Matthew Buechner

Tubes and the single C. elegans excretory cell

The formation and regulation of tubule shape and size is fundamental to the development and function of many tissues and organs in metazoan organisms. The excretory canals of the nematode Caenorhabditis elegans are a fascinating example of cell morphogenesis, as the tiny worm manages to create a complicated set of tubular epithelia within a single cell. In addition to the inherent attraction of studying this cytoengineering feat, the excretory cell provides a simple genetically tractable model for studying tubule formation and regulation of tubule diameter. Mutations in the exc genes alter the diameter of the lumenal surface of these tubules. Cloning of these genes reveals a set of proteins that both control tubule diameter and regulate the comparative growth of the apical and basal tubular surfaces.

Intracellular lumen extension requires ERM-1-dependent apical membrane expansion and AQP-8-mediated flux.

Many unicellular tubes such as capillaries form lumens intracellularly, a process that is not well understood. Here we show that the cortical membrane organizer ERM-1 is required to expand the intracellular apical/lumenal membrane and its actin undercoat during single-cell Caenorhabditis elegans excretory canal morphogenesis. We characterize AQP-8, identified in an ERM-1-overexpression (ERM-1[++]) suppressor screen, as a canalicular aquaporin that interacts with ERM-1 in lumen extension in a mercury-sensitive manner, implicating water-channel activity. AQP-8 is transiently recruited to the lumen by ERM-1, co-localizing in peri-lumenal cuffs interspaced along expanding canals. An ERM-1[++]-mediated increase in the number of lumen-associated canaliculi is reversed by AQP-8 depletion. We propose that the ERM-1/AQP-8 interaction propels lumen extension by translumenal flux, suggesting a direct morphogenetic effect of water-channel-regulated fluid pressure.

A putative GDP–GTP exchange factor is required for development of the excretory cell in Caenorhabditis elegans

The Caenorhabditis elegans excretory cell extends tubular processes, called canals, along the basolateral surface of the epidermis. Mutations in the exc‐5 gene cause tubulocystic defects in this canal. Ultrastructural analysis suggests that exc‐5 is required for the proper placement of cytoskeletal elements at the apical epithelial surface. exc‐5 encodes a protein homologous to guanine nucleotide exchange factors and contains motif architecture similar to that of FGD1, which is responsible for faciogenital dysplasia. exc‐5 interacts genetically with mig‐2, which encodes Rho GTPase. These results suggest that EXC‐5 controls the structural organization of the excretory canal by regulating Rho family GTPase activities.

The role of the ELAV homologue EXC-7 in the development of the Caenorhabditis elegans excretory canals.

The exc mutations of Caenorhabditis elegans alter the position and shape of the apical cytoskeleton in polarized epithelial cells. Mutants in exc-7 form small cysts throughout the tubular excretory canals that regulate organismal osmolarity. We have cloned the exc-7 gene, the closest nematode homologue to the neural RNA-binding protein ELAV. EXC-7 is expressed in the canal for a short time midway through embryogenesis. Cysts in exc-7 mutants do not develop until several hours later, beginning at the time of hatching. We find that the first larval period is when the canal completes the majority of its outgrowth, and adds new apical cytoskeleton at a rapid rate. Ultrastructural studies show that exc-7 mutant defects resemble loss of beta(H)-spectrin (encoded by sma-1) at the distal ends of the excretory canals. In addition, exc-7 mutants exhibit synergistic excretory canal defects with mutations in sma-1, and EXC-7 binds sma-1 mRNA. These data imply that EXC-7 protein may affect expression of sma-1 and other genes to effect proper development of the excretory canals.

Towards understanding the polycystins

Autosomal dominant polycystic kidney disease (ADPKD) is a very common inherited disease caused by mutations in PKD1 or PKD2 genes characterized by progressive enlargement of fluid-filled cysts and loss of renal function [1]. Previous studies proposed a role for human polycystin-1 in renal morphogenesis acting as a matrix receptor in focal adhesions and for polycystin-2 as a putative calcium channel [2, 3]. The genome of Caenorhabditis elegans contains 2 new members of the polycystin family: lov-1, the homolog for PKD1; and pkd-2, the homolog for PKD2 [4; this paper]. Mutation analysis in C. elegans showed similarly compromised male mating behaviors in all single and double lov-1 and pkd-2 mutants, indicating their participation in a single genetic pathway. Expression analysis localized LOV-1 and PKD-2 to the ends of sensory neurons in male tails and to the tips of CEM neurons in the head, consistent with functions as chemo- or mechanosensors. Human and C. elegans PKD1 and PKD2 homologs, transfected into mammalian renal epithelial cells, co-localized with paxillin in focal adhesions suggesting function in a single biological pathway. Based on the role of polycystins in C. elegans sensory neuron function and the conservation of PKD pathways we suggest that polycystins act as sensors of the extracellular environment, initiating, via focal adhesion assembly, intracellular transduction events in neuronal or morphogenetic processes.

