Cell Polarity Protein Research Articles

Wnt/planar cell polarity signaling controls morphogenetic movements of gastrulation and neural tube closure.

Gastrulation and neurulation are successive morphogenetic processes that play key roles in shaping the basic embryonic body plan. Importantly, they operate through common cellular and molecular mechanisms to set up the three spatially organized germ layers and to close the neural tube. During gastrulation and neurulation, convergent extension movements driven by cell intercalation and oriented cell division generate major forces to narrow the germ layers along the mediolateral axis and elongate the embryo in the anteroposterior direction. Apical constriction also makes an important contribution to promote the formation of the blastopore and the bending of the neural plate. Planar cell polarity proteins are major regulators of asymmetric cell behaviors and critically involved in a wide variety of developmental processes, from gastrulation and neurulation to organogenesis. Mutations of planar cell polarity genes can lead to general defects in the morphogenesis of different organs and the co-existence of distinct congenital diseases, such as spina bifida, hearing deficits, kidney diseases, and limb elongation defects. This review outlines our current understanding of non-canonical Wnt signaling, commonly known as Wnt/planar cell polarity signaling, in regulating morphogenetic movements of gastrulation and neural tube closure during development and disease. It also attempts to identify unanswered questions that deserve further investigations.

Subcellular localization of type IV pili regulates bacterial multicellular development

In mammals, subcellular protein localization of factors like planar cell polarity proteins is a key driver of the multicellular organization of tissues. Bacteria also form organized multicellular communities, but these patterns are largely thought to emerge from regulation of whole-cell processes like growth, motility, cell shape, and differentiation. Here we show that a unique intracellular patterning of appendages known as type IV pili (T4P) can drive multicellular development of complex bacterial communities. Specifically, dynamic T4P appendages localize in a line along the long axis of the cell in the bacterium Acinetobacter baylyi. This long-axis localization is regulated by a functionally divergent chemosensory Pil-Chp system, and an atypical T4P protein homologue (FimV) bridges Pil-Chp signaling and T4P positioning. We further demonstrate through modeling and empirical approaches that subcellular T4P localization controls how individual cells interact with one another, independently of T4P dynamics, with different patterns of localization giving rise to distinct multicellular architectures. Our results reveal how subcellular patterning of single cells regulates the development of multicellular bacterial communities.

Par3 promotes breast cancer invasion and migration through pull tension and protein nanoparticle-induced osmotic pressure

Cancer cell invasion and metastasis are closely related to intracellular tension. The cell-polarity protein, Par3, is a mechanical transmitter that affects cytoskeletal forces and determines breast cancer aggressiveness. Increased Par3 tension caused by aPKC inactivation is involved in filopodia and lamellipodia formation. Blocking the connection between Par3 and aPKC increases breast cancer aggressiveness both in vitro and in vivo. Meanwhile, aPKC-induced Par3 cytoplasmic translocation results in JAM-A phase separation and microfilament depolymerization, which is associated with increased intracellular protein nanoparticle-induced osmotic pressure. This study demonstrated the effects of aPKC on Par3 tension and osmotic pressure in breast cancer metastasis, and introduced Par3-associated mechanical mechanisms as potential targets for breast cancer treatment.

Circular RNA Circ_0001777 Suppresses Lung Adenocarcinoma Progression In Vitro and In Vivo.

Circular RNA_0001777 (circ_0001777) is reported to be down-regulated in lung cancer. Nevertheless, the function of circ_0001777 in lung adenocarcinoma is largely unclear. We explored the role of circ_0001777 in lung adenocarcinoma progression and the underlying molecular mechanism. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot assay were conducted to determine the expression of RNAs and proteins. 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, 5-ethynyl-20-deoxyuridine, and colony formation assays were conducted to analyze cell proliferation ability. Flow cytometry was carried out to assess cell apoptosis rate. Cell migration and invasion abilities were analyzed by wound healing assay and transwell assays. Cell glycolytic metabolism was measured using a fluorescence-based glucose assay kit and a lactate oxidase-based colorimetric assay kit. Dual-luciferase reporter assay and RNA immunoprecipitation assay were implemented to verify the intermolecular interactions. Circ_0001777 expression was reduced in lung adenocarcinoma tissues and cell lines. Circ_0001777 overexpression restrained the proliferation, migration, invasion, and glycolysis and promoted the apoptosis of lung adenocarcinoma cells. Circ_0001777 could directly bind to microRNA-942-5p (miR-942-5p). The anti-tumor effects of circ_0001777 overexpression in lung adenocarcinoma cells were reversed after miR-942-5p accumulation. miR-942-5p directly bound to the 3' untranslated region (3'UTR) of prickle planar cell polarity protein 2 (PRICKLE2), and PRICKLE2 silencing reversed the anti-tumor effects of miR-942-5p knockdown in lung adenocarcinoma cells. Circ_0001777 could regulate PRICKLE2 expression by absorbing miR-942-5p. Circ_0001777 overexpression markedly hampered tumor progression in vivo. Circ_0001777 suppressed the progression of lung adenocarcinoma by binding to miR-942-5p to induce PRICKLE2 expression.

