A comparative study of stress-mediated immunological functions with the adjuvanticity of alum

The efficacy of a vaccine is generally dependent on an adjuvant, which enhances the immune functions and alum has been widely used in human immunization. Alum activates the intracellular stress sensors inflammasomes, but whether these are responsible for the adjuvanticity is controversial. The objectives of this investigation were to examine the hypothesis that alum-mediated adjuvanticity is a function of stress and conversely that stress agents will elicit adjuvanticity. The investigation was carried out in BALB/c mice by SC immunization with ovalbumin (OVA) mixed with alum. This elicited inflammasomes, with significant activation of caspase 1, production of IL-1β, and adjuvanticity, demonstrated by enhancing OVA-specific serum IgG antibodies, CD4(+) T cells, and proliferation. The novel finding that alum induced HSP70 suggests that stress is involved in the mechanism of adjuvanticity. This was confirmed by inhibition studies with PES (phenylethynesulfonamide), which disrupts inducible HSP70 function, and inhibited both inflammasomes and the adjuvant function. Parallel studies were pursued with an oxidative agent (sodium arsenite), K-releasing agent (Gramicidin) and a metal ionophore (dithiocarbamate). All 3 stress agents induced HSP70, inflammasomes, and the adjuvant functions. Furthermore, up-regulation of membrane associated IL-15 on DC and CD40L on T cells in the animals treated with alum or the stress agents mediate the interactions between splenic CD11c DC and CD4(+) or CD8(+) T cells. The results suggest that the three stress agents elicit HSP70, a hallmark of stress, as well as inflammasomes and adjuvanticity, commensurate with those of alum, which may provide an alternative strategy in developing novel adjuvants.     

Microdomains of the C-type lectin DC-SIGN are portals for virus entry into dendritic cells

The C-type lectin dendritic cell (DC)-specific intercellular adhesion molecule grabbing non-integrin (DC-SIGN; CD209) facilitates binding and internalization of several viruses, including HIV-1, on DCs, but the underlying mechanism for being such an efficient phagocytic pathogen-recognition receptor is poorly understood. By high resolution electron microscopy, we demonstrate a direct relation between DC-SIGN function as viral receptor and its microlocalization on the plasma membrane. During development of human monocyte-derived DCs, DC-SIGN becomes organized in well-defined microdomains, with an average diameter of 200 nm. Biochemical experiments and confocal microscopy indicate that DC-SIGN microdomains reside within lipid rafts. Finally, we show that the organization of DC-SIGN in microdomains on the plasma membrane is important for binding and internalization of virus particles, suggesting that these multimolecular assemblies of DC-SIGN act as a docking site for pathogens like HIV-1 to invade the host.     

ICAM-2 and lipid rafts disappear from the basal plasma membrane of uterine epithelial cells during early pregnancy in rats

Adhesion molecules are redistributed in rat uterine epithelial cells (UECs) during early pregnancy for endometrial receptivity and implantation. Intercellular adhesion molecule-2 (ICAM-2) is located as an oligomer on the basal plasma membrane of non-receptive UECs on day 1 of pregnancy and colocalizes with the lipid raft marker flotillin-2. At the time of implantation in rats and in ovariectomized rats primed with progesterone, ICAM-2 disappears from the basal plasma membrane and lipid rafts redistribute to the apical membrane. The loss of ICAM-2 might render UECs less adherent to the underlying basal lamina and more prone to apoptosis. Flotillin-2 in the apical plasma membrane at the time of implantation might provide an anchoring point for several adhesion molecules that are known to localize to this region at this time. We suggest that flotillin-2 is involved with adhesion between UECs and the implanting blastocyst, whereas ICAM-2 is associated with the ability for UECs to be removed at the time of implantation.     

