Mechanisms involved in maintaining plasma membrane domains in fully polarized epithelial cells are known, but when and how directed protein sorting and trafficking occur to initiate cell surface polarity are not. We tested whether establishment of the basolateral membrane domain and E-cadherin–mediated epithelial cell–cell adhesion are mechanistically linked. We show that the basolateral membrane aquaporin (AQP)-3, but not the equivalent apical membrane AQP5, is delivered in post-Golgi structures directly to forming cell–cell contacts where it co-accumulates precisely with E-cadherin. Functional disruption of individual components of a putative lateral targeting patch (e.g., microtubules, the exocyst, and soluble N-ethylmaleimide–sensitive factor attachment protein receptors) did not inhibit cell–cell adhesion or colocalization of the other components with E-cadherin, but each blocked AQP3 delivery to forming cell–cell contacts. Thus, components of the lateral targeting patch localize independently of each other to cell–cell contacts but collectively function as a holocomplex to specify basolateral vesicle delivery to nascent cell–cell contacts and immediately initiate cell surface polarity.
In epithelial cells, Sec3 associates with Exocyst complexes enriched at desmosomes and centrosomes, distinct from Sec6/8 complexes at the apical junctional complex. RNAi-mediated suppression of Sec3 alters trafficking of desmosomal cadherins and impairs desmosome morphology and function, without noticeable effect on adherens junctions.
The Exocyst is a conserved multisubunit complex involved in the docking of post-Golgi transport vesicles to sites of membrane remodeling during cellular processes such as polarization, migration, and division. In mammalian epithelial cells, Exocyst complexes are recruited to nascent sites of cell–cell contact in response to E-cadherin–mediated adhesive interactions, and this event is an important early step in the assembly of intercellular junctions. Sec3 has been hypothesized to function as a spatial landmark for the development of polarity in budding yeast, but its role in epithelial cells has not been investigated. Here, we provide evidence in support of a function for a Sec3-containing Exocyst complex in the assembly or maintenance of desmosomes, adhesive junctions that link intermediate filament networks to sites of strong intercellular adhesion. We show that Sec3 associates with a subset of Exocyst complexes that are enriched at desmosomes. Moreover, we found that membrane recruitment of Sec3 is dependent on cadherin-mediated adhesion but occurs later than that of the known Exocyst components Sec6 and Sec8 that are recruited to adherens junctions. RNA interference-mediated suppression of Sec3 expression led to specific impairment of both the morphology and function of desmosomes, without noticeable effect on adherens junctions. These results suggest that two different exocyst complexes may function in basal–lateral membrane trafficking and will enable us to better understand how exocytosis is spatially organized during development of epithelial plasma membrane domains.
The tumor suppressor lethal giant larvae (Lgl) plays a critical role in epithelial cell polarization. However, the molecular mechanism by which Lgl carries out its functions is unclear. In this study, we report that the yeast Lgl proteins Sro7p and Sro77p directly interact with Exo84p, which is a component of the exocyst complex that is essential for targeting vesicles to specific sites of the plasma membrane for exocytosis, and that this interaction is important for post-Golgi secretion. Genetic analyses demonstrate a molecular pathway from Rab and Rho GTPases through the exocyst and Lgl to SNAREs, which mediate membrane fusion. We also found that overexpression of Lgl and t-SNARE proteins not only improves exocytosis but also rescues polarity defects in exocyst mutants. We propose that, although Lgl is broadly distributed in the cells, its localized interaction with the exocyst and kinetic activation are important for the establishment and reenforcement of cell polarity.
Epithelial cyst and tubule formation are critical processes that
involve transient, highly choreographed changes in cell polarity.
Factors controlling these changes in polarity are largely unknown. One
candidate factor is the highly conserved eight-member protein complex
called the exocyst. We show that during tubulogenesis in an in vitro
model system the exocyst relocalized along growing tubules consistent
with changes in cell polarity. In yeast, the exocyst subunit Sec10p is
a crucial component linking polarized exocytic vesicles with the rest
of the exocyst complex and, ultimately, the plasma membrane. When the
exocyst subunit human Sec10 was exogenously expressed in
epithelial Madin-Darby canine kidney cells, there was a selective
increase in the synthesis and delivery of apical and basolateral
secretory proteins and a basolateral plasma membrane protein, but not
an apical plasma membrane protein. Overexpression of human Sec10
resulted in more efficient and rapid cyst formation and increased
tubule formation upon stimulation with hepatocyte growth factor. We
conclude that the exocyst plays a central role in the development of
epithelial cysts and tubules.
