Myosin VI is required for sorting of AP-1B-dependent cargo to the basolateral domain in polarized MDCK cells

In polarized epithelial cells, newly synthesized membrane proteins are delivered on specific pathways to either the apical or basolateral domains, depending on the sorting motifs present in these proteins. Because myosin VI has been shown to facilitate secretory traffic in nonpolarized cells, we investigated its role in biosynthetic trafficking pathways in polarized MDCK cells. We observed that a specific splice isoform of myosin VI with no insert in the tail domain is required for the polarized transport of tyrosine motif containing basolateral membrane proteins. Sorting of other basolateral or apical cargo, however, does not involve myosin VI. Site-directed mutagenesis indicates that a functional complex consisting of myosin VI, optineurin, and probably the GTPase Rab8 plays a role in the basolateral delivery of membrane proteins, whose sorting is mediated by the clathrin adaptor protein complex (AP) AP-1B. Our results suggest that myosin VI is a crucial component in the AP-1B-dependent biosynthetic sorting pathway to the basolateral surface in polarized epithelial cells.     

Selective alterations in biosynthetic and endocytic protein traffic in Madin-Darby canine kidney epithelial cells expressing mutants of the small GTPase Rac1

Madin-Darby canine kidney (MDCK) cells expressing constitutively active Rac1 (Rac1V12) accumulate a large central aggregate of membranes beneath the apical membrane that contains filamentous actin, Rac1V12, rab11, and the resident apical membrane protein GP-135. To examine the roles of Rac1 in membrane traffic and the formation of this aggregate, we analyzed endocytic and biosynthetic trafficking pathways in MDCK cells expressing Rac1V12 and dominant inactive Rac1 (Rac1N17). Rac1V12 expression decreased the rates of apical and basolateral endocytosis, whereas Rac1N17 expression increased those rates from both membrane domains. Basolateral-to-apical transcytosis of immunoglobulin A (IgA) (a ligand for the polymeric immunoglobulin receptor [pIgR]), apical recycling of pIgR-IgA, and accumulation of newly synthesized GP-135 at the apical plasma membrane were all decreased in cells expressing Rac1V12. These effects of Rac1V12 on trafficking pathways to the apical membrane were the result of the delivery and trapping of these proteins in the central aggregate. In contrast to abnormalities in apical trafficking events, basolateral recycling of transferrin, degradation of EGF internalized from the basolateral membrane, and delivery of newly synthesized pIgR from the Golgi to the basolateral membrane were all relatively unaffected by Rac1V12 expression. Rac1N17 expression had little or no effect on these postendocytic or biosynthetic trafficking pathways. These results show that in polarized MDCK cells activated Rac1 may regulate the rate of endocytosis from both membrane domains and that expression of dominant active Rac1V12 specifically alters postendocytic and biosynthetic membrane traffic directed to the apical, but not the basolateral, membrane.     

Regulation of membrane trafficking in polarized epithelial cells

Polarized epithelial cells continuously sort transmembrane proteins to either apical or basolateral plasma membrane domains. Research in recent years has made tremendous progress in understanding the molecular mechanisms of the major pathways to either basolateral or apical domain. This understanding will help us elucidating how these pathways are interconnected in ensuring maintenance of cell polarity and integrity of epithelial monolayers.     

The role of membranes and membrane trafficking in RNA localization

Eukaryotic cells possess highly sophisticated membrane trafficking pathways that define specific membrane domains and provide a means for moving vesicles between them (Mostov, Su, and ter Beest, 2003, Nat. Cell Biol. 5, 287-293). Here, I review recent data that indicate a role for membrane trafficking in mRNA localization. Specifically, I review evidence that some localized mRNAs are anchored to specific membrane domains and/or transported on membranous organelles or vesicles to specific subcellular sites. This review is not intended as a discussion on indirect influences of membrane trafficking on mRNA localization. I will not, for example, discuss the role of membrane trafficking in the regulation of extracellular signalling events that could indirectly influence mRNA localization through polarization of the actin or microtubule cytoskeleton (for examples, see reviews by Drubin and Nelson, 1996, Cell 84, 335-344; Shulman and St Johnston, 1999, Trends Cell Biol. 9, M60-M64).     