Dendritic cells acquire antigens from live cells for cross-presentation to CTL

Dendritic cells (DC) can readily capture Ag from dead and dying cells for presentation to MHC class I-restricted CTL. We now show by using a primate model that DC also acquire Ag from healthy cells, including other DC. Coculture assays showed that fluorescently labeled plasma membrane was rapidly and efficiently transferred between DC, and transfer of intracellular proteins was observed to a lesser extent. Acquisition of labeled plasma membrane and intracellular protein was cell contact-dependent and was primarily a function of immature DC, whereas both immature and CD40L-matured DC could serve as donors. Moreover, immature DC could acquire labeled plasma membrane and intracellular proteins from a wide range of hemopoietic cells, including macrophages, B cells, and activated T cells. Notably, macrophages, which readily phagocytose apoptotic bodies, were very inefficient at acquiring labeled plasma membrane and intracellular proteins from other live macrophages or DC. With live-cell imaging techniques, we demonstrate that individual DC physically extract plasma membrane from other DC, generating endocytic vesicles of up to 1 microm in diameter. Finally, DC but not macrophages acquired an endogenous melanoma Ag expressed by live DC and cross-presented Ag to MHC class I-restricted CTL, demonstrating the immunological relevance of our finding. These data show for the first time that DC readily acquire Ag from other live cells. We suggest that Ag acquisition from live cells may provide a novel mechanism whereby DC can present Ag in the absence of direct infection, and may serve to expand and regulate the immune response in vivo.     

A novel approach to examining compositional heterogeneity of detergent-resistant lipid rafts

Lipid rafts play an important role in cell signalling, cell adhesion and other cellular functions. Compositional heterogeneity of lipid rafts provides one mechanism of how lipid rafts provide the spatial and temporal regulation of cell signalling and cell adhesion. The constitutive presence of some signalling receptors/molecules and accumulation of others in the lipid raft allows them to interact with each other and thereby facilitate relay of signals from the plasma membrane to the cell interior. Devising a method that can analyze these lipid microdomains for the presence of signalling receptors/molecules on an individual raft basis is required to address the issue of lipid raft heterogeneity. SDS-PAGE analysis, currently used for analyses of detergent-resistant lipid rafts, does not address this question. We have designed a cell-free assay that captures detergent-resistant lipid rafts with an antibody against a raft-resident molecule and detects the presence of another lipid raft molecule. Our results suggest that detergent-resistant lipid rafts, also known as detergent-resistant membranes, are heterogeneous populations on an immortalized mouse T-cell plasma membrane with respect to antigen receptor/signalling complex and other signalling/adhesion proteins. This cell-free assay provides a simple and quick way to examine the simultaneous presence of two proteins in the lipid rafts and has the potential to estimate trafficking of molecules in and out of the lipid microdomains during cell signalling on a single detergent-resistant lipid raft basis.     

Lipid Rafts Disruption Increases Ochratoxin A Cytotoxicity to Hepatocytes

Lipid rafts are microdomains in plasma membrane and can mediate cytotoxicity. In this study, the role of lipid rafts in ochratoxin A‐induced toxicity was investigated using Hepatoblastoma Cell Line HepG‐2 cells. Disruption of cholesterol‐containing lipid rafts enhanced Ochratoxin A (OTA) toxicity, as shown by increased lactate dehydrogenase leakage, increased reactive oxygen species level and reduction of superoxide dismutase activity in a time‐dependent manner. Isobaric tags for relative and absolute quantitation‐based proteomics of the cell membranes showed that nearly 85.5% proteins were downregulated by OTA, indicating that OTA inhibited the membrane protein synthesis. Most of altered proteins were involved in Gene Ontology “transport”, “cell adhesion” and “vesicle‐mediated transport”. In conclusion, lipid rafts play a key role in OTA‐induced cytotoxicity. This study provides insight into how OTA toxicity is regulated by the plasma membrane, especially the lipid rafts.     

Lipid Sorting by Ceramide Structure from Plasma Membrane to ER for the Cholera Toxin Receptor Ganglioside GM1

The glycosphingolipid GM1 binds cholera toxin (CT) on host cells and carries it retrograde from the plasma membrane (PM) through endosomes, the trans-Golgi (TGN), and the endoplasmic reticulum (ER) to induce toxicity. To elucidate how a membrane?lipid can specify trafficking in these pathways, we synthesized GM1 isoforms with alternate ceramide domains and imaged their trafficking in live cells.?Only GM1 with unsaturated acyl chains sorted efficiently from PM to TGN and ER. Toxin binding, which effectively crosslinks GM1 lipids, was dispensable, but membrane cholesterol and the lipid raft-associated proteins actin and flotillin were required. The results implicate a protein-dependent mechanism of lipid sorting by ceramide structure and provide a molecular explanation for the diversity?and specificity of retrograde trafficking by CT in?host cells.     

