Heiman M G et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Electron Microscopic Reconstruction of Sensory Dendrite Formation"

Lu Y, Oikonomou G, Shaham S, Heiman M G

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

One of the oldest questions in neuroscience is how a neuron establishes its proper shape and connectivity. The stereotyped anatomy of C. elegans offers an extraordinary opportunity to answer this question. We have focused on the major sense organ of C. elegans, called the amphid, which consists of 12 ciliated sensory neurons that extend unbranched dendrites to the nose, as well as two glial cells that associate with these dendrites and surround their ciliated endings. Specifically, we have asked how the ciliated endings of the dendrites become properly positioned at the nose tip. Previously, we showed that these neurons are born near the nose, anchor a short projection there, and then the cell body migrates away, stretching the dendrite out behind it. In mutants lacking the secreted matrix proteins DEX-1 or DYF-7, the dendrite fails to anchor at the nose and is dragged along with the migrating cell body, giving rise to a short dendritic stub. These results suggested that extracellular matrix proteins can act as local anchors to determine neuronal shape and wiring. The physical structure of these anchors and how they interact with the dendrite surface have remained unclear. To address these points, we used electron microscopy of staged embryos to visualize intermediates in dendrite development. We found that dendrites develop at a stage when the neurons appear to be covered by a cellular cap, possibly the amphid socket glial cell. At this stage, dendrites do not exhibit cilia, although we observed potentially ciliogenic cytoplasmic structures, oriented as would be expected for basal bodies, within ~0.5 micron of almost every dendrite tip. Finally, and most importantly, we observed thin extracellular filaments emanating from the tips of the developing dendrites. These filaments are numerous, tightly packed, and uniformly oriented so as to appear like bushy bundles extending from the dendrite tip to the cellular cap that overlays the dendrites. Meanwhile, we identified a genetic requirement for the fibrillin homolog FBN-1 in dendrite extension. Fibrillin is an extracellular matrix protein mutated in Marfan syndrome and, as its name suggests, has the propensity to form fibrils. In other systems, fibrillin confers tissue elasticity. FBN-1 may thus be a component of extracellular filaments that contact dendrite tips, possibly acting as a spring to help anchor the dendrite against the force of cell migration.

Lillis, M. et al. (2019) International Worm Meeting "A multi-compartment neuron reveals differences between apical-basal and axon-dendrite sorting signals."

Lillis, M., Heiman, M.

[International Worm Meeting, 2019]

Cells are highly organized machines with functionally specialized compartments, such as the dendrites and axon of a neuron or the apical and basolateral surfaces of an epithelial cell. To carry out their functions, each compartment requires different proteins. A fundamental question is how proteins are sorted to the correct compartment. We recently showed that C. elegans amphid sensory neurons have a multi-compartment organization. Like other neurons, they have axons and dendrites but, like epithelial cells, they also have apical and basolateral surfaces. The apical surface consists of the distal ~5 m of the dendrite and sensory cilium, is exposed to the environment, and is delimited by tight junctions with a glial cell. The basolateral surface consists of the proximal ~95 m of the dendrite, cell body, and axon, and is not exposed to the environment. We reasoned that these neurons could thus be used to identify differences between apical-basal and axon-dendrite sorting signals. To this end, we focused on the neuronal adhesion molecule SAX-7/L1CAM as a model single-pass transmembrane protein. We expressed sfGFP-tagged SAX-7 in a single amphid neuron (ASER) and found that it localizes exclusively basolaterally. Constructs in which we replaced the extracellular region with sfGFP also localized basolaterally; in contrast, constructs replacing the cytoplasmic region with sfGFP localized exclusively apically. Thus, the SAX-7 cytoplasmic region is necessary and sufficient for basolateral localization. To further define the relevant sorting signals, we generated a series of constructs consisting solely of extracellular sfGFP, a transmembrane segment, and fragments of the SAX-7 cytoplasmic region. Using this approach, we identified two sorting signals that are sufficient for basolateral localization. One sequence, EPIL, resembles a dileucine sorting signal (ExxxLL) which has previously been characterized in epithelia as a basolateral motif. Interestingly, we show that the SAX-7 signal is distinct from that used in epithelia, as replacing it with a canonical ExLL or ExxxLL motif changes its localization from basolateral to "axon-only." The second sequence has not been described before, but is conserved across SAX-7/L1CAM family members. In summary, we identified signals that distinguish apical-basal from axon-dendrite sorting, thus enabling these cells to maintain their complex multi-compartment organization.

