Saarikangas J et al. (2009) Curr Biol "Molecular mechanisms of membrane deformation by I-BAR domain proteins."

Butcher SJ, Zhao H, Lappalainen P, Kinnunen PK, Mattila PK, Saarikangas J, Pykalainen A, Laurinmaki P

[Curr Biol, 2009]

BACKGROUND: Generation of membrane curvature is critical for the formation of plasma membrane protrusions and invaginations and for shaping intracellular organelles. Among the central regulators of membrane dynamics are the BAR superfamily domains, which deform membranes into tubular structures. In contrast to the relatively well characterized BAR and F-BAR domains that promote the formation of plasma membrane invaginations, I-BAR domains induce plasma membrane protrusions through a poorly understood mechanism. RESULTS: We show that I-BAR domains induce strong PI(4,5)P(2) clustering upon membrane binding, bend the membrane through electrostatic interactions, and remain dynamically associated with the inner leaflet of membrane tubules. Thus, I-BAR domains induce the formation of dynamic membrane protrusions to the opposite direction than do BAR and F-BAR domains. Strikingly, comparison of different I-BAR domains revealed that they deform PI(4,5)P(2)-rich membranes through distinct mechanisms. IRSp53 and IRTKS I-BARs bind membranes mainly through electrostatic interactions, whereas MIM and ABBA I-BARs additionally insert an amphipathic helix into the membrane bilayer, resulting in larger tubule diameter in vitro and more efficient filopodia formation in vivo. Furthermore, FRAP analysis revealed that whereas the mammalian I-BAR domains display dynamic association with filopodia, the C. elegans I-BAR domain forms relatively stable structures inside the plasma membrane protrusions. CONCLUSIONS: These data define I-BAR domain as a functional member of the BAR domain superfamily and unravel the mechanisms by which I-BAR domains deform membranes to induce filopodia in cells. Furthermore, our work reveals unexpected divergence in the mechanisms by which evolutionarily distinct groups of I-BAR domains interact with PI(4,5)P(2)-rich membranes.

Snead WT et al. (2019) J Cell Biol "BAR scaffolds drive membrane fission by crowding disordered domains."

Lafer EM, Zhao C, Kago G, Stachowiak JC, Richter JB, Zeno WF, Perkins RW, Snead WT

[J Cell Biol, 2019]

Cellular membranes are continuously remodeled. The crescent-shaped bin-amphiphysin-rvs (BAR) domains remodel membranes in multiple cellular pathways. Based on studies of isolated BAR domains in vitro, the current paradigm is that BAR domain-containing proteins polymerize into cylindrical scaffolds that stabilize lipid tubules. But in nature, proteins that contain BAR domains often also contain large intrinsically disordered regions. Using in vitro and live cell assays, here we show that full-length BAR domain-containing proteins, rather than stabilizing membrane tubules, are instead surprisingly potent drivers of membrane fission. Specifically, when BAR scaffolds assemble at membrane surfaces, their bulky disordered domains become crowded, generating steric pressure that destabilizes lipid tubules. More broadly, we observe this behavior with BAR domains that have a range of curvatures. These data suggest that the ability to concentrate disordered domains is a key driver of membrane remodeling and fission by BAR domain-containing proteins.

Maro GS et al. (2009) PLoS One "A beta-catenin-dependent Wnt pathway mediates anteroposterior axon guidance in C. elegans motor neurons."

Maro GS, Klassen MP, Shen K

[PLoS One, 2009]

BACKGROUND: Wnts are secreted glycoproteins that regulate diverse aspects of development, including cell proliferation, cell fate specification and differentiation. More recently, Wnts have been shown to direct axon guidance in vertebrates, flies and worms. However, little is known about the intracellular signaling pathways downstream of Wnts in axon guidance. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that the posterior C. elegans Wnt protein LIN-44 repels the axons of the adjacent D-type motor neurons by activating its receptor LIN-17/Frizzled on the neurons. Moreover, mutations in mig-5/Disheveled, gsk-3, pry-1/Axin, bar-1/beta-catenin and pop-1/TCF, also cause disrupted D-type axon pathfinding. Reduced BAR-1/beta-catenin activity in D-type axons leads to undergrowth of axons, while stabilization of BAR-1/beta-catenin in a lin-23/SCF(beta-TrCP) mutant results in an overextension phenotype. CONCLUSIONS/SIGNIFICANCE: Together, our data provide evidence that Wnt-mediated axon guidance can be transduced through a beta-catenin-dependent pathway.