The C. elegans EMAP-like protein, ELP-1 is required for touch sensation and associates with microtubules and adhesion complexes

BackgroundThe founding member of the EMAP-like protein family is the Echinoderm Microtubule-Associated Protein (EMAP), so-named for its abundance in sea urchin, starfish, and sand dollar eggs. The EMAP-like protein family has five members in mammals (EML1 through EML5) and only one in both Drosophila (ELP-1) and C. elegans (ELP-1). Biochemical studies of sea urchin EMAP and vertebrate EMLs implicate these proteins in the regulation of microtubule stability. So far, however, the physiological function of this protein family remains unknown.ResultsWe examined the expression pattern of C. elegans ELP-1 by means of transgenic gene expression in living embryos and adults, and by immunolocalization with an ELP-1-specific antibody in fixed tissues. In embryos, ELP-1 is expressed in the hypodermis. In larvae and adults, ELP-1 is expressed in the body wall, spermatheca and vulval muscles, intestine, and hypodermal seam cells. In muscle, ELP-1 is associated with adhesion complexes near the cell surface and is bound to a criss-crossing network of microtubules in the cytoplasm. ELP-1 is also expressed in a subset of mechanoreceptor neurons, including the ray neurons in the male tail, microtubule-rich touch receptor neurons, and the six ciliated IL1 neurons. This restricted localization in the nervous system implies that ELP-1 plays a role in mechanotransmission. Consistent with this idea, decreasing ELP-1 expression decreases sensitivity to gentle touch applied to the body wall.ConclusionThese data imply that ELP-1 may play an important role during the transmission of forces and signals between the body surface and both muscle cells and touch receptor neurons.

The FGD homologue EXC-5 regulates apical trafficking in C. elegans tubules.

Maintenance of the shape of biological tubules is critical for development and physiology of metazoan organisms. Loss of function of the Caenorhabditis elegans FGD protein EXC-5 allows large fluid-filled cysts to form in the lumen of the single-cell excretory canal tubules, while overexpression of exc-5 causes defects at the tubules basolateral surface. We have examined the effects of altering expression levels of exc-5 on the distribution of fluorescently-marked subcellular organelles. In exc-5 mutants, early endosomes build up in the cell, especially in areas close to cysts, while recycling endosomes are depleted. Endosome morphology changes prior to cyst formation. Conversely, when exc-5 is overexpressed, recycling endosomes are enriched. Since FGD proteins activate the small GTPases CDC42 and Rac, these results support the hypothesis that EXC-5 acts through small GTPases to move material from apical early endosomes to recycling endosomes, and that loss of such movement is likely the cause of tubule deformation both in nematodes and in tissues affected by FGD dysfunction such as Charcot-Marie-Tooth Syndrome type 4H.

The Caenorhabditis elegans Excretory System: A Model for Tubulogenesis, Cell Fate Specification, and Plasticity.

The excretory system of the nematode Caenorhabditis elegans is a superb model of tubular organogenesis involving a minimum of cells. The system consists of just three unicellular tubes (canal, duct, and pore), a secretory gland, and two associated neurons. Just as in more complex organs, cells of the excretory system must first adopt specific identities and then coordinate diverse processes to form tubes of appropriate topology, shape, connectivity, and physiological function. The unicellular topology of excretory tubes, their varied and sometimes complex shapes, and the dynamic reprogramming of cell identity and remodeling of tube connectivity that occur during larval development are particularly fascinating features of this organ. The physiological roles of the excretory system in osmoregulation and other aspects of the animal’s life cycle are only beginning to be explored. The cellular mechanisms and molecular pathways used to build and shape excretory tubes appear similar to those used in both unicellular and multicellular tubes in more complex organs, such as the vertebrate vascular system and kidney, making this simple organ system a useful model for understanding disease processes.

CRIP homologues maintain apical cytoskeleton to regulate tubule size in C. elegans

Maintenance of the shape and diameter of biological tubules is a critical task in the development and physiology of all metazoan organisms. We have cloned the exc-9 gene of Caenorhabditis elegans, which regulates the diameter of the single-cell excretory canal tubules. exc-9 encodes a homologue of the highly expressed mammalian intestinal LIM-domain protein CRIP, whose function has not previously been determined. A second well-conserved CRIP homologue functions in multiple valves of C. elegans. EXC-9 shows genetic interactions with other EXC proteins, including the EXC-5 guanine exchange factor that regulates CDC-42 activity. EXC-9 and its nematode homologue act in polarized epithelial cells that must maintain great flexibility at their apical surface; our results suggest that CRIPs function to maintain cytoskeletal flexibility at the apical surface.

Facilitation of Endosomal Recycling by an IRG Protein Homolog Maintains Apical Tubule Structure in Caenorhabditis elegans.

Determination of luminal diameter is critical to the function of small single-celled tubes. A series of EXC proteins, including EXC-1, prevent swelling of the tubular excretory canals in Caenorhabditis elegans. In this study, cloning of exc-1 reveals it to encode a homolog of mammalian IRG proteins, which play roles in immune response and autophagy and are associated with Crohn’s disease. Mutants in exc-1 accumulate early endosomes, lack recycling endosomes, and exhibit abnormal apical cytoskeletal structure in regions of enlarged tubules. EXC-1 interacts genetically with two other EXC proteins that also affect endosomal trafficking. In yeast two-hybrid assays, wild-type and putative constitutively active EXC-1 binds to the LIM-domain protein EXC-9, whose homolog, cysteine-rich intestinal protein, is enriched in mammalian intestine. These results suggest a model for IRG function in forming and maintaining apical tubule structure via regulation of endosomal recycling.