Localization Of Polarity Proteins Research Articles

Opposing polarity domains provide direction and play a role in cell division in plant growth

Polar localization of proteins is important for plant growth and development. Identifying the interactors of polarized proteins provides spatial information and cell-type functions. In this issue of Developmental Cell, Wallner etal. (2024) utilize opposing polarity domain proteins to identify interactors and their functions during cell division in Arabidopsis stomata.

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.

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.

Growth asymmetry precedes differential auxin response during apical hook initiation in Arabidopsis.

The development of a hook-like structure at the apical part of the soil-emerging organs has fascinated botanists for centuries, but how it is initiated remains unclear. Here, we demonstrate with high-throughput infrared imaging and 2-D clinostat treatment that, when gravity-induced root bending is absent, apical hook formation still takes place. In such scenarios, hook formation begins with a de novo growth asymmetry at the apical part of a straightly elongating hypocotyl. Remarkably, such de novo asymmetric growth, but not the following hook enlargement, precedes the establishment of a detectable auxin response asymmetry, and is largely independent of auxin biosynthesis, transport and signaling. Moreover, we found that functional cortical microtubule array is essential for the following enlargement of hook curvature. When microtubule array was disrupted by oryzalin, the polar localization of PIN proteins and the formation of an auxin maximum became impaired at the to-be-hook region. Taken together, we propose a more comprehensive model for apical hook initiation, in which the microtubule-dependent polar localization of PINs may mediate the instruction of growth asymmetry that is either stochastically taking place, induced by gravitropic response, or both, to generate a significant auxin gradient that drives the full development of the apical hook.

Mapping Asymmetry in Collective Cell Migration: Lessons from Border Cells in Drosophila Oogenesis.

Asymmetry in the migrating group of cells is critical for efficient directed movement observed in normal development and in pathological conditions like tumor cell metastasis. This is conspicuously detected at the level of polarized protrusions and differential localization of various polarity proteins in collectively moving clusters. Over the years, border cell migration in Drosophila oogenesis has emerged as an excellent model system for studying polarity in the migrating group of cells. Here we report two protocols employing live cell imaging and tissue immunohistochemistry to evaluate the polarity in migrating border cell clusters.

Recent developments in the understanding of PIN polarity.

Polar localization of PIN-FORMED proteins (PINs) at the plasma membrane is essential for plant development as they direct the transport of phytohormone auxin between cells. PIN polar localization to certain sides of a given cell is dynamic, strictly regulated and provides directionality to auxin flow. Signals that act upstream to control subcellular PIN localization modulate auxin distribution, thereby regulating diverse aspects of plant development. Here I summarize the current understanding of mechanisms by which PIN polarity is established, maintained and rearranged to provide a glimpse into the complexity of PIN polarity.

Key Role for CRB2 in the Maintenance of Apicobasal Polarity in Retinal Pigment Epithelial Cells.

Apicobasal polarity is essential for epithelial cell function, yet the roles of different proteins in its completion is not fully understood. Here, we have studied the role of the polarity protein, CRB2, in human retinal pigment epithelial (RPE) cells during polarization in vitro, and in mature murine RPE cells in vivo. After establishing a simplified protocol for the culture of human fetal RPE cells, we studied the temporal sequence of the expression and localization of polarity and cell junction proteins during polarization in these epithelial cells. We found that CRB2 plays a key role in tight junction maintenance as well as in cell cycle arrest. In addition, our studies in vivo show that the knockdown of CRB2 in the RPE affects to the distribution of different apical polarity proteins and results in perturbed retinal homeostasis, manifested by the invasion of activated microglial cells into the subretinal space. Together our results demonstrate that CRB2 is a key protein for the development and maintenance of a polarized epithelium.

Plant cell biology: PIN polarity maintained

PIN-FORMED (PIN) polar protein localization directs transport of the growth and developmental regulator auxin in plants. Once established after cytokinesis, PIN polarity requires maintenance. Now, direct interactions between PIN, MAB4/MEL and PID proteins suggest self-reinforced maintenance of PIN polarity through limiting lateral diffusion.