Patel MR et al. (2009) Cell "Neurite extension: starting at the finish line."

Patel MR, Shen K

[Cell, 2009]

The outgrowth of axons and dendrites from neuronal cell bodies to their appropriate targets is the canonical means of creating new processes. Heiman and Shaham (2009) now show that neuronal processes can also be made by anchoring dendrite tips at their target locations while the cell body pulls away, a process termed retrograde extension.

Wypijewska A et al. (2012) Biochemistry "7-methylguanosine diphosphate (m(7)GDP) is not hydrolyzed but strongly bound by decapping scavenger (DcpS) enzymes and potently inhibits their activity."

Wypijewska A, Darzynkiewicz E, Davis RE, Jemielity J, Bojarska E, Lukaszewicz M, Stepinski J

[Biochemistry, 2012]

Decapping scavenger (DcpS) enzymes catalyze the cleavage of a residual cap structure following 3' 5' mRNA decay. Some previous studies suggested that both m(7)GpppG and m(7)GDP were substrates for DcpS hydrolysis. Herein, we show that mononucleoside diphosphates, m(7)GDP (7-methylguanosine diphosphate) and m(3)(2,2,7)GDP (2,2,7-trimethylguanosine diphosphate), resulting from mRNA decapping by the Dcp1/2 complex in the 5' 3' mRNA decay, are not degraded by recombinant DcpS proteins (human, nematode, and yeast). Furthermore, whereas mononucleoside diphosphates (m(7)GDP and m(3)(2,2,7)GDP) are not hydrolyzed by DcpS, mononucleoside triphosphates (m(7)GTP and m(3)(2,2,7)GTP) are, demonstrating the importance of a triphosphate chain for DcpS hydrolytic activity. m(7)GTP and m(3)(2,2,7)GTP are cleaved at a slower rate than their corresponding dinucleotides (m(7)GpppG and m(3)(2,2,7)GpppG, respectively), indicating an involvement of the second nucleoside for efficient DcpS-mediated digestion. Although DcpS enzymes cannot hydrolyze m(7)GDP, they have a high binding affinity for m(7)GDP and m(7)GDP potently inhibits DcpS hydrolysis of m(7)GpppG, suggesting that m(7)GDP may function as an efficient DcpS inhibitor. Our data have important implications for the regulatory role of m(7)GDP in mRNA metabolic pathways due to its possible interactions with different cap-binding proteins, such as DcpS or eIF4E.

O'Connell EM et al. (2015) J Infect Dis "Targeting Filarial Abl-like Kinases: Orally Available, Food and Drug Administration-Approved Tyrosine Kinase Inhibitors Are Microfilaricidal and Macrofilaricidal."

Nutman TB, O'Connell EM, Bennuru S, Steel C, Dolan MA

[J Infect Dis, 2015]

BACKGROUND: Elimination of onchocerciasis and lymphatic filariasis is targeted for 2020. Given the coincident Loa loa infections in Central Africa and the potential for drug resistance development, the need for new microfilaricides and macrofilaricides has never been greater. With the genomes of L. loa, Onchocerca volvulus, Wuchereria bancrofti, and Brugia malayi available, new drug targets have been identified. METHODS: The effects of the tyrosine kinase inhibitors imatinib, nilotinib, and dasatinib on B. malayi adult males, adult females, L3 larvae, and microfilariae were assessed using a wide dose range (0-100 M) in vitro. RESULTS: For microfilariae, median inhibitory concentrations (IC50 values) on day 6 were 6.06 M for imatinib, 3.72 M for dasatinib, and 81.35 M for nilotinib; for L3 larvae, 11.27 M, 13.64 M, and 70.98 M, respectively; for adult males, 41.6 M, 3.87 M, and 68.22 M, respectively; and for adult females, 42.89 M, 9.8 M, and >100 M, respectively. Three-dimensional modeling suggests how these tyrosine kinase inhibitors bind and inhibit filarial protein activity. CONCLUSIONS: Given the safety of imatinib in humans, plans are underway for pilot clinical trials to assess its efficacy in patients with filarial infections.

McLachlan, I. G. et al. (2017) International Worm Meeting "MAPK-15 regulates dendrite size in the URX sensory neuron."

McLachlan, I. G., Heiman, M. G.

