Asymmetric Protein Localization Research Articles

Polar targeting of proteins - a green perspective.

Cell polarity - the asymmetric distribution of molecules and cell structures within the cell - is a feature that almost all cells possess. Even though the cytoskeleton and other intracellular organelles can have a direction and guide protein distribution, the plasma membrane is, in many cases, essential for the asymmetric localization of proteins because it helps to concentrate proteins and restrict their localization. Indeed, many proteins that exhibit asymmetric or polarized localization are either embedded in the PM or located close to it in the cellular cortex. Such proteins, which we refer to here as 'polar proteins', use various mechanisms of membrane targeting, including vesicle trafficking, direct phospholipid binding, or membrane anchoring mediated by post-translational modifications or binding to other proteins. These mechanisms are often shared with non-polar proteins, yet the unique combinations of several mechanisms or protein-specific factors assure the asymmetric distribution of polar proteins. Although there is a relatively detailed understanding of polar protein membrane targeting mechanisms in animal and yeast models, knowledge in plants is more fragmented and focused on a limited number of known polar proteins in different contexts. In this Review, we combine the current knowledge of membrane targeting mechanisms and factors for known plant transmembrane and cortical proteins and compare these with the mechanisms elucidated in non-plant systems. We classify the known factors as general or polarity specific, and we highlight areas where more knowledge is needed to construct an understanding of general polar targeting mechanisms in plants or to resolve controversies.

Role of BicDR in bristle shaft construction and support of BicD functions.

Cell polarization requires asymmetric localization of numerous mRNAs, proteins and organelles. The movement of cargo towards the minus end of microtubules mostly depends on cytoplasmic dynein motors. In the dynein-dynactin-Bicaudal-D transport machinery, Bicaudal-D (BicD) links the cargo to the motor. Here, we focus on the role of Drosophila BicD-related (BicDR, CG32137) in the development of the long bristles. Together with BicD, it contributes to the organization and stability of the actin cytoskeleton in the not-yet-chitinized bristle shaft. BicD and BicDR also support the stable expression and distribution of Rab6 and Spn-F in the bristle shaft, including the distal tip localization of Spn-F, pointing to the role of microtubule-dependent vesicle trafficking for bristle construction. BicDR supports the function of BicD, and we discuss the hypothesis whereby BicDR might transport cargo more locally, with BicD transporting cargo over long distances, such as to the distal tip. We also identified embryonic proteins that interact with BicDR and appear to be BicDR cargo. For one of them, EF1γ (also known as eEF1γ), we show that the encoding gene EF1γ interacts with BicD and BicDR in the construction of the bristles.

Notch-dependent Abl signaling regulates cell motility during ommatidial rotation in Drosophila.

A collective cell motility event that occurs during Drosophila eye development, ommatidial rotation (OR), serves as a paradigm for signaling-pathway-regulated directed movement of cell clusters. OR is instructed by the EGFR and Notch pathways and Frizzled/planar cell polarity (Fz/PCP) signaling, all of which are associated with photoreceptor R3 and R4 specification. Here, we show that Abl kinase negatively regulates OR through its activity in the R3/R4 pair. Abl is localized to apical junctional regions in R4, but not in R3, during OR, and this apical localization requires Notch signaling. We demonstrate that Abl and Notch interact genetically during OR, and Abl co-immunoprecipitates in complexes with Notch in eye discs. Perturbations of Abl interfere with adherens junctional organization of ommatidial preclusters, which mediate the OR process. Together, our data suggest that Abl kinase acts directly downstream of Notch in R4 to fine-tune OR via its effect on adherens junctions.

