. Specifically, by analyzing bleaching traces obtained with a high frame rate in live Drosophila wing cells imaged using total internal reflection microscopy, we quantify the intensity of single fluorophores in each complex and thereby calculate the molecular number in the corresponding PCP complex. Using this approach, we find that the molecular size distribution of PCP complexes follows an exponential function, indicating a single underlying growth mechanism. We report that specifically, the number of Vang molecules in PCP complexes increases from 15 hours after puparium formation until reaching a steady state around 28 hours. Besides, we measure the asymmetry as a function of cluster size and test the dependence of the observed relationship by introducing gain and loss of function mutations that perturb cluster assembly. In conclusion, our findings provide a detailed understanding of how the PCP mechanism generates stable polarization during embryonic development.

Cell polarity signalling at the birth of multicellularity: What can we learn from the first animals.

The innovation of multicellularity has driven the unparalleled evolution of animals (Metazoa). But how is a multicellular organism formed and how is its architecture maintained faithfully? The defining properties and rules required for the establishment of the architecture of multicellular organisms include the development of adhesive cell interactions, orientation of division axis, and the ability to reposition daughter cells over long distances. Central to all these properties is the ability to generate asymmetry (polarity), coordinated by a highly conserved set of proteins known as cell polarity regulators. The cell polarity complexes, Scribble, Par and Crumbs, are considered to be a metazoan innovation with apicobasal polarity and adherens junctions both believed to be present in all animals. A better understanding of the fundamental mechanisms regulating cell polarity and tissue architecture should provide key insights into the development and regeneration of all animals including humans. Here we review what is currently known about cell polarity and its control in the most basal metazoans, and how these first examples of multicellular life can inform us about the core mechanisms of tissue organisation and repair, and ultimately diseases of tissue organisation, such as cancer.

Aberrant Mucin Expression Profiles Associate With Pediatric Inflammatory Bowel Disease Presentation and Activity.

Intestinal mucosal healing is nowadays preferred as the therapeutic endpoint in inflammatory bowel disease (IBD), but objective measurements at the molecular level are lacking. Because dysregulated mucin expression is suggested to be involved in mucosal barrier dysfunction in IBD, we investigated mucin expression in association with barrier mediators and clinical characteristics in colonic tissue of a pediatric IBD population. In this cross-sectional monocentric study, we quantified messenger RNA (mRNA) expression of mucins, intercellular junctions, and cell polarity complexes in inflamed and noninflamed colonic biopsies from pediatric IBD (n = 29) and non-IBD (n = 15) patients. We then validated mucin expression at protein level and correlated mucin mRNA expression with expression of barrier mediators and clinical data. The expression of MUC1, MUC3A, MUC4, and MUC13 was increased in the inflamed colon of pediatric IBD patients compared with the noninflamed colon of non-IBD control subjects. Especially MUC13 mRNA expression associated with the expression of barrier mediators, including CDH1, OCLN, and TJP2. MUC1 and MUC3B mRNA expression in combination with calprotectin levels most accurately discriminated IBD patients from non-IBD control subjects (90.6% area under the receiver-operating characteristic curve [AUCROC], 92.0% sensitivity, 73.7% specificity), whereas aberrant mRNA expression of MUC1, MUC3A, MUC4, and MUC13 was distinctive for ulcerative colitis and of MUC3B for Crohn's disease. Furthermore, expression of MUC3A, MUC3B, and MUC4 correlated with clinical disease activity (ie, Pediatric Ulcerative Colitis Activity Index and Pediatric Crohn's Disease Activity Index), and of MUC1, MUC2, MUC4, and MUC13 with endoscopic colitis severity in ulcerative colitis patients. Colonic mucin expression is disturbed in pediatric IBD patients and associates with disease activity and presentation, suggesting its use as molecular marker to aid in disease diagnosis and management.