The Sec6 subunit of the multisubunit exocyst tethering complex interacts with the Sec1/Munc18 protein Sec1 and with the t-SNARE Sec9. Assembly of the exocyst upon vesicle arrival at sites of secretion is proposed to release Sec9 for SNARE complex assembly and to recruit Sec1 for interaction with SNARE complexes to facilitate fusion.
Trafficking of protein and lipid cargo through the secretory pathway in eukaryotic cells is mediated by membrane-bound vesicles. Secretory vesicle targeting and fusion require a conserved multisubunit protein complex termed the exocyst, which has been implicated in specific tethering of vesicles to sites of polarized exocytosis. The exocyst is directly involved in regulating soluble N-ethylmaleimide–sensitive factor (NSF) attachment protein receptor (SNARE) complexes and membrane fusion through interactions between the Sec6 subunit and the plasma membrane SNARE protein Sec9. Here we show another facet of Sec6 function—it directly binds Sec1, another SNARE regulator, but of the Sec1/Munc18 family. The Sec6–Sec1 interaction is exclusive of Sec6–Sec9 but compatible with Sec6–exocyst assembly. In contrast, the Sec6–exocyst interaction is incompatible with Sec6–Sec9. Therefore, upon vesicle arrival, Sec6 is proposed to release Sec9 in favor of Sec6–exocyst assembly and to simultaneously recruit Sec1 to sites of secretion for coordinated SNARE complex formation and membrane fusion.
Sec6/8 (exocyst) complex regulates vesicle delivery and polarized membrane growth in a variety of cells, but mechanisms regulating Sec6/8 localization are unknown. In epithelial cells, Sec6/8 complex is recruited to cell-cell contacts with a mixture of junctional proteins, but then sorts out to the apex of the lateral membrane with components of tight junction and nectin complexes. Sec6/8 complex fractionates in a high molecular mass complex with tight junction proteins and a portion of E-cadherin, and co-immunoprecipitates with cell surface-labeled E-cadherin and nectin-2α. Recruitment of Sec6/8 complex to cell-cell contacts can be achieved in fibroblasts when E-cadherin and nectin-2α are co-expressed. These results support a model in which localized recruitment of Sec6/8 complex to the plasma membrane by specific cell-cell adhesion complexes defines a site for vesicle delivery and polarized membrane growth during development of epithelial cell polarity.
Cell polarity; Cell membrane; Intercellular junctions; Intracellular membranes; Metabolism
Epithelial tubes represent fundamental building blocks of metazoan organisms; however, the mechanisms responsible for their formation and maintenance are not well understood. Here we show that unique and evolutionary conserved coiled-coil MAGUK protein, Dlg5, is required for epithelial tube maintenance in mammalian brain and kidneys. We demonstrate that Dlg5-/- mice develop fully penetrant hydrocephalus and kidney cysts caused by deficiency in membrane delivery of cadherin-catenin adhesion complexes and loss of cell polarity. Dlg5 travels with cadherin-containing vesicles and binds to syntaxin 4, a t-SNARE protein that regulates fusion of transport vesicles with the lateral membrane domain. We propose that Dlg5 functions in plasma membrane delivery of cadherins by linking cadherin-containing transport vesicles with the t-SNARE targeting complex. These findings identify a novel protein causally involved in hydrocephalus and renal cysts and reveal that targeted membrane delivery of cadherin-catenin adhesion complexes is critical for cell polarity and epithelial tube maintenance.