Molecular mechanism of an oncogenic mutation that alters membrane targeting: Glu17Lys modifies the PIP lipid specificity of the AKT1 PH domain

The protein kinase AKT1 regulates multiple signaling pathways essential for cell function. Its N-terminal PH domain (AKT1 PH) binds the rare signaling phospholipid phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)], resulting in plasma membrane targeting and phosphoactivation of AKT1 by a membrane-bound kinase. Recently, it was discovered that the Glu17Lys mutation in the AKT1 PH domain is associated with multiple human cancers. This mutation constitutively targets the AKT1 PH domain to the plasma membrane by an unknown mechanism, thereby promoting constitutive AKT1 activation and oncogenesis. To elucidate the molecular mechanism underlying constitutive plasma membrane targeting, this work compares the membrane docking reactions of the isolated wild-type and E17K AKT1 PH domains. In vitro studies reveal that the E17K mutation dramatically increases the affinity for the constitutive plasma membrane lipid PI(4,5)P(2). The resulting PI(4,5)P(2) equilibrium affinity is indistinguishable from that of the standard PI(4,5)P(2) sensor, PLCdelta1 PH domain. Kinetic studies indicate that the effects of E17K on PIP lipid binding arise largely from electrostatic modulation of the dissociation rate. Membrane targeting analysis in live cells confirms that the constitutive targeting of E17K AKT1 PH to plasma membrane, like PLCdelta1 PH, stems from PI(4,5)P(2) binding. Overall, the evidence indicates that the molecular mechanism underlying E17K oncogenesis is a broadened target lipid selectivity that allows high-affinity binding to PI(4,5)P(2). Moreover, the findings strongly implicate the native Glu17 side chain as a key element of PIP lipid specificity in the wild-type AKT1 PH domain. Other PH domains may employ an analogous anionic residue to control PIP specificity.     

Plasma membrane lipid packing and leukocyte function-associated antigen-1-dependent aggregation of lymphocytes

A simple model system for study of adhesion mediated by leukocyte function-associated antigen-1 (LFA-1) is aggregation of lymphocytes stimulated in vitro. Although aggregation is blocked by monoclonal antibodies to LFA-1, not all lymphocytes expressing LFA-1 aggregate, indicating that LFA-1 is necessary but not sufficient for aggregation. To investigate whether the lipid bilayer plays a role in the functional activation of LFA-1, human peripheral blood lymphocytes and murine splenic lymphocytes were stimulated in culture, and measurements made of aggregation vs. packing of plasma membrane lipids. Progression of cells into aggregates was paralleled by a decrease in lipid packing of the population as a whole, as monitored by increased staining with the fluorescent probe merocyanine 540. Cells from aggregates stained more intensely than nonaggregated cells from the same population, indicating that aggregates are preferentially formed from cells in the population with the loosest packed membrane. In contrast, aggregated cells were found to express equivalent or even lower amounts of LFA-1 than nonaggregated cells. Looser lipid packing is therefore associated with the development of LFA-1-dependent aggregation, and might be involved in the functional activation of this cell adhesion molecule. © 1993 Wiley-Liss, Inc.     

Interaction of heat shock protein 70 with membranes depends on the lipid environment

Heat shock proteins (hsp) are well recognized for their protein folding activity. Additionally, hsp expression is enhanced during stress conditions to preserve cellular homeostasis. Hsp are also detected outside cells, released by an active mechanism independent of cell death. Extracellular hsp appear to act as signaling molecules as part of a systemic response to stress. Extracellular hsp do not contain a consensus signal for their secretion via the classical ER-Golgi compartment. Therefore, they are likely exported by an alternative mechanism requiring translocation across the plasma membrane. Since Hsp70, the major inducible hsp, has been detected on surface of stressed cells, we propose that membrane interaction is the first step in the export process. The question that emerges is how does this charged cytosolic protein interact with lipid membranes? Prior studies have shown that Hsp70 formed ion conductance pathways within artificial lipid bilayers. These early observations have been extended herewith using a liposome insertion assay. We showed that Hsp70 selectively interacted with negatively charged phospholipids, particularly phosphatidyl serine (PS), within liposomes, which was followed by insertion into the lipid bilayer, forming high-molecular weight oligomers. Hsp70 displayed a preference for less fluid lipid environments and the region embedded into the lipid membrane was mapped toward the C-terminus end of the molecule. The results from our studies provide evidence of an unexpected ability of a large, charged protein to become inserted into a lipid membrane. This observation provides a new paradigm for the interaction of proteins with lipid environments. In addition, it may explain the export mechanism of an increasing number of proteins that lack the consensus secretory signals.     

