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Introduction: opening up tight junctions
seminars in CELL & DEVELOPMENTAL BIOLOGY, Vol. 11, 2000: pp. 277–279 doi: 10.1006/scdb.2000.0176, available online at http://www.idealibrary.com on
Introduction: opening up tight junctions Sandra Citi
TJ display several characteristic features. Similarly to other cell–cell contacts (for example, adherens and gap junctions), TJ comprise transmembrane proteins which mediate the interaction between adjoining cells. However, the TJ is peculiar because it forms pores which, as far as we know, are not involved in the passage of molecules between cells (such as gap junctions), but only between two compartments of the extracellular space (hence the term ‘paracellular’). How do TJ achieve this? The first paper in this issue, by Balda and Matter, addresses this question, by describing the current state of knowledge about the structure, function and molecular interactions of three known membrane components of TJ: occludin, claudins and JAM. Several lines of evidence indicate that claudins are the key components of TJ pores and are responsible for building the characteristic TJ strands visualized by freeze-fracture electron microscopy. Occludin can be incorporated into the strands, but its main role may be regulatory rather than structural. On the other hand, JAM appears to be more directly involved in interaction with cells which cross the TJ barrier (such as for example leukocytes transmigrating through endothelial cell sheets). Another interesting feature of TJ is the fact that their paracellular permeability properties must fit the physiological requirements of each specialized tissue. For example, epithelial cells of the proximal kidney tubule have TJ with high permeability, to allow maximal recovery of useful substances present in the glomerular filtrate, whereas epithelial cells of the distal kidney tubule have TJ with low permeability, to reduce leakage of urine components back into the blood. It is now clear, from work of the Tsukita laboratory, that such functional heterogeneity may be due in large part to the differential expression of claudin isoforms in different tissues. Finally, the paper by Balda and Matter addresses other key questions. How does occludin reach its ultimate destination, and what are the sequences involved in TJ targeting? How do TJ membrane proteins contribute to the ‘fence’ function of TJ, and to the pathogenesis of diseases?
This issue of seminars in cell and developmental biology is about Tight Junctions (TJ), specialized sites of cell–cell contact present in epithelial and some endothelial cells, that have been characterized mainly in vertebrate systems. The functional counterparts of TJ in insect cells are called septate junctions. The primary role of TJ and septate junctions is to seal together the apicolateral surfaces of adjoining cells in an epithelial (or endothelial) tissue, thus allowing these tissues to form a barrier separating different compartments (apical versus basolateral) of the extracellular space. This barrier is formed by so-called paracellular ‘pores’, which function as selective and semipermeable filters for the passage of ions, molecules and cells across the space separated by an epithelial cell sheet. In addition, TJ are the structural boundary between two distinct domains of the plasma membrane (apical and basolateral), which display different protein/lipid composition and functions. Thus, TJ are crucial to enable polarized cells to carry out directional transport. In the last few years, the increase in our knowledge about the molecular basis of TJ function has been remarkable, both at a quantitative and qualitative level. This issue of SCDB does not attempt to be a comprehensive review of the field, it focuses on some areas of investigation where progress has been particularly informative, such as: (i) the characterization of specific transmembrane TJ proteins and the MAGUK family of cytoplasmic plaque proteins of TJ; (ii) the study of TJ assembly during early development, and (iii) the role of physiological and pathological signaling pathways in controlling TJ function.