Distinct overlapping functions for Prickle1 and Prickle2 in the polarization of the airway epithelium.

Planar cell polarity (PCP) signaling polarizes cells within the plane of an epithelium. In the airways, planar cell polarity signaling orients the directional beating of motile cilia required for effective mucociliary clearance. The planar cell polarity signaling mechanism is best understood from work in Drosophila, where it has been shown to both coordinate the axis of polarity between cells and to direct the morphological manifestations of polarization within cells. The ‘core’ planar cell polarity signaling mechanism comprises two protein complexes that segregate to opposite sides of each cell and interact with the opposite complex in neighboring cells. Proper subcellular localization of core planar cell polarity proteins correlates with, and is almost certainly responsible for, their ability to direct polarization. This mechanism is highly conserved from Drosophila to vertebrates, though for most of the core genes, mammals have multiple paralogs whereas Drosophila has only one. In the mouse airway epithelium, the core protein Prickle2 segregates asymmetrically, as is characteristic for core proteins, but is only present in multiciliated cells and is absent from other cell types. Furthermore, Prickle2 mutant mice show only modest ciliary polarity defects. These observations suggest that other Prickle paralogs might contribute to polarization. Here, we show that Prickle1 segregates asymmetrically in multiciliated and nonciliated airway epithelial cell types, that compared to Prickle2, Prickle1 has different spatial and temporal expression dynamics and a stronger ciliary polarity phenotype, and that Prickle1 and Prickle2 mutants genetically interact. We propose distinct and partially overlapping functions for the Prickle paralogs in polarization of the airway epithelium.

GPR125 (ADGRA3) is an autocleavable adhesion GPCR that traffics with Dlg1 to the basolateral membrane and regulates epithelial apicobasal polarity

The adhesion family of G protein–coupled receptors (GPCRs) is defined by an N-terminal large extracellular region that contains various adhesion-related domains and a highly-conserved GPCR-autoproteolysis-inducing (GAIN) domain, the latter of which is located immediately before a canonical seven-transmembrane domain. These receptors are expressed widely and involved in various functions including development, angiogenesis, synapse formation, and tumorigenesis. GPR125 (ADGRA3), an orphan adhesion GPCR, has been shown to modulate planar cell polarity in gastrulating zebrafish, but its biochemical properties and role in mammalian cells have remained largely unknown. Here, we show that human GPR125 likely undergoes cis-autoproteolysis when expressed in canine kidney epithelial MDCK cells and human embryonic kidney HEK293 cells. The cleavage appears to occur at an atypical GPCR proteolysis site within the GAIN domain during an early stage of receptor biosynthesis. The products, i.e., the N-terminal and C-terminal fragments, seem to remain associated after self-proteolysis, as observed in other adhesion GPCRs. Furthermore, in polarized MDCK cells, GPR125 is exclusively recruited to the basolateral domain of the plasma membrane. The recruitment likely requires the C-terminal PDZ-domain–binding motif of GPR125 and its interaction with the cell polarity protein Dlg1. Knockdown of GPR125 as well as that of Dlg1 results in formation of aberrant cysts with multiple lumens in Matrigel 3D culture of MDCK cells. Consistent with the multilumen phenotype, mitotic spindles are incorrectly oriented during cystogenesis in GPR125-KO MDCK cells. Thus, the basolateral protein GPR125, an autocleavable adhesion GPCR, appears to play a crucial role in apicobasal polarization in epithelial cells.

Dual Function of Par3 in Tumorigenesis.

