Jentsch TJ (1994) Nature "Trinity of cation channels."

Jentsch TJ

[Nature, 1994]

One of the most satisfying moments in science is when different lines of investigation converge to yield a beautiful picture that opens up new perspectives. This happened last year when expression cloning of an epithelial sodium channel subunit revealed that the DNA encoding it was significantly similar in sequence to that of certain nematode genes, mutations in which lead to insensitivity to touch, neurodegeneration or both. Three reports on pages 463, 467 and 470 of this issue now suggest that at least three distinct subunits are used to build channel complexes in both mammals and the nematode Caenorhabditis elegans. Further, the new work provides insights into the relationship between subunit structure and function, and demonstrates a remarkable degree of functional conservation between vertebrates and invertebrates.

Jentsch TJ et al. (2018) Physiol Rev "CLC Chloride Channels and Transporters: Structure, Function, Physiology, and Disease."

Jentsch TJ, Pusch M

[Physiol Rev, 2018]

CLC anion transporters are found in all phyla and form a gene family of eight members in mammals. Two CLC proteins, each of which completely contains an ion translocation parthway, assemble to homo- or heteromeric dimers that sometimes require accessory -subunits for function. CLC proteins come in two flavors: anion channels and anion/proton exchangers. Structures of these two CLC protein classes are surprisingly similar. Extensive structure-function analysis identified residues involved in ion permeation, anion-proton coupling and gating and led to attractive biophysical models. In mammals, ClC-1, -2, -Ka/-Kb are plasma membrane Cl^- channels, whereas ClC-3 through ClC-7 are 2Cl^-/H⁺-exchangers in endolysosomal membranes. Biological roles of CLCs were mostly studied in mammals, but also in plants and model organisms like yeast and Caenorhabditis elegans. CLC Cl^- channels have roles in the control of electrical excitability, extra- and intracellular ion homeostasis, and transepithelial transport, whereas anion/proton exchangers influence vesicular ion composition and impinge on endocytosis and lysosomal function. The surprisingly diverse roles of CLCs are highlighted by human and mouse disorders elicited by mutations in their genes. These pathologies include neurodegeneration, leukodystrophy, mental retardation, deafness, blindness, myotonia, hyperaldosteronism, renal salt loss, proteinuria, kidney stones, male infertility, and osteopetrosis. In this review, emphasis is laid on biophysical structure-function analysis and on the cell biological and organismal roles of mammalian CLCs and their role in disease.

Schriever AM et al. (1999) J Biol Chem "CLC chloride channels in Caenorhabditis elegans."

Jentsch TJ, Friedrich T, Schriever AM, Pusch M

[J Biol Chem, 1999]

The genome of the nematode Caenorhabditis elegans encodes six putative chloride channels (CeCLC-1 through CeCLC-6) that represent all three known branches of the mammalian CLC gene family. Using promoter fragments to drive the expression of the green fluorescent protein, CeCLC-2, -3, and -4 expression was studied in transgenic C. elegans. CeCLC-4 was specifically expressed in the large H-shaped excretory cell, where it was co-expressed with CeCLC-3, which is also expressed in other cells, including neurons, muscles, and epithelial cells. Also, CeCLC-2 was expressed in several cells of the nervous system, intestinal cells, and vulval muscle cells. Similar to mammalian CLC proteins, only two nematode CLC channels elicited detectable plasma membrane currents in Xenopus oocytes. CeCLC-3 currents were inwardly rectifying and were activated by positive prepulses. Its complex gating behavior can be explained by two gates, at least one of which depends on extracellular anions. In this respect it resembles some mammalian chloride channels with which it also shares a preference of chloride over iodide. C. elegans thus provides new opportunities to understand common mechanisms underlying structure and function in CLC channels and will allow for a genetic dissection of chloride channels in this simple model organism.

Gao L et al. (2004) J Biol Chem "Isoforms of the polarity protein par6 have distinct functions."