Reprogramming Intestinal Epithelial Cell Polarity by Interleukin-22.

Background: Interleukin-22 (IL-22) impacts the integrity of intestinal epithelia and has been associated with the development of colitis-associated cancer and inflammatory bowel diseases (IBD). Previous data suggest that IL-22 protects the mucosal barrier and promotes wound healing and barrier defect. We hypothesized, that IL-22 modulates cell polarity of intestinal epithelial cells (IECs) acting on tight junction assembly. The aim of the study was to investigate IL-22-dependent mechanisms in the reprogramming of intestinal epithelia.Methods: IECs were exposed to IL-22 at various concentrations. IECs in Matrigel® were grown to 3-dimensional cysts in the presence or absence of IL-22 and morphology and expression of polarity proteins were analyzed by confocal microscopy. Epithelial cell barrier (TER and sandwich assay) and TJ assembly analysis (calcium-switch assay) were performed. TJ and cell polarity protein expression were assessed by western blotting and confocal microscopy. Cell migration and invasion assays were performed. Induction of epithelial-mesenchymal transition (EMT) was assessed by RT-qPCR analysis and western blotting. Signaling pathway analyses were performed by phosphoblotting and functional assays after blocking STAT3 and ERK signaling pathways. Using the toxoplasma-model of terminal ileitis, IL-22-knock-out mice were compared to wild-type littermates, analyzed for barrier function using one-path-impedance-analysis and macromolecular flux (H3-mannitol, Ussing-chambers).Results: IECs exhibited a barrier defect after IL-22 exposure. TJ protein distribution and expression were severely impaired. Delayed recovery in the calcium-switch assay was observed suggesting a defect in TJ assembly. Analyzing the 3D-cyst model, IL-22 induced multi-lumen and aberrant cysts, and altered the localization of cell polarity proteins. Cell migration and invasion was caused by IL-22 as well as induction of EMT. Interestingly, only inhibition of the MAPK pathway, rescued the TJal barrier defect, while blocking STAT3 was relevant for cell survival. In addition, ileal mucosa of IL-22 deficient mice was protected from the barrier defect seen in Toxoplasma gondii-induced ileitis in wild type mice shown by significantly higher Re values and correspondingly lower macromolecule fluxes.Conclusion: IL-22 impairs intestinal epithelial cell barrier by inducing EMT, causing defects in epithelial cell polarity and increasing cell motility and cell invasion. IL-22 modulates TJ protein expression and mediates tight junctional (TJal) barrier defects via ERK pathway.

Dual β-Catenin and γ-Catenin Loss in Hepatocytes Impacts Their Polarity through Altered Transforming Growth Factor-β and Hepatocyte Nuclear Factor 4α Signaling

Hepatocytes are highly polarized epithelia. Loss of hepatocyte polarity is associated with various liver diseases, including cholestasis. However, the molecular underpinnings of hepatocyte polarization remain poorly understood. Loss of β-catenin at adherens junctions is compensated by γ-catenin and dual loss of both catenins in double knockouts (DKOs) in mice liver leads to progressive intrahepatic cholestasis. However, the clinical relevance of this observation, and further phenotypic characterization of the phenotype, is important. Herein, simultaneous loss of β-catenin and γ-catenin was identified in a subset of liver samples from patients of progressive familial intrahepatic cholestasis and primary sclerosing cholangitis. Hepatocytes in DKO mice exhibited defects in apical-basolateral localization of polarity proteins, impaired bile canaliculi formation, and loss of microvilli. Loss of polarity in DKO livers manifested as epithelial-mesenchymal transition, increased hepatocyte proliferation, and suppression of hepatocyte differentiation, which was associated with up-regulation of transforming growth factor-β signaling and repression of hepatocyte nuclear factor 4α expression and activity. In conclusion, concomitant loss of the two catenins in the liver may play a pathogenic role in subsets of cholangiopathies. The findings also support a previously unknown role of β-catenin and γ-catenin in the maintenance of hepatocyte polarity. Improved understanding of the regulation of hepatocyte polarization processes by β-catenin and γ-catenin may potentially benefit development of new therapies for cholestasis.