To form epithelial organs cells must polarize and generate de novo an apical domain and lumen. Epithelial polarization is masterminded by polarity complexes, which are thought to direct downstream events such as polarized membrane traffic, though this interconnection is not well understood. We report that Rab11a regulates apical traffic and lumen formation via the Rab GEF Rabin8, and its target Rab8a. Rab8a/11a act via the exocyst to target Par3 to the apical surface, and control apical Cdc42 activation via the Cdc42 GEF, Tuba. These components assemble at a transient apical membrane initiation site to form the lumen. This Rab11a-directed network directs Cdc42-dependent apical exocytosis during lumen formation, revealing a novel interplay of the machineries of vesicular transport and polarization.
Most epithelial cells contain two AP-1 clathrin adaptor complexes. AP-1A is ubiquitously expressed and involved in transport between the TGN and endosomes. AP-1B is expressed only in epithelia and mediates the polarized targeting of membrane proteins to the basolateral surface. Both AP-1 complexes are heterotetramers and differ only in their 50-kD μ1A or μ1B subunits. Here, we show that AP-1A and AP-1B, together with their respective cargoes, define physically and functionally distinct membrane domains in the perinuclear region. Expression of AP-1B (but not AP-1A) enhanced the recruitment of at least two subunits of the exocyst complex (Sec8 and Exo70) required for basolateral transport. By immunofluorescence and cell fractionation, the exocyst subunits were found to selectively associate with AP-1B–containing membranes that were both distinct from AP-1A–positive TGN elements and more closely apposed to transferrin receptor–positive recycling endosomes. Thus, despite the similarity of the two AP-1 complexes, AP-1A and AP-1B exhibit great specificity for endosomal transport versus cell polarity.
epithelial cells; basolateral sorting; exocytosis; TGN; exocyst
Type Iγ phosphatidylinositol-4-phosphate 5-kinase and Exo70 cooperate in the directed targeting of E-cadherin on the plasma membrane to newly formed adherens junctions. This promotes the regional accumulation of E-cadherin, expansion and maturation of adherens junctions, and differentiation of the lateral membrane domain.
E-Cadherin–mediated formation of adherens junctions (AJs) is essential for the morphogenesis of epithelial cells. However, the mechanisms underlying E-cadherin clustering and AJ maturation are not fully understood. Here we report that type Iγ phosphatidylinositol-4-phosphate 5-kinase (PIPKIγ) associates with the exocyst via a direct interaction with Exo70, the exocyst subunit that guides the polarized targeting of exocyst to the plasma membrane. By means of this interaction, PIPKIγ mediates the association between E-cadherin and Exo70 and determines the targeting of Exo70 to AJs. Further investigation revealed that Exo70 is necessary for clustering of E-cadherin on the plasma membrane and extension of nascent E-cadherin adhesions, which are critical for the maturation of cohesive AJs. In addition, we observed phosphatidylinositol-4,5-bisphosphate (PI4,5P2) accumulation at E-cadherin clusters during the assembly of E-cadherin adhesions. PIPKIγ-generated PI4,5P2 is required for recruiting Exo70 to newly formed E-cadherin junctions and facilitates the assembly and maturation of AJs. These results support a model in which PIPKIγ and PIPKIγ-generated PI4,5P2 pools at nascent E-cadherin contacts cue Exo70 targeting and orient the tethering of exocyst-associated E-cadherin. This could be an important mechanism that regulates E-cadherin clustering and AJ maturation, which is essential for the establishment of solid, polarized epithelial structures.
The exocyst is an essential protein complex required for targeting and fusion of secretory vesicles to sites of exocytosis at the plasma membrane. To study the function of the exocyst complex, we performed a structure-based mutational analysis of the Saccharomyces cerevisiae exocyst subunit Sec6p. Two “patches” of highly conserved residues are present on the surface of Sec6p; mutation of either patch does not compromise protein stability. Nevertheless, replacement of SEC6 with the patch mutants results in severe temperature-sensitive growth and secretion defects. At nonpermissive conditions, although trafficking of secretory vesicles to the plasma membrane is unimpaired, none of the exocyst subunits are polarized. This is consistent with data from other exocyst temperature-sensitive mutants, which disrupt the integrity of the complex. Surprisingly, however, these patch mutations result in mislocalized exocyst complexes that remain intact. Our results indicate that assembly and polarization of the exocyst are functionally separable events, and that Sec6p is required to anchor exocyst complexes at sites of secretion.