Macara IG, Gao L

[J Biol Chem, 2004]

PAR-6 is essential for asymmetric division of the Caenorhabditis elegans zygote. It is also critical for cell polarization in many other contexts, throughout the Metazoa. The Par6 protein contains a PDZ domain and a partial CRIB (Cdc42/Rac interactive binding) domain, which mediate interactions with other polarity proteins such as Par3, Cdc42, Pals1 and Lgl. A family of mammalian Par6 isoforms (Par6A-D) has been described, but the significance of this diversification has been unclear. Here, we demonstrate that Par6 family members localize differently when expressed in MDCK epithelial cells and have distinct effects on tight junction (TJ) assembly. Par6B localizes to the cytosol and inhibits TJ formation, but Par6A co-localizes predominantly with the TJ marker ZO-1 at cell-cell contacts and does not affect junctions. These functional differences correlate with differences in Pals1 binding - Par6B interacts strongly with Pals1, whereas Par6A binds very weakly to Pals1 even in the presence of active Cdc42. Pals1 has a low affinity for the isolated CRIB+PDZ domain of Par6A, but analysis of chimeras showed that in addition, Pals1 binding is blocked by an inhibitory property of the N-terminus of Par6A. Unexpectedly, the localization of Par6A to cell-cell contacts is Cdc42-independent.

Kim MR et al. (2022) Phytomedicine "Schisandrin C improves leaky gut conditions in intestinal cell monolayer, organoid, and nematode models by increasing tight junction protein expression."

Cho SY, Kim JY, Kim JH, Nguyen UTT, Cha KH, Choi KY, Kim MR, Kim WK, Ha NM, Kang K, Lee HJ

[Phytomedicine, 2022]

Background: Leaky gut symptoms and inflammatory bowel disease (IBD) are associated with damaged intestinal mucosa, intestinal permeability dysfunction by epithelial cell cytoskeleton contraction, disrupted intercellular tight junction (TJ) protein expression, and abnormal immune responses and are intractable diseases.Purpose: We evaluated the effects of schisandrin C, a dibenzocyclooctadiene lignan from Schisandra chinensis, on intestinal inflammation and permeability dysfunction in gut mimetic systems: cultured intestinal cells, intestinal organoids, and a Caenorhabditis elegans model.Methods: Schisandrin C was selected from 9 lignan compounds from S. chinensis based on its anti-inflammatory effects in HT-29 human intestinal cells. IL-1β and Pseudomonas aeruginosa supernatants were used to disrupt intestinal barrier formation in vitro and in C. elegans, respectively. The effects of schisandrin C on transepithelial electrical resistance (TEER) and intestinal permeability were evaluated in intestinal cell monolayers, and its effect on intestinal permeability dysfunction was tested in mouse intestinal organoids and C. elegans by measuring fluorescein isothiocyanate (FITC)-dextran efflux. The effect of schisandrin C on TJ protein expression was investigated by western blotting and fluorescence microscopy. The signaling pathway underlying these effects was also elucidated.Results: Schisandrin C ameliorated intestinal permeability dysfunction in three IBD model systems and enhanced epithelial barrier formation via upregulation of ZO-1 and occludin in intestinal cell monolayers and intestinal organoids. In Caco-2 cells, schisandrin C restored IL-1β-mediated increases in MLCK and p-MLC expression, in turn blocking cytoskeletal contraction and subsequent intestinal permeabilization. Schisandrin C inhibited NF-ĸB and p38 MAPK signaling, which regulates MLCK expression and structural reorganization of the TJ complex in Caco-2 cells. Schisandrin C significantly improved abnormal FITC-dextran permeabilization in both intestinal organoids and C. elegans.Conclusion: Schisandrin C significantly improves abnormal intestinal permeability and regulates the expression of TJ proteins, long MLCK, p-MLC, and inflammation-related proteins, which are closely related to leaky gut symptoms and IBD development. Therefore, schisandrin C is a candidate to treat leaky gut symptoms and IBDs.

Suzuki A et al. (2002) J Cell Sci "aPKC kinase activity is required for the asymmetric differentiation of the premature junctional complex during epithelial cell polarization."