Differential Expression of the Genes Coding for Adipokines and Epithelial Cell Polarity Components in Women With Low and High Mammographic Density

BackgroundWomen with extensive mammographic density (MD) are more likely to develop breast cancer than women with low MD because of a high epithelial component associated with a high proportion of stromal cells. To elucidate the biological association between high MD and risk of breast cancer, we compared the expression of a panel of genes coding for leptin, adiponectin, and some component of cell polarity and adherens junction complexes in dense and non-dense breast tissue. MethodsWe interrogated a public dataset composed by 120 specimens of normal breast tissue with MD evaluation. The differential expression of the selected genes in the 2 MD subgroups was assessed by the Wilcoxon test, whereas Kruskal-Wallis test evaluated the differential expression of single genes in the fatty, epithelium, or nonfatty compartment. Spearman's correlation measured the relationship among genes in the subset with the highest epithelium proportion. ResultsIn high MD, the expression level of PARD6B, CRB3, PATJ, LLGL2, CDH1, and MARVELD2 significantly lowered in tissues with the highest epithelium proportion, whereas, in low MD, the expression level of the genes increased with the increasing of the epithelium proportion. In the low MD subgroup, LEP correlated negatively with PRKCZ and DLG3, whereas, in high MD, such correlation was not observed. ConclusionsThe expression of the genes governing cell polarity establishment and cell-cell adhesion assembly differed significantly in the epithelial component of dense and non-dense breasts. The correlation pattern between LEP and PRKCZ or DLG3 agrees with the role of leptin in cell polarity disruption.

Genomic analysis of paired IDHwt glioblastomas reveals recurrent alterations of MPDZ at relapse after radiotherapy and chemotherapy

PurposeWe aimed to identify genomic drivers of glioblastoma inevitable recurrence. MethodsTen pairs of initial and recurrent frozen IDHwt glioblastoma samples were screened by CGH Array. Next Generation Sequencing (NGS) was then performed on an enriched cohort of 19 pairs. MPDZ alterations were analyzed using TCGA dataset. ResultsNineteen IDHwt glioblastoma patients were included. Median age was 54.5 y/o (37.2–72.8). Using CGH array, unsupervised analysis aggregated the cohort by paired initial and recurrent tumors. Only 44% of CGH Array alterations were conserved at recurrence (amplifications: 55%; deletions: 30%). Two regions (including FPR1, 2 and 3) were lost at relapse: 19q13.33 and 19q13.41. MPDZ and 25 other genes were altered in ≥20% of recurrent tumors. NGS analysis of 29 candidate genes revealed 4 genes with pathogenic mutations: (FPR2, REL, TYRP1 and MPDZ). MPDZ (Multiple PDZ Domain Crumbs Cell Polarity Complex Component) was altered by two pathogenic mutations occurring at relapse. Using TCGA dataset we observed that a lower MPDZ mRNA expression was associated with IDHwt (p < 0.001) and grade IV (p < 0.001) gliomas. Finally, a low mRNA MPDZ expression was significantly correlated to poor overall survival in both IDHwt and IDH mutated gliomas, reinforcing the potential pejorative impact of MPDZ loss. ConclusionOur results suggest that MPDZ is more frequently altered at relapse after radio-chemotherapy in glioblastoma IDHwt patients, suggesting that MPDZ impairment could contribute to the systematic resistance of these tumors opening new therapeutic perspectives.

PCP Protein Inversin Regulates Testis Function Through Changes in Cytoskeletal Organization of Actin and Microtubules.

Inversin is an integrated component of the Frizzled (Fzd)/Dishevelled (Dvl)/Diversin planar cell polarity (PCP) complex that is known to work in concert with the Van Gogh-like protein (eg, Vangl2)/Prickle PCP complex to support tissue and organ development including the brain, kidney, pancreas, and others. These PCP protein complexes are also recently shown to confer developing haploid spermatid PCP to support spermatogenesis in adult rat testes. However, with the exception of Dvl3 and Vangl2, other PCP proteins have not been investigated in the testis. Herein, we used the technique of RNA interference (RNAi) to examine the role of inversin (Invs) in Sertoli cell (SC) and testis function by corresponding studies in vitro and in vivo. When inversin was silenced by RNAi using specific small interfering RNA duplexes by transfecting primary cultures of SCs in vitro or testes in vivo, it was shown that inversin knockdown (KD) perturbed the SC tight junction-barrier function in vitro and in vivo using corresponding physiological and integrity assays. More important, inversin exerted its regulatory effects through changes in the organization of the actin and microtubule cytoskeletons, including reducing the ability of their polymerization. These changes, in turn, induced defects in spermatogenesis by loss of spermatid polarity, disruptive distribution of blood-testis barrier-associated proteins at the SC-cell interface, appearance of multinucleated round spermatids, and defects in the release of sperm at spermiation.

