Crosstalk of cell polarity signaling pathways

Cell polarity, the asymmetric organization of cellular components along one or multiple axes, is present in most cells. From budding yeast cell polarization induced by pheromone signaling, oocyte polarization at fertilization to polarized epithelia and neuronal cells in multicellular organisms, similar mechanisms are used to determine cell polarity. Crucial role in this process is played by signaling lipid molecules, small Rho family GTPases and Par proteins. All these signaling circuits finally govern the cytoskeleton, which is responsible for oriented cell migration, cell shape changes, and polarized membrane and organelle trafficking. Thus, typically in the process of cell polarization, most cellular constituents become polarized, including plasma membrane lipid composition, ion concentrations, membrane receptors, and proteins in general, mRNA, vesicle trafficking, or intracellular organelles. This review gives a brief overview how these systems talk to each other both during initial symmetry breaking and within the signaling feedback loop mechanisms used to preserve the polarized state.     

Regulation of the polarity of protein trafficking by phosphorylation

The asymmetry of environmental stimuli and the execution of developmental programs at the organism level require a corresponding polarity at the cellular level, in both unicellular and multicellular organisms. In plants, cell polarity is important in major developmental processes such as cell division, cell enlargement, cell morphogenesis, embryogenesis, axis formation, organ development, and defense. One of the most important factors controlling cell polarity is the asymmetric distribution of polarity determinants. In particular, phosphorylation is implicated in the polar distribution of the determinant protein factors, a mechanism conserved in both prokaryotes and eukaryotes. In plants, formation of local gradients of auxin, the morphogenic hormone, is critical for plant developmental processes exhibiting polarity. The auxin efflux carriers PIN-FORMEDs (PINs) localize asymmetrically in the plasma membrane and cause the formation of local auxin gradients throughout the plant. The asymmetry of PIN distribution in the plasma membrane is determined by phosphorylationmediated polar trafficking of PIN proteins. This review discusses recent studies on the role of phosphorylation in polar PIN trafficking.     

Conserved function of Rho-related Rop/RAC GTPase signaling in regulation of cell polarity in Physcomitrella patens

Cell polarity is fundamentally important to growth and development in higher plants, from pollen tubes to root hairs. Basal land plants (mosses and ferns) also have cell polarity, developing protonemal apical cells that show polar tip growth. Flowering plants have a distinct group of Rho GTPases that regulate polarity in polarized cell growth. Rop/RAC signaling module components have been identified in non-flowering plants, but their roles remain unclear. To understand the importance and evolution of Rop/RAC signaling in polarity regulation in land plants, we examined the functions of PpRop and PpRopGEF in protonemal apical cells of the moss Physcomitrella patens. Inducible overexpression of PpRop2 or PpRopGEF3 caused depolarized growth of tip-growing apical cells. PpRop2 overexpression also caused aberrant cross wall formation. Fluorescent protein-tagged PpRop2 localized to the plasma membrane, including the cross wall membrane, and fluorescent-tagged PpRopGEF3 showed polarized localization to the tip region in apical cells. Thus, our results suggest common functions of PpRop and PpRopGEF in the tip-growing apical cells and the importance of a conserved Rop/RAC signaling module in the control of cell polarity in land plants.     

Polar-localized NPH3-like proteins regulate polarity and endocytosis of PIN-FORMED auxin efflux carriers

PIN-FORMED (PIN)-dependent auxin transport is essential for plant development and its modulation in response to the environment or endogenous signals. A NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3)-like protein, MACCHI-BOU 4 (MAB4), has been shown to control PIN1 localization during organ formation, but its contribution is limited. The Arabidopsis genome contains four genes, MAB4/ENP/NPY1-LIKE1 (MEL1), MEL2, MEL3 and MEL4, highly homologous to MAB4. Genetic analysis disclosed functional redundancy between MAB4 and MEL genes in regulation of not only organ formation but also of root gravitropism, revealing that NPH3 family proteins have a wider range of functions than previously suspected. Multiple mutants showed severe reduction in PIN abundance and PIN polar localization, leading to defective expression of an auxin responsive marker DR5rev::GFP. Pharmacological analyses and fluorescence recovery after photo-bleaching experiments showed that mel mutations increase PIN2 internalization from the plasma membrane, but affect neither intracellular PIN2 trafficking nor PIN2 lateral diffusion at the plasma membrane. Notably, all MAB4 subfamily proteins show polar localization at the cell periphery in plants. The MAB4 polarity was almost identical to PIN polarity. Our results suggest that the MAB4 subfamily proteins specifically retain PIN proteins in a polarized manner at the plasma membrane, thus controlling directional auxin transport and plant development.     