Effects of integrin-mediated cell adhesion on plasma membrane lipid raft components and signaling

Anchorage dependence of cell growth, which is mediated by multiple integrin-regulated signaling pathways, is a key defense against cancer metastasis. Detachment of cells from the extracellular matrix triggers caveolin-1-dependent internalization of lipid raft components, which mediates suppression of Rho GTPases, Erk, and phosphatidylinositol 3-kinase in suspended cells. Elevation of cyclic adenosine monophosphate (cAMP) following cell detachment is also implicated in termination of growth signaling in suspended cells. Studies of integrins and lipid rafts, however, examined mainly ganglioside GM1 and glycosylphosphatidylinositol-linked proteins as lipid raft markers. In this study, we examine a wider range of lipid raft components. Whereas many raft components internalized with GM1 following cell detachment, flotillin2, connexin43, and Gα(s) remained in the plasma membrane. Loss of cell adhesion caused movement of many components from the lipid raft to the nonraft fractions on sucrose gradients, although flotillin2, connexin43, and H-Ras were resistant. Gα(s) lost its raft association, concomitant with cAMP production. Modification of the lipid tail of Gα(s) to increase its association with ordered domains blocked the detachment-induced increase in cAMP. These data define the effects of that integrin-mediated adhesion on the localization and behavior of a variety of lipid raft components and reveal the mechanism of the previously described elevation of cAMP after cell detachment.     

Targeting Src homology 2 domain-containing tyrosine phosphatase (SHP-1) into lipid rafts inhibits CD3-induced T cell activation

To study the mechanism by which protein tyrosine phosphatases (PTPs) regulate CD3-induced tyrosine phosphorylation, we investigated the distribution of PTPs in subdomains of plasma membrane. We report here that the bulk PTP activity associated with T cell membrane is present outside the lipid rafts, as determined by sucrose density gradient sedimentation. In Jurkat T cells, approximately 5--10% of Src homology 2 domain-containing tyrosine phosphatase (SHP-1) is constitutively associated with plasma membrane, and nearly 50% of SHP-2 is translocated to plasma membrane after vanadate treatment. Similar to transmembrane PTP, CD45, the membrane-associated populations of SHP-1 and SHP-2 are essentially excluded from lipid rafts, where other signaling molecules such as Lck, linker for activation of T cells, and CD3 zeta are enriched. We further demonstrated that CD3-induced tyrosine phosphorylation of these substrates is largely restricted to lipid rafts, unless PTPs are inhibited. It suggests that a restricted partition of PTPs among membrane subdomains may regulate protein tyrosine phosphorylation in T cell membrane. To test this hypothesis, we targeted SHP-1 into lipid rafts by using the N-terminal region of Lck (residues 1--14). The results indicate that the expression of Lck/SHP-1 chimera inside lipid rafts profoundly inhibits CD3-induced tyrosine phosphorylation of CD3 zeta/epsilon, IL-2 generation, and nuclear mobilization of NF-AT. Collectively, these results suggest that the exclusion of PTPs from lipid rafts may be a mechanism that potentiates TCR/CD3 activation.     

CD1 molecules efficiently present antigen in immature dendritic cells and traffic independently of MHC class II during dendritic cell maturation

Upon exposure to Ag and inflammatory stimuli, dendritic cells (DCs) undergo a series of dynamic cellular events, referred to as DC maturation, that involve facilitated peptide Ag loading onto MHC class II molecules and their subsequent transport to the cell surface. Besides MHC molecules, human DCs prominently express molecules of the CD1 family (CD1a, -b, -c, and -d) and mediate CD1-dependent presentation of lipid and glycolipid Ags to T cells, but the impact of DC maturation upon CD1 trafficking and Ag presentation is unknown. Using monocyte-derived immature DCs and those stimulated with TNF-alpha for maturation, we observed that none of the CD1 isoforms underwent changes in intracellular trafficking that mimicked MHC class II molecules during DC maturation. In contrast to the striking increase in surface expression of MHC class II on mature DCs, the surface expression of CD1 molecules was either increased only slightly (for CD1b and CD1c) or decreased (for CD1a). In addition, unlike MHC class II, DC maturation-associated transport from lysosomes to the plasma membrane was not readily detected for CD1b despite the fact that both molecules were prominently expressed in the same MIIC lysosomal compartments before maturation. Consistent with this, DCs efficiently presented CD1b-restricted lipid Ags to specific T cells similarly in immature and mature DCs. Thus, DC maturation-independent pathways for lipid Ag presentation by CD1 may play a crucial role in host defense, even before DCs are able to induce maximum activation of peptide Ag-specific T cells.     