Cell maintenance and the establishment of cell polarity involve complicated interactions among multiple protein complexes as well as the regulation of different signaling pathways. As an important cell polarity protein, Par3 is evolutionarily conserved and involved in tight junction formation as well as tumorigenesis. In this review, we aimed to explore the function of Par3 in tumorigenesis. Research has shown that Par3 exhibits dual functions in human cancers, both tumor-promoting and tumor-suppressive. Here, we focus on the activities of Par3 in different stages and types of tumors, aiming to offer a new perspective on the molecular mechanisms that regulate the functions of Par3 in tumor development. Tumor origin, tumor microenvironment, tumor type, cell density, cell–cell contact, and the synergistic effect of Par3 and other tumor-associated signaling pathways may be important reasons for the dual function of Par3. The important role of Par3 in mammalian tumorigenesis and potential signaling pathways is context dependent.

The cell polarity protein Vangl2 in the muscle shapes the neuromuscular synapse by binding to and regulating the tyrosine kinase MuSK.

The development of the neuromuscular junction (NMJ) requires dynamic trans-synaptic coordination orchestrated by secreted factors, including Wnt family morphogens. To investigate how these synaptic cues in NMJ development are transduced, particularly in the regulation of acetylcholine receptor (AChR) accumulation in the postsynaptic membrane, we explored the function of Van Gogh-like protein 2 (Vangl2), a core component of Wnt planar cell polarity signaling. We found that conditional, muscle-specific ablation of Vangl2 in mice reproduced the NMJ differentiation defects seen in mice with global Vangl2 deletion. These alterations persisted into adulthood and led to NMJ disassembly, impaired neurotransmission, and deficits in motor function. Vangl2 and the muscle-specific receptor tyrosine kinase MuSK were functionally associated in Wnt signaling in the muscle. Vangl2 bound to and promoted the signaling activity of MuSK in response to Wnt11. The loss of Vangl2 impaired RhoA activation in cultured mouse myotubes and caused dispersed, rather than clustered, organization of AChRs at the postsynaptic or muscle cell side of NMJs in vivo. Our results identify Vangl2 as a key player of the core complex of molecules shaping neuromuscular synapses and thus shed light on the molecular mechanisms underlying NMJ assembly.

Cell cortex regulation by the planar cell polarity protein Prickle1.

The planar cell polarity pathway regulates cell polarity, adhesion, and rearrangement. Its cytoplasmic core components Prickle (Pk) and Dishevelled (Dvl) often localize as dense puncta at cell membranes to form antagonizing complexes and establish cell asymmetry. In vertebrates, Pk and Dvl have been implicated in actomyosin cortex regulation, but the mechanism of how these proteins control cell mechanics is unclear. Here we demonstrate that in Xenopus prechordal mesoderm cells, diffusely distributed, cytoplasmic Pk1 up-regulates the F-actin content of the cortex. This counteracts cortex down-regulation by Dvl2. Both factors act upstream of casein kinase II to increase or decrease cortical tension. Thus, cortex modulation by Pk1 and Dvl2 is translated into mechanical force and affects cell migration and rearrangement during radial intercalation in the prechordal mesoderm. Pk1 also forms puncta and plaques, which are associated with localized depletion of cortical F-actin, suggesting opposite roles for diffuse and punctate Pk1.

Polarity protein SCRIB interacts with SLC3A2 to regulate proliferation and tamoxifen resistance in ER+ breast cancer

Estrogen receptor (ER) positive breast cancer represents 75% of all breast cancers in women. Although patients with ER+ cancers receive endocrine therapies, more than 30% develop resistance and succumb to the disease, highlighting the need to understand endocrine resistance. Here we show an unexpected role for the cell polarity protein SCRIB as a tumor-promoter and a regulator of endocrine resistance in ER-positive breast cancer cells. SCRIB expression is induced by estrogen signaling in a MYC-dependent manner. SCRIB interacts with SLC3A2, a heteromeric component of leucine amino acid transporter SLC7A5. SLC3A2 binds to the N-terminus of SCRIB to facilitate the formation of SCRIB/SLC3A2/LLGL2/SLC7A5 quaternary complex required for membrane localization of the amino acid transporter complex. Both SCRIB and SLC3A2 are required for cell proliferation and tamoxifen resistance in ER+ cells identifying a new role for the SCRIB/SLC3A2 complex in ER+ breast cancer.

In preprints: buckling under pressure during gastrulation.

In preprints: buckling under pressure during gastrulation.

The Nab2 RNA-binding protein patterns dendritic and axonal projections through a planar cell polarity-sensitive mechanism.