Shimizu M, Ebnet K, Hashiba K, Ohno S, Ishiyama C, Suzuki A

[J Cell Sci, 2002]

We have previously shown that aPKC interacts with cell polarity proteins PAR-3 and PAR-6 and plays an indispensable role in cell polarization in the C. elegans one-cell embryo as well as in mammalian epithelial cells. Here, to clarify the molecular basis underlying this aPKC function in mammalian epithelial cells, we analyzed the localization of aPKC and PAR-3 during the cell repolarization process accompanied by wound healing of MTD1-A epithelial cells. Immunofluorescence analysis revealed that PAR-3 and aPKClambda translocate to cell-cell contact regions later than the formation of the primordial spot-like adherens junctions (AJs) containing E-cadherin and ZO-1. Comparison with three tight junction (TJ) membrane proteins, JAM, occludin and claudin-1, further indicates that aPKClambda is one of the last TJ components to be recruited. Consistently, the expression of a dominant-negative mutant of aPKClambda (aPKClambdakn) in wound healing cells does not inhibit the formation of the spot-like AJs; rather, it blocks their development into belt-like AJs. These persistent spot-like AJs in aPKClambda-expressing cells contain all TJ membrane proteins and PAR-3, indicating that aPKC kinase activity is not required for their translocation to these premature junctional complexes but is indispensable for their further differentiation into belt-like AJs and TJs. Cortical bundle formation is also blocked at the intermediate step where fine actin bundles emanating from premature cortical bundles link the persistent spot-like AJs at apical tips of columnar cells. These results suggest that aPKC contributes to the establishment of epithelial cell polarity by promoting the transition of fibroblastic junctional structures into epithelia-specific asymmetric ones.

Gerami B et al. (1997) International C. elegans Meeting "MAPPING QUANTITATIVE TRAIT LOCI FOR CHEMOTAXIS: CORRESPONDENCE BETWEEN QTL AND odr CANDIDATE LOCI"

Phillips PC, Gerami B, Morphew JJ

[International C. elegans Meeting, 1997]

Understanding the genetics of complex traits is challenging because it can be difficult to separate out the effects of the numerous genes affecting the trait. C. elegans can serve as a useful model system for studying complex traits because of the wealth of genetic and functional information available. Chemotaxis provides an ideal trait for this type of study because it ecologically important to the nematodes, as well as likely to be influenced by many genes (a number of which are known). In collaboration with Thomas Johnson and David Shook [1] and Robert Shmookler Reis and Robert Ebert [2], who provided genotyped lines generated in a cross between N2 and BO, we have assayed the chemotaxic response of 180 recombinant inbred lines to benzaldehyde at two concentrations. These concentrations, 50 nl and 500 nl, respectively lead to an attraction or repulsion response. Separate analysis of the lines from the two labs yield different results. The SR lines show a QTL mapping to the fourth chromosome, whereas the TJ lines show several QTL, primarily on the X chromosome. The QTL on X map to the same locations as two mutants known to block the response to benzaldehyde, odr-1 and odr-5 [3]. We are in the process of evaluating these candidate loci as the actual QTL using backcrossing and complementation testing approaches. Our hope is to create a model system for studying a variety of quantitative genetic questions. [1] Genetics 142:801 (1996), [2] Genetics 135:1003 (1993), [3] Bargmann et al., Cell 74:515 (1993)

Su WH et al. (2012) Adv Exp Med Biol "Polarity protein complex Scribble/Lgl/Dlg and epithelial cell barriers."

Mruk DD, Wong EW, Cheng CY, Su WH, Lui WY

[Adv Exp Med Biol, 2012]

The Scribble polarity complex or module is one of the three polarity modules that regulate cell polarity in multiple epithelia including blood-tissue barriers. This protein complex is composed of Scribble, Lethal giant larvae (Lgl) and Discs large (Dlg), which are well conserved across species from fruitflies and worms to mammals. Originally identified in Drosophila and C. elegans where the Scribble complex was found to work with the Par-based and Crumbs-based polarity modules to regulate apicobasal polarity and asymmetry in cells and tissues during embryogenesis, their mammalian homologs have all been identified in recent years. Components of the Scribble complex are known to regulate multiple cellular functions besides cell polarity, which include cell proliferation, assembly and maintenance of adherens junction (AJ) and tight junction (TJ), and they are also tumor suppressors. Herein, we provide an update on the Scribble polarity complex and how this protein complex modulates cell adhesion with some emphasis on its role in Sertoli cell blood-testis barrier (BTB) function. It should be noted that this is a rapidly developing field, in particular the role of this protein module in blood-tissue barriers, and this short chapter attempts to provide the information necessary for investigators studying reproductive biology and blood-tissue barriers to design future studies. We also include results of recent studies from flies and worms since this information will be helpful in planning experiments for future functional studies in the testis to understand how Scribble-based proteins regulate BTB dynamics and spermatogenesis.