Natarajan L et al. (2000) East Coast Worm Meeting "Structure-function characterization of BAR-1 and other b -catenins in C. elegans"

Eisenmann DM, Natarajan L

[East Coast Worm Meeting, 2000]

C. elegans vulval development is co-ordinated by conserved RTK/Ras and Notch signaling pathways, so that six Vulval Precursor Cells (VPCs) adopt cell fates in the pattern 3/F 3 2 1 2 3 (F stands for Fused fate). In a screen for mutants affecting vulval development, bar-1 was identified. bar-1 mutants show defects in VPC and P12 cell fate specification and QL progeny migration. In each case, bar-1 is required to maintain Hox gene expression in the affected cells. bar-1 encodes a C. elegans b -catenin/Armadillo homolog. These proteins function in both cell adhesion and Wnt signal transduction. Wnt pathways regulate Hox genes in other systems and Wnt pathway mutants in C. elegans show similar phenotypes as bar-1 mutants which suggests that bar-1 acts in a Wnt pathway in the vulva. We would like to further understand BAR-1 structure and function. To identify transcriptional activation domains in BAR-1, regions of BAR-1 were made as GAL4 DBD fusions and tested in yeast. N-terminal and the C-terminal regions of BAR-1 possess transcriptional activating function, similar to other b -catenins. The effects of deletion of different BAR-1 domains on BAR-1 activity in the worm will be analyzed. A single region containing Armadillo repeats (1-9) did not activate transcription on its own and was used to identify BAR-1 interacting factors (143) by the yeast two hybrid method. The roles of potential interactors in vulval development are being investigated. BAR-1 is also being tested for interaction with other known Wnt pathway components in C. elegans. APR-1 and POP-1 interact with BAR-1 and reducing the activity of these genes results in vulval defects (work from our lab and Hajnal lab). BAR-1 does not interact with EGL-27, LIN-25 and HMP-1 (a -catenin) in yeast. C. elegans has three b -catenins, HMP-2, WRM-1 and BAR-1, as opposed to one (Armadillo) in Drosophila. WRM-1 functions in Wnt signaling in embryogenesis and HMP-2 in cell adhesion/morphogenesis. We are testing if BAR-1 function in the vulva can be substituted by fly Armadillo and other b -catenins in C. elegans. We would also like to know if BAR-1 interacting proteins can interact with WRM-1 and HMP-2. These experiments will help us understand if the functions of b-catenins in C. elegans have been conserved.

Sakoguchi H et al. (2016) Biosci Biotechnol Biochem "Screening of biologically active monosaccharides: growth inhibitory effects of d-allose, d-talose, and l-idose against the nematode Caenorhabditis elegans."

Izumori K, Yoshihara A, Sakoguchi H, Sato M

[Biosci Biotechnol Biochem, 2016]

We compared the growth inhibitory effects of all aldohexose stereoisomers against the model animal Caenorhabditis elegans. Among the tested compounds, the rare sugars d-allose (d-All), d-talose (d-Tal), and l-idose (l-Ido) showed considerable growth inhibition under both monoxenic and axenic culture conditions. 6-Deoxy-d-All had no effect on growth, which suggests that C6-phosphorylation by hexokinase is essential for inhibition by d-All.

Sakoguchi H et al. (2016) Bioorg Med Chem Lett "Growth inhibitory effect of d-arabinose against the nematode Caenorhabditis elegans: Discovery of a novel bioactive monosaccharide."

Shintani T, Izumori K, Sato M, Sakoguchi H, Okuma K, Yoshihara A

[Bioorg Med Chem Lett, 2016]

Biological activities of unusual monosaccharides (rare sugars) have largely remained unstudied until recently. We compared the growth inhibitory effects of aldohexose stereoisomers against the animal model Caenorhabditis elegans cultured in monoxenic conditions with Escherichia coli as food. Among these stereoisomers, the rare sugar d-arabinose (d-Ara) showed particularly strong growth inhibition. The IC50 value for d-Ara was estimated to be 7.5mM, which surpassed that of the potent glycolytic inhibitor 2-deoxy-d-glucose (19.5mM) used as a positive control. The inhibitory effect of d-Ara was also observed in animals cultured in axenic conditions using a chemically defined medium; this excluded the possible influence of E. coli. To our knowledge, this is the first report of biological activity of d-Ara. The d-Ara-induced inhibition was recovered by adding either d-ribose or d-fructose, but not d-glucose. These findings suggest that the inhibition could be induced by multiple mechanisms, for example, disturbance of d-ribose and d-fructose metabolism.

Yoshihara A et al. (2019) Bioorg Med Chem Lett "Growth inhibition by 1-deoxy-d-allulose, a novel bioactive deoxy sugar, screened using Caenorhabditis elegans assay."

Sakoguchi H, Fleet GWJ, Izumori K, Shintani T, Sato M, Yoshihara A

[Bioorg Med Chem Lett, 2019]

The biological activities of deoxy sugars (deoxy monosaccharides) have remained largely unstudied until recently. We compared the growth inhibition by all 1-deoxyketohexoses using the animal model Caenorhabditis elegans. Among the eight stereoisomers, 1-deoxy-d-allulose (1d-d-Alu) showed particularly strong growth inhibition. The 50% inhibition of growth (GI₅₀) concentration by 1d-d-Alu was estimated to be 5.4mM, which is approximately 10 times lower than that of d-allulose (52.7mM), and even lower than that of the potent glycolytic inhibitor, 2-deoxy-d-glucose (19.5mM), implying that 1d-d-Alu has a strong growth inhibition. In contrast, 5-deoxy- and 6-deoxy-d-allulose showed no growth inhibition of C. elegans. The inhibition by 1d-d-Alu was alleviated by the addition of d-ribose or d-fructose. Our findings suggest that 1d-d-Alu-mediated growth inhibition could be induced by the imbalance in d-ribose metabolism. To our knowledge, this is the first report of biological activity of 1d-d-Alu which may be considered as an antimetabolite drug candidate.