From the Department of Molecular Biology, University of Geneva, 30 Quai Ernest Ansermet, 1211 Geneva 4, Switzerland and Dipartimento di Biologia, Universita’ degli Studi di Padova, Viale G. Colombo 3, 35121 Padova, Italy. E-mail: [email protected] . c
2000 Academic Press 1084–9521 / 00 / 000277+ 03 / $35.00/0 / 0
277
S. Citi
The first TJ-associated molecule to be identified was called ZO-1 (from the latin name for TJ, zonula occludens = small occluding zone), and soon thereafter it was discovered that ZO-1 was part of an important family of proteins which share three different types of protein modules, called PDZ, SH3 and GUK domains. These proteins are collectively known as ‘MAGUK’ proteins, and include proteins localized in insect septate junctions and in neurons. The second paper in this issue, by Gonzalez-Mariscal and colleagues, addresses the domain organization of TJ MAGUK proteins (ZO-1, ZO-2 and ZO-3), the functions of the PDZ, SH3 and GUK domains, and the phosphorylation and protein interactions of these three proteins. In addition, recent evidence concerning the possible roles of TJ MAGUK in directly or indirectly regulating nuclear transcription is discussed. Interestingly, ZO-1 and ZO-2 are not the only TJ proteins that can be detected in the nucleus, raising the possibility that signaling from the TJ to the nucleus may occur by multiple pathways. Finally, this paper illustrates how the functional heterogeneity of TJ in normal and neoplastic cells, and the growth potential of the cells, may depend, at least in part, on the differential expression of ZO-1 and ZO-2 isoforms. It should also be noted that ZO-1 and ZO-2 are known to be expressed in non-epithelial cells and tissues, where they are associated with components of cadherin-based junctions, suggesting that MAGUK proteins may function as structural and signaling components which can be adapted to different types of cell–cell contact. A critical event in the development of vertebrate organisms is the formation of compartments and the differentiation of cells into specialized tissues and organs. Since TJ create boundaries between tissue and body compartments, the biogenesis of TJ in early development would be expected to be closely related, in space and time, to these key events in embryonic organization. Indeed, as illustrated by the paper by Fleming and collaborators, studies on TJ assembly in early mouse and Xenopus development have shown that TJ formation closely parallels the establishment of cell polarity and the appearance of body compartments such as the blastocoel in the mouse. Studying early embryos also provides an excellent means of understanding the stepwise assembly of distinct TJ proteins into the nascent TJ, and of determining the role of different protein isoforms, protein domains and post-translational modifications in the maturation of TJ from early sites of cell–cell to fully mature TJ. Finally, embryo
models have been used to address a long-standing question in the cell biology of TJ: do TJ require the prior formation of cadherin-based adhesion sites in order to assemble? Perhaps not surprisingly, different models provide different answers. In the mouse, cadherin adhesion is necessary for compaction, polarization and blastocyst formation, and temporally precedes TJ assembly, suggesting that TJ may arise from cadherin-based adhesion sites by a maturation process which entails the redistribution of proteins such as ZO-1 from the cadherin-based adhesion sites to the new TJ sites following the expression of specific TJ transmembrane proteins such as occludin. Such a model is not supported by studies on Xenopus TJ assembly, which show that even in the absence of basolateral cell–cell adhesion, TJ protein assembly occurs normally at the apicolateral cell–cell contact sites. The nature of the molecules and mechanisms which specify the location of the initial site of TJ assembly in Xenopus remains a fascinating puzzle. Perhaps the key to understanding how TJ are formed and regulated lies in the cytoskeleton. The fact that actin filaments are present in the cytoplasmic face of TJ, and that their integrity is required for TJ function has long been established. However, only recently has the connection between actomyosin regulation and TJ modulation been addressed in biochemical and molecular detail. The fourth paper in this issue, by Turner, reviews a number of studies where the structure and physiology of the TJ have been correlated with the organization and contractility of the actomyosin cytoskeleton. An elegant model of TJ regulation has been developed, where stimulation of Na+ -glucose co-transport in intestinal epithelial cells (in an isolated intestine or in culture) leads to an increase in the permeability of TJ to ions and small molecules. This physiological response has been analyzed in ultrastructural and molecular detail, leading to the hypothesis that TJ regulation depends on a cascade of signals involving myosin light chain kinase activation, myosin light chain phosphorylation, and contraction of the perijunctional actomyosin ring. Besides Na+ -glucose co-transport, other physiological and pathological stimuli, from outside or inside the cell, may regulate TJ permeability through the same pathway. Since many TJ proteins are now known to interact with actin and associated proteins, one of the challenges of future research will be to link actomyosin regulation to TJ function through a molecular chain of interactions of TJ proteins. Finally, it is important to note that TJ are one hallmark of polarized epithelial cells, and that most 278