Where sterols are required for endocytosis

Sterols are essential membrane components of eukaryotic cells. Interacting closely with sphingolipids, they provide the membrane surrounding required for membrane sorting and trafficking processes. Altering the amount and/or structure of free sterols leads to defects in endocytic pathways in mammalian cells and yeast. Plasma membrane structures functioning in the internalization step in mammalian cells, caveolae and clathrin-coated pits, are affected by cholesterol depletion. Accumulation of improper plasma membrane sterols prevents hyperphosphorylation of a plasma membrane receptor in yeast. Once internalized, sterols still interact with sphingolipids and are recycled to the plasma membrane to keep an intracellular sterol gradient with the highest amount of free sterols at the cell periphery. Interestingly, cells from patients suffering from sphingolipid storage diseases show high intracellular amounts of free cholesterol. We propose that the balanced interaction of sterols and sphingolipids is responsible for protein recruitment to specialized membrane domains and their functionality in the endocytic pathway.     

MYADM regulates Rac1 targeting to ordered membranes required for cell spreading and migration

Membrane organization into condensed domains or rafts provides molecular platforms for selective recruitment of proteins. Cell migration is a general process that requires spatiotemporal targeting of Rac1 to membrane rafts. The protein machinery responsible for making rafts competent to recruit Rac1 remains elusive. Some members of the MAL family of proteins are involved in specialized processes dependent on this type of membrane. Because condensed membrane domains are a general feature of the plasma membrane of all mammalian cells, we hypothesized that MAL family members with ubiquitous expression and plasma membrane distribution could be involved in the organization of membranes for cell migration. We show that myeloid-associated differentiation marker (MYADM), a protein with unique features within the MAL family, colocalizes with Rac1 in membrane protrusions at the cell surface and distributes in condensed membranes. MYADM knockdown (KD) cells had altered membrane condensation and showed deficient incorporation of Rac1 to membrane raft fractions and, similar to Rac1 KD cells, exhibited reduced cell spreading and migration. Results of rescue-of-function experiments by expression of MYADM or active Rac1L61 in cells knocked down for Rac1 or MYADM, respectively, are consistent with the idea that MYADM and Rac1 act on parallel pathways that lead to similar functional outcomes.     

Phosphoinositides and engulfment

Cellular engulfment of particles, cells or solutes displaces large domains of plasma membrane into intracellular membranous vacuoles. This transfer of membrane is accompanied by major transitions of the phosphoinositide (PI) species that comprise the cytoplasmic face of membrane bilayers. Mapping of membrane PIs during engulfment reveals distinct patterns of protein and PI distributions associated with each stage of engulfment, which correspond with activities that regulate the actin cytoskeleton, membrane movements and vesicle secretion. Experimental manipulation of PI chemistry during engulfment indicates that PIs integrate organelle identity and orient signal transduction cascades within confined subdomains of membrane. These pathways are exploited by microbial pathogens to direct or redirect the engulfment process.     

Polarized Sphingolipid Transport from the Subapical Compartment: Evidence for Distinct Sphingolipid Domains

In polarized HepG2 cells, the sphingolipids glucosylceramide and sphingomyelin (SM), transported along the reverse transcytotic pathway, are sorted in subapical compartments (SACs), and subsequently targeted to either apical or basolateral plasma membrane domains, respectively. In the present study, evidence is provided that demonstrates that these sphingolipids constitute separate membrane domains at the luminal side of the SAC membrane. Furthermore, as revealed by the use of various modulators of membrane trafficking, such as calmodulin antagonists and dibutyryl-cAMP, it is shown that the fate of these separate sphingolipid domains is regulated by different signals, including those that govern cell polarity development. Thus under conditions that stimulate apical plasma membrane biogenesis, SM is rerouted from a SAC-to-basolateral to a SAC-to-apical pathway. The latter pathway represents the final leg in the transcytotic pathway, followed by the transcytotic pIgR-dIgA protein complex. Interestingly, this pathway is clearly different from the apical recycling pathway followed by glucosylceramide, further indicating that randomization of these pathways, which are both bound for the apical membrane, does not occur. The consequence of the potential coexistence of separate sphingolipid domains within the same compartment in terms of "raft" formation and apical targeting is discussed.     