Invadopodia are actin-based membrane protrusions formed at contact sites between invasive tumor cells and the extracellular matrix with matrix proteolytic activity. Actin regulatory proteins participate in invadopodia formation, whereas matrix degradation requires metalloproteinases (MMPs) targeted to invadopodia. In this study, we show that the vesicle-tethering exocyst complex is required for matrix proteolysis and invasion of breast carcinoma cells. We demonstrate that the exocyst subunits Sec3 and Sec8 interact with the polarity protein IQGAP1 and that this interaction is triggered by active Cdc42 and RhoA, which are essential for matrix degradation. Interaction between IQGAP1 and the exocyst is necessary for invadopodia activity because enhancement of matrix degradation induced by the expression of IQGAP1 is lost upon deletion of the exocyst-binding site. We further show that the exocyst and IQGAP1 are required for the accumulation of cell surface membrane type 1 MMP at invadopodia. Based on these results, we propose that invadopodia function in tumor cells relies on the coordination of cytoskeletal assembly and exocytosis downstream of Rho guanosine triphosphatases.
The octameric exocyst complex is associated with the junctional complex and recycling endosomes and is proposed to selectively tether cargo vesicles directed toward the basolateral surface of polarized Madin-Darby canine kidney (MDCK) cells. We observed that the exocyst subunits Sec6, Sec8, and Exo70 were localized to early endosomes, transferrin-positive common recycling endosomes, and Rab11a-positive apical recycling endosomes of polarized MDCK cells. Consistent with its localization to multiple populations of endosomes, addition of function-blocking Sec8 antibodies to streptolysin-O–permeabilized cells revealed exocyst requirements for several endocytic pathways including basolateral recycling, apical recycling, and basolateral-to-apical transcytosis. The latter was selectively dependent on interactions between the small GTPase Rab11a and Sec15A and was inhibited by expression of the C-terminus of Sec15A or down-regulation of Sec15A expression using shRNA. These results indicate that the exocyst complex may be a multipurpose regulator of endocytic traffic directed toward both poles of polarized epithelial cells and that transcytotic traffic is likely to require Rab11a-dependent recruitment and modulation of exocyst function, likely through interactions with Sec15A.
E-cadherin plays an essential role in cell polarity and cell-cell adhesion; however, the pathway for delivery of E-cadherin to the basolateral membrane of epithelial cells has not been fully characterized. We first traced the post-Golgi, exocytic transport of GFP-tagged E-cadherin (Ecad-GFP) in unpolarized cells. In live cells, Ecad-GFP was found to exit the Golgi complex in pleiomorphic tubulovesicular carriers, which, instead of moving directly to the cell surface, most frequently fused with an intermediate compartment, subsequently identified as a Rab11-positive recycling endosome. In MDCK cells, basolateral targeting of E-cadherin relies on a dileucine motif. Both E-cadherin and a targeting mutant, ΔS1-E-cadherin, colocalized with Rab11 and fused with the recycling endosome before diverging to basolateral or apical membranes, respectively. In polarized and unpolarized cells, coexpression of Rab11 mutants disrupted the cell surface delivery of E-cadherin and caused its mistargeting to the apical membrane, whereas apical ΔS1-E-cadherin was unaffected. We thus demonstrate a novel pathway for Rab11 dependent, dileucine-mediated, μ1B-independent sorting and basolateral trafficking, exemplified by E-cadherin. The recycling endosome is identified as an intermediate compartment for the post-Golgi trafficking and exocytosis of E-cadherin, with a potentially important role in establishing and maintaining cadherin-based adhesion.
The closely related GTPases RalA and RalB are required for assembly of tight junction gate, but not fence, function. These activities depend on direct binding to the exocyst complex. Whereas RalA–exocyst complexes are required for exocytosis of junction proteins, RalB–exocyst complexes are required for endocytosis of these components.