Cell polarity in plants: a PARspective on PINs

Plants have acquired the ability for organized multicellular development independent from animals. Because of this, they represent an independent example in nature for the development of coordinated, complex cell polarity from the simple polarity found in unicellular eukaryotes. Plants display a striking array of polarized cell types, with different axes of polarity being defined in one cell. The most investigated and best understood aspect of plant polarity is the apical-basal polarity of the PIN family of auxin efflux facilitators, which are of crucial importance for the organization of the entire plant body. Striking differences exist between the PAR-polarity modules known in animals and the ways PINs polarize plant cells. Nonetheless, a common regulatory logic probably applies to all polarizing eukaryotic cells, which includes self-reinforcing, positive feedback loops, intricate interactions between membrane-attached proteins, lipid signatures, and the targeting of transmembrane proteins to the correct domains of the plasma membrane.     

Selective control of basolateral membrane protein polarity by cdc42

The rho GTPase cdc42 is implicated in several aspects of cell polarity. A recent study (Kroschewski R, Hall A, Mellman I. Nat Cell Biol 1999;1:8–13) demonstrated that a dominant negative mutant of cdc42 abolishes the polarity of basolateral membrane proteins in MDCK cells, but did not elucidate whether this effect was selective for basolateral proteins or nonselective for all secreted proteins. To answer this question, we analyzed the polarity of newly synthesized membrane and soluble proteins in MDCK cell lines previously induced to overexpress mutant forms of cdc42. GTPase-deficient and dominant negative cdc42 did not affect the apical targeting of a newly synthesized apical membrane protein, but reversed to apical the distribution of two exogenous basolateral membrane proteins. In striking contrast, GTPase-deficient cdc42 did not affect polarized exocytosis of endogenous soluble proteins, either apical or basolateral. The exquisitely selective regulation of polarized protein targeting by cdc42 may allow cells to fine-tune their membrane composition in response to extracellular signals during development, migration and in response to injury.     

Polar delivery in plants; commonalities and differences to animal epithelial cells

Although plant and animal cells use a similar core mechanism to deliver proteins to the plasma membrane, their different lifestyle, body organization and specific cell structures resulted in the acquisition of regulatory mechanisms that vary in the two kingdoms. In particular, cell polarity regulators do not seem to be conserved, because genes encoding key components are absent in plant genomes. In plants, the broad knowledge on polarity derives from the study of auxin transporters, the PIN-FORMED proteins, in the model plant Arabidopsis thaliana. In animals, much information is provided from the study of polarity in epithelial cells that exhibit basolateral and luminal apical polarities, separated by tight junctions. In this review, we summarize the similarities and differences of the polarization mechanisms between plants and animals and survey the main genetic approaches that have been used to characterize new genes involved in polarity establishment in plants, including the frequently used forward and reverse genetics screens as well as a novel chemical genetics approach that is expected to overcome the limitation of classical genetics methods.     

Cell polarity in plants: when two do the same, it is not the same...

In unicellular and multicellular organisms, cell polarity is essential for a wide range of biological processes. An important feature of cell polarity is the asymmetric distribution of proteins in or at the plasma membrane. In plants such polar localized proteins play various specific roles ranging from organizing cell morphogenesis, asymmetric cell division, pathogen defense, nutrient transport and establishment of hormone gradients for developmental patterning. Moreover, flexible respecification of cell polarities enables plants to adjust their physiology and development to environmental changes. Having evolved multicellularity independently and lacking major cell polarity mechanisms of animal cells, plants came up with alternative solutions to generate and respecify cell polarity as well as to regulate polar domains at the plasma membrane.     