[International Worm Meeting, 2017]

The morphology of a neuronal dendrite determines what signals it receives and how those signals are integrated. For example, the positions and shapes of dendritic spines determine synaptic partners and synapse strength. In C. elegans, many sensory neurons extend dendrites that terminate at the nose in a sensory cilium ensheathed by glia, and we previously identified genes required for their morphogenesis. In contrast, the oxygen-sensing neuron URX extends a dendrite to the nose that lacks a cilium, is not ensheathed by glia, and does not require any of the genes we previously identified. Therefore, to identify novel regulators of dendrite morphogenesis, we performed an unbiased visual genetic screen for mutants with defects in URX dendrite morphogenesis. We isolated 17 recessive mutants exhibiting four classes of phenotype: (I) short dendrites; (II) ectopic branching; (III) disorganized dendrites, and (IV) dendrite overgrowth, in which the URX dendrite extends up to ~150% of its normal length. We identified two genes that cause this overgrowth phenotype: C05D10.2/mapk-15, a previously uncharacterized gene encoding a kinase with homology to mammalian MAPK15/ERK8, and sma-1, a homolog of beta-H spectrin. Both mapk-15 and sma-1 phenotypes appear during L4 stage and become increasingly pronounced as the animal ages. We determined via mosaic and rescue experiments that MAPK-15 acts cell-autonomously in URX neurons and requires its kinase activity to function. The URX dendrite ending is enriched in signaling molecules including the receptor-type guanylyl cyclase GCY-35, and we found that MAPK-15 also localizes to the dendrite ending in wild-type animals. Further, in mapk-15 mutants, GCY-35 becomes localized throughout the region of dendrite overgrowth, suggesting the additional dendrite length reflects expansion of this sensory compartment. Surprisingly, overexpression of wild-type, but not inactive, GCY-35 suppresses the mapk-15 phenotype, suggesting that the size of this sensory compartment may be regulated by sensory signaling in a manner epistatic to mapk-15. Together, these results suggest that mapk-15 may mediate activity-dependent size regulation of a dendritic sensory compartment, possibly analogous to regulation of dendritic spine size and shape.

Wood WB (1976) Worm Breeder's Gazette "maternal effects among ts zygote-defective (zyg) mutants."

Wood WB

[Worm Breeder's Gazette, 1976]

We have studied maternal effects in 23 zyg ts mutants to estimate the times of expression of genes whose products are required in embryogenesis. We have used the following three tests, called arbitrarily A, B, and C. A test: Heterozygous (m/+) L4's are shifted to 25 C and allowed to self-fertilize. If 100% of their eggs yield larvae (25% of which express the mutant phenotype as adults), then the mutant is scored as maternal (M). If 25% of the F1 eggs fail to hatch, then the mutant is scored as non-maternal (N). An M result indicates that expression of the + allele in the parent allows m/m zygotes to hatch and grow to adulthood. A result of N indicates the opposite: that the + allele must be expressed in the zygote for hatching to occur. Out of 23 zyg mutants tested, 3 were scored N and 20 were scored M in the A test. Therefore, for most of the genes defined by these mutants, expression in the parent is sufficient for zygote survival, even if the gene is not expressed in the zygote. B test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with N2 (+/+) males. If eggs fail to hatch at 25 C, but mated hermaphrodites shifted to 16 C produce cross progeny to give proof of mating, then the mutant is scored M. If cross progeny appear in the 25 C mating, then the mutant is scored N. An M result indicates that expression of the + allele in the zygote is not sufficient to allow m/+ progeny of an m/m hermaphrodite to survive. Conversely an N result indicates either that zygotic expression of the + allele is sufficient for survival, or that a sperm function or factor needed for early embryogenesis can be supplied paternally (see C test below). Out of the 23 zyg mutants tested, 11 were scored M and 12 were scored N. The combined results of A and B tests and their simplest interpretation are as follows. Ten mutants are M,M; the genes defined by these mutants must be expressed in the hermaphrodite parent for the zygote to survive. Ten mutants are M,N; these genes can be expressed either in the parent or in the zygote. Two mutants are N,N; these genes must be expressed in the zygote. One mutant is N,M; this gene must be expressed both in the maternal parent and in the zygote. C test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with heterozygous (m/+) males. If rescue by a +/+ male in the B test depends on the + allele, then only half the cross progeny zygotes of a C test mating (m/+ male x m/m hermaphrodite) should survive. However, if rescue depends on a function or cytoplasmic component from the male sperm, then all the cross progeny zygotes in a C test should survive. Of the 10 M,N mutants, 6 have been C tested; one exhibited paternal rescue independent of the + allele. The A and B tests also were carried out on 16 mutants that arrest before the L3 molt (acc mutants). In the A test on 2 of these mutants, all m/m progeny of m/+ parents grew to adulthood at 25 C. Therefore, parental contributions are sufficient to overcome a progeny mutational block as late as the L2 stage. All 16 acc mutants scored N in the B test.