Chris Link et al. (2007) International Worm Meeting "Using C. elegans for in vivo structure/function analysis of beta amyloid peptide toxicity."

Chris Link, Christine Roberts, David Merin, Gin Fonte

[International Worm Meeting, 2007]

Expression of human beta amyloid peptide (Abeta), causally implicated in Alzheimers disease, in C. elegans body wall muscle results in a robust paralysis phenotype that provides a convenient readout for Abeta toxicity. We have engineered C. elegans to express wild type Abeta 1-42 or single amino acid variants thereof to test models of Abeta toxicity. One such model is the formation of membrane pores via Abeta oligomerization driven by glycine zipper interactions between alpha helical regions of Abeta monomers (Kim S, Jeon TJ, Oberai A, Yang D, Schmidt JJ, Bowie JU. 2005. Proc Natl Acad Sci U S A 102: 14278-83). Transgenic worms expressing Abeta with a Gly37-to-Leu substitution, predicted to disrupt glycine zipper interactions, show a dramatic reduction in paralysis relative to worms expressing wild type Abeta. In addition to this reduction in paralysis, Abeta G37L worms did not show the muscle cell calcium perturbations or abnormal mitochondria observed in worms expressing wt Abeta. Immunoblot and immunohistochemical analysis demonstrates that the Abeta G37L strain has Abeta levels and cellular distribution indistinguishable from control wt Abeta strains, indicating that this reduced toxicity is due to a qualitative difference in the Abeta G37L peptide. Studies with transgenic worms co-expressing wt and G37L Abeta reveal that the Abeta G37L peptide can be anti-toxic, suggesting a dominant negative effect consistent with the variant peptide interfering with toxic oligomerization of wt Abeta. To test the oligomer model more directly, we engineered second site substitutions in the Abeta G37L peptide that based on the proposed structural model could potentially restore the predicted glycine zipper interface. Two of these second site mutations (N27G and M35G) restore toxicity to the G37L variant, strongly supporting the toxic oligomer model. These studies support the oligomer pore mechanism and identify the glycine zipper interface as a potential target for therapeutic drugs.

Onken, Brian D. et al. (2011) International Worm Meeting "Insulin Signaling and Dietary Restriction Differentially Regulate Glucose Metabolism to Impact C. elegans Healthspan."

Onken, Brian D., Driscoll, Monica

[International Worm Meeting, 2011]

A major goal of aging research is to understand the underlying relationship between nutritional intake, metabolism, and healthy aging. Low-glycemic index diets have been shown to reduce risk of age-related metabolic diseases such as diabetes and cardiovascular disease, and reduced caloric intake via dietary restriction increases healthspan across species. One potential approach for supporting healthy aging is via interventions that engage healthspan-promoting metabolism. In Caenorhabditis elegans, adding excess glucose to the growth medium shortens lifespan [1, 2], while inhibiting the glycolytic enzyme hexokinase with the glucose analog 2-deoxyglucose increases lifespan [1]. We have shown that disrupting genes encoding two other glycolytic enzymes that catalyze unidirectional, irreversible reactions lengthens C. elegans median lifespan, induces large gains in youthful locomotory ability, and triggers a fluorescent biomarker that distinguishes a healthy metabolic state. Conversely, disrupting counterpart gluconeogenic genes decreases nematode healthspan. In investigating potential longevity-related pathways that might impinge upon glucose metabolism, we found that disrupting glycolytic genes increases healthspan through the FOXO transcription factor DAF-16, which is also required for the increased lifespan seen with lowered levels of insulin signaling, and which is downregulated by increased glucose availability [2]. Strikingly, we also found that gluconeogenic activity is absolutely and specifically required for increased healthspan under dietary restriction. These results provide evidence for an intriguing new paradigm: breakdown of glucose via glycolysis negatively impacts healthy aging through insulin signaling and DAF-16, while dietary restriction engages the reciprocal gluconeogenic pathway to promote healthspan. Our observations support that healthspan might be optimized via dietary, pharmacological, or genetic interventions that increase gluconeogenic activity or decrease glycolysis. 1. Schulz TJ, Zarse K, Voigt A, Urban N, Birringer M, et al. (2007). Cell Metab 6: 280-293. 2. Lee SJ, Murhpy CT, Kenyon C (2009). Cell Metab 10: 379-391.