Recruitment of CD40 and tumor necrosis factor receptor-associated factors 2 and 3 to membrane microdomains during CD40 signaling

Signals delivered to antigen-presenting cells through CD40 are critical for the activation of immune responses. Intracellular tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are key elements of the signal transduction pathways of many TNF receptor family members, including CD40. We show for the first time that engagement of CD40 in intact B cells induces the rapid translocation of TRAF2 from the cytoplasm to the plasma membrane. We found that CD40 engagement also results in its recruitment, together with TRAF2 and TRAF3, to membrane microdomains, regions of the plasma membrane enriched in signaling molecules such as the Src family kinases. Using a membrane-permeable chelator of zinc or a mutant TRAF2 molecule, we show that the putative zinc-binding domains of TRAFs contribute to their recruitment to microdomains and to the downstream activation of c-Jun N-terminal kinase. We suggest that the zinc RING and zinc finger domains of TRAFs are required for communication between CD40 and microdomain-associated signaling molecules and may serve a similar role in the signal transduction pathways of other TNF receptor family members.     

Compartmentalized IgE Receptor–mediated Signal Transduction in Living Cells

Several receptor-mediated signal transduction pathways, including EGF and IgE receptor pathways, have been proposed to be spatially restricted to plasma membrane microdomains. However, the experimental evidence for signaling events in these microdomains is largely based on biochemical fractionation and immunocytochemical studies and only little is known about their spatial dynamics in living cells. Here we constructed green fluorescent protein–tagged SH2 domains to investigate where and when IgE receptor (FcεRI)–mediated tyrosine phosphorylation occurs in living tumor mast cells. Strikingly, within minutes after antigen addition, tandem SH2 domains from Syk or PLC-γ1 translocated from a uniform cytosolic distribution to punctuate plasma membrane microdomains. Colocalization experiments showed that the microdomains where tyrosine phosphorylation occurred were indistinguishable from those stained by cholera toxin B, a marker for glycosphingolipids. Competitive binding studies with coelectroporated unlabeled Syk, PLC-γ1, and other SH2 domains selectively suppressed the induction of IgE receptor–mediated calcium signals as well as the binding of the fluorescent SH2 domains. This supports the hypothesis that PLC-γ1 and Syk SH2 domains selectively bind to Syk and IgE receptors, respectively. Unlike the predicted prelocalization of EGF receptors to caveolae microdomains, fluorescently labeled IgE receptors were found to be uniformly distributed in the plasma membrane of unstimulated cells and only transiently translocated to glycosphingolipid rich microdomains after antigen addition. Thus, these in vivo studies support a plasma membrane signaling mechanism by which IgE receptors transiently associate with microdomains and induce the spatially restricted activation of Syk and PLC-γ1.     

Cdc42-dependent modulation of tight junctions and membrane protein traffic in polarized Madin-Darby canine kidney cells