Localization of stomatal lineage proteins reveals contrasting planar polarity patterns in Arabidopsis cotyledons

Many plant cells exhibit polarity, revealed by asymmetric localization of specific proteins within each cell.1,2,3,4,5,6 Polarityis typically coordinated between cells across a tissue, raising the question of how coordination is achieved. One hypothesis is that mechanical stresses provide cues.7 This idea gains support from experiments in which cotyledons were mechanically stretched transversely to their midline.8 These previously published results showed that without applied tension, the stomatal lineage cell polarity marker, BREVIS RADIX-LIKE 2 (BRXL2), exhibited no significant excess in the transverse orientation. By contrast, 7h after stretching, BRXL2 polarity distribution exhibited transverse excess, aligned with the stretch direction. These stretching experiments involved statistical comparisons between snapshots of stretched and unstretched cotyledons, with different specimens being imaged in each case.8 Here, we image the same cotyledon before and after stretching and find no evidence for reorientation of polarity. Instead, statistical analysis shows that cotyledons contain a pre-existing transverse excess in BRXL2 polarity orientation that is not significantly modified by applied tension. The transverse excess reflects BRLX2 being preferentially localized toward the medial side of the cell, nearer to the cotyledon midline, creating a weak medial bias. A second polarity marker, BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL), also exhibits weak medial bias in stomatal lineages, whereas ectopic expression of BASL in non-stomatal cells exhibits strong proximal bias, as previously observed in rosette leaves. This proximal bias is also unperturbed by applied tension. Our findings therefore show that cotyledons contain two near-orthogonal coordinated biases in planar polarity: mediolateral and proximodistal.

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.

Phase Separation and Mechanical Forces in Regulating Asymmetric Cell Division of Neural Stem Cells.

Asymmetric cell division (ACD) of neural stem cells and progenitors not only renews the stem cell population but also ensures the normal development of the nervous system, producing various types of neurons with different shapes and functions in the brain. One major mechanism to achieve ACD is the asymmetric localization and uneven segregation of intracellular proteins and organelles into sibling cells. Recent studies have demonstrated that liquid-liquid phase separation (LLPS) provides a potential mechanism for the formation of membrane-less biomolecular condensates that are asymmetrically distributed on limited membrane regions. Moreover, mechanical forces have emerged as pivotal regulators of asymmetric neural stem cell division by generating sibling cell size asymmetry. In this review, we will summarize recent discoveries of ACD mechanisms driven by LLPS and mechanical forces.

Adaptor protein-3 complex is required for Vangl2 trafficking and planar cell polarity of the inner ear.

Planar cell polarity (PCP) regulates coordinated cellular polarity among neighboring cells to establish a polarity axis parallel to the plane of the tissue. Disruption in PCP results in a range of developmental anomalies and diseases. A key feature of PCP is the polarized and asymmetric localization of several membrane PCP proteins, which is essential to establish the polarity axis to orient cells coordinately. However, the machinery that regulates the asymmetric partition of PCP proteins remains largely unknown. In the present study, we show Van gogh-like 2 (Vangl2) in early and recycling endosomes as made evident by colocalization with diverse endosomal Rab proteins. Vangl2 biochemically interacts with adaptor protein-3 complex (AP-3). Using short hairpin RNA knockdown, we found that Vangl2 subcellular localization was modified in AP-3–depleted cells. Moreover, Vangl2 membrane localization within the cochlea is greatly reduced in AP-3–deficient mocha mice, which exhibit profound hearing loss. In inner ears from AP-3–deficient mocha mice, we observed PCP-dependent phenotypes, such as misorientation and deformation of hair cell stereociliary bundles and disorganization of hair cells characteristic of defects in convergent extension that is driven by PCP. These findings demonstrate a novel role of AP-3–mediated sorting mechanisms in regulating PCP proteins.

Actin-binding domains mediate the distinct distribution of two Dictyostelium Talins through different affinities to specific subsets of actin filaments during directed cell migration.