Analysis of Planar Cell Polarity Complexes by Proximity Biotinylation in Xenopus Embryos.

Understanding signaling processes operating in cells during development and disease requires extensive knowledge of protein interactions. Proximity-dependent biotinylation mediated by a promiscuous bacterial biotin ligase is a sensitive approach for evaluating protein interactions under physiological conditions. This technique allows for assessing protein association when conventional pull-down assays are not applicable due to high background or transient nature of the interaction. In contrast to many studies of proximity biotinylation in cultured cells, this protocol has been adapted to detect protein interactions in Xenopus embryos. Here, we apply this technique to evaluate planar cell polarity (PCP) complexes formed by Prickle3 and Vangl2, and show that Prickle3 fused to the N-terminal fragment of the biotin ligase from Aquifex aeolicus efficiently biotinylates Vangl2 in vivo. We present our step-by-step proximity biotinylation protocol that provides a reliable semiquantitative assay for protein interactions and highlights the use of Xenopus embryos as a model for biochemical studies.

Prickle promotes the formation and maintenance of glutamatergic synapses by stabilizing the intercellular planar cell polarity complex.

Whether there exists a common signaling mechanism that assembles all glutamatergic synapses is unknown. We show here that knocking out Prickle1 and Prickle2 reduced the formation of the PSD-95–positive glutamatergic synapses in the hippocampus and medial prefrontal cortex in postnatal development by 70–80%. Prickle1 and Prickle2 double knockout in adulthood lead to the disassembly of 70 to 80% of the postsynaptic-density(PSD)-95–positive glutamatergic synapses. PSD-95–positive glutamatergic synapses in the hippocampus of Prickle2E8Q/E8Q mice were reduced by 50% at postnatal day 14. Prickle2 promotes synapse formation by antagonizing Vangl2 and stabilizing the intercellular complex of the planar cell polarity (PCP) components, whereas Prickle2 E8Q fails to do so. Coculture experiments show that the asymmetric PCP complexes can determine the presynaptic and postsynaptic polarity. In summary, the PCP components regulate the assembly and maintenance of a large number of glutamatergic synapses and specify the direction of synaptic transmission.

CRB1-Associated Retinal Dystrophies: A Prospective Natural History Study in Anticipation of Future Clinical Trials

To investigate the natural disease course of retinal dystrophies associated with crumbs cell polarity complex component 1 (CRB1) and identify clinical end points for future clinical trials. Single-center, prospective case series. An investigator-initiated nationwide collaborative study that included 22 patients with CRB1-associated retinal dystrophies. Patients underwent ophthalmic assessment at baseline and 2 years after baseline. Clinical examination included best-corrected visual acuity (BCVA) using Early Treatment Diabetic Retinopathy Study charts, Goldmann kinetic perimetry (V4e isopter seeing retinal areas), microperimetry, full-field electroretinography, full-field stimulus threshold (FST), fundus photography, spectral-domain optical coherence tomography, and fundus autofluorescence imaging. Based on genetic, clinical, and electrophysiological data, patients were diagnosed with retinitis pigmentosa (19 [86%]), cone-rod dystrophy (2 [9%]), or isolated macular dystrophy (1 [5%]). Analysis of the entire cohort at 2 years showed no significant changes in BCVA (P=.069) or V4e isopter seeing retinal areas (P=.616), although signs of clinical progression were present in individual patients. Macular sensitivity measured on microperimetry revealed a significant reduction at the 2-year follow-up (P < .001). FST responses were measurable in patients with nonrecordable electroretinograms. On average, FST responses remained stable during follow-up. In CRB1-associated retinal dystrophies, visual acuity and visual field measures remain relatively stable over the course of 2 years. Microperimetry showed a significant decrease in retinal sensitivity during follow-up and may be a more sensitive progression marker. Retinal sensitivity on microperimetry may serve as a functional clinical end point in future human treatment trials for CRB1-associated retinal dystrophies.