Ali Khan L et al. (1994) Worm Breeder's Gazette "cej-1 Encodes a Novel Protein With Poly-Threonine Motif"

Siddiqui SS, Fukushige T, Ali Khan L, Tsukita Sh, Tabish M, Itoh M

[Worm Breeder's Gazette, 1994]

cej-1 Encodes a Novel Protein with Poly-Threonine Motif M. L. A. Khanl, M. Tabish, T. Fukushigel1 S. Tsukita2, M. Itoh , Sh. Tsukita , and S. S. Siddiqui. (1): Lab. of Molecular Biology, Dept of Ecological Engg. Toyohashi Univ. Technology, Toyohashi 441, and (2). National Institute for Physiological Sciences, Okazaki 444, Japan.

Lemire BD et al. (2009) Mech Ageing Dev "C. elegans longevity pathways converge to decrease mitochondrial membrane potential."

Behrendt M, Decorby A, Lemire BD, Gaskova D

[Mech Ageing Dev, 2009]

Energy production via oxidative phosphorylation generates a mitochondrial membrane potential (DeltaPsi(m)) across the inner membrane. In this work, we show that a lower DeltaPsi(m) is associated with increased lifespan in Caenorhabditis elegans. The long-lived mutants daf-2(e1370), age-1(hx546), clk-1(qm30), isp-1(qm150) and eat-2(ad465) all have a lower DeltaPsi(m) than wild type animals. The lower DeltaPsi(m) of daf-2(e1370) is daf-16 dependent, indicating that the insulin-like signaling pathway not only regulates lifespan but also mitochondrial energetics. RNA interference (RNAi) against 17 genes shown to extend lifespan also decrease DeltaPsi(m). Furthermore, lifespan can be significantly extended with the uncoupler carbonylcyanide-3-chlorophenylhydrazone (CCCP), which dissipates DeltaPsi(m). We conclude that longevity pathways converge on the mitochondria and lead to a decreased DeltaPsi(m). Our results are consistent with the 'uncoupling to survive' hypothesis, which states that dissipation of the DeltaPsi(m) will extend lifespan.

Seo J et al. (2005) Arch Environ Contam Toxicol "Biodegradation of the insecticide N,N-diethyl-m-toluamide by fungi: identification and toxicity of metabolites."

Kim SD, Cha CJ, Hur HG, Lee YG, Seo J, Ahn JH

[Arch Environ Contam Toxicol, 2005]

Fungi (Cunninghamella elegans ATCC 9245, Mucor ramannianus R-56, Aspergillus niger VKMF-1119, and Phanerochaete chrysosporium BKMF-1767) were tested to elucidate the biologic fate of the topical insect repellent N,N-diethyl-m-toluamide (DEET). The elution profile obtained from analysis by high-pressure liquid chromatography equipped with a reverse-phase C-18 column, showed that three peaks occurred after incubation of C. elegans, with which 1 mM DEET was combined as a final concentration. The peaks were not detected in the control experiments with either DEET alone or tested fungus alone. The metabolites produced by C. elegans exhibited a molecular mass of 207 with a fragment ion (m/z) at 135, a molecular mass of 179 with an m/z at 135, and a molecular mass of 163 with an m/z at 119, all of which correspond to N,N-diethyl-m-toluamide-N-oxide, N-ethyl-m-toluamide-N-oxide, and N-ethyl-m-toluamide, respectively. M. ramannianus R-56 also produced N, N-diethyl-m-toluamide-N-oxide and N-ethyl-m-toluamide but did not produce N-ethyl-m-toluamide-N-oxide. For the biologic toxicity test with DEET and its metabolites, the freshwater zooplankton Daphnia magna was used. The biologic sensitivity in decreasing order was DEET > N-ethyl-m-toluamide > N,N-diethyl-m-toluamide-N-oxide. Although DEET and its fungal metabolites showed relatively low mortality compared with other insecticides, the toxicity was increased at longer exposure periods. These are the first reports of the metabolism of DEET by fungi and of the biologic toxicity of DEET and its fungal metabolites to the freshwater zooplankton D. magna.