Deformation-induced lipid trafficking in alveolar epithelial cells

Mechanical ventilation with a high tidal volume results in lung injury that is characterized by blebbing and breaks both between and through alveolar epithelial cells. We developed an in vitro model to simulate ventilator-induced deformation of the alveolar basement membrane and to investigate, in a direct manner, epithelial cell responses to deforming forces. Taking advantage of the novel fluorescent properties of BODIPY lipids and the fluorescent dye FM1-43, we have shown that mechanical deformation of alveolar epithelial cells results in lipid transport to the plasma membrane. Deformation-induced lipid trafficking (DILT) was a vesicular process, rapid in onset, and was associated with a large increase in cell surface area. DILT could be demonstrated in all cells; however, only a small percentage of cells developed plasma membrane breaks that were reversible and nonlethal. Therefore, DILT was not only involved in site-directed wound repair but might also have served as a cytoprotective mechanism against plasma membrane stress failure. This study suggests that DILT is a regulatory mechanism for membrane trafficking in alveolar epithelia and provides a novel biological framework within which to consider alveolar deformation injury and repair.     

Changes in membrane lipid composition cause alterations in epithelial cell-cell adhesion structures in renal papillary collecting duct cells

In epithelial tissues, adherens junctions (AJ) mediate cell-cell adhesion by using proteins called E-cadherins, which span the plasma membrane, contact E-cadherin on other cells and connect with the actin cytoskeleton inside the cell. Although AJ protein complexes are inserted in detergent-resistant membrane microdomains, the influence of membrane lipid composition in the preservation of AJ structures has not been extensively addressed. In the present work, we studied the contribution of membrane lipids to the preservation of renal epithelial cell-cell adhesion structures. We biochemically characterized the lipid composition of membranes containing AJ complexes. By using lipid membrane-affecting agents, we found that such agents induced the formation of new AJ protein-containing domains of different lipid composition. By using both biochemical approaches and fluorescence microscopy we demonstrated that the membrane phospholipid composition plays an essential role in the in vivo maintenance of AJ structures involved in cell-cell adhesion structures in renal papillary collecting duct cells.     

Lipid Domain Structure of the Plasma Membrane Revealed by Patching of Membrane Components

Lateral assemblies of glycolipids and cholesterol, “rafts,” have been implicated to play a role in cellular processes like membrane sorting, signal transduction, and cell adhesion. We studied the structure of raft domains in the plasma membrane of non-polarized cells. Overexpressed plasma membrane markers were evenly distributed in the plasma membrane. We compared the patching behavior of pairs of raft markers (defined by insolubility in Triton X-100) with pairs of raft/non-raft markers. For this purpose we cross-linked glycosyl-phosphatidylinositol (GPI)-anchored proteins placental alkaline phosphatase (PLAP), Thy-1, influenza virus hemagglutinin (HA), and the raft lipid ganglioside GM1 using antibodies and/or cholera toxin. The patches of these raft markers overlapped extensively in BHK cells as well as in Jurkat T–lymphoma cells. Importantly, patches of GPI-anchored PLAP accumulated src-like protein tyrosine kinase fyn, which is thought to be anchored in the cytoplasmic leaflet of raft domains. In contrast patched raft components and patches of transferrin receptor as a non-raft marker were sharply separated. Taken together, our data strongly suggest that coalescence of cross-linked raft elements is mediated by their common lipid environments, whereas separation of raft and non-raft patches is caused by the immiscibility of different lipid phases. This view is supported by the finding that cholesterol depletion abrogated segregation. Our results are consistent with the view that raft domains in the plasma membrane of non-polarized cells are normally small and highly dispersed but that raft size can be modulated by oligomerization of raft components.     