Regulation, Diversity and Evolution of Boron Transporters in Plants.

Boron (B) is an essential trace element in plants, and borate cross-linking of pectic polysaccharide rhamnogalacturonan-II (RG-II) in cell walls is required for normal cell growth. High concentrations of B are toxic to cells. Therefore, plants need to control B transport to respond to B conditions in the environment. Over the past two decades, genetic analyses of Arabidopsis thaliana have revealed that B transport is governed by two types of membrane transport molecules: NIPs (nodulin-26-like intrinsic proteins), which facilitate boric acid permeation, and BORs, which export borate from cells. In this article, we review recent findings on the (i) regulation at the cell level, (ii) diversity among plant species and (iii) evolution of these B transporters in plants. We first describe the systems regulating these B transporters at the cell level, focusing on the molecular mechanisms underlying the polar localization of proteins and B-dependent expression, as well as their physiological significance in A. thaliana. Then, we examine the presence of homologous genes and characterize the functions of NIPs and BORs in B homeostasis, in a wide range of plant species, including Brassica napus, Oryza sativa and Zea mays. Finally, we discuss the evolutionary aspects of NIPs and BORs as B transporters, and the possible relationship between the diversification of B transport and the occurrence of RG-II in plants. This review considers the sophisticated systems of B transport that are conserved among various plant species, which were established to meet mineral nutrient requirements.

Recruitment of Polarity Complexes and Tight Junction Proteins to the Site of Apical Bulk Endocytosis.

Background & AimsThe molecular motor, Myosin Vb (MYO5B), is well documented for its role in trafficking cargo to the apical membrane of epithelial cells. Despite its involvement in regulating apical proteins, the role of MYO5B in cell polarity is less clear. Inactivating mutations in MYO5B result in microvillus inclusion disease (MVID), a disorder characterized by loss of key apical transporters and the presence of intracellular inclusions in enterocytes. We previously identified that inclusions in Myo5b knockout (KO) mice form from invagination of the apical brush border via apical bulk endocytosis. Herein, we sought to elucidate the role of polarity complexes and tight junction proteins during the formation of inclusions.MethodsIntestinal tissue from neonatal control and Myo5b KO littermates was analyzed by immunofluorescence to determine the localization of polarity complexes and tight junction proteins.ResultsProteins that make up the apical polarity complexes—Crumbs3 and Pars complexes—were associated with inclusions in Myo5b KO mice. In addition, tight junction proteins were observed to be concentrated over inclusions that were present at the apical membrane of Myo5b-deficient enterocytes in vivo and in vitro. Our mouse findings are complemented by immunostaining in a large animal swine model of MVID genetically engineered to express a human MVID-associated mutation that shows an accumulation of Claudin-2 over forming inclusions. The findings from our swine model of MVID suggest that a similar mechanism of tight junction accumulation occurs in patients with MVID.ConclusionsThese data show that apical bulk endocytosis involves the altered localization of apical polarity proteins and tight junction proteins after loss of Myo5b.

A Conditional Mutation in SCD1 Reveals Linkage Between PIN Protein Trafficking, Auxin Transport, Gravitropism, and Lateral Root Initiation.

Auxin is transported in plants with distinct polarity, defined by transport proteins of the PIN-formed (PIN) family. Components of the complex trafficking machinery responsible for polar PIN protein localization have been identified by genetic approaches, but severe developmental phenotypes of trafficking mutants complicate dissection of this pathway. We utilized a temperature sensitive allele of Arabidopsis thaliana SCD1 (stomatal cytokinesis defective1) that encodes a RAB-guanine nucleotide exchange factor. Auxin transport, lateral root initiation, asymmetric auxin-induced gene expression after gravitropic reorientation, and differential gravitropic growth were reduced in the roots of the scd1-1 mutant relative to wild type at the restrictive temperature of 25°C, but not at the permissive temperature of 18°C. In scd1-1 at 25°C, PIN1- and PIN2-GFP accumulated in endomembrane bodies. Transition of seedlings from 18 to 25°C for as little as 20 min resulted in the accumulation of PIN2-GFP in endomembranes, while gravitropism and root developmental defects were not detected until hours after transition to the non-permissive temperature. The endomembrane compartments that accumulated PIN2-GFP in scd1-1 exhibited FM4-64 signal colocalized with ARA7 and ARA6 fluorescent marker proteins, consistent with PIN2 accumulation in the late or multivesicular endosome. These experiments illustrate the power of using a temperature sensitive mutation in the gene encoding SCD1 to study the trafficking of PIN2 between the endosome and the plasma membrane. Using the conditional feature of this mutation, we show that altered trafficking of PIN2 precedes altered auxin transport and defects in gravitropism and lateral root development in this mutant upon transition to the restrictive temperature.