Apical-basal polarity in Drosophila neuroblasts is independent of vesicular trafficking

The possession of apical-basal polarity is a common feature of epithelia and neural stem cells, so-called neuroblasts (NBs). In Drosophila, an evolutionarily conserved protein complex consisting of atypical protein kinase C and the scaffolding proteins Bazooka/PAR-3 and PAR-6 controls the polarity of both cell types. The components of this complex localize to the apical junctional region of epithelial cells and form an apical crescent in NBs. In epithelia, the PAR proteins interact with the cellular machinery for polarized exocytosis and endocytosis, both of which are essential for the establishment of plasma membrane polarity. In NBs, many cortical proteins show a strongly polarized subcellular localization, but there is little evidence for the existence of distinct apical and basolateral plasma membrane domains, raising the question of whether vesicular trafficking is required for polarization of NBs. We analyzed the polarity of NBs mutant for essential regulators of the main exocytic and endocytic pathways. Surprisingly, we found that none of these mutations affected NB polarity, demonstrating that NB cortical polarity is independent of plasma membrane polarity and that the PAR proteins function in a cell type-specific manner.     

Membrane domains and polarized trafficking of sphingolipids

The plasma membrane of polarized cells consists of distinct domains, the apical and basolateral membrane, that are characterized by a distinct lipid and protein content. Apical protein transport is largely mediated by (glyco)sphingolipid--cholesterol enriched membrane microdomains, so called rafts. In addition changes in the direction of polarized sphingolipid transport appear instrumental in cell polarity development. Knowledge is therefore required of the mechanisms that mediate sphingolipid sorting and the complexity of the trafficking pathways that are involved in polarized transport of both sphingolipids and proteins. Here we summarize specific biophysical properties that underly mechanisms relevant to sphingolipid sorting, cargo recruitment and polarized trafficking, and discuss the central role of a subapical compartment, SAC or common endosome (CE), as a major intracellular site involved in polarized sorting of sphingolipids, and in development and maintenance of membrane polarity.     

The exocyst complex in exocytosis and cell migration

Exocytosis is a fundamental membrane trafficking event in eukaryotic cells in which membrane proteins or lipids are incorporated into the plasma membrane and vesicle contents are secreted to the exterior of the cell. The exocyst, an evolutionarily conserved octameric protein complex, plays a crucial role in the targeting of secretory vesicles to the plasma membrane during exocytosis. The exocyst has been shown to be involved in diverse cellular processes requiring polarized exocytosis such as yeast budding, epithelial polarity establishment, and neurite outgrowth. Recently, the exocyst has also been implicated in cell migration through mechanisms independent of its role in exocytosis. In this review, we will first summarize our knowledge on the exocyst complex at a molecular and structural level. Then, we will discuss the specific functions of the exocyst in exocytosis in various cell types. Finally, we will review the emerging roles of the exocyst during cell migration and tumor cell invasion.     

Catch the KIF5B train to the apical surface

As epithelial cells become polarized, they develop new pathways to send proteins to the apical or basolateral domains of their plasma membrane. In this issue of Developmental Cell, Jaulin et al. (2007) show that as polarity develops, there is a shift in the kinesin motor protein used to transport an apical protein to the cell surface.     

Recycling,clustering, and endocytosis jointly maintain PIN auxin carrier polarity at the plasma membrane

Cell polarity reflected by asymmetric distribution of proteins at the plasma membrane is a fundamental feature of unicellular and multicellular organisms. It remains conceptually unclear how cell polarity is kept in cell wall‐encapsulated plant cells. We have used super‐resolution and semi‐quantitative live‐cell imaging in combination with pharmacological, genetic, and computational approaches to reveal insights into the mechanism of cell polarity maintenance in Arabidopsis thaliana. We show that polar‐competent PIN transporters for the phytohormone auxin are delivered to the center of polar domains by super‐polar recycling. Within the plasma membrane, PINs are recruited into non‐mobile membrane clusters and their lateral diffusion is dramatically reduced, which ensures longer polar retention. At the circumventing edges of the polar domain, spatially defined internalization of escaped cargos occurs by clathrin‐dependent endocytosis. Computer simulations confirm that the combination of these processes provides a robust mechanism for polarity maintenance in plant cells. Moreover, our study suggests that the regulation of lateral diffusion and spatially defined endocytosis, but not super‐polar exocytosis have primary importance for PIN polarity maintenance.     