Introduction: opening up tight junctions
human tumors are of epithelial origins. Malignant cancers are characterized by a loss of the polarized epithelial phenotype, and transformed epithelial cells tend to undergo morphological and functional changes associated with an epithelial-mesenchymal transition, such as an increase in motility and the loss of cell–cell junctions. A large percentage of human tumors show mutations in a Ras gene. So, what is the relationship between tumorigenesis, Ras and cell–cell junctions? The last paper in this issue, by Mercer, addresses this question by summarizing the present evidence about the complex interactions between Ras, cytoskeletal organization and protein components of adherens junctions and TJ. One of the many molecules involved in signaling between Ras and intercellular junctions is AF-6, which has been localized both at TJ and adherens junctions, and which interacts with ZO-1 in a Ras-dependent manner. Mice lacking AF-6 die during embryonic development due to abnormal polarization of cells of neuroectodermal origin. In addition, studies in Drosophila suggest that the AF-6 homolog Canoe can act upstream of activated Ras. Thus, although large
multidomain proteins such as ZO-1 and AF-6 are probably directly involved in epithelial development, the molecular details of their multiple functions and interactions still needs to be clarified. In summary, the molecular mechanisms underlying the assembly and function of TJ are no longer an insurmountable barrier, they are beginning to open up to our understanding. Furthermore, the role of some TJ proteins and their Drosophila homologs in modulating cell growth and nuclear transcription suggests that TJ may also function to transmit information from cell–cell contacts to the nucleus.
Acknowledgements I am very grateful to many colleagues and collaborators for discussions and advice, to David Shore for support and comments on the manuscript, and to the State of Geneva, the Swiss National Fonds, Ministero dell’Universita’ e della Ricerca Scientifica e Tecnologica, and Consiglio Nazionale delle Ricerche for support.
279
Introduction: opening up tight junctions Sandra Citi
TJ display several characteristic features. Similarly to other cell–cell contacts (for example, adherens and gap junctions), TJ comprise transmembrane proteins which mediate the interaction between adjoining cells. However, the TJ is peculiar because it forms pores which, as far as we know, are not involved in the passage of molecules between cells (such as gap junctions), but only between two compartments of the extracellular space (hence the term ‘paracellular’). How do TJ achieve this? The first paper in this issue, by Balda and Matter, addresses this question, by describing the current state of knowledge about the structure, function and molecular interactions of three known membrane components of TJ: occludin, claudins and JAM. Several lines of evidence indicate that claudins are the key components of TJ pores and are responsible for building the characteristic TJ strands visualized by freeze-fracture electron microscopy. Occludin can be incorporated into the strands, but its main role may be regulatory rather than structural. On the other hand, JAM appears to be more directly involved in interaction with cells which cross the TJ barrier (such as for example leukocytes transmigrating through endothelial cell sheets). Another interesting feature of TJ is the fact that their paracellular permeability properties must fit the physiological requirements of each specialized tissue. For example, epithelial cells of the proximal kidney tubule have TJ with high permeability, to allow maximal recovery of useful substances present in the glomerular filtrate, whereas epithelial cells of the distal kidney tubule have TJ with low permeability, to reduce leakage of urine components back into the blood. It is now clear, from work of the Tsukita laboratory, that such functional heterogeneity may be due in large part to the differential expression of claudin isoforms in different tissues. Finally, the paper by Balda and Matter addresses other key questions. How does occludin reach its ultimate destination, and what are the sequences involved in TJ targeting? How do TJ membrane proteins contribute to the ‘fence’ function of TJ, and to the pathogenesis of diseases?