Tight junctions (TJs) are structures indispensable to epithelial cells and are responsible for regulation of paracellular diffusion and maintenance of cellular polarity. Although many interactions between TJ constituents have been identified, questions remain concerning how specific functions of TJs are established and regulated. Here we investigated the roles of Ral GTPases and their common effector exocyst complex in the formation of nascent TJs. Unexpectedly, RNA interference–mediated suppression of RalA or RalB caused opposing changes in TJ development. RalA reduction increased paracellular permeability and decreased incorporation of components into TJs, whereas RalB reduction decreased paracellular permeability and increased incorporation of components into TJs. Activities of both Ral GTPases were mediated through the exocyst. Finally, we show that TJ-mediated separation of apical–basal membrane domains is established prior to equilibration of barrier function and that it is unaffected by Ral knockdown or specific composition of TJs.
The E-cadherin/catenin complex regulates Ca++-dependent cell–cell adhesion and is localized to the basal-lateral membrane of polarized epithelial cells. Little is known about mechanisms of complex assembly or intracellular trafficking, or how these processes might ultimately regulate adhesion functions of the complex at the cell surface. The cytoplasmic domain of E-cadherin contains two putative basal-lateral sorting motifs, which are homologous to sorting signals in the low density lipoprotein receptor, but an alanine scan across tyrosine residues in these motifs did not affect the fidelity of newly synthesized E-cadherin delivery to the basal-lateral membrane of MDCK cells. Nevertheless, sorting signals are located in the cytoplasmic domain since a chimeric protein (GP2CAD1), comprising the extracellular domain of GP2 (an apical membrane protein) and the transmembrane and cytoplasmic domains of E-cadherin, was efficiently and specifically delivered to the basal-lateral membrane. Systematic deletion and recombination of specific regions of the cytoplasmic domain of GP2CAD1 resulted in delivery of <10% of these newly synthesized proteins to both apical and basal-lateral membrane domains. Significantly, >90% of each mutant protein was retained in the ER. None of these mutants formed a strong interaction with β-catenin, which normally occurs shortly after E-cadherin synthesis. In addition, a simple deletion mutation of E-cadherin that lacks β-catenin binding is also localized intracellularly. Thus, β-catenin binding to the whole cytoplasmic domain of E-cadherin correlates with efficient and targeted delivery of E-cadherin to the lateral plasma membrane. In this capacity, we suggest that β-catenin acts as a chauffeur, to facilitate transport of E-cadherin out of the ER and the plasma membrane.
polarity; epithelia; adhesion; protein targeting; membrane domains
The exocyst complex plays a critical role in targeting and tethering vesicles to specific sites of the plasma membrane. These events are crucial for polarized delivery of membrane components to the cell surface, which is critical for cell motility and division. Though Rho GTPases are involved in regulating actin dynamics and membrane trafficking, their role in exocyst-mediated vesicle targeting is not very clear. Herein, we present evidence that depletion of GEF-H1, a guanine nucleotide exchange factor for Rho proteins, affects vesicle trafficking. Interestingly, we found that GEF-H1 directly binds to exocyst component Sec5 in a Ral GTPase-dependent manner. This interaction promotes RhoA activation, which then regulates exocyst assembly/localization and exocytosis. Taken together, our work defines a mechanism for RhoA activation in response to RalA-Sec5 signaling and involvement of GEF-H1/RhoA pathway in the regulation of vesicle trafficking.
The epithelial cadherin (E-cadherin)–catenin complex binds to cytoskeletal components and regulatory and signaling molecules to form a mature adherens junction (AJ). This dynamic structure physically connects neighboring epithelial cells, couples intercellular adhesive contacts to the cytoskeleton, and helps define each cell’s apical–basal axis. Together these activities coordinate the form, polarity, and function of all cells in an epithelium. Several molecules regulate AJ formation and integrity, including Rho family GTPases and Par polarity proteins. However, only recently, with the development of live-cell imaging, has the extent to which E-cadherin is actively turned over at junctions begun to be appreciated. This turnover contributes to junction formation and to the maintenance of epithelial integrity during tissue homeostasis and remodeling.