Linkage between cell membrane proteins and actin-based cytoskeleton: the cytoskeletal-driven cellular functions

Asymmetric organization of the plasma membrane and cytosolic organelles is fundamental for a variety of cells, including bacteria, yeast and eukaryotic cells (Nelson, 1992). The degree into which cells polarize is characterized by their ability to create and maintain morphologically and biochemically distinct plasma membrane domains. The generation and maintenance of polarized distribution of membrane components (proteins and lipids) is thus critical to the ability of cells to perform complex activities such as cell-to-cell interactions, vectorial transport and secretion, cellular immunity, development and morphogenesis. Modification of cellular polarity may potentially lead to abnormal cellular activities and various pathological disorders (Molitoris, 1991; Carone et al., 1994; Chen et al., 1995). Our review shows the complex interplay between membrane proteins and the cytoskeletal network in determining the "polarized phenotype" in the cell. We provide evidence that membrane/cytoskeleton interaction is the key to regulation of the vast majority of cellular functions.     

SPIKE1 signals originate from and assemble specialized domains of the endoplasmic reticulum

In the leaf epidermis, intricately lobed pavement cells use Rho of plants (ROP) small GTPases to integrate actin and microtubule organization with trafficking through the secretory pathway. Cell signaling occurs because guanine nucleotide exchange factors (GEFs) promote ROP activation and their interactions with effector proteins that direct the cell growth machineries. In Arabidopsis, SPIKE1 (SPK1) is the lone DOCK family GEF. SPK1 promotes polarized growth and cell-cell adhesion in the leaf epidermis; however, its mode of action in cells is not known. Vertebrate DOCK proteins are deployed at the plasma membrane. Likewise, current models place SPK1 activity and/or active ROP at the plant plasma membrane and invoke the localized patterning of the cortical cytoskeleton as the mechanism for shape control. In this paper, we find that SPK1 is a peripheral membrane protein that accumulates at, and promotes the formation of, a specialized domain of the endoplasmic reticulum (ER) termed the ER exit site (ERES). SPK1 signals are generated from a distributed network of ERES point sources and maintain the homeostasis of the early secretory pathway. The ERES is the location for cargo export from the ER. Our findings open up unexpected areas of plant G protein biology and redefine the ERES as a subcellular location for signal integration during morphogenesis.     

Identification of a membrane-cytoskeletal complex containing the cell adhesion molecule uvomorulin (E-cadherin), ankyrin, and fodrin in Madin- Darby canine kidney epithelial cells

