- Email: [email protected]
A perspective on inversin
Cell Biology International 28 (2004) 119–124
Cell Biology International www.elsevier.com/locate/cellbi
Review
A perspective on inversin Lorraine Eley a, Lee Turnpenny b, Laura M. Yates a, A. Scott Craighead a, David Morgan a, Catherine Whistler a, Judith A. Goodship a*, Tom Strachan a,* a
Institute of Human Genetics, University of Newcastle, International Centre for Life, Central Parkway, Newcastle upon Tyne NE1 3BZ, UK b Division of Human Genetics, University of Southampton, Duthie Building, Southampton General Hospital, Tremona Road, Southampton SO16 6YD, UK Received 2 October 2003; accepted 4 November 2003
Abstract Over the past 5 years, there has been increasing evidence for the role of primary (9+0) cilia in renal physiology and in establishing the left–right axis. The cilia in the renal tract are immotile and thought to have a sensory function. Cilia at the murine embryonic node have a vortical movement that sets up a leftward flow. Inversin, the protein defective in the inv mouse and in patients with type-2 nephronophthisis, localizes to both renal and node primary cilia. However, we present evidence that it is also expressed before the node forms and that its subcellular localization in renal tubular cells is not confined to the cilia. Its role in both the pathway determining left–right axis and renal function remains to be elucidated. 2004 Elsevier Ltd. All rights reserved. Keywords: Inversin; Situs inversus; Primary cilia; Nephronopthisis
1. Background The inv (inversion of embryo turning) mouse is an insertional mutant with a complex phenotype: homozygotes exhibit consistent situs abnormalities (unlike other mouse models of laterality defects where situs is randomized), and also severe cystic changes of the kidney and pancreas (Yokoyama et al., 1993). The phenotype resulted from insertion of a tyrosinase minigene, the integration event being accompanied by deletion of a small segment of chromosome 4 and duplication of a short region which in the wild type chromosome is located about 6 cm distal to the region deleted in inv (Yokoyama et al., 1993, 1995). We and others used positional cloning approaches to identify a novel gene which lacks nine internal exons as a result of the deletion event in the inv mouse and whose coding sequence is sufficient to rescue the inv phenotype (Mochizuki et al., 1998; Morgan et al., 1998). The novel
* Corresponding authors. Tel.: +44-191-241-8616; fax: +44-191-2418699 E-mail addresses: [email protected] (J.A. Goodship), [email protected] (T. Strachan). 1065-6995/04/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2003.11.009
gene (official symbol: Invs) was predicted to encode a 1062 amino acid protein which we named inversin. 2. Structural features of Invs and inversin The Invs gene spans over 150 kb and is organized into 17 exons. Exons 2 through to 17 specify the coding sequence but since the inv mutation results in deletion of exons 4 through to 12, a profound effect on gene expression can be expected, with at a minimum the loss of the coding potential for the region spanning amino acids 92–642. Evolutionary analyses have shown that almost all of this region, and indeed the N-terminal half of inversin, is very highly conserved (Morgan et al., 2002b), and its biological importance has been underscored in phenotype rescue experiments (Watanabe et al., 2003). The C-terminal half, by contrast, is comparatively poorly conserved. Within the conserved N-terminal half are 16 tandem ankyrin repeats spanning amino acids 13–557 followed by a central lysine-rich domain spanning amino acids 558–604 (Fig. 1). The predicted protein lacks an N-terminal signal peptide or transmembrane domain but three nuclear localization signals are predicted: a standard signal in
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L. Eley et al. / Cell Biology International 28 (2004) 119–124
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' Fig. 1. Structural features of mouse inversin. The pale blue shading refers to the generally very highly conserved N-terminal half of inversin and encompasses a tandem series of ankyrin repeats (circles labelled as A, 13–557 aa) followed directly by a lysine-rich domain (558–604 aa). The yellow shading denotes the generally poorly conserved C-terminal half. Other abbreviations are: D—D-box (490–498 and 907–915 aa); IQ—IQ domain (562–575 and 921–934 aa); NLS—standard nuclear localization sequence (735–738 aa); b-NLS—bipartite nuclear localization sequence (589–604 and 782–798 aa). Red bars at bottom indicate the regions that are deleted in the S1 and S2 isoforms with loss respectively of 120 amino acids (731–850 aa) or 165 amino acids (731–895 aa).