Although the distinct distribution of certain molecules along the anterior or posterior edge is essential for directed cell migration, the mechanisms to maintain asymmetric protein localization have not yet been fully elucidated. Here, we studied a mechanism for the distinct localizations of two Dictyostelium talin homologues, talin A and talin B, both of which play important roles in cell migration and adhesion. Using GFP fusion, we found that talin B, as well as its C-terminal actin-binding region, which consists of an I/LWEQ domain and a villin headpiece domain, was restricted to the leading edge of migrating cells. This is in sharp contrast to talin A and its C-terminal actin-binding domain, which co-localized with myosin II along the cell posterior cortex, as reported previously. Intriguingly, even in myosin II-null cells, talin A and its actin-binding domain displayed a specific distribution, co-localizing with stretched actin filaments. In contrast, talin B was excluded from regions rich in stretched actin filaments, although a certain amount of its actin-binding region alone was present in those areas. When cells were sucked by a micro-pipette, talin B was not detected in the retracting aspirated lobe where acto-myosin, talin A, and the actin-binding regions of talin A and talin B accumulated. Based on these results, we suggest that talin A predominantly interacts with actin filaments stretched by myosin II through its C-terminal actin-binding region, while the actin-binding region of talin B does not make such distinctions. Furthermore, talin B appears to have an additional, unidentified mechanism that excludes it from the region rich in stretched actin filaments. We propose that these actin-binding properties play important roles in the anterior and posterior enrichment of talin B and talin A, respectively, during directed cell migration.

Spatial and temporal analysis of PCP protein dynamics during neural tube closure.

Planar cell polarity (PCP) controls convergent extension and axis elongation in all vertebrates. Although asymmetric localization of PCP proteins is central to their function, we understand little about PCP protein localization during convergent extension. Here, we use quantitative live imaging to simultaneously monitor cell intercalation behaviors and PCP protein dynamics in the Xenopus laevis neural plate epithelium. We observed asymmetric enrichment of PCP proteins, but more interestingly, we observed tight correlation of PCP protein enrichment with actomyosin-driven contractile behavior of cell-cell junctions. Moreover, we found that the turnover rates of junctional PCP proteins also correlated with the contractile behavior of individual junctions. All these dynamic relationships were disrupted when PCP signaling was manipulated. Together, these results provide a dynamic and quantitative view of PCP protein localization during convergent extension and suggest a complex and intimate link between the dynamic localization of core PCP proteins, actomyosin assembly, and polarized junction shrinking during cell intercalation in the closing vertebrate neural tube.

873 Planar cell polarity-disrupting mutations alter Celsr1-mediated cell adhesion and dynamics

Planar cell polarity (PCP), the collective and organized polarization of cells along a tissue plane, is fundamental to early embryonic development and tissue organization in complex, multicellular organisms. A striking example of PCP is the ordered alignment of body hairs on mammalian skin. Deficiencies in PCP components leads to neural tube defects, cardiomyopathies, as well as, abnormal hair patterning in the skin. Atypical cadherin Celsr is the principle component of an asymmetric junctional complex that regulates PCP. How Celsr mediates adhesive interactions to coordinate asymmetric PCP protein localization and function remains poorly understood. Using the mammalian epidermis as a model system, along with junctional recruitment and cell aggregation assays, we confirmed that Celsr1 mediates calcium-dependent, homophilic cell adhesion. Interestingly, disease-associated PCP variants Celsr1Crsh and Celsr1Scy, which harbor mutations in the extracellular cadherin domains, still mediated cell aggregation. These mutants lack the ability to enrich at epithelial junctions, suggesting that intercellular adhesion alone cannot explain Celsr1 functions. Surprisingly, Celsr1Crsh aggregates segregated from Celsr1WT clusters when mixed, demonstrating this point mutation generates a novel Celsr variant with altered homophilic binding properties. Further, Celsr1Crsh displayed broader cell surface distribution and reduced junction localization relative to WT even when in adhesive aggregates. Stable association of Celsr1 at the membrane was also substantially reduced in Celsr1Crsh. These observations indicate that perturbing Celsrs homophilic adhesive properties compromises functional PCP. We hypothesize Celsr1Crsh is defective in lateral clustering and that this mutation affects a region required to stabilize and reinforce the adhesive complex. Domain mapping, biochemistry and advanced imaging are ongoing to elucidate how Celsr1 adhesion mediates PCP complex assembly, maturation and functional asymmetry.

Neural Progenitor Cell Polarity and Cortical Development.