Cell-cell contact is an important determinant in the formation of functionally distinct plasma membrane domains during the development of epithelial cell polarity. In cultures of Madin-Darby canine kidney (MDCK) epithelial cells, cell-cell contact induces the assembly and accumulation of the Na+,K+-ATPase and elements of the membrane-cytoskeleton (ankyrin and fodrin) at the regions of cell-cell contact. Epithelial cell-cell contact appears to be regulated by the cell adhesion molecule uvomorulin (E-cadherin) which also becomes localized at the lateral plasma membrane of polarized cells. We have sought to determine whether the colocalization of these proteins reflects direct molecular interactions which may play roles in coordinating cell-cell contact and the assembly of the basal-lateral domain of the plasma membrane. Recently, we identified a complex of proteins containing the Na+,K+-ATPase, ankyrin, and fodrin in extracts of whole MDCK cells (Nelson, W.J., and R. W. Hammerton. 1989. J. Cell Biol. 108:893-902). We have now examined cell extracts for protein complexes containing the cell adhesion molecule uvomorulin. Proteins were solubilized from whole MDCK cells and fractionated in sucrose gradients. The sedimentation profile of solubilized uvomorulin is well separated from the majority of cell surface proteins, suggesting that uvomorulin occurs in a protein complex. A distinct portion of uvomorulin (30%) cosediments with ankyrin and fodrin (approximately 10.5S). Further fractionation of cosedimenting proteins in nondenaturing polyacrylamide gels reveals a discrete band of proteins that binds antibodies specific for uvomorulin, Na+,K+-ATPase, ankyrin, and fodrin. Significantly, ankyrin and fodrin, but not Na+K+-ATPase, coimmunoprecipitate in a complex with uvomorulin using uvomorulin antibodies. This result indicates that separate complexes exist containing ankyrin and fodrin with either uvomorulin or Na+,K+-ATPase. These results are discussed in the context of the possible roles of uvomorulin-induced cell-cell contact in the assembly of the membrane-cytoskeleton and associated membrane proteins (e.g., Na+,K+-ATPase) at the contact zone and in the development of cell polarity.     

Membrane Organization and Dynamics in Cell Polarity

The establishment and maintenance of cell polarity is important to a wide range of biological processes ranging from chemotaxis to embryogenesis. An essential feature of cell polarity is the asymmetric organization of proteins and lipids in the plasma membrane. In this article, we discuss how polarity regulators such as small GTP-binding proteins and phospholipids spatially and kinetically control vesicular trafficking and membrane organization. Conversely, we discuss how membrane trafficking contributes to cell polarization through delivery of polarity determinants and regulators to the plasma membrane.Cell polarity is essential in most if not all eukaryotes for their development and physiological functions at the tissue and organism level. Although there are significant differences in gross morphology and function among various tissues and organisms, at the cellular level, the establishment and maintenance of cell polarity tend to follow common themes.A basic feature of cell polarity is the asymmetric organization of the plasma membrane (see McCaffrey and Macara 2009; Nelson 2009). This is mostly achieved through membrane trafficking along cytoskeleton tracks under the control of signaling molecules. In general, membrane trafficking occurs through sequential budding, transport, and fusion of vesicles from donor membranes to acceptor membranes (for recent reviews, see Bonifacino and Glick 2004; Cai et al. 2007). During budding, protein complexes interact with phospholipids to induce membrane curvature and generate vesicular carriers that capture different cargos from the donor compartments. After vesicles form, they are delivered to their acceptor compartments, most often along the cytoskeletons. Vesicle fusion at the acceptor membrane is mediated by the assembly of SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) complexes. Before membrane fusion, proteins or protein complexes tether the vesicles to the acceptor membranes and likely promote SNARE assembly. The Arf and Rab family of small GTPases are localized to different membrane compartments and regulate various stages of membrane trafficking.Polarized distribution of proteins at the plasma membrane often results from a balance of vesicle delivery and fusion with the plasma membrane (“exocytosis”), two-dimensional spread through the plasma membrane (“diffusion”), and internalization and membrane recycling (“endocytosis”). There are two main layers of regulation that control polarized protein transport and incorporation to the plasma membrane. The first involves sorting at the trans-Golgi network (TGN) and endosomal compartments, such as the recycling endosomes. Protein sorting involves recognition of sorting signals in the cargo proteins by the adaptor protein (AP) complexes. There are a number of different AP complexes, and each is localized to different membrane compartments and captures distinct sets of cargo proteins before targeting to their correct destination. Protein sorting before delivery to different domains of the plasma membrane has been best characterized in epithelial cells, which have distinctive basolateral and apical domains separated by junctional complexes. This layer of regulation has been discussed in a recent review (Mellman and Nelson 2008) and is further discussed by Nelson (Nelson 2009), so it will not be discussed further here. The second layer of regulation of membrane protein polarization is through the polarized tethering and docking of vesicles at specific domains of the plasma membrane (Fig. 1). Tethering proteins (i.e., the exocyst) target secretory vesicles to specific domains of the plasma membrane and SNARE assembly eventually drives membrane fusion. Proteins at the plasma membrane can be retrieved back into the cell via endocytosis. These proteins are internalized via clathrin-coated pits, and transported through different endosomal compartments either for degradation in the lysosomes or for recycling back to the plasma membrane. The endosomal compartment that mediates the transport of internalized plasma membrane proteins back to the cell surface is called the “recycling endosome.” Recycling endosomes are major sources of cargo destined to the plasma membrane for exocytosis in many types of cells.Open in a separate windowFigure 1.Membrane trafficking to the plasma membrane. Schematic of the endocytic and exocytic routes involving trans-Golgi network (TGN), endosomal compartments, and the plasma membrane. During exocytosis, cargo leaves the TGN or recycling endosomes in vesicular carriers to the plasma membrane. Once on the membrane, proteins can be internalized and transported to early endosomes, and then either travel through late endosomes to the lysosome to be degraded or return to the plasma membrane through the recycling endosomes. Early endosomes may serve as sorting stations for the next stages of cargo transport.Signaling molecules such as the Rho family of small GTPases spatially and kinetically regulate membrane trafficking during cell polarization (see McCaffrey and Macara 2009; Slaughter et al. 2009). Reversely, vesicular trafficking is required for the polarized deposition and accrual of these regulators. In the first part of this article, we examine the membrane organization and dynamics of cell polarity, focusing on the polarized tethering and docking of vesicles at the plasma membrane. We highlight key components and regulators of polarized exocytosis including the exocyst, small GTPases, and phospholipids. We also use different organisms and systems to show analogous mechanisms during cell polarization. In the second part of this article, we focus on the aforementioned reciprocal effects of cell polarity and membrane trafficking using two representative examples, one from yeast (Cdc42 polarization) and one in mammalian epithelial cells (E-cadherin trafficking).     