Polarized epithelial cells maintain the asymmetric composition of their apical and basolateral membrane domains by at least two different processes. These include the regulated trafficking of macromolecules from the biosynthetic and endocytic pathway to the appropriate membrane domain and the ability of the tight junction to prevent free mixing of membrane domain-specific proteins and lipids. Cdc42, a Rho family GTPase, is known to govern cellular polarity and membrane traffic in several cell types. We examined whether this protein regulated tight junction function in Madin-Darby canine kidney cells and pathways that direct proteins to the apical and basolateral surface of these cells. We used Madin-Darby canine kidney cells that expressed dominant-active or dominant-negative mutants of Cdc42 under the control of a tetracycline-repressible system. Here we report that expression of dominant-active Cdc42V12 or dominant-negative Cdc42N17 altered tight junction function. Expression of Cdc42V12 slowed endocytic and biosynthetic traffic, and expression of Cdc42N17 slowed apical endocytosis and basolateral to apical transcytosis but stimulated biosynthetic traffic. These results indicate that Cdc42 may modulate multiple cellular pathways required for the maintenance of epithelial cell polarity.     

Intracellular redirection of plasma membrane trafficking after loss of epithelial cell polarity

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.     

Membrane microdomains and caveolae.

Glycosphingolipid- and cholesterol-enriched microdomains, or rafts, within the plasma membrane of eukaryotic cells have been implicated in many important cellular processes, such as polarized sorting of apical membrane proteins in epithelial cells and signal transduction. Until recently, however, the existence of such domains remained controversial. The past year has brought compelling evidence that microdomains indeed exist in living cells. In addition, several recent papers have suggested that caveolae, which are considered to be a specific form of raft, and caveolins, the major membrane proteins of caveolae, are involved in the dynamic cholesterol-dependent regulation of specific signal transduction pathways.     

The effect of membrane domains on the G protein–phospholipase Cβ signaling pathway

The plasma membrane serves as a barrier to limit the exit and entry of components into and out of the cell, offering protection from the external environment. Communication between the cell and the external environment is mediated by multiple signaling pathways. While the plasma membrane was historically viewed as a lipid bilayer with freely diffusing proteins, the last decade has shown that the lipids and proteins in the plasma membrane are organized in a non-random manner, and that this organization can direct and modify various signaling pathways in the cell. In this review, we qualitatively discuss the ways that membrane domains can affect cell signaling. We then focus on how membrane domains can affect a specific signaling pathway – the G protein–phospholipase Cβ pathway and show how membrane domains can play an active role in directing or redirecting G protein signals.     

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.     

Effect of nocodazole on vesicular traffic to the apical and basolateral surfaces of polarized MDCK cells

A polarized cell, to maintain distinct basolateral and apical membrane domains, must tightly regulate vesicular traffic terminating at either membrane domain. In this study we have examined the extent to which microtubules regulate such traffic in polarized cells. Using the polymeric immunoglobulin receptor expressed in polarized MDCK cells, we have examined the effects of nocodazole, a microtubule-disrupting agent, on three pathways that deliver proteins to the apical surface and two pathways that deliver proteins to the basolateral surface. The biosynthetic and transcytotic pathways to the apical surface are dramatically altered by nocodazole in that a portion of the protein traffic on each of these two pathways is misdirected to the basolateral surface. The apical recycling pathway is slowed in the presence of nocodazole but targeting is not disrupted. In contrast, the biosynthetic and recycling pathways to the basolateral surface are less affected by nocodazole and therefore appear to be more resistant to microtubule disruption.     

Protein secretion through autotransporter and two-partner pathways

Two distinct protein secretion pathways, the autotransporter (AT) and the two-partner secretion (TPS) pathways are characterized by their apparent simplicity. Both are devoted to the translocation across the outer membrane of mostly large proteins or protein domains. As implied by their name, AT proteins contain their own transporter domain, covalently attached to the C-terminal extremity of the secreted passenger domain, while TPS systems are composed of two separate proteins, with TpsA being the secreted protein and TpsB its specific transporter. In both pathways, the secreted proteins are exported in a Sec-dependent manner across the inner membrane, after which they cross the outer membrane with the help of their cognate transporters. The AT translocator domains and the TpsB proteins constitute distinct families of protein-translocating, outer membrane porins of Gram-negative bacteria. Both types of transporters insert into the outer membrane as beta-barrel proteins possibly forming oligomeric pores in the case of AT and serve as conduits for their cognate secreted proteins or domains across the outer membrane. Translocation appears to be folding-sensitive in both pathways, indicating that AT passenger domains and TpsA proteins cross the periplasm and the outer membrane in non-native conformations and fold progressively at the cell surface. A major difference between AT and TPS pathways arises from the manner by which specificity is established between the secreted protein and its transporter. In AT, the covalent link between the passenger and the translocator domains ensures the translocation of the former without the need for a specific molecular recognition between the two modules. In contrast, the TPS pathway has solved the question of specific recognition between the TpsA proteins and their transporters by the addition to the TpsA proteins of an N-proximal module, the conserved TPS domain, which represents a hallmark of the TPS pathway.     