Targeting PKCθ Promotes Satellite Cell Self-Renewal.

Skeletal muscle regeneration following injury depends on the ability of satellite cells (SCs) to proliferate, self-renew, and eventually differentiate. The factors that regulate the process of self-renewal are poorly understood. In this study we examined the role of PKCθ in SC self-renewal and differentiation. We show that PKCθ is expressed in SCs, and its active form is localized to the chromosomes, centrosomes, and midbody during mitosis. Lack of PKCθ promotes SC symmetric self-renewal division by regulating Pard3 polarity protein localization, without affecting the overall proliferation rate. Genetic ablation of PKCθ or its pharmacological inhibition in vivo did not affect SC number in healthy muscle. By contrast, after induction of muscle injury, lack or inhibition of PKCθ resulted in a significant expansion of the quiescent SC pool. Finally, we show that lack of PKCθ does not alter the inflammatory milieu after acute injury in muscle, suggesting that the enhanced self-renewal ability of SCs in PKCθ-/- mice is not due to an alteration in the inflammatory milieu. Together, these results suggest that PKCθ plays an important role in SC self-renewal by stimulating their expansion through symmetric division, and it may represent a promising target to manipulate satellite cell self-renewal in pathological conditions.

Assorted Pastries at the South Pole.

Plant cells use polarity cues to confine membrane proteins to specific localizations. In this issue of Developmental Cell, Marhava etal. (2020) describe a biochemical feedforward mechanism that reinforces polar protein localization and regulates membrane composition and endocytosis.

Microtubules are required for the maintenance of planar cell polarity in monociliated floorplate cells

The asymmetric localization of planar cell polarity (PCP) proteins is essential for the establishment of many planar polarized cellular processes, but the mechanisms that maintain these asymmetric distributions remain poorly understood. A body of evidence has tied oriented subapical microtubules (MTs) to the establishment of PCP protein polarity, yet recent studies have suggested that the MT cytoskeleton is later dispensable for the maintenance of this asymmetry. As MTs underlie the vesicular trafficking of membrane-bound proteins within cells, the requirement for MTs in the maintenance of PCP merited further investigation. We investigated the complex interactions between PCP proteins and the MT cytoskeleton in the polarized context of the floorplate of the zebrafish neural tube. We demonstrated that the progressive posterior polarization of the primary cilia of floorplate cells requires not only Vangl2 but also Fzd3a. We determined that GFP-Vangl2 asymmetrically localizes to anterior membranes whereas Fzd3a-GFP does not polarize on anterior or posterior membranes but maintains a cytosolic enrichment at the base of the primary cilium. Vesicular Fzd3a-GFP is rapidly trafficked along MTs primarily toward the apical membrane during a period of PCP maintenance, whereas vesicular GFP-Vangl2 is less frequently observed. Nocodazole-induced loss of MT polymerization disrupts basal body positioning as well as GFP-Vangl2 localization and reduces cytosolic Fzd3a-GFP movements. Removal of nocodazole after MT disruption restores MT polymerization but does not restore basal body polarity. Interestingly, GFP-Vangl2 repolarizes to anterior membranes and vesicular Fzd3a-GFP dynamics recover after multiple hours of recovery, even in the context of unpolarized basal bodies. Together our findings challenge previous work by revealing an ongoing role for MT-dependent transport of PCP proteins in maintaining both cellular and PCP protein asymmetry during development.

Two Pathways for Trafficking the Borate Receptor BOR1.