The effect of membrane-fluidizing agents on the adhesion of CHO cells

Treatment of CHO cells with drugs which are known to increase membrane lipid fluidity reduced the cells' ability to adhere to protein coated substrates, The concentrations of local anesthetics, nonionic detergents or aliphatic alcohols required to reduce CHO cell adhesion by 50% were similar to those reported to block nerve conduction, indicating that these drugs can affect the membrane at physiologically significant concentrations. Nonionic detergents and aliphatic alcohols, but not local anesthetics, caused increases in the fluidity of CHO plasma membranes (measured by fluorescence polarization) at concentrations which inhibited cell adhesion. The adhesion versus temperature profile had a sigmoidal shape, suggesting that a temperature dependent cooperative process such as a lipid phase transition, might be involved. However, the temperature profile for CHO membrane fluidity manifested no discontinuities, indicating the absence of any discrete phase transitions of the lipid matrix. This observation, coupled with the result that the inhibition of CHO cell adhesion produced by low temperatures was not relieved by drugs which can increase membrane fluidity, suggests that the reduced adhesion seen at low temperature is probably not due to reduced lipid fluidity.     

Dynamic clustering and dispersion of lipid rafts contribute to fusion competence of myogenic cells

Recent research indicates that the leading edge of lamellipodia of myogenic cells (myoblasts and myotubes) contains presumptive fusion sites, yet the mechanisms that render the plasma membrane fusion-competent remain largely unknown. Here we show that dynamic clustering and dispersion of lipid rafts contribute to both cell adhesion and plasma membrane union during myogenic cell fusion. Adhesion-complex proteins including M-cadherin, β-catenin, and p120-catenin accumulated at the leading edge of lamellipodia, which contains the presumptive fusion sites of the plasma membrane, in a lipid raft-dependent fashion prior to cell contact. In addition, disruption of lipid rafts by cholesterol depletion directly prevented the membrane union of myogenic cell fusion. Time-lapse recording showed that lipid rafts were laterally dispersed from the center of the lamellipodia prior to membrane fusion. Adhesion proteins that had accumulated at lipid rafts were also removed from the presumptive fusion sites when lipid rafts were laterally dispersed. The resultant lipid raft- and adhesion complex-free area at the leading edge fused with the opposing plasma membrane. These results demonstrate a key role for dynamic clustering/dispersion of lipid rafts in establishing fusion-competent sites of the myogenic cell membrane, providing a novel mechanistic insight into the regulation of myogenic cell fusion.     

Lipid rafts control human melanoma cell migration by regulating focal adhesion disassembly

Tumor cell migration is a crucial step in the metastatic cascade, and interruption of this step is considered to be logically effective in preventing tumor metastasis. Lipid rafts, distinct liquid ordered plasma membrane microdomains, have been shown to influence cancer cell migration, but the underlying mechanisms are still not well understood. Here, we report that lipid rafts regulate the dynamics of actin cytoskeleton and focal adhesion in human melanoma cell migration. Disrupting the integrity of lipid rafts with methyl-β cyclodextrin enhances actin stress fiber formation and inhibits focal adhesion disassembly, accompanied with alterations in cell morphology. Furthermore, actin cytoskeleton, rather than microtubules, mediates the lipid raft-dependent focal adhesion disassembly by regulating the dephosphorylation of focal adhesion proteins and the internalization of β3 integrin. We also show that Src–RhoA–Rho kinase signaling pathway is responsible for lipid raft disruption-induced stress fiber formation. Taken together, these observations provide a new mechanism to further explain how lipid rafts regulate the migration of melanoma cell and suggest that lipid rafts may be novel and attractive targets for cancer therapy.     

Cell Adhesion Structures in Epithelial Cells Are Formed in Dynamic and Cooperative Ways

There are many morphologically distinct membrane structures with different functions at the surface of epithelial cells. Among these, adherens junctions (AJ) and tight junctions (TJ) are responsible for the mechanical linkage of epithelial cells and epithelial barrier function, respectively. In the process of new cell–cell adhesion formation between two epithelial cells, such as after wounding, AJ form first and then TJ form on the apical side of AJ. This process is very complicated because AJ formation triggers drastic changes in the organization of actin cytoskeleton, the activity of Rho family of small GTPases, and the lipid composition of the plasma membrane, all of which are required for subsequent TJ formation. In this review, the authors focus on the relationship between AJ and TJ as a representative example of specialization of plasma membrane regions and introduce recent findings on how AJ formation promotes the subsequent formation of TJ.