HG
HG
HG
When we cloned Invs we obtained evidence from cDNA library screening and RT-PCR experiments for the existence of three inversin isoforms in exon 14 (Morgan et al., 1998). In addition to a full-length isoform (F), specifying a 1062 amino acid protein, two shorter forms had in frame deletions that would be expected to produce proteins of 942 (S1 isoform) and 897 (S2 isoform) amino acids (Fig. 1). The biological importance is underscored by evolutionary conservation. Thus although exon 14 of the human inversin gene specifies five more amino acids than the mouse exon 14, several human cDNA clones show a sequence corresponding to the S2 isoform and with precise evolutionary conservation of the alternative splice sites used to generate the mouse S2 isoform. We have further validated the existence of the exon 14 isoforms by using RT–PCR and primers designed to prime from exons flanking mouse exon 14 (Fig. 2). During development there is a predominance of F (764 bp) and S2 (269 bp) transcripts. The minority S1 isoform cannot be detected at very early stages but appears to be produced by the 7.5 embryonic day (ed) stage. The subcellular fractionation data from Phillips’ group also identifies three inversin isoforms of 140, 125 and 90 kD (Nu¨rnberger et al.,
HG
3. Inversin isoforms
(6 FHOOV
the C-terminal region and two bipartite signals, one located in the lysine-rich central domain and the other in the C-terminal region (Fig. 1). Two other types of conserved motifs have been identified. A calmodulinbinding IQ domain is represented at two locations: at the beginning of the lysine-rich central domain and within the C-terminal region. In addition there are two copies of the destruction box (D-box) motif, which is found in most targets of the anaphasepromoting complex (APC), where it is important for ubiquitination-mediated destruction of the target protein.
) ES 6 ES 6 ES Fig. 2. RT–PCR-detected expression of inversin isoforms. Primers designed across the region of exon 14 demonstrated to be alternatively spliced shows the existence of 3 inversin isoforms in mouse embryos from embryonic day (ed) 6.5 to 10 and ES cells. The transcripts 764, 404 and 269 bp represent F, S1 and S2 isoforms respectively.
2002). These may possibly correspond to the above isoforms—which are predicted to be 118 kD (F), 104 kD (S1) and 99 kD (S2) respectively. Differences in sizes may be due to post-translational modification.
4. Expression and subcellular localization of inversin It has been proposed that the node is involved in the initial event that breaks left–right asymmetry (Hamada et al., 2002). However, there is evidence that gap junctions/ion channels pre node may also play a role (Levin and Mercola, 1999; Levin et al., 2002). Inversin plays a pivotal role in the breaking of left–right symmetry since all inv mice have situs inversus. Thus determining its function is of great importance. Here we show that inversin RNA is detectable as early as the two cell stage by RT–PCR (Fig. 3). Subcellular localization has been studied in cell lines derived from wild type mice and in an inv:GFP mouse (Morgan et al., 2002a; Phillips et al., 2001; Watanabe et al., 2003). In the Inv:GFP mouse inversin is present in 9+0 cilia of the node, renal tubules, pituitary gland,
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5. Proteins interacting with inversin
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Fig. 3. RT–PCR-detected expression of inversin in earlier-stage embryos. RT–PCR was used to demonstrate the expression of inversin in 2-cell, 4-cell and blastocyst stage mouse embryos. The primer pair used was designed to exon 7 (forward) and exon 9 (reverse); separated at the genomic level by two introns, thus verifying products as mRNA transcript-specific.
retina and primary fibroblast cultures. Inversin was not present in 9+2 cilia of the trachea, oviducts or ependyma (Watanabe et al., 2003). The primary cilium arises from one of the two centrioles, always the mother centriole. In the inv:GFP mouse signal is seen in the mother centriole before the extension of the cilium. Watanabe and colleagues studied expression at the embryonic node. In the figures that they show all cilia, visualized using an acetylated tubulin antibody, stain for inversin. In their figure only the periphery of the node is in the plane of focus. McGrath and colleagues have recently suggested that there are two populations of cilia at the node, motile cila in the centre of the node and immotile cilia around the periphery (McGrath et al., 2003). It is therefore important to address whether inversin is present in all cilia across the node. Inversin localization has been studied in MDCK II, mIMCD-3 and S1 cultures. The S1 line was derived from the early segment of the murine renal proximal tubule. In subconfluent S1 cultures inversin localizes in the nuclei with additional weak perinuclear and membrane staining. When S1 cells are grown to confluence inversin localizes to the membranes between the cells (Fig. 4; Nu¨rnberger et al., 2002). MDCK-II and mIMCD-3 were derived from canine collecting duct and murine inner medullary collecting duct respectively. We found that inversin localization changed through the cell cycle. In confluent, ciliated MDCK-II and mIMCD-3 cells inversin is seen in the cilia. Like Nu¨rnberger and colleagues we also observed generalized nuclear staining. In prophase we observed staining at the centrosomes, at metaphase and anaphase we observed staining at the spindle poles. In cells at late telophase we have observed localization at the midbody, a region of microtubule overlap (Fig. 4; Morgan et al., 2002a).