This issue of seminars in cell and developmental biology is about Tight Junctions (TJ), specialized sites of cell–cell contact present in epithelial and some endothelial cells, that have been characterized mainly in vertebrate systems. The functional counterparts of TJ in insect cells are called septate junctions. The primary role of TJ and septate junctions is to seal together the apicolateral surfaces of adjoining cells in an epithelial (or endothelial) tissue, thus allowing these tissues to form a barrier separating different compartments (apical versus basolateral) of the extracellular space. This barrier is formed by so-called paracellular ‘pores’, which function as selective and semipermeable filters for the passage of ions, molecules and cells across the space separated by an epithelial cell sheet. In addition, TJ are the structural boundary between two distinct domains of the plasma membrane (apical and basolateral), which display different protein/lipid composition and functions. Thus, TJ are crucial to enable polarized cells to carry out directional transport. In the last few years, the increase in our knowledge about the molecular basis of TJ function has been remarkable, both at a quantitative and qualitative level. This issue of SCDB does not attempt to be a comprehensive review of the field, it focuses on some areas of investigation where progress has been particularly informative, such as: (i) the characterization of specific transmembrane TJ proteins and the MAGUK family of cytoplasmic plaque proteins of TJ; (ii) the study of TJ assembly during early development, and (iii) the role of physiological and pathological signaling pathways in controlling TJ function.
From the Department of Molecular Biology, University of Geneva, 30 Quai Ernest Ansermet, 1211 Geneva 4, Switzerland and Dipartimento di Biologia, Universita’ degli Studi di Padova, Viale G. Colombo 3, 35121 Padova, Italy. E-mail: [email protected] . c
2000 Academic Press 1084–9521 / 00 / 000277+ 03 / $35.00/0 / 0
277
S. Citi
The first TJ-associated molecule to be identified was called ZO-1 (from the latin name for TJ, zonula occludens = small occluding zone), and soon thereafter it was discovered that ZO-1 was part of an important family of proteins which share three different types of protein modules, called PDZ, SH3 and GUK domains. These proteins are collectively known as ‘MAGUK’ proteins, and include proteins localized in insect septate junctions and in neurons. The second paper in this issue, by Gonzalez-Mariscal and colleagues, addresses the domain organization of TJ MAGUK proteins (ZO-1, ZO-2 and ZO-3), the functions of the PDZ, SH3 and GUK domains, and the phosphorylation and protein interactions of these three proteins. In addition, recent evidence concerning the possible roles of TJ MAGUK in directly or indirectly regulating nuclear transcription is discussed. Interestingly, ZO-1 and ZO-2 are not the only TJ proteins that can be detected in the nucleus, raising the possibility that signaling from the TJ to the nucleus may occur by multiple pathways. Finally, this paper illustrates how the functional heterogeneity of TJ in normal and neoplastic cells, and the growth potential of the cells, may depend, at least in part, on the differential expression of ZO-1 and ZO-2 isoforms. It should also be noted that ZO-1 and ZO-2 are known to be expressed in non-epithelial cells and tissues, where they are associated with components of cadherin-based junctions, suggesting that MAGUK proteins may function as structural and signaling components which can be adapted to different types of cell–cell contact. A critical event in the development of vertebrate organisms is the formation of compartments and the differentiation of cells into specialized tissues and organs. Since TJ create boundaries between tissue and body compartments, the biogenesis of TJ in early development would be expected to be closely related, in space and time, to these key events in embryonic organization. Indeed, as illustrated by the paper by Fleming and collaborators, studies on TJ assembly in early mouse and Xenopus development have shown that TJ formation closely parallels the establishment of cell polarity and the appearance of body compartments such as the blastocoel in the mouse. Studying early embryos also provides an excellent means of understanding the stepwise assembly of distinct TJ proteins into the nascent TJ, and of determining the role of different protein isoforms, protein domains and post-translational modifications in the maturation of TJ from early sites of cell–cell to fully mature TJ. Finally, embryo
