A C. elegans CLIC-like Protein Required for Intracellular Tube Formation and Maintenance

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Science  19 Dec 2003:
Vol. 302, Issue 5653, pp. 2134-2137
DOI: 10.1126/science.1087667


The Caenorhabditis elegans excretory canal is composed of a single elongated and branched cell that is tunneled by an inner lumen of apical character. Loss of the exc-4 gene causes a cystic enlargement of this intracellular tube. exc-4 encodes a member of the chloride intracellular channel (CLIC) family of proteins. EXC-4 protein localizes to various tubular membranes in distinct cell types, including the lumenal membrane of the excretory tubes. A conserved 55–amino acid domain enables EXC-4 translocation from the cytosol to the lumenal membrane. The tubular architecture of this membrane requires EXC-4 for both its formation and maintenance.

The morphogenesis of biological tubes is central to the development of a wide variety of metazoan structures, from the simplest Cnidarian body plans to the vertebrate respiratory, excretory, and circulatory systems. Although biological tubes form by such distinct processes as the hollowing of single cells and the folding of epithelial sheets, in each case an inner lumen is surrounded by a surface of apical character generated by the polarized movement or growth of vesicles or vacuoles (1, 2). However, the molecular mechanisms of tube formation and subsequent maintenance are not well understood.

A simple model of unicellular tube morphogenesis is provided by the Caenorhabditis elegans excretory cell, a single cell that forms the major tubular component of the four-cell nematode excretory system (3, 4). This cell extends branched processes, termed canals, along the length of the body on the basolateral surface of the epidermis (Fig. 1A). These processes are tunneled by an inner lumen that is closed at its four endings and is presumed to collect fluids and waste, which it then empties into the excretory duct (5). In exc (for excretory canal abnormal) mutants, the tubular structure of the excretory cell lumen is disrupted by swellings termed cysts that have been proposed to model tubulocystic kidney disease (6). The disruption of tubular morphology in exc-4 mutants has been previously described as a cystic enlargement of the canal's interior lumen (6). Using green fluorescent protein (GFP) markers that specifically label either the cytoplasm or the apical surface of the canal, we confirmed that in the exc-4 null allele rh133, the morphology of the apical surface is altered from a single, long, narrow tube to a set of large, closely packed cystic enlargements, some of which may be disconnected spheres (Fig. 1B) (7).

Fig. 1.

The intracellular tube of the excretory cell is disrupted upon loss of EXC-4, a CLIC-type putative chloride channel. (A) Schematic depiction of the excretory system (4). (B) The apical surface of the excretory lumen, visualized in wild-type and exc-4 null mutant animals with an apically localized, nonfunctional EXC-4(R202Stop)::GFP reporter construct. (C) Predicted secondary structure of EXC-4. Structural elements are conserved in the experimentally determined human CLIC1 structure (7). Location of mutant alleles and points of fusion with GFP used to analyze EXC-4 localization are indicated. (D) Dendrogram of CLIC sequences from human (black), Drosophila (blue), and C. elegans (red). GSTΩ sequences were included as outliers because of their distant similarity to CLIC channel protein (8). Genbank accession numbers: EXC-4, AAQ75554; GST-44, T20806; EXL-1, T21429.

We used single-nucleotide polymorphisms to map the exc-4 locus to a 130-kb interval on chromosome I and found that a polymerase chain reaction product encompassing the coding region of a predicted intracellular ion channel protein fully rescued the mutant phenotype (7). Molecular lesions in this putative channel protein are present in all three exc-4 mutant alleles— rh133, n561, and n2400 (Fig. 1C)—which appear similar in the severity of the excretory phenotype (94 to 100% of animals were defective; n > 30). The rh133 allele has an amber mutation in its sixth residue and therefore presumably eliminates all protein function.

The protein encoded by the exc-4 locus is one of two C. elegans orthologs of the human CLIC family of proteins (Fig. 1D). Members of this family are small proteins that have the unconventional property of translocating from a globular cytosolic form (8) to an integral membrane form with chloride channel activity (9, 10). Although it cannot formally be excluded that CLIC proteins are channel-associated rather than pore-forming proteins, the observations that they confer chloride conductance to transfected cells (8, 1116) and that they are alone sufficient to self-insert into microsomes to form chloride-conducting ion channels (1719) indicate that CLIC proteins are important integral components of a chloride channel. In cell and tissue culture systems, different CLIC family members localize to distinct sets of intracellular membranes, including the endoplasmic reticulum, Golgi apparatus, nuclear membrane, large dense core vesicles, secretory vesicles, and sometimes the plasma membrane, and they are expressed in many different tissue types (9, 20). An understanding of the physiological role of CLICs has been lacking because of the previous absence of animal models. Such a model is now provided by exc-4 mutant animals.