Several proteins of varying function and subcellular localization have been identified as interacting directly or in a multiprotein complex with inversin (Table 1). Calmodulin and Apc2 are two proteins within the former category that bind specifically to corresponding inversin motifs. Although calmodulin binding has been demonstrated to occur at both IQ domains (Morgan et al., 2002b; Yasuhiko et al., 2001), Yasuhiko et al identified IQ2 as being critical for inversin to randomize left–right asymmetry in Xenopus. Calmodulin is ubiquitously distributed within cells and functions as a major calcium sensor that regulates numerous target proteins in either a Ca2+-independent or Ca2+-dependent manner (Rhoads and Friedberg, 1997). As expected of proteins containing IQ motifs, calmodulin binding to IQ2 appears to occur only in the absence of calcium (Yasuhiko et al., 2001). Of note, calmodulin is present in the 9+0 cilia of mIMCD-3 cells and shows partial colocalization with inversin (Fig. 5). As mentioned previously, inversin contains two destruction box (D-box) motifs and shows dynamic expression in mitotic cells. A conserved destruction box (D-box) is important for the ubiquitination-mediated destruction of most anaphase promoting complex (APC; also called cyclosome) targets. Yeast two-hybrid identification of Apc2 as an interacting partner further supports a role for inversin in the cell cycle (Morgan et al., 2002a). Apc2 is one of at least ten subunits of the vertebrate APC, which is responsible for the ubiquitination and subsequent destruction of cell regulators at the metaphase–anaphase and mitosis–G1 transitions (Glotzer et al., 1991; Zachariae and Nasmyth, 1999). Of the two D-boxes, the N-terminal D-box (no. 1) is extremely highly conserved and site directed mutation of D-box no. 1 abrogated the inversin-Apc2 interaction, whereas the same changes to D-box no. 2 did not prevent Apc2 binding (Morgan et al., 2002a). The recent identification of inversin as the gene mutated in nephronopthisis (NPHP) type 2, prompted a search for an interaction between inversin and nephrocystin (Otto et al., 2003). NHPH2 is an autosomal recessive cystic kidney disease in which a subset of patients demonstrates situs abnormalities. Nephrocystin, the product of the gene mutated in NPHP type 1, is a novel docking protein that interacts with components of cell–cell and cell–matrix signalling, and nephrocystin4. Although no direct interaction between inversin and nephrocystin has been demonstrated, both proteins coprecipitate and colocalize to the primary cilia of renal cells (Otto et al., 2003). Localization to the lateral cell membrane suggests a further role for inversin in cell–cell signalling (Nu¨rnberger et al., 2002). Phillips et al demonstrated that 90- and 125-kDa inversin isoforms coprecipitate
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(a)
(b)
Fig. 4. Subcellular localization of inversin. (a) Inversin expression at the cell membranes and nucleus. Confluent S1 cells were double-labelled with antibodies to inversin and analyzed by confocal microscopy as described previously (Nu¨rnberger et al., 2002). Inversin stains the cell membranes, nuclei and perinuclear compartment. (b) Dynamic expression of inversin in cultured kidney cells. Immunofluorescence staining of MDCK-II cells with rabbit polyclonal antiinversin antibody (green) shows a dynamic expression throughout the cell cycle. Cells in early prophase (A) show inversin expression at the centrosomes whereas in metaphase (B) and anaphase (C) cells inversin localizes to the spindle poles. In cells undergoing cytokinesis (D) expression was observed at the midbody, a region of microtubule overlap. Throughout these stages general nuclear expression is also observed, which becomes ubiquitous upon breakdown of the nuclear envelope. The plasma membranes were counterstained with TRITC-wheat germ agglutinin (red) (Morgan et al., 2002a). Reproduced with permission from Oxford University Press.
with -catenin and N-cadherin in the membrane but not the cytosolic fraction of confluent cultured kidney cells. Of these, the 125-kDa band was the most enriched in the cell membrane fractions. These proteins also colocalize at the plasma membrane where it is postulated they form a complex involved in regulating the architecture of cell–cell junctions in a calcium dependent manner (Nu¨rnberger et al., 2002).
6. Inversin and the event(s) that break left–right asymmetry It is postulated that primary (9+0) cilia at the node are involved in generating the asymmetrical expression patterns at the node and hence determining the left–right axis (Hamada et al., 2002). Several models showing
randomization of the left–right axis lack nodal cilia (Brody et al., 2000; Marszalek et al., 1999; Murcia et al., 2000; Takeda et al., 1999), or in the case of the iv mouse (Supp et al., 1999), have immotile cilia. More recent work by Hirokawa’s group led to a major change in thinking about the node (Nonaka et al., 1998). Studying normal mice they found that nodal cilia rotated in a clockwise direction and they demonstrated a leftward flow of perinodal fluid. The nodal flow hypothesis proposed that rotation of nodal cilia generated the leftwards flow of perinodal fluid, and this was supported by studies where mouse embryos were cultured under an artificial fluid flow, where left–right patterning was shown to be reversed by reversing the fluid flow. Recent evidence shows that there are two types of cilia at the node, motile and immotile. It is proposed that motile cilia generate the leftward flow and the immotile cilia
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Fig. 5. Inversin and calmodulin show partial colocalization to the primary cilia of kidney cells. Confocal microscopy of paraformaldehyde fixed confluent mIMCD-3 cells double-stained with mouse monoclonal anti-calmodulin (Upstate biotechnology) (A and D) and rabbit polyclonal anti-inversin (Morgan et al., 2002a) (B and E) show that both proteins localize to the primary cilia. Composite images (C and F) demonstrate a partial overlap (yellow) in expression. The scale bar represents 5 µm.