Polarized exocytosis is important for morphogenesis and cell growth. The exocyst is a multiprotein complex implicated in tethering secretory vesicles at specific sites of the plasma membrane for exocytosis. In the budding yeast, the exocyst is localized to sites of bud emergence or the tips of small daughter cells, where it mediates secretion and cell surface expansion. To understand how exocytosis is spatially controlled, we systematically analyzed the localization of Sec15p, a member of the exocyst complex and downstream effector of the rab protein Sec4p, in various mutants. We found that the polarized localization of Sec15p relies on functional upstream membrane traffic, activated rab protein Sec4p, and its guanine exchange factor Sec2p. The initial targeting of both Sec4p and Sec15p to the bud tip depends on polarized actin cable. However, different recycling mechanisms for rab and Sec15p may account for the different kinetics of polarization for these two proteins. We also found that Sec3p and Sec15p, though both members of the exocyst complex, rely on distinctive targeting mechanisms for their localization. The assembly of the exocyst may integrate various cellular signals to ensure that exocytosis is tightly controlled. Key regulators of cell polarity such as Cdc42p are important for the recruitment of the exocyst to the budding site. Conversely, we found that the proper localization of these cell polarity regulators themselves also requires a functional exocytosis pathway. We further report that Bem1p, a protein essential for the recruitment of signaling molecules for the establishment of cell polarity, interacts with the exocyst complex. We propose that a cyclical regulatory network contributes to the establishment and maintenance of polarized cell growth in yeast.
The keratin intermediate filament network is abundant in epithelial cells, but its function in the establishment and maintenance of cell polarity is unclear. Here, we show that Albatross complexes with Par3 to regulate formation of the apical junctional complex (AJC) and maintain lateral membrane identity. In nonpolarized epithelial cells, Albatross localizes with keratin filaments, whereas in polarized epithelial cells, Albatross is primarily localized in the vicinity of the AJC. Knockdown of Albatross in polarized cells causes a disappearance of key components of the AJC at cell–cell borders and keratin filament reorganization. Lateral proteins E-cadherin and desmoglein 2 were mislocalized even on the apical side. Although Albatross promotes localization of Par3 to the AJC, Par3 and ezrin are still retained at the apical surface in Albatross knockdown cells, which retain intact microvilli. Analysis of keratin-deficient epithelial cells revealed that keratins are required to stabilize the Albatross protein, thus promoting the formation of AJC. We propose that keratins and the keratin-binding protein Albatross are important for epithelial cell polarization.
The accurate targeting of secretory vesicles to distinct sites on
the plasma membrane is necessary to achieve polarized growth and to
establish specialized domains at the surface of eukaryotic cells.
Members of a protein complex required for exocytosis, the exocyst, have
been localized to regions of active secretion in the budding yeast
Saccharomyces cerevisiae where they may function to
specify sites on the plasma membrane for vesicle docking and fusion. In
this study we have addressed the function of one member of the exocyst
complex, Sec10p. We have identified two functional domains of Sec10p
that act in a dominant-negative manner to inhibit cell growth upon
overexpression. Phenotypic and biochemical analysis of the
dominant-negative mutants points to a bifunctional role for Sec10p. One
domain, consisting of the amino-terminal two-thirds of Sec10p directly
interacts with Sec15p, another exocyst component. Overexpression of
this domain displaces the full-length Sec10 from the exocyst complex,
resulting in a block in exocytosis and an accumulation of secretory
vesicles. The carboxy-terminal domain of Sec10p does not interact with
other members of the exocyst complex and expression of this domain does
not cause a secretory defect. Rather, this mutant results in the
formation of elongated cells, suggesting that the second domain of
Sec10p is required for morphogenesis, perhaps regulating the
reorientation of the secretory pathway from the tip of the emerging
daughter cell toward the mother–daughter connection during cell cycle
Epithelial cells in vivo form tight cell-cell associations that spatially separate distinct apical and basolateral domains. These domains provide discrete cellular processes essential for proper tissue and organ development. Using confocal imaging and selective plasma membrane domain activation, the type I and type II transforming growth factor-β (TGFβ) receptors were found to be localized specifically at the basolateral surfaces of polarized Madin-Darby canine kidney (MDCK) cells. Receptors concentrated predominantly at the lateral sites of cell-cell contact, adjacent to the gap junctional complex. Cytoplasmic domain truncations for each receptor resulted in the loss of specific lateral domain targeting and dispersion to both the apical and basal domains. Whereas receptors concentrate basolaterally in regions of direct cell-cell contact in nonpolarized MDCK cell monolayers, receptor staining was absent from areas of noncell contact. In contrast to the defined basolateral polarity observed for the TGFβ receptor complex, TGFβ ligand secretion was found to be from the apical surfaces. Confocal imaging of MDCK cells with an antibody to TGFβ1 confirmed a predominant apical localization, with a stark absence at the basal membrane. These findings indicate that cell adhesion regulates the localization of TGFβ receptors in polarized epithelial cultures and that the response to TGFβ is dependent upon the spatial distribution and secretion of TGFβ receptors and ligand, respectively.