Cholesterol content drives distinct pharmacological behaviours of µ-opioid receptor in different microdomains of the CHO plasma membrane

Cholesterol in the plasma membrane of eukaryotic cells contributes to modulating the functions and signalling pathways of numerous transmembrane proteins, including G protein Coupled Receptors (GPCRs). We have previously shown that the function of the human µ-opioid receptor (hMOR) expressed in Saccharomyces cerevisiae is modulated by sterols including cholesterol. Here, we investigated the effects of cholesterol content on hMOR pharmacology and on hMOR partitioning in cholesterol-poor and -rich domains in eukaryotic mammalian cells (CHO). We show that cholesterol is required for the stabilization of a receptor conformation with high agonist affinity and for triggering G-protein activation after agonist binding to the receptor. Biochemical analysis of untreated and cholesterol-depleted membranes in cells expressing hMOR indicated that the receptor is only present in cholesterol poor domains, in the basal state. After agonist binding to untreated CHO membranes, two distinct populations of receptor were found in cholesterol-rich and -poor domains. Cholesterol depletion or treatment of CHO membranes with the G-protein-decoupling agent GppNHp prevented the redistribution, indicating that receptor activated states localized into cholesterol-rich domains. Pharmacological data and biochemical analysis indicate that distinct activated conformations of hMOR exist in CHO plasma membrane and correspond to microdomains differing by thickness and proportions of lipid components, including cholesterol.     

Cholesterol content drives distinct pharmacological behaviours of micro-opioid receptor in different microdomains of the CHO plasma membrane

Cholesterol in the plasma membrane of eukaryotic cells contributes to modulating the functions and signalling pathways of numerous transmembrane proteins, including G protein Coupled Receptors (GPCRs). We have previously shown that the function of the human micro-opioid receptor (hMOR) expressed in Saccharomyces cerevisiae is modulated by sterols including cholesterol. Here, we investigated the effects of cholesterol content on hMOR pharmacology and on hMOR partitioning in cholesterol-poor and -rich domains in eukaryotic mammalian cells (CHO). We show that cholesterol is required for the stabilization of a receptor conformation with high agonist affinity and for triggering G-protein activation after agonist binding to the receptor. Biochemical analysis of untreated and cholesterol-depleted membranes in cells expressing hMOR indicated that the receptor is only present in cholesterol poor domains, in the basal state. After agonist binding to untreated CHO membranes, two distinct populations of receptor were found in cholesterol-rich and -poor domains. Cholesterol depletion or treatment of CHO membranes with the G-protein-decoupling agent GppNHp prevented the redistribution, indicating that receptor activated states localized into cholesterol-rich domains. Pharmacological data and biochemical analysis indicate that distinct activated conformations of hMOR exist in CHO plasma membrane and correspond to microdomains differing by thickness and proportions of lipid components, including cholesterol.     

The multiple faces of caveolae

Caveolae are a highly abundant but enigmatic feature of mammalian cells. They form remarkably stable membrane domains at the plasma membrane but can also function as carriers in the exocytic and endocytic pathways. The apparently diverse functions of caveolae, including mechanosensing and lipid regulation, might be linked to their ability to respond to plasma membrane changes, a property that is dependent on their specialized lipid composition and biophysical properties.