Table 1 Interacting partners of inversin and supporting evidence Supporting evidence Interacting protein
Y2H
Gel overlay
Co-IP Colocalize
Calmodulin Apc2 Nephrocystin -catenin N-cadherin
U U – – –
U – – – –
U U U U U
cilia – cilia cell junction cell junction
sense the flow, thus breaking bilateral symmetry and polarizing the L–R axis (McGrath et al., 2003). However, in the inv mouse the vast majority of the mice have situs inversus and yet the perinodal fluid moves in the same leftwards direction as in wild type mice, albeit more slowly and showing more turbulence (Okada et al., 1999). It is difficult to reconcile this observation with the principles of the nodal flow model. One possibility is that inversin is important in the sensing cilia, hence the importance of clarifying whether inversin is expressed in all nodal cilia or only peripheral nodal cilia. However,
it may be that inversin is implicated in asymmetry preceding nodal flow. Levin and Mercola have presented data for gap junction-mediated transfer of left–right patterning signals in the chick blastoderm preceding asymmetry at the node (Levin and Mercola, 1999). Levin and co-workers have subsequently shown that there is an endogenous H+/K+-ATPase-dependent difference in membrane potential between the left and right side of the primitive streak in chick preceding node asymmetry and that in Xenopus there is asymmetric expression of the H+/K+ATPase 2 h post fertilization (Levin et al., 2002). They postulate that this H+/K+-ATPase asymmetry could generate unidirectional flow through the gap junctions. In chick and Xenopus, therefore, left–right differences appear to exist before the signalling cascade is set up from the node but it is not known if this is the case in mice or humans. The shape of the node and the distribution and length of the cilia differs between mouse, chick, Xenopus and zebrafish, and it is not clear at this stage how well the pathways are conserved between different model organisms (Essner et al., 2002).
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Inversin could potentially be involved in the above mentioned gap junction/ion flux mechanisms. In support of such a role (a) it has been previously shown that inversin localized to the cell membrane (b) inv mice exhibit polycystic kidney disease, a phenotype associated with reversals in electric polarity and ion pump localization (c) overexpression of inv on the right side of Xenopus embryos randomizes the left–right axis, while overexpression on the left side does not (Yasuhiko et al., 2001). Here we have shown that inversin transcripts are expressed as early as the two-cell stage thus supporting such a role for inversin. Also inversin expression in the node cilia has been shown (Watanabe et al., 2003). Thus it is still not clear whether situs inversus in the inv mouse results from perturbation at the embryo node or an earlier stage or both.
Acknowledgements This work was supported by the Biotechnology and Biological Sciences Research Council, the British Heart Foundation, the Lily Ross Fund, the Medical Research Council and the National Kidney Research Fund. Fig. 4 (i) kindly provided by Carrie L. Phillips. References Brody SL, Yan XH, Wuerffel MK, Song SK, Shapiro SD. Ciliogenesis and left-right axis defects in forkhead factor HFH-4-null mice. Am J Respir Cell Mol Biol 2000;23:45–51. Essner JJ, Vogan KJ, Wagner MK, Tabin CJ, Yost HJ, Brueckner M. Conserved function for embryonic nodal cilia. Nature 2002; 418:37–8. Glotzer M, Murray AW, Kirschner MW. Cyclin is degraded by the ubiquitin pathway. Nature 1991;349:132–8. Hamada H, Meno C, Watanabe D, Saijoh Y. Establishment of vertebrate left-right asymmetry. Nat Rev Genet 2002;3:103–13. Levin M, Mercola M. Gap junction-mediated transfer of left-right patterning signals in the early chick blastoderm is upstream of Shh asymmetry in the node. Development 1999;126:4703–14. Levin M, Thorlin T, Robinson KR, Nogi T, Mercola M. Asymmetries in H+/K+-ATPase and cell membrane potentials comprise a very early step in left-right patterning. Cell 2002;111:77–89. Marszalek JR, Ruiz-Lozano P, Roberts E, Chien KR, Goldstein LS. Situs inversus and embryonic ciliary morphogenesis defects in mouse mutants lacking the KIF3A subunit of kinesin-II. Proc Natl Acad Sci U S A 1999;96:5043–8. McGrath J, Somlo S, Makova S, Tian X, Brueckner M. Two populations of node monocilia initiate left-right asymmetry in the mouse. Cell 2003;114:61–73. Mochizuki T, Saijoh Y, Tsuchiya K, Shirayoshi Y, Takai S, Taya C et al. Cloning of inv, a gene that controls left/right asymmetry and kidney development. Nature 1998;395:177–81.