Katane M et al. (2020) Biochim Biophys Acta Proteins Proteom "Biochemical characterization of d-aspartate oxidase from Caenorhabditis elegans: Its potential use in the determination of free D-glutamate in biological samples."

Katane M, Sekine M, Nakayama K, Homma H, Kuwabara H, Saitoh Y, Miyamoto T

[Biochim Biophys Acta Proteins Proteom, 2020]

d-Aspartate oxidase (DDO) is a flavin adenine dinucleotide (FAD)-containing flavoprotein that stereospecifically acts on acidic D-amino acids (i.e., free d-aspartate and D-glutamate). Mammalian DDO, which exhibits higher activity toward d-aspartate than D-glutamate, is presumed to regulate levels of d-aspartate in the body and is not thought to degrade D-glutamate in vivo. By contrast, three DDO isoforms are present in the nematode Caenorhabditis elegans, DDO-1, DDO-2, and DDO-3, all of which exhibit substantial activity toward D-glutamate as well as d-aspartate. In this study, we optimized the Escherichia coli culture conditions for production of recombinant C. elegans DDO-1, purified the protein, and showed that it is a flavoprotein with a noncovalently but tightly attached FAD. Furthermore, C. elegans DDO-1, but not mammalian (rat) DDO, efficiently and selectively degraded D-glutamate in addition to d-aspartate, even in the presence of various other amino acids. Thus, C. elegans DDO-1 might be a useful tool for determining these acidic D-amino acids in biological samples.

Natarajan L et al. (2004) Dev Biol "Identification of evolutionarily conserved promoter elements and amino acids required for function of the C. elegans beta-catenin homolog BAR-1."

Szyleyko E, Natarajan L, Jackson MB, Eisenmann DM

[Dev Biol, 2004]

beta-catenins are conserved transcription factors regulated posttranslationally by Wnt signaling. bar-1 encodes a Caenorhabditis elegans beta-catenin acting in multiple Wnt-mediated processes, including cell fate specification by vulval precursor cells (VPCs) and migration of the Q(L) neuroblast progeny. We took two approaches to extend our knowledge of bar-1 function. First, we undertook a bar-1 promoter analysis using transcriptional GFP reporter fusions and found that bar-1 expression is regulated in specific cells at the transcriptional level. We identified promoter elements necessary for bar-1 expression in several cell types, including a 321-bp element sufficient for expression in ventral cord neurons (VCNs) and a 1.1-kb element sufficient for expression in the developing vulva and adult seam cells. Expression of bar-l from the 321-bp element rescued the Uncoordinated (Unc) phenotype of bar-1 mutants, but not the vulval phenotype, suggesting that a Writ pathway may act in ventral cord neurons to mediate proper locomotion. By comparison of the 1.1-kb element to homologous sequences from Caenorhabditis briggsae, we identified evolutionarily conserved sequences necessary for expression in vulval or seam cells. Second, we analyzed 24 mutations in bar-1 and identified several residues required for BAR-1 activity in C. elegans. By phylogenetic comparison, we found that most of these residues are conserved and may identify amino acids necessary for beta-catenin function in all species.

Eisenmann DM et al. (1998) Development "The beta-catenin homolog BAR-1 and LET-60 Ras coordinately regulate the Hox gene lin-39 during Caenorhabditis elegans vulval development."

Kenyon CJ, Kim SK, Eisenmann DM, Maloof JN, Simske JS

[Development, 1998]

In C. elegans, the epithelial Pn.p cells adopt either a vulval precursor cell fate or fuse with the surrounding hypodermis (the F fate). Our results suggest that a Wnt signal transduced through a pathway involving the beta-catenin homolog BAR-1 controls whether P3.p through P8.p adopt the vulval precursor cell fate. In bar-1 mutants, P3.p through P8.p can adopt F fates instead of vulval precursor cell fates. The Wnt/bar-1 signaling pathway acts by regulating the expression of the Hox gene lin-39, since bar-1 is required for LIN-39 expression and forced lin-39 expression rescues the bar-1 mutant phenotype. LIN-39 activity is also regulated by the anchor cell signal/let-23 receptor tyrosine kinase/let-60 Ras signaling pathway. Our genetic and molecular experiments show that the vulval precursor cells can integrate the input from the BAR-1 and LET-60 Ras signaling pathways by coordinately regulating activity of the common target LIN-39 Hox.