RNA-binding proteins support neurodevelopment by modulating numerous steps in post-transcriptional regulation, including splicing, export, translation, and turnover of mRNAs that can traffic into axons and dendrites. One such RNA-binding protein is ZC3H14, which is lost in an inherited intellectual disability. The Drosophila melanogaster ZC3H14 ortholog, Nab2, localizes to neuronal nuclei and cytoplasmic ribonucleoprotein granules and is required for olfactory memory and proper axon projection into brain mushroom bodies. Nab2 can act as a translational repressor in conjunction with the Fragile-X mental retardation protein homolog Fmr1 and shares target RNAs with the Fmr1-interacting RNA-binding protein Ataxin-2. However, neuronal signaling pathways regulated by Nab2 and their potential roles outside of mushroom body axons remain undefined. Here, we present an analysis of a brain proteomic dataset that indicates that multiple planar cell polarity proteins are affected by Nab2 loss, and couple this with genetic data that demonstrate that Nab2 has a previously unappreciated role in restricting the growth and branching of dendrites that elaborate from larval body-wall sensory neurons. Further analysis confirms that Nab2 loss sensitizes sensory dendrites to the genetic dose of planar cell polarity components and that Nab2-planar cell polarity genetic interactions are also observed during Nab2-dependent control of axon projection in the central nervous system mushroom bodies. Collectively, these data identify the conserved Nab2 RNA-binding protein as a likely component of post-transcriptional mechanisms that limit dendrite growth and branching in Drosophila sensory neurons and genetically link this role to the planar cell polarity pathway. Given that mammalian ZC3H14 localizes to dendritic spines and controls spine density in hippocampal neurons, these Nab2-planar cell polarity genetic data may highlight a conserved path through which Nab2/ZC3H14 loss affects morphogenesis of both axons and dendrites in diverse species.

Planar cell polarity protein-dependent basal cell height in the later stage embryonic mouse epidermis impacts on the shape of overlying suprabasal cells.

Background: Complex organ formation requires the coordinated morphogenesis of adjacent tissue layers. Here, a role for the planar cell polarity (PCP) proteins Fz6 and Celsr1 in generating squamous basal cells in the later stage embryonic epidermis of the mouse is reported, which impacts upon the shape of overlying suprabasal cells. Methods: The depth of the epidermis and basal layer as well as cell proliferation index was scored from immunostained wax sections taken from different mouse embryos mutant in planar cell polarity signalling and their wild-type littermates. Orientation of epidermal cell division in Celsr1 Crash/Crash mutants was determined from thick frozen immunostained sections. Immunostained wax sections of wild-type skin explants cultured using the Lumox method enabled any changes in epidermal and basal layer depth to be measured following the release of surface tension upon dissection of skin away from the whole embryo. Results: Increased numbers of columnar and cuboidal basal epidermal cells were observed in fz6 and Celsr1 mouse mutants including Celsr1 Crash/Crash which correlated with more rounded suprabasal cells and a thicker epidermis. Conclusions: Altogether these data support tissue intrinsic roles for PCP proteins in 'outside-in' (radial) skin architecture.

PDZ Proteins SCRIB and DLG1 Regulate Myeloma Cell Surface CD86 Expression, Growth, and Survival.

These findings demonstrate the importance of proper trafficking of CD86 to the cell surface in myeloma cell survival and may provide a new therapeutic target in this disease.

“Loss of polarity of basal cells” – The term revisited in the light of genomics

The concept of polarity in development, homeostasis and pathological alteration of tissues has emerged as an interesting aspect of the concerned biology. The epithelial cells exhibit apicobasal polarity which is maintained by Crumbs complex located at apical region of tight junction, ‘PAR’ complex at sub-apical region of tight junction and Scribble complex at adherens junction. Any functional perturbation of these proteins cause alteration of normal epithelial physiology en-route to epithelial pathology. In this maiden scientific exercise, we have tried to explore the association of expression of cell polarity proteins in OPMD and OSCC. Here, we have chosen DLG1 as a representative of polarity protein. RNA was isolated from the tissue samples. Then cDNA was prepared by RTPCR technique. qPCR was performed on cDNA samples. Expression data was analysed on the basis of Ct values. Paired t-test was performed with normalized Ct values of disease and normal tissue to determine whether there was any significant difference in expression between them. The statistical tests were done using SPSS software. Results of this study reflected increase in DLG1 expression in high grade dysplasia. There was no significant alteration in expression of DLG1 in WDSCC where there is formation of cluster of neoplastic cells ultimately producing epithelial islands and keratin pearls. But, in case of MDSCC, when the same neoplastic cells keep on invading with minimal keratin pearl formation, they again gain the mesenchymal character in full potential. And this phenomenon supports the upregulation of DLG1 in MDSCC in our study.