The role of Transcription Factor Hepatocyte Nuclear Factor 1β in a Transcriptional Network Regulating Cell Polarity in Epithelial Kidney Cells

Hepatocyte nuclear factor 1β (HNF1β) is an essential transcription factor in development and function of the kidney, the pancreas and the liver. Mutations or deletions in HNF1β cause a heterogenous phenotype including renal cysts and/or malformation, maturity-onset diabetes of the young, liver malfunction and hypomagnesemia. The spectrum of HNF1β–regulated genes and pathways is only partially deciphered. Here, we aim to identify the HNF1β target genes and pathways that explain the multifaceted role of HNF1β in the kidney. RNA-sequencing was performed in a mouse distal convoluted tubule (DCT) cell line transfected with siRNAs targeting HNF1β or non-targeting siRNAs. A total number of 989 differential expressed genes was identified. Subsequently, HNF1β binding sites in the promoter regions (defined as <5-kb from the transcription start site) of these genes were identified using ChIP-sequencing data from the same cell line. Only 9,3% (92 genes) of the differentially expressed genes contained a HNF1β-binding site within their promoter region. These genes included known targets of HNF1β including Pkhd1, Kif12 and Kcnj16 and unknown targets. GO-term pathway analysis demonstrated that regulation of cell polarity, cell-cell junction complexes and cytoskeleton organization were regulated by HNF1β. Indeed, transepithelial electrical resistance measurements were reduced upon expression of dominant negative (DN) HNF1β. Nine HNF1 β targets that are part of the apical cell polarity complex were selected for further analysis. To study the importance of HNF1β in regulating cell polarity, the integrity of this apical cell polarity complex will be analyzed in kidney cells expressing wild-type HNF1β and DN HNF1β using immunocytochemistry. Together these findings suggest a role for HNF1β in a transcriptional network regulating cell polarity in adult kidney cells by transcriptional control of the apical polarity complex in epithelial cells.

Multimodal Retinal Imaging and Microperimetry Reveal a Novel Phenotype and Potential Trial End Points in CRB1-Associated Retinopathies.

PurposeBiallelic crumbs cell polarity complex component 1 (CRB1) mutations can present as Leber congenital amaurosis (LCA), retinitis pigmentosa (RP), or cystic maculopathy. This study reports a novel phenotype of asymptomatic fenestrated slit maculopathy (AFSM) and examines macular volume profile and microperimetry as clinical trial end points in CRB1-associated retinopathies.MethodsTwelve patients from nine families with CRB1 mutation were recruited. Ultra-widefield (UWF) color fundus photography and autofluorescence (AF), spectral-domain optical coherence tomography (SD-OCT), microperimetry, and adaptive optics (AO) imaging were performed. Macular volume profiles were compared with age-matched healthy controls. Genotyping was performed using APEX genotyping microarrays, targeted next-generation sequencing, and Sanger sequencing.ResultsWe identified one patient with LCA, five patients with RP, and four patients with macular dystrophy (MD) with biallelic CRB1 mutations. Two siblings with compound heterozygote genotype (c.[2843G>A]; [498_506del]) had AFSM characterized by localized outer retinal disruption on SD-OCT and parafoveal cone loss on AO imaging despite normal fundus appearance, visual acuity, and foveal sensitivity. UWF AF demonstrated preserved para-arteriolar retinal pigment epithelium (PPRPE) in all patients with RP. Microperimetry documented preserved central retinal function in six patients. The ratio of perifoveal-to-foveal retinal volume was greater than controls in 89% (8/9) of patients with RP or MD, whereas central subfield and total macular volume were outside normal limits in 67% (6/9).ConclusionsAO imaging was helpful in detecting parafoveal cone loss in asymptomatic patients. Macular volume profile and microperimetry parameters may have utility as CRB1 trials end points.Translational RelevanceMacular volume and sensitivity can be used as structural and functional end points for trials on CRB1-associated RP and MD.