Morgan D, Eley L, Sayer J, Strachan T, Yates LM, Craighead AS et al. Expression analyses and interaction with Apc2 suggest a role for inversin in primary cilia and in the cell cycle. Hum Mol Genet 2002a;11:3345–50. Morgan D, Goodship J, Essner JJ, Vogan KJ, Turnpenny L, Yost J et al. The left-right determinant inversin has highly conserved ankyrin repeat and IQ domains and interacts with calmodulin. Hum Genet 2002b;110:377–84. Morgan D, Turnpenny L, Goodship J, Dai W, Majumder K, Matthews L et al. Inversin, a novel gene in the vertebrate left-right axis pathway, is partially deleted in the inv mouse. Nat Genet 1998; 20:149–56. Murcia NS, Richards WG, Yoder BK, Mucenski ML, Dunlap JR, Woychik RP. The Oak Ridge Polycystic Kidney (orpk) disease gene is required for left-right axis determination. Development 2000;127:2347–55. Nonaka S, Tanaka Y, Okada Y, Takeda S, Harada A, Kanai Y et al. Randomization of left-right asymmetry due to loss of nodal cilia generating leftward flow of extraembryonic fluid in mice lacking KIF3B motor protein. Cell 1998;95:829–37. Nu¨rnberger J, Bacallao RL, Phillips CL. Inversin forms a complex with catenins and N-cadherin in polarized epithelial cells. Mol Biol Cell 2002;13:3096–106. Okada Y, Nonaka S, Tanaka Y, Saijoh Y, Hamada H, Hirokawa N. Abnormal nodal flow precedes situs inversus in iv and inv mice. Mol Cell 1999;4:459–68. Otto EA, Schermer B, Obara T, O’Toole JF, Hiller KS, Mueller AM et al. Mutations in INVS encoding inversin cause nephronophthisis type 2, linking renal cystic disease to the function of primary cilia and left-right axis determination. Nat Genet 2003;34:413–20. Phillips CL, Arend LJ, Filson AJ, Kojetin DJ, Clendenon JL, Fang S et al. Three-dimensional imaging of embryonic mouse kidney by two-photon microscopy. Am J Pathol 2001;158:49–55. Rhoads AR, Friedberg F. Sequence motifs for calmodulin recognition. FASEB J 1997;11:331–40. Supp DM, Brueckner M, Kuehn MR, Witte DP, Lowe LA, McGrath J et al. Targeted deletion of the ATP binding domain of left-right dynein confirms its role in specifying development of left-right asymmetries. Development 1999;126:5495–504. Takeda S, Yonekawa Y, Tanaka Y, Okada Y, Nonaka S, Hirokawa N. Left-right asymmetry and kinesin superfamily protein KIF3A: new insights in determination of laterality and mesoderm induction by kif3A-/- mice analysis. J Cell Biol 1999;145:825–36. Watanabe D, Saijoh Y, Nonaka S, Sasaki G, Ikawa Y, Yokoyama T et al. The left-right determinant Inversin is a component of node monocilia and other 9+0 cilia. Development 2003;130:1725–34. Yasuhiko Y, Imai F, Ookubo K, Takakuwa Y, Shiokawa K, Yokoyama T. Calmodulin binds to inv protein: implication for the regulation of inv function. Dev Growth Differ 2001;43:671–81. Yokoyama T, Copeland NG, Jenkins NA, Montgomery CA, Elder FF, Overbeek PA. Reversal of left-right asymmetry: a situs inversus mutation. Science 1993;260:679–82. Yokoyama T, Harrison WR, Elder FFB, Overbeek PA. Molecular analysis of the inv insertional mutation. In: Clark EB, Markwald RR, Takao A, editors. Developmental mechanisms of heart disease. New York: Futura Publishing Company, 1995;513–20. Zachariae W, Nasmyth K. Whose end is destruction: cell division and the anaphase-promoting complex. Genes Dev 1999;13:2039–58.
Cell Biology International www.elsevier.com/locate/cellbi
Review
A perspective on inversin Lorraine Eley a, Lee Turnpenny b, Laura M. Yates a, A. Scott Craighead a, David Morgan a, Catherine Whistler a, Judith A. Goodship a*, Tom Strachan a,* a
Institute of Human Genetics, University of Newcastle, International Centre for Life, Central Parkway, Newcastle upon Tyne NE1 3BZ, UK b Division of Human Genetics, University of Southampton, Duthie Building, Southampton General Hospital, Tremona Road, Southampton SO16 6YD, UK Received 2 October 2003; accepted 4 November 2003
Abstract Over the past 5 years, there has been increasing evidence for the role of primary (9+0) cilia in renal physiology and in establishing the left–right axis. The cilia in the renal tract are immotile and thought to have a sensory function. Cilia at the murine embryonic node have a vortical movement that sets up a leftward flow. Inversin, the protein defective in the inv mouse and in patients with type-2 nephronophthisis, localizes to both renal and node primary cilia. However, we present evidence that it is also expressed before the node forms and that its subcellular localization in renal tubular cells is not confined to the cilia. Its role in both the pathway determining left–right axis and renal function remains to be elucidated. 2004 Elsevier Ltd. All rights reserved. Keywords: Inversin; Situs inversus; Primary cilia; Nephronopthisis