Cdc42 localization and cell polarity depend on membrane traffic

Cell polarity is essential for cell division, cell differentiation, and most differentiated cell functions including cell migration. The small G protein Cdc42 controls cell polarity in a wide variety of cellular contexts. Although restricted localization of active Cdc42 seems to be important for its distinct functions, mechanisms responsible for the concentration of active Cdc42 at precise cortical sites are not fully understood. In this study, we show that during directed cell migration, Cdc42 accumulation at the cell leading edge relies on membrane traffic. Cdc42 and its exchange factor βPIX localize to intracytosplasmic vesicles. Inhibition of Arf6-dependent membrane trafficking alters the dynamics of Cdc42-positive vesicles and abolishes the polarized recruitment of Cdc42 and βPIX to the leading edge. Furthermore, we show that Arf6-dependent membrane dynamics is also required for polarized recruitment of Rac and the Par6-aPKC polarity complex and for cell polarization. Our results demonstrate influence of membrane dynamics on the localization and activation of Cdc42 and consequently on directed cell migration.     

Pole position: How plant cells polarize along the axes

Having a sense of direction is a fundamental cellular trait that can determine cell shape, division orientation, or function, and ultimately the formation of a functional, multicellular body. Cells acquire and integrate directional information by establishing discrete subcellular domains along an axis with distinct molecular profiles, a process known as cell polarization. Insight into the principles and mechanisms underlying cell polarity has been propelled by decades of extensive research mostly in yeast and animal models. Our understanding of cell polarity establishment in plants, which lack most of the regulatory molecules identified in other eukaryotes, is more limited, but significant progress has been made in recent years. In this review, we explore how plant cells coordinately establish stable polarity axes aligned with the organ axes, highlighting similarities in the molecular logic used to polarize both plant and animal cells. We propose a classification system for plant cell polarity events and nomenclature guidelines. Finally, we provide a deep phylogenetic analysis of polar proteins and discuss the evolution of polarity machineries in plants.

This review discusses the principles, mechanisms, and evolution of plant cell polarity, an essential cellular feature for plant development and physiology that endows cells with a sense of direction.