Membrane ratcheting directs progressive cell shape changes in a pulsatile system through PIP3 lipid cues

In epithelial sheets, processes that drive cells to change topological relationships can be harnessed by developmental mechanisms to effect changes in tissue architecture. Organisms ranging from humans to Drosophila lengthen their anterior-posterior axis during development. An essential driver of tissue elongation is oriented cell intercalation. In the Drosophila embryonic epithelium, individual cells are able to either consolidate cell-cell contacts or direct neighbor exchange movements through the asymmetric localization of cell adhesion and polarity proteins.

Analysis of the Ubiquitination and Phosphorylation of Vangl Proteins.

The core planar cell polarity (PCP) protein Vang/Vangl, including Vangl1 and Vangl2 in vertebrates, is indispensable during development. Our previous studies showed that the activity of Vangl is tightly controlled by two important posttranslational modifications, ubiquitination and phosphorylation. Vangl is ubiquitinated through an endoplasmic reticulum-associated degradation (ERAD) pathway and is phosphorylated by casein kinase 1 (CK1) in response to Wnt. Here, we present step-by-step procedures to analyze Vangl ubiquitination and phosphorylation, including cell culture, transfection, sample preparation, and signal detection, as well as the use of newly available phospho-specific antibodies to detect Wnt-induced Vangl2 phosphorylation. The protocol described here can be applicable to the analysis of posttranslational modifications of other membrane proteins.

Colocalization Analysis for Cryosectioned and Immunostained Tissue Samples with or without Label Retention Expansion Microscopy (LR-ExM) by JACoP.

Asymmetric cell division (ACD) is fundamental for balancing cell proliferation and differentiation in metazoans. During active neurogenesis in the developing zebrafish forebrain, radial glia progenitors (RGPs) mainly undergo ACD to produce one daughter with high activity of Delta/Notch signaling (proliferative cell fate) and another daughter with low Delta/Notch signaling (differentiative cell fate). The cell polarity protein partitioning-defective 3 (Par-3) is critical for regulating this process. To understand how polarized Par-3 on the cell cortex can lead to differential Notch activity in the nuclei of daughter cells, we combined an anti-Delta D (Dld) -atto 647N antibody uptake assay with label retention expansion microscopy (LR-ExM), to obtain high resolution immunofluorescent images of Par-3, dynein light intermediate chain 1 (Dlic1), and Dld endosomes in mitotic RGPs. We then developed a protocol for analyzing the colocalization of Par-3, Dlic1, and endosomal DeltaD, using JACoP (Just Another Co-localization Plugin) in ImageJ software (Bolte and Cordelières, 2006). Through such analyses, we have shown that cytosolic Par-3 is associated with Dlic1 on Dld endosomes. Our work demonstrates a direct involvement of Par-3 in dynein-mediated polarized transport of Notch signaling endosomes. This bio-protocol may be generalizable for analysis of protein co-localization in any cryosectioned and immunostained tissue samples.

Regulation of Cell Polarity by Posttranslational Protein Palmitoylation.

Cell polarity is a common feature of many living cells, especially epithelial cells, and plays important roles in development, tissue homeostasis, and diseases. Therefore, the signaling pathways involved in establishing and maintaining cell polarity are tightly controlled. Protein S-palmitoylation has been recently recognized as an important posttranslational modification involved in cell polarity, via dynamic covalent attachment of fatty acyl groups to the cysteine residues of cell polarity proteins. Here, we describe the methods to study the function and regulation of S-palmitoylation of cell polarity proteins.

The Celsr3-Kif2a axis directs neuronal migration in the postnatal brain.

The tangential migration of immature neurons in the postnatal brain involves consecutive migration cycles and depends on constant remodeling of the cell cytoskeleton, particularly in the leading process (LP). Despite the identification of several proteins with permissive and empowering functions, the mechanisms that specify the direction of migration remain largely unknown. Here, we report that planar cell polarity protein Celsr3 orients neuroblasts migration from the subventricular zone (SVZ) to olfactory bulb (OB). In Celsr3-forebrain conditional knockout mice, neuroblasts loose directionality and few can reach the OB. Celsr3-deficient neuroblasts exhibit aberrant branching of LP, de novo LP formation, and decreased growth rate of microtubules (MT). Mechanistically, we show that Celsr3 interacts physically with Kif2a, a MT depolymerizing protein and that conditional inactivation of Kif2a in the forebrain recapitulates the Celsr3 knockout phenotype. Our findings provide evidence that Celsr3 and Kif2a cooperatively specify the directionality of neuroblasts tangential migration in the postnatal brain.