1. Background The inv (inversion of embryo turning) mouse is an insertional mutant with a complex phenotype: homozygotes exhibit consistent situs abnormalities (unlike other mouse models of laterality defects where situs is randomized), and also severe cystic changes of the kidney and pancreas (Yokoyama et al., 1993). The phenotype resulted from insertion of a tyrosinase minigene, the integration event being accompanied by deletion of a small segment of chromosome 4 and duplication of a short region which in the wild type chromosome is located about 6 cm distal to the region deleted in inv (Yokoyama et al., 1993, 1995). We and others used positional cloning approaches to identify a novel gene which lacks nine internal exons as a result of the deletion event in the inv mouse and whose coding sequence is sufficient to rescue the inv phenotype (Mochizuki et al., 1998; Morgan et al., 1998). The novel
* Corresponding authors. Tel.: +44-191-241-8616; fax: +44-191-2418699 E-mail addresses: [email protected] (J.A. Goodship), [email protected] (T. Strachan). 1065-6995/04/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2003.11.009
gene (official symbol: Invs) was predicted to encode a 1062 amino acid protein which we named inversin. 2. Structural features of Invs and inversin The Invs gene spans over 150 kb and is organized into 17 exons. Exons 2 through to 17 specify the coding sequence but since the inv mutation results in deletion of exons 4 through to 12, a profound effect on gene expression can be expected, with at a minimum the loss of the coding potential for the region spanning amino acids 92–642. Evolutionary analyses have shown that almost all of this region, and indeed the N-terminal half of inversin, is very highly conserved (Morgan et al., 2002b), and its biological importance has been underscored in phenotype rescue experiments (Watanabe et al., 2003). The C-terminal half, by contrast, is comparatively poorly conserved. Within the conserved N-terminal half are 16 tandem ankyrin repeats spanning amino acids 13–557 followed by a central lysine-rich domain spanning amino acids 558–604 (Fig. 1). The predicted protein lacks an N-terminal signal peptide or transmembrane domain but three nuclear localization signals are predicted: a standard signal in
120
L. Eley et al. / Cell Biology International 28 (2004) 119–124
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' Fig. 1. Structural features of mouse inversin. The pale blue shading refers to the generally very highly conserved N-terminal half of inversin and encompasses a tandem series of ankyrin repeats (circles labelled as A, 13–557 aa) followed directly by a lysine-rich domain (558–604 aa). The yellow shading denotes the generally poorly conserved C-terminal half. Other abbreviations are: D—D-box (490–498 and 907–915 aa); IQ—IQ domain (562–575 and 921–934 aa); NLS—standard nuclear localization sequence (735–738 aa); b-NLS—bipartite nuclear localization sequence (589–604 and 782–798 aa). Red bars at bottom indicate the regions that are deleted in the S1 and S2 isoforms with loss respectively of 120 amino acids (731–850 aa) or 165 amino acids (731–895 aa).
HG
HG
HG
When we cloned Invs we obtained evidence from cDNA library screening and RT-PCR experiments for the existence of three inversin isoforms in exon 14 (Morgan et al., 1998). In addition to a full-length isoform (F), specifying a 1062 amino acid protein, two shorter forms had in frame deletions that would be expected to produce proteins of 942 (S1 isoform) and 897 (S2 isoform) amino acids (Fig. 1). The biological importance is underscored by evolutionary conservation. Thus although exon 14 of the human inversin gene specifies five more amino acids than the mouse exon 14, several human cDNA clones show a sequence corresponding to the S2 isoform and with precise evolutionary conservation of the alternative splice sites used to generate the mouse S2 isoform. We have further validated the existence of the exon 14 isoforms by using RT–PCR and primers designed to prime from exons flanking mouse exon 14 (Fig. 2). During development there is a predominance of F (764 bp) and S2 (269 bp) transcripts. The minority S1 isoform cannot be detected at very early stages but appears to be produced by the 7.5 embryonic day (ed) stage. The subcellular fractionation data from Phillips’ group also identifies three inversin isoforms of 140, 125 and 90 kD (Nu¨rnberger et al.,
HG
3. Inversin isoforms
(6 FHOOV
the C-terminal region and two bipartite signals, one located in the lysine-rich central domain and the other in the C-terminal region (Fig. 1). Two other types of conserved motifs have been identified. A calmodulinbinding IQ domain is represented at two locations: at the beginning of the lysine-rich central domain and within the C-terminal region. In addition there are two copies of the destruction box (D-box) motif, which is found in most targets of the anaphasepromoting complex (APC), where it is important for ubiquitination-mediated destruction of the target protein.
) ES 6 ES 6 ES Fig. 2. RT–PCR-detected expression of inversin isoforms. Primers designed across the region of exon 14 demonstrated to be alternatively spliced shows the existence of 3 inversin isoforms in mouse embryos from embryonic day (ed) 6.5 to 10 and ES cells. The transcripts 764, 404 and 269 bp represent F, S1 and S2 isoforms respectively.
2002). These may possibly correspond to the above isoforms—which are predicted to be 118 kD (F), 104 kD (S1) and 99 kD (S2) respectively. Differences in sizes may be due to post-translational modification.
