Caenorhabditis Elegans 2

Chapter 11 1 [AU1] Claudin Family Proteins in Caenorhabditis elegans 2 Jeffrey S. Simske and Jeff Hardin 3 Abstract 4 In the last decade, the claudin family of integral membrane proteins has been identified as the major 5 protein component of the tight junctions in all vertebrates. The claudin superfamily proteins also function 6 to regulate channel activity, intercellular signaling, and cell morphology. Subsequently, claudin homo- 7 logues have been identified in invertebrates, including Drosophila and Caenorhabditis elegans. Recent 8 studies demonstrate that the C. elegans claudins, clc-1 to clc-5, and similar proteins in the greater PMP22/ 9 EMP/claudin/calcium channel g subunit family, including nsy-1-nsy-4 and vab-9, while highly divergent 10 at a sequence level from each other and from the vertebrate claudins, in some cases play roles similar to 11 those traditionally assigned to their vertebrate homologues. These include regulating cell adhesion and 12 passage of small molecules through the paracellular space. The claudin superfamily proteins also function 13 to regulate channel activity, intercellular signaling, and cell morphology. Study of claudin superfamily 14 proteins in C. elegans should continue to provide clues as to how core claudin protein function can be 15 modified to serve various specific roles at regions of cell–cell contact in metazoans. 16 Key words: Claudin, Junctions, Caenorhabditis elegans, Epithelia, Morphogenesis, Actomyosin, 17 Neuronal symmetry, VAB-9, CLC-1, NSY-4 18 1. Introduction: Claudins Regulate Barrier Functions 19 in Vertebrate Epithelia function as regulated barriers in tissue and organ 20 Epithelia architecture, defining compartment boundaries within organisms 21 and forming boundaries apposed to the external environment. 22 The proper physiological state of epithelia depend on cellular 23 contacts, or junctions, that mediate cell–cell adhesion, function as 24 selective gates for the ingress or egress of specific ions and small 25 molecules, and maintain unique membrane identities on apical and 26 basal sides of the cellular layer, thus contributing to epithelial 27 polarity. The tight junction is the most apical of cellular junctions 28 in vertebrate epithelia. Freeze fracture TEM reveals one of the 29 most striking features of tight junctions, a network of intertwined 30 Kursad Turksen (ed.), Claudins: Methods and Protocols, Methods in Molecular Biology, vol. 762, DOI 10.1007/978-1-61779-185-7_11, © Springer Science+Business Media, LLC 2011 J.S. Simske and J. Hardin 31 strands that occlude the intercellular or paracellular space at points 32 of contact that often circumscribe cells. In this way, tight junction 33 strands function as gaskets that separate and isolate the two 34 sides of an epithelium (Fig. 1). The major protein component of 35 the tight junction has been identified in the last decade as the 36 claudin family of proteins. Claudins were originally identified in a 37 search for the protein component of tight junction strands in 38 vertebrate epithelia following a painstaking purification scheme 39 (1, 2). Claudins 1 and 2 were shown to localize to tight junctions, 40 to generate tight junction strands when expressed in fibroblasts, 41 and to mediate homotypic and heterotypic cell–cell adhesion 42 (1, 2). Consistent with these findings, loss of claudin activity 43 results in paracellular barrier dysfunction (3–8). Overall tissue 44 polarity does not appear to be heavily dependent on claudin activity, [AU2] Fig. 1. Schematic of various cell junction arrangements from C. elegans, Drosophila, and vertebrates. Corresponding cell junction regions either in relative location, function, and/or molecular make-up are indicated with similar colors. The SAR-like region in C. elegans, the subapical region (SAR) in Drosophila, and tight junction in vertebrates, respectively, are indicated with black shading. AJ indicates the cadherin-based adherens junction. SJ identifies the septate junction in Drosophila, and AD identifies the AJM-1/ DLG-1 region in C. elegans. Vertebrates have no similar structure in epithelia, but share a molecularly similar barrier at the paranodal junction (see text). Vertebrate desmosomes are shown (d). Lateral views (cutaways) show the nature of the junctional structures in the membranes. Adherens junctions appear as solid bands in the membrane, Pleated Septate junctions are characterized by regular wave-like strands, and tight junctions appear as irregular but connected anastomosing strands. For simplicity, only the composition of cell junction components in the paracellular space is shown. 11 C. elegans Claudins since gross cell polarity is maintained following the loss of 45 claudin function (6). The vertebrate claudin family has expanded 46 over the years to include about 24 members all of which are 47 predicted four-pass integral membrane proteins with cytoplasmic 48 N and C termini, two extracellular loops, and one short intracel- 49 lular loop (9–11). 