Polar localization of plasma membrane proteins is an important way cells regulate signaling pathways, transport across membranes, and growth. Polarization has obvious cellular effects, but localization of membrane components also contributes significantly to processes such as tip growth in root

Retromer Controls Planar Polarity Protein Levels and Asymmetric Localization at Intercellular Junctions

SummaryThe coordinated polarization of cells in the plane of a tissue, termed planar polarity, is a characteristic feature of epithelial tissues [1]. In the fly wing, trichome positioning is dependent on the core planar polarity proteins adopting asymmetric subcellular localizations at apical junctions, where they form intercellular complexes that link neighboring cells [1, 2, 3]. Specifically, the seven-pass transmembrane protein Frizzled and the cytoplasmic proteins Dishevelled and Diego localize to distal cell ends, the four-pass transmembrane protein Strabismus and the cytoplasmic protein Prickle localize proximally, and the seven-pass transmembrane spanning atypical cadherin Flamingo localizes both proximally and distally. To establish asymmetry, these core proteins are sorted from an initially uniform distribution; however, the mechanisms underlying this polarized trafficking remain poorly understood. Here, we describe the identification of retromer, a master controller of endosomal recycling [4, 5, 6], as a key component regulating core planar polarity protein localization in Drosophila. Through generation of mutants, we verify that loss of the retromer-associated Snx27 cargo adaptor, but notably not components of the Wash complex, reduces junctional levels of the core proteins Flamingo and Strabismus in the developing wing. We establish that Snx27 directly associates with Flamingo via its C-terminal PDZ binding motif, and we show that Snx27 is essential for normal Flamingo trafficking. We conclude that Wash-independent retromer function and the Snx27 cargo adaptor are important components in the endosomal recycling of Flamingo and Strabismus back to the plasma membrane and thus contribute to the establishment and maintenance of planar polarization.

Cadherin-26 (CDH26) regulates airway epithelial cell cytoskeletal structure and polarity

Polarization of the airway epithelial cells (AECs) in the airway lumen is critical to the proper function of the mucociliary escalator and maintenance of lung health, but the cellular requirements for polarization of AECs are poorly understood. Using human AECs and cell lines, we demonstrate that cadherin-26 (CDH26) is abundantly expressed in differentiated AECs, localizes to the cell apices near ciliary membranes, and has functional cadherin domains with homotypic binding. We find a unique and non-redundant role for CDH26, previously uncharacterized in AECs, in regulation of cell–cell contact and cell integrity through maintaining cytoskeletal structures. Overexpression of CDH26 in cells with a fibroblastoid phenotype increases contact inhibition and promotes monolayer formation and cortical actin structures. CDH26 expression is also important for localization of planar cell polarity proteins. Knockdown of CDH26 in AECs results in loss of cortical actin and disruption of CRB3 and other proteins associated with apical polarity. Together, our findings uncover previously unrecognized functions for CDH26 in the maintenance of actin cytoskeleton and apicobasal polarity of AECs.

Perspective: challenges and opportunities for the study of cardiolipin, a key player in bacterial cell structure and function.

Cardiolipin (CL) is a key player in bacterial cell biology. CL accumulates at the poles of rod-shaped cells; the polar localization and function of diverse bacterial proteins are CL-dependent. Cardiolipin (CL) is an unusual phospholipid comprised of a glycerol headgroup coupled with two phosphatidate moieties. CL-rich membrane domains are often visualized with the fluorescent indicator 10-N-nonyl-acridine orange (NAO). Recent data show that NAO can also indicate phosphatidylglycerol localization under different experimental conditions, in the absence of CL. The formation of CL-rich membrane domains at bacterial cell poles was predicted to occur spontaneously, by lipid microphase separation arising from the conical CL shape. New data reveal that membrane-anchored cardiolipin synthase A is targeted to the cytoplasmic membrane surface at bacterial cell poles. Thus, localized CL synthesis, interaction of CL with ClsA, and membrane curvature could all contribute to retention of CL at cell poles. These observations provide new insight regarding the mechanism for assembly of CL-rich membrane domains in prokaryotes and eukaryotes.