Neurons populating the cerebral cortex are generated during embryonic development from neural stem and progenitor cells in a process called neurogenesis. Neural stem and progenitor cells are classified into several classes based on the different location of mitosis (apical or basal) and polarity features (bipolar, monopolar and non-polar). The polarized architecture of stem cells is linked to the asymmetric localization of proteins, mRNAs and organelles, such as the centrosome and the Golgi apparatus (GA). Polarity affects stem cell function and allows stem cells to integrate environmental cues from distinct niches in the developing cerebral cortex. The crucial role of polarity in neural stem and progenitor cells is highlighted by the fact that impairment of cell polarity is linked to neurodevelopmental disorders such as Down syndrome, Fragile X syndrome, autism spectrum disorders (ASD) and schizophrenia.

Er:YAG Laser and Cyclosporin A Effect on Cell Cycle Regulation of Human Gingival Fibroblast Cells.

Introduction: Periodontitis is a set of inflammatory disorders characterized by periodontal attachment loss and alveolar bone resorption. Because of deficiency in periodontitis mechanical therapy, this study was aimed to explore the molecular influence of the erbiumdoped: yttrium aluminum garnet (Er:YAG) laser and cyclosporin A (CsA) on human gingival fibroblasts (HGFs) for improvement in periodontal diseases therapy. Methods: We focused on articles that studied the proteome profiles of HGFs after treatment with laser irradiation and application of CsA. The topological features of differentially expressed proteins were analyzed using Cytoscape Version 3.4.0 followed by module selection from the protein-protein interaction (PPI) network using Cluster ONE plugin. In addition, we performed gene ontology (GO) enrichment analysis for the densely connected region and key proteins in both PPI networks. Results: Analysis of PPI network of Er:YAG laser irradiation on HGFs lead to introducing YWHAZ, VCP, HNRNPU, YWHAE, UBA52, CLTC, FUS and IGHG1 as key proteins while similar analysis revealed that ACAT1, CTSD, ALDOA, ANXA2, PRDX1, LGALS3, ARHGDI and EEF1A1 are the crucial proteins related to the effect of drug. GO enrichment analysis of hubbottleneck proteins of the 2 networks showed the different significant biological processes and cellular components. The functional enrichments of module of Er:YAG laser network are included as fatty acid transmembrane transport, cytokinesis, regulation of RNA splicing and asymmetric protein localization. There are not any significant clusters in network of HGF treated by CsA. Conclusion: The results indicate that there are 2 separate biomarker panels for the 2 treatment methods.

Messenger RNA transport in the opportunistic fungal pathogen Candida albicans.

Candida albicans, a common commensal fungus, can cause disease in immunocompromised hosts ranging from mild mucosal infections to severe bloodstream infections with high mortality rates. The ability of C. albicans cells to switch between a budding yeast form and an elongated hyphal form is linked to pathogenicity in animal models. Hyphal-specific proteins such as cell-surface adhesins and secreted hydrolases facilitate tissue invasion and host cell damage, but the specific mechanisms leading to asymmetric protein localization in hyphae remain poorly understood. In many eukaryotes, directional cytoplasmic transport of messenger RNAs that encode asymmetrically localized proteins allows efficient local translation at the site of protein function. Over the past two decades, detailed mechanisms for polarized mRNA transport have been elucidated in the budding yeast Saccharomyces cerevisiae and the filamentous fungus Ustilago maydis. This review highlights recent studies of RNA-binding proteins in C. albicans that have revealed intriguing similarities to and differences from known fungal mRNA transport systems. I also discuss outstanding questions that will need to be answered to reach an in-depth understanding of C. albicans mRNA transport mechanisms and the roles of asymmetric mRNA localization in polarized growth, hyphal function, and virulence of this opportunistic pathogen.

Robust Asymmetric Localization of Planar Polarity Proteins Is Associated with Organization into Signalosome-like Domains of Variable Stoichiometry

SummaryIn developing epithelia, the core planar polarity proteins physically interact with each other and localize asymmetrically at opposite cell ends, forming intercellular complexes that link the polarity of neighboring cells. Using quantitative imaging to examine the composition of the core protein complex in vivo, we find that complex composition is unexpectedly plastic. The transmembrane proteins Frizzled and Flamingo form a stoichiometric nucleus in the complex, while the relative levels of the other four core proteins can vary independently. Exploring the functional consequences of this, we show that robust cell polarization is achieved over a range of complex stoichiometries but is dependent on maintaining appropriate levels of the components Frizzled and Strabismus. We propose that the core proteins assemble into signalosome-like structures, where stable association is not dependent on one-to-one interactions with binding partners, and signaling functions can act over a wide range of complex compositions.