models have been used to address a long-standing question in the cell biology of TJ: do TJ require the prior formation of cadherin-based adhesion sites in order to assemble? Perhaps not surprisingly, different models provide different answers. In the mouse, cadherin adhesion is necessary for compaction, polarization and blastocyst formation, and temporally precedes TJ assembly, suggesting that TJ may arise from cadherin-based adhesion sites by a maturation process which entails the redistribution of proteins such as ZO-1 from the cadherin-based adhesion sites to the new TJ sites following the expression of specific TJ transmembrane proteins such as occludin. Such a model is not supported by studies on Xenopus TJ assembly, which show that even in the absence of basolateral cell–cell adhesion, TJ protein assembly occurs normally at the apicolateral cell–cell contact sites. The nature of the molecules and mechanisms which specify the location of the initial site of TJ assembly in Xenopus remains a fascinating puzzle. Perhaps the key to understanding how TJ are formed and regulated lies in the cytoskeleton. The fact that actin filaments are present in the cytoplasmic face of TJ, and that their integrity is required for TJ function has long been established. However, only recently has the connection between actomyosin regulation and TJ modulation been addressed in biochemical and molecular detail. The fourth paper in this issue, by Turner, reviews a number of studies where the structure and physiology of the TJ have been correlated with the organization and contractility of the actomyosin cytoskeleton. An elegant model of TJ regulation has been developed, where stimulation of Na+ -glucose co-transport in intestinal epithelial cells (in an isolated intestine or in culture) leads to an increase in the permeability of TJ to ions and small molecules. This physiological response has been analyzed in ultrastructural and molecular detail, leading to the hypothesis that TJ regulation depends on a cascade of signals involving myosin light chain kinase activation, myosin light chain phosphorylation, and contraction of the perijunctional actomyosin ring. Besides Na+ -glucose co-transport, other physiological and pathological stimuli, from outside or inside the cell, may regulate TJ permeability through the same pathway. Since many TJ proteins are now known to interact with actin and associated proteins, one of the challenges of future research will be to link actomyosin regulation to TJ function through a molecular chain of interactions of TJ proteins. Finally, it is important to note that TJ are one hallmark of polarized epithelial cells, and that most 278
Introduction: opening up tight junctions
human tumors are of epithelial origins. Malignant cancers are characterized by a loss of the polarized epithelial phenotype, and transformed epithelial cells tend to undergo morphological and functional changes associated with an epithelial-mesenchymal transition, such as an increase in motility and the loss of cell–cell junctions. A large percentage of human tumors show mutations in a Ras gene. So, what is the relationship between tumorigenesis, Ras and cell–cell junctions? The last paper in this issue, by Mercer, addresses this question by summarizing the present evidence about the complex interactions between Ras, cytoskeletal organization and protein components of adherens junctions and TJ. One of the many molecules involved in signaling between Ras and intercellular junctions is AF-6, which has been localized both at TJ and adherens junctions, and which interacts with ZO-1 in a Ras-dependent manner. Mice lacking AF-6 die during embryonic development due to abnormal polarization of cells of neuroectodermal origin. In addition, studies in Drosophila suggest that the AF-6 homolog Canoe can act upstream of activated Ras. Thus, although large
multidomain proteins such as ZO-1 and AF-6 are probably directly involved in epithelial development, the molecular details of their multiple functions and interactions still needs to be clarified. In summary, the molecular mechanisms underlying the assembly and function of TJ are no longer an insurmountable barrier, they are beginning to open up to our understanding. Furthermore, the role of some TJ proteins and their Drosophila homologs in modulating cell growth and nuclear transcription suggests that TJ may also function to transmit information from cell–cell contacts to the nucleus.
Acknowledgements I am very grateful to many colleagues and collaborators for discussions and advice, to David Shore for support and comments on the manuscript, and to the State of Geneva, the Swiss National Fonds, Ministero dell’Universita’ e della Ricerca Scientifica e Tecnologica, and Consiglio Nazionale delle Ricerche for support.
279