We examined the expression pattern and subcellular localization of exc-4 with fluorescent reporter gene technology (7). The gfp-tagged genomic exc-4 locus, which is capable of rescuing the exc-4 mutant phenotype (7), shows expression in the excretory system, hypodermis, vulva, pharyngeal muscle, rectal gland, tubular rectal epithelial cells, and tubular neuronal support cells in the head and tail (Fig. 2) (21). EXC-4 protein showed highly specific patterns of subcellular localization. In the excretory system, EXC-4 localizes to the lumenal membrane of the excretory cell, the duct, and the pore cell (Fig. 2A). Apical lumenal localization is also observed in the tube-forming cells generating the anus (7). In the lateral seam cells of the hypodermis, which contain membranes that extensively grow and eventually fuse to form a cylindrically shaped syncytium along each side of the animal, EXC-4 localizes directly adjacent to the adherens junction marker AJM-1 (22) to a compact subapical belt of plasma membrane called the apical junction (Fig. 2B). In phasmid and labial sheath cells, EXC-4 selectively localizes to the extreme tip of the sheath cell membranes, which enwrap the dendritic ending of sensory neurons and fuse with themselves to form an elongated tubular structure exposed to the sensory environment (Fig. 2C). Taken together, EXC-4 localizes to very distinct membranes that undergo substantial remodeling (growth and fusion) in order to create diverse, often tubular membrane structures.

Fig. 2.

Expression and localization of EXC-4 to membrane domains in different cell types. (A) Red fluorescent protein (RFP)–tagged EXC-4 protein localizes to the apical membrane of the excretory canal lumen. The cytoplasm is labeled with the GFP reporter transgene bgIs312. (B) RFP-tagged wild-type EXC-4 protein localizes to the apical junction of the seam cells; localization is directly adjacent to AJM-1::GFP. (C) EXC-4::RFP localizes to the tip (arrow) of sensory neuron sheath cells in the tail. The cytoplasm is labeled with a GFP expressed under control of the exc-4 promoter. SH, sheath cells. Neurons are labeled in blue. A similar localization of EXC-4::RFP can be observed in labial sheath cells in the head (21).

We found that the only other CLIC family member predicted to exist in the C. elegans genome (Fig. 1D), which we term EXL-1 (for EXC-4–like), also localizes to intracellular membranes (7). Mirroring the differential localization of individual vertebrate CLIC proteins to distinct intracellular membranes, we observed that EXL-1::GFP localizes to lysosomal membranes in the intestine and to the Golgi apparatus in neurons and muscle (7). Thus, distinct sets of tissues express EXC-4 and EXL-1.

Glutathione S-transferase (GST) proteins of the Ω subclass have a three-dimensional (3D) fold that is similar to the globular form of CLIC proteins (8). We observed that GFP-tagging of the sole C. elegans member of the GSTΩ class (Fig. 1D), which we term GST-44, reveals a strictly cytoplasmic localization in the excretory cell and neuronal support cells (7), the same cells in which EXC-4 localizes to specific membrane compartments. Membrane localization is thus a specific property of the CLIC family rather than a generic property of the GSTΩ-type fold.

Whereas the 3D structure of the soluble form of human CLIC1 has been experimentally determined (8), the transmembrane topology of CLIC proteins is unclear, as is the nature and regulation of the conformational change between their soluble and integral membrane conformations. On the basis of the structural homology of a C-terminal helix bundle to bacterial poreforming toxins, α helix 6 has been proposed to insert into the membrane (23). Rendering this possibility unlikely, we found that a truncation of EXC-4 in the middle of the α helix 6 does not affect localization of EXC-4 to the lumenal membrane in wild-type or in exc-4 mutant backgrounds (#3 in Fig. 3). Further narrowing the membrane localization domain, we found that the first 55 amino acids of EXC-4 confer lumenal membrane localization to the attached GFP reporter protein, both in wild-type and exc-4 mutant backgrounds (#4 in Fig. 3). Deletion of β sheet 2 from this construct resulted in patchy membrane localization and a strong cytosolic partition (#5 in Fig. 3). Moreover, if the highly conserved leucine 46 in the middle of α helix 1 is mutated to a potentially helix-breaking proline in the context of a full-length protein, membrane localization is disrupted (#6 in Fig. 3). In summary, a 55–amino acid domain containing conserved secondary structure elements is both necessary and sufficient for translocation of a CLIC-type protein from the cytosol to the membrane.

Fig. 3.