4. Expression and subcellular localization of inversin It has been proposed that the node is involved in the initial event that breaks left–right asymmetry (Hamada et al., 2002). However, there is evidence that gap junctions/ion channels pre node may also play a role (Levin and Mercola, 1999; Levin et al., 2002). Inversin plays a pivotal role in the breaking of left–right symmetry since all inv mice have situs inversus. Thus determining its function is of great importance. Here we show that inversin RNA is detectable as early as the two cell stage by RT–PCR (Fig. 3). Subcellular localization has been studied in cell lines derived from wild type mice and in an inv:GFP mouse (Morgan et al., 2002a; Phillips et al., 2001; Watanabe et al., 2003). In the Inv:GFP mouse inversin is present in 9+0 cilia of the node, renal tubules, pituitary gland,
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5. Proteins interacting with inversin
ES
Fig. 3. RT–PCR-detected expression of inversin in earlier-stage embryos. RT–PCR was used to demonstrate the expression of inversin in 2-cell, 4-cell and blastocyst stage mouse embryos. The primer pair used was designed to exon 7 (forward) and exon 9 (reverse); separated at the genomic level by two introns, thus verifying products as mRNA transcript-specific.
retina and primary fibroblast cultures. Inversin was not present in 9+2 cilia of the trachea, oviducts or ependyma (Watanabe et al., 2003). The primary cilium arises from one of the two centrioles, always the mother centriole. In the inv:GFP mouse signal is seen in the mother centriole before the extension of the cilium. Watanabe and colleagues studied expression at the embryonic node. In the figures that they show all cilia, visualized using an acetylated tubulin antibody, stain for inversin. In their figure only the periphery of the node is in the plane of focus. McGrath and colleagues have recently suggested that there are two populations of cilia at the node, motile cila in the centre of the node and immotile cilia around the periphery (McGrath et al., 2003). It is therefore important to address whether inversin is present in all cilia across the node. Inversin localization has been studied in MDCK II, mIMCD-3 and S1 cultures. The S1 line was derived from the early segment of the murine renal proximal tubule. In subconfluent S1 cultures inversin localizes in the nuclei with additional weak perinuclear and membrane staining. When S1 cells are grown to confluence inversin localizes to the membranes between the cells (Fig. 4; Nu¨rnberger et al., 2002). MDCK-II and mIMCD-3 were derived from canine collecting duct and murine inner medullary collecting duct respectively. We found that inversin localization changed through the cell cycle. In confluent, ciliated MDCK-II and mIMCD-3 cells inversin is seen in the cilia. Like Nu¨rnberger and colleagues we also observed generalized nuclear staining. In prophase we observed staining at the centrosomes, at metaphase and anaphase we observed staining at the spindle poles. In cells at late telophase we have observed localization at the midbody, a region of microtubule overlap (Fig. 4; Morgan et al., 2002a).
Several proteins of varying function and subcellular localization have been identified as interacting directly or in a multiprotein complex with inversin (Table 1). Calmodulin and Apc2 are two proteins within the former category that bind specifically to corresponding inversin motifs. Although calmodulin binding has been demonstrated to occur at both IQ domains (Morgan et al., 2002b; Yasuhiko et al., 2001), Yasuhiko et al identified IQ2 as being critical for inversin to randomize left–right asymmetry in Xenopus. Calmodulin is ubiquitously distributed within cells and functions as a major calcium sensor that regulates numerous target proteins in either a Ca2+-independent or Ca2+-dependent manner (Rhoads and Friedberg, 1997). As expected of proteins containing IQ motifs, calmodulin binding to IQ2 appears to occur only in the absence of calcium (Yasuhiko et al., 2001). Of note, calmodulin is present in the 9+0 cilia of mIMCD-3 cells and shows partial colocalization with inversin (Fig. 5). As mentioned previously, inversin contains two destruction box (D-box) motifs and shows dynamic expression in mitotic cells. A conserved destruction box (D-box) is important for the ubiquitination-mediated destruction of most anaphase promoting complex (APC; also called cyclosome) targets. Yeast two-hybrid identification of Apc2 as an interacting partner further supports a role for inversin in the cell cycle (Morgan et al., 2002a). Apc2 is one of at least ten subunits of the vertebrate APC, which is responsible for the ubiquitination and subsequent destruction of cell regulators at the metaphase–anaphase and mitosis–G1 transitions (Glotzer et al., 1991; Zachariae and Nasmyth, 1999). Of the two D-boxes, the N-terminal D-box (no. 1) is extremely highly conserved and site directed mutation of D-box no. 1 abrogated the inversin-Apc2 interaction, whereas the same changes to D-box no. 2 did not prevent Apc2 binding (Morgan et al., 2002a). The recent identification of inversin as the gene mutated in nephronopthisis (NPHP) type 2, prompted a search for an interaction between inversin and nephrocystin (Otto et al., 2003). NHPH2 is an autosomal recessive cystic kidney disease in which a subset of patients demonstrates situs abnormalities. Nephrocystin, the product of the gene mutated in NPHP type 1, is a novel docking protein that interacts with components of cell–cell and cell–matrix signalling, and nephrocystin4. Although no direct interaction between inversin and nephrocystin has been demonstrated, both proteins coprecipitate and colocalize to the primary cilia of renal cells (Otto et al., 2003). Localization to the lateral cell membrane suggests a further role for inversin in cell–cell signalling (Nu¨rnberger et al., 2002). Phillips et al demonstrated that 90- and 125-kDa inversin isoforms coprecipitate
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(a)
(b)
Fig. 4. Subcellular localization of inversin. (a) Inversin expression at the cell membranes and nucleus. Confluent S1 cells were double-labelled with antibodies to inversin and analyzed by confocal microscopy as described previously (Nu¨rnberger et al., 2002). Inversin stains the cell membranes, nuclei and perinuclear compartment. (b) Dynamic expression of inversin in cultured kidney cells. Immunofluorescence staining of MDCK-II cells with rabbit polyclonal antiinversin antibody (green) shows a dynamic expression throughout the cell cycle. Cells in early prophase (A) show inversin expression at the centrosomes whereas in metaphase (B) and anaphase (C) cells inversin localizes to the spindle poles. In cells undergoing cytokinesis (D) expression was observed at the midbody, a region of microtubule overlap. Throughout these stages general nuclear expression is also observed, which becomes ubiquitous upon breakdown of the nuclear envelope. The plasma membranes were counterstained with TRITC-wheat germ agglutinin (red) (Morgan et al., 2002a). Reproduced with permission from Oxford University Press.