Cellular mechanisms for cargo delivery and polarity maintenance at different polar domains in plant cells.

The asymmetric localization of proteins in the plasma membrane domains of eukaryotic cells is a fundamental manifestation of cell polarity that is central to multicellular organization and developmental patterning. In plants, the mechanisms underlying the polar localization of cargo proteins are still largely unknown and appear to be fundamentally distinct from those operating in mammals. Here, we present a systematic, quantitative comparative analysis of the polar delivery and subcellular localization of proteins that characterize distinct polar plasma membrane domains in plant cells. The combination of microscopic analyses and computational modeling revealed a mechanistic framework common to diverse polar cargos and underlying the establishment and maintenance of apical, basal, and lateral polar domains in plant cells. This mechanism depends on the polar secretion, constitutive endocytic recycling, and restricted lateral diffusion of cargos within the plasma membrane. Moreover, our observations suggest that polar cargo distribution involves the individual protein potential to form clusters within the plasma membrane and interact with the extracellular matrix. Our observations provide insights into the shared cellular mechanisms of polar cargo delivery and polarity maintenance in plant cells.

Novel protein Callipygian defines the back of migrating cells

Asymmetric protein localization is essential for cell polarity and migration. We report a novel protein, Callipygian (CynA), which localizes to the lagging edge before other proteins and becomes more tightly restricted as cells polarize; additionally, it accumulates in the cleavage furrow during cytokinesis. CynA protein that is tightly localized, or "clustered," to the cell rear is immobile, but when polarity is disrupted, it disperses throughout the membrane and responds to uniform chemoattractant stimulation by transiently localizing to the cytosol. These behaviors require a pleckstrin homology-domain membrane tether and a WD40 clustering domain, which can also direct other membrane proteins to the back. Fragments of CynA lacking the pleckstrin homology domain, which are normally found in the cytosol, localize to the lagging edge membrane when coexpressed with full-length protein, showing that CynA clustering is mediated by oligomerization. Cells lacking CynA have aberrant lateral protrusions, altered leading-edge morphology, and decreased directional persistence, whereas those overexpressing the protein display exaggerated features of polarity. Consistently, actin polymerization is inhibited at sites of CynA accumulation, thereby restricting protrusions to the opposite edge. We suggest that the mutual antagonism between CynA and regions of responsiveness creates a positive feedback loop that restricts CynA to the rear and contributes to the establishment of the cell axis.

The RNA binding protein MEX-3 retains asymmetric activity in the early Caenorhabditis elegans embryo in the absence of asymmetric protein localization

The RNA binding protein MEX-3 is required to restrict translation of pal-1, the Caenorhabditis elegans caudal homolog, to the posterior of the early embryo. MEX-3 is present uniformly throughout the newly fertilized embryo, but becomes depleted in the posterior by the 4-cell stage. This MEX-3 patterning requires the CCCH zinc-finger protein MEX-5, the RNA Recognition Motif protein SPN-4, and the kinase PAR-4. Genetic and biochemical evidence suggests that MEX-5 binds to MEX-3 in the anterior of the embryo, protecting MEX-3 from degradation and allowing it to bind the pal-1 3′UTR and repress translation. MEX-3 that is not bound to MEX-5 becomes inactivated by par-4, then targeted for spn-4 dependent degradation. After the 4-cell stage, residual MEX-3 is degraded in somatic cells, and only persists in the germline precursors.To better understand regulation of mex-3, GFP was fused to MEX-3 or regions of MEX-3 and expressed in developing oocytes. GFP::MEX-3 expressed in this manner can replace endogenous MEX-3, but surprisingly is not asymmetrically localized at the 4-cell stage. These results indicate that GFP::MEX-3 retains asymmetric activity even in the absence of asymmetric protein localization. Neither the mex-3 3′UTR nor protein degradation at the 4-cell stage is strictly required. A region of MEX-3 containing a glutamine-rich region and potential ubiquitination and phosphorylation sites is sufficient for soma-germline asymmetry. Results from mex-5/6 and spn-4(RNAi) suggest two pathways for MEX-3 degradation, an early spn-4 dependent pathway and a later spn-4 independent pathway. These results indicate that mex-3 activity is regulated at multiple levels, leading to rapid and robust regulation in the quickly developing early embryo.