50 Claudins have been shown to form oligomers in vitro, with 51 hexamers being favored (12–14). This organization suggests that 52 claudins, like gap junctions, might generate pores through the 53 lipid bilayer, although there is no clear evidence at present that 54 this is the case. Rather, claudins appear to establish charged 55 pores within the paracellular space that regulate the traffic of 56 charged ions across the epithelial barrier. Charged residues in the 57 extracellular domain are responsible for regulating charge selec- 58 tivity. As a result, claudins with different charged amino acids can, 59 by virtue of their expression in overlapping and nonoverlapping 60 patterns, define the paracellular charge selectivity of a wide variety 61 of tissues (15). For example, in the vertebrate kidney, different 62 claudins are expressed along the length of the nephron, suggesting 63 that the differential expression correlates and may help generate 64 that different charge selectivity among the distinct regions of 65 the nephron (16). Supporting this hypothesis, claudin 16 maintains 66 a negative charge required for the reuptake of Mg2+ and Ca2+ ions 67 in the thick ascending part of Henle’s loop in the nephron and 68 loss of claudin 16 activity results in magnesium and calcium wasting 69 (17). Similarly, in the longitudinal axis of the intestine as well as 70 the crypt to villus axis of the intestine, the claudins are expressed 71 in complex patterns (18). Charge selectivity of claudins is most 72 dramatically illustrated by experiments in which the charge of 73 the extracellular loops is reversed by swapping amino acids in 74 this loop; in this case, the charge selectivity is reversed, as was 75 demonstrated for claudins 10 and 15 (19, 20). In general, amino 76 acid charge in extracellular loop 1 dramatically influences para- 77 cellular charge selectivity. 78 Claudins are also targets of viruses, and binding of viruses to 79 extracellular claudin loops can disrupt epithelial barrier, open the 80 paracellular space, and allow viral entry (21–23). The claudin C 81 terminus functions in trafficking of the claudin protein, binding 82 to junctional plaque proteins through PDZ domain binding 83 motifs, or regulation of the paracellular space through phospho- 84 rylation (24–29). While the PDZ-binding peptide is found in 85 most claudins, this domain is absent from many of the C termini 86 in related protein families of both vertebrates and invertebrates, 87 suggesting that there may be as many functions as there are unique 88 C-terminal domains. 89 While much is now known about the function of vertebrate 90 claudins, less is known about similar molecules in invertebrates. 91 Since tight junction structures in invertebrates are uncommon, 92 J.S. Simske and J. Hardin 93 claudin-like molecules in invertebrates will necessarily have 94 functions distinct from those of their vertebrate relatives (30). 95 This review will detail the known functions of this class of proteins 96 in Caenorhabditis elegans. 97 2. Sequence Conservation Among Claudin 98 Family Proteins The sequence similarity of C. elegans and vertebrate claudin family 99 proteins so low that it was proposed that sequence similarity was 100 insufficient to establish homology or that claudins are present 101 only in chordates (9, 31). Despite these initial assessments, claudin 102 family proteins were ultimately identified inC. elegans by sequence 103 homology searches and also through the identification of claudin- 104 related proteins participating in diverse processes. Here we will 105 review what is known about vab-9, nsy-4, clc-1–5, and related 106 claudin superfamily genes. The comparison of C. elegans sequences 107 with each other and with claudin family proteins from vertebrates 108 indicates that the sequences are indeed highly divergent (Fig. 2). 109 A similar observation was made when Drosophila claudins, including 110 Sinuous and Megatrachea were placed into a claudin family 111 phylogenetic tree (32). A previous analysis of Stargazin indicated 112 that there were at least three distinct clades within this greater 113 tetraspan family: PMP22/EMP, claudin, and the gamma subunits 114 of voltage-gated calcium channels (33). Generally, PMP22/ 115 EMP/MP/Claudin-related proteins belong to pfam00822 (15). 116 Analysis of these protein groupings, in conjunction with VAB-9 117 and VAB-9 orthologs from Drosophila and vertebrates, indicates 118 that a separate VAB-9 clade exists, which is distinct from other 119 subgroups (Fig. 2). Previously PERP, a tetraspan protein required 120 for desmosomal structure and epithelial integrity, was recognized 121 as the closest ortholog to TM4SF10 (or TMEM47) in the mouse 122 (34, 35) (JSS and L. Attardi, unpublished observations). In our 123 phylogenetic analysis, PERP associated with the VAB-9 clade 124 (Fig. .2). Despite being closer to VAB-9 in homology, expression 125 of mouse PERP in C. elegans epithelia is incapable of rescuing 126 vab-9 phenotypes, while similar expression of TM4SF10 rescues 127 vab-9 (36) (JSS and L. Attardi, unpublished observations).

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