with -catenin and N-cadherin in the membrane but not the cytosolic fraction of confluent cultured kidney cells. Of these, the 125-kDa band was the most enriched in the cell membrane fractions. These proteins also colocalize at the plasma membrane where it is postulated they form a complex involved in regulating the architecture of cell–cell junctions in a calcium dependent manner (Nu¨rnberger et al., 2002).
6. Inversin and the event(s) that break left–right asymmetry It is postulated that primary (9+0) cilia at the node are involved in generating the asymmetrical expression patterns at the node and hence determining the left–right axis (Hamada et al., 2002). Several models showing
randomization of the left–right axis lack nodal cilia (Brody et al., 2000; Marszalek et al., 1999; Murcia et al., 2000; Takeda et al., 1999), or in the case of the iv mouse (Supp et al., 1999), have immotile cilia. More recent work by Hirokawa’s group led to a major change in thinking about the node (Nonaka et al., 1998). Studying normal mice they found that nodal cilia rotated in a clockwise direction and they demonstrated a leftward flow of perinodal fluid. The nodal flow hypothesis proposed that rotation of nodal cilia generated the leftwards flow of perinodal fluid, and this was supported by studies where mouse embryos were cultured under an artificial fluid flow, where left–right patterning was shown to be reversed by reversing the fluid flow. Recent evidence shows that there are two types of cilia at the node, motile and immotile. It is proposed that motile cilia generate the leftward flow and the immotile cilia
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Fig. 5. Inversin and calmodulin show partial colocalization to the primary cilia of kidney cells. Confocal microscopy of paraformaldehyde fixed confluent mIMCD-3 cells double-stained with mouse monoclonal anti-calmodulin (Upstate biotechnology) (A and D) and rabbit polyclonal anti-inversin (Morgan et al., 2002a) (B and E) show that both proteins localize to the primary cilia. Composite images (C and F) demonstrate a partial overlap (yellow) in expression. The scale bar represents 5 µm.
Table 1 Interacting partners of inversin and supporting evidence Supporting evidence Interacting protein
Y2H
Gel overlay
Co-IP Colocalize
Calmodulin Apc2 Nephrocystin -catenin N-cadherin
U U – – –
U – – – –
U U U U U
cilia – cilia cell junction cell junction
sense the flow, thus breaking bilateral symmetry and polarizing the L–R axis (McGrath et al., 2003). However, in the inv mouse the vast majority of the mice have situs inversus and yet the perinodal fluid moves in the same leftwards direction as in wild type mice, albeit more slowly and showing more turbulence (Okada et al., 1999). It is difficult to reconcile this observation with the principles of the nodal flow model. One possibility is that inversin is important in the sensing cilia, hence the importance of clarifying whether inversin is expressed in all nodal cilia or only peripheral nodal cilia. However,
it may be that inversin is implicated in asymmetry preceding nodal flow. Levin and Mercola have presented data for gap junction-mediated transfer of left–right patterning signals in the chick blastoderm preceding asymmetry at the node (Levin and Mercola, 1999). Levin and co-workers have subsequently shown that there is an endogenous H+/K+-ATPase-dependent difference in membrane potential between the left and right side of the primitive streak in chick preceding node asymmetry and that in Xenopus there is asymmetric expression of the H+/K+ATPase 2 h post fertilization (Levin et al., 2002). They postulate that this H+/K+-ATPase asymmetry could generate unidirectional flow through the gap junctions. In chick and Xenopus, therefore, left–right differences appear to exist before the signalling cascade is set up from the node but it is not known if this is the case in mice or humans. The shape of the node and the distribution and length of the cilia differs between mouse, chick, Xenopus and zebrafish, and it is not clear at this stage how well the pathways are conserved between different model organisms (Essner et al., 2002).
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Inversin could potentially be involved in the above mentioned gap junction/ion flux mechanisms. In support of such a role (a) it has been previously shown that inversin localized to the cell membrane (b) inv mice exhibit polycystic kidney disease, a phenotype associated with reversals in electric polarity and ion pump localization (c) overexpression of inv on the right side of Xenopus embryos randomizes the left–right axis, while overexpression on the left side does not (Yasuhiko et al., 2001). Here we have shown that inversin transcripts are expressed as early as the two-cell stage thus supporting such a role for inversin. Also inversin expression in the node cilia has been shown (Watanabe et al., 2003). Thus it is still not clear whether situs inversus in the inv mouse results from perturbation at the embryo node or an earlier stage or both.
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