In polarized Madin-Darby canine kidney epithelial cells, components
of the plasma membrane fusion machinery, the t-SNAREs syntaxin 2, 3,
and 4 and SNAP-23, are differentially localized at the apical and/or
basolateral plasma membrane domains. Here we identify syntaxin 11 as a
novel apical and basolateral plasma membrane t-SNARE. Surprisingly, all
of these t-SNAREs redistribute to intracellular locations when
Madin-Darby canine kidney cells lose their cellular polarity. Apical
SNAREs relocalize to the previously characterized vacuolar apical
compartment, whereas basolateral SNAREs redistribute to a novel
organelle that appears to be the basolateral equivalent of the vacuolar
apical compartment. Both intracellular plasma membrane compartments
have an associated prominent actin cytoskeleton and receive membrane
traffic from cognate apical or basolateral pathways, respectively.
These findings demonstrate a fundamental shift in plasma membrane
traffic toward intracellular compartments while protein sorting is
preserved when epithelial cells lose their cell polarity.
Polarized delivery of signaling and adhesion molecules to the leading edge is required for directional migration of cells. Here, we describe a role for the PIP2 synthesizing enzyme, PIPKIγi2, in regulation of exocyst complex control of cell polarity and polarized integrin trafficking during migration. Loss of PIPKIγi2 impaired directional migration, formation of cell polarity, and integrin trafficking to the leading edge. Upon initiation of directional migration PIPKIγi2 via PIP2 generation controls the integration of the exocyst complex into an integrin-containing trafficking compartment(s) that requires the talin-binding ability of PIPKIγi2, and talin for integrin recruitment to the leading edge. A PIP2 requirement is further emphasized by inhibition of PIPKIγi2-regulated directional migration by an Exo70 mutant deficient in PIP2 binding. These results reveal how phosphoinositide generation orchestrates polarized trafficking of integrin in coordination with talin that links integrins to the actin cytoskeleton, processes that are required for directional migration.
Cell Migration; Phosphatidylinositol-4; 5-biphosphate; Cell Polarity; Integrin; Exocyst
E-cadherin forms calcium-dependent homophilic intercellular adhesions between epithelial cells. These contacts regulate multiple aspects of cell behavior, including the organization of intercellular tight junctions (TJs). To distinguish between the roles of E-cadherin in formation versus maintenance of junctions, Madin-Darby canine kidney (MDCK) cells were depleted of E-cadherin by RNA interference. Surprisingly, reducing E-cadherin expression had little effect on the protein levels or localization of adherens junction (AJ) or TJ markers. The cells underwent morphological changes, as the normally flat apical surface swelled into a dome. However, apical–basal polarity was not compromised, transmembrane resistance was normal, and zonula occludin protein 1 dynamics at the TJs were unchanged. Additionally, an E-cadherin/Cadherin-6 double knockdown also failed to disrupt established TJs, although β-catenin was lost from the cell cortex. Nevertheless, cells depleted of E-cadherin failed to properly reestablish cell polarity after junction disassembly. Recovery of cell–cell adhesion, transepithelial resistance, and the localization of TJ and AJ markers were all delayed. In contrast, depletion of α-catenin caused long-term disruption of junctions. These results indicate that E-cadherin and Cadherin-6 function as a scaffold for the construction of polarized structures, and they become largely dispensable in mature junctions, whereas α-catenin is essential for the maintenance of functional junctions.