The cell biology of planar cell polarity.

Planar cell polarity (PCP) refers to the coordinated alignment of cell polarity across the tissue plane. Key to the establishment of PCP is asymmetric partitioning of cortical PCP components and intercellular communication to coordinate polarity between neighboring cells. Recent progress has been made toward understanding how protein transport, endocytosis, and intercellular interactions contribute to asymmetric PCP protein localization. Additionally, the functions of gradients and mechanical forces as global cues that bias PCP orientation are beginning to be elucidated. Together, these findings are shedding light on how global cues integrate with local cell interactions to organize cellular polarity at the tissue level.

The Conserved Discs-large Binding Partner Banderuola Regulates Asymmetric Cell Division in Drosophila

Asymmetric cell division (ACD) is a key process that allows different cell types to be generated at precisely defined times and positions. In Drosophila, neural precursor cells rely heavily on ACD to generate the different cell types in the nervous system. A conserved protein machinery that regulates ACD has been identified in Drosophila, but how this machinery acts to allow the establishment of differential cell fates is not entirely understood. To identify additional proteins required for ACD, we have carried out an invivo live imaging RNAi screen for genes affecting the asymmetric segregation of Numb in Drosophila sensory organ precursor cells. We identify Banderuola (Bnd), an essential regulator of cell polarization, spindle orientation, and asymmetric protein localization in Drosophila neural precursor cells. Genetic and biochemical experiments show that Bnd acts together with the membrane-associated tumor suppressor Discs-large (Dlg) to establish antagonistic cortical domains during ACD. Inhibiting Bnd strongly enhances the dlg phenotype, causing massive brain tumors upon knockdown of both genes. Because the mammalian homologs of Bnd and Dlg are interacting as well, Bnd function might be conserved in vertebrates, and it might also regulate cell polarity in higher organisms. Bnd is a novel regulator of ACD in different types of cells. Our data place Bnd at the top of the hierarchy of the factors involved in ACD, suggesting that its main function is to mediate the localization and function of the Dlg tumor suppressor. Bnd has an antioncogenic function that is redundant with Dlg, and the physical interaction between the two proteins is conserved in evolution.

Arabidopsis UNHINGED encodes a VPS51 homolog and reveals a role for the GARP complex in leaf shape and vein patterning.

Asymmetric localization of PIN proteins controls directionality of auxin transport and many aspects of plant development. Directionality of PIN1 within the marginal epidermis and the presumptive veins of developing leaf primordia is crucial for establishing leaf vein pattern. One mechanism that controls PIN protein distribution within the cell membranes is endocytosis and subsequent transport to the vacuole for degradation. The Arabidopsis mutant unhinged-1 (unh-1) has simpler leaf venation with distal non-meeting of the secondary veins and fewer higher order veins, a narrower leaf with prominent serrations, and reduced root and shoot growth. We identify UNH as the Arabidopsis vacuolar protein sorting 51 (VPS51) homolog, a member of the Arabidopsis Golgi-associated retrograde protein (GARP) complex, and show that UNH interacts with VPS52, another member of the complex and colocalizes with trans Golgi network and pre-vacuolar complex markers. The GARP complex in yeast and metazoans retrieves vacuolar sorting receptors to the trans-Golgi network and is important in sorting proteins for lysosomal degradation. We show that vacuolar targeting is reduced in unh-1. In the epidermal cells of unh-1 leaf margins, PIN1 expression is expanded. The unh-1 leaf phenotype is partially suppressed by pin1 and cuc2-3 mutations, supporting the idea that the phenotype results from expanded PIN1 expression in the marginal epidermis. Our results suggest that UNH is important for reducing expression of PIN1 within margin cells, possibly by targeting PIN1 to the lytic vacuole.