Determinants of apical membrane localization of the EXC-4 protein. (A) GFP reporter fusion constructs (7). α helix 1 (orange) and β sheets 2 (blue) are indicated within the exons (boxes). (B) EXC-4::GFP reporter fusion localization in transgenic animals. n, excretory cell nucleus; l, intralumenal space; L, left; P, posterior; R, right; A, anterior. Dashed lines show approximate location of the excretory cell perimeter. For all mutant proteins, localization was assessed both in wild-type and in exc-4(rh133) mutant backgrounds to eliminate a potential contribution of wild-type channel proteins to the localization of the GFP construct. Expression levels of most of the mutant exc-4::gfp reporters are down-regulated compared with wild-type protein.

To elucidate the timing of exc-4 function, we sought to rescue the exc phenotype at specific stages during development of the excretory canal by using a heat-inducible promoter to drive expression of exc-4 (7). To this end, we first characterized the initial stages of excretory lumen formation in wild-type animals through the reconstruction of electron micrographs of serial-sectioned embryos of different stages. We found that lumen formation is preceded by the pinocytotic generation of a large vacuole, which extends tubular arms that eventually collapse and remodel to form a mature lumen (7) (Fig. 4A). Heat-shock induction of exc-4 expression in an exc-4 mutant background before or during the time at which the developing vacuoles extend tubular arms is able to fully rescue the exc phenotype (Fig. 4B). In contrast, heat-induced expression during or after the time of tubule flattening is not able to fully rescue the exc phenotype (Fig. 4B). The timing of exc-4 function as well as the successful articulation of canaliculi around cysts in exc-4 mutants indicates that exc-4 functions in early lumen formation rather than in late lumen remodeling. The existence of lumen in exc-4 mutants, albeit of aberrant morphology and topology (7), indicates that the initial process of vacuole formation is probably not disrupted. We conclude that exc-4 acts during the stage of extension of tubular arms from the initial vacuole(s).

Fig. 4.

Timing of exc-4 function. (A) Schematic representation of excretory cell and canal development as determined from electron micrographs at different developmental stages (fig. S4). Time below each panel indicates time post-fertilization. (B) Heat shock–induced rescue of exc-4 cystic phenotype by exc-4 cDNA. The horizontal axis represents the time of heat-shock initiation, and the vertical axis represents percentage of rescued animals. n = 22 to 107. (C) Inverse correlation of fraction of animals that show exc-4::gfp expression in the L1 and L4 stages (white bars) with fraction of animals with cystic phenotype (black bars) (23). n = 22. Error bars in (B) and (C) show standard error of proportion.

Because exc-4 is continuously expressed throughout the life of an animal, we tested whether exc-4 also has a role in postdevelopmental maintenance of tube structure. We provided exc-4 mutant animals with a very short pulse of GFP-tagged EXC-4 during mid-embryonic development (a 10-min heat shock at the 400-min stage), such that EXC-4::GFP rescued the developmental defects but started to subside postembryonically. Transgenic animals that express EXC-4::GFP at the first larval stage (L1) but not the fourth larval stage (L4) show virtually no cysts at the L1 stage but reveal a cystic phenotype by the L4 stage (Fig. 4C) (24). We conclude that EXC-4 not only functions during tube formation but is continuously required to maintain tubular architecture after the termination of excretory canal development and outgrowth.

We can envision several mechanisms by which EXC-4 enables tube formation as a putative chloride channel protein. Intracellular membrane growth, and hence outgrowth of the tubular arms, is likely achieved through the directed fusion of vesicles, which may require acidification of the fusing compartments (2, 25). Acidification necessitates the existence of an electric shunt, potentially provided through a CLIC-type channel such as EXC-4, to dissipate the electrical potential generated by proton pumps. Alternatively, given that chloride channels regulate water transport across membrane and cell swelling (20), EXC-4 may be required to precisely control the correct diameter of the growing tubule and/or the thinning of the vacuole that accompanies the outgrowth of the tubes from the vacuole. Loss of exc-4 may cause parts of the vacuole or the developing lumen to collapse or overswell, resulting in a rupture of the structure and the production of cysts. Finally, because mature tubes may recycle through the addition of fresh membranes from vesicle pools and are also likely to regulate their swelling state, EXC-4 may have a similar function in the development and maintenance of tubular architecture. Because, for example, the development of unicellular capillaries from individual vertebrate endothelial cells also occurs through pinocytotic vacuole formation, extension, and remodeling into a tubular structure (26), the role of EXC-4 in tubular development may reflect an evolutionarily conserved mechanism for shaping a vacuolar lumen into tubular form.

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Materials and Methods

Figs. S1 to S5


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