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Groisman, Alex, Carvalho, Ana, Olson, Sara, Gutierrez, Edgar, Desai, Arshad, Noble, Lisa, Zhang, Kelly, Oegema, Karen
[
International Worm Meeting,
2011]
Small molecule inhibitors are a valuable tool for the analysis of fundamental cellular functions and an entry point for the development of therapeutic agents. Their primary advantage is temporal control, which is especially powerful when combined with live imaging. In C. elegans embryos the use of small molecule inhibitors has been limited by eggshell impermeability. C. elegans eggshells have been permeabilized with laser puncturing, pushing against a coverslip, and by passage through a narrow mouth pipette after weakening by sequential treatment with bleach and chitinase. However, these techniques suffer from problems with consistency, non-uniform permeability, and disruption of embryonic processes. To address these problems, we identified a gene whose inhibition renders the eggshell permeable without disrupting events during the early embryonic division and built a specialized microdevice for in situ worm dissection and high resolution imaging of embryos. The microdevice has a rectangular well (8x6x3mm) with an array of microwells (0.3x0.3x0.15mm) at the bottom and an integrated dissection board near the microwell array. Worms are soaked in dsRNA to inhibit the gene and make the embryos permeable. Treated worms are placed on the dissection board, cut with a scalpel and the fragile permeable embryos are gently swept towards the microwell array using an eyelash tool. Embryos settle onto the bottom of the microwells, where they rest on a coverslip, being accessible to high-resolution imaging and protected from flow by the microwell walls. Permeabilized embryos are acutely exposed to drugs after the medium in the microwells is exchanged by aspiration of the existing medium from the macroscopic well and dispensing of a medium with the drug into the well with a pipette. Embryos remain motionless and within ~7 sec are exposed to a drug concentration that is ~60% of that in the added medium. 90 sec is sufficient for ~99% medium exchange, including removal of a drug from the embryo environment. The technique was tested with 4 common inhibitors: the microtubule inhibitor nocodazole, actin inhibitor latrunculin A, myosin inhibitor blebbistatin, and proteasome inhibitor c-lactocystin-b-lactone. All 4 inhibitors had the expected effects on permeable embryos and no effect on control impermeable embryos. These results establish that our method can be used to combine precisely controlled inhibitor treatments with live imaging in the early C. elegans embryo.
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Lee, K.Y.*, Desai, A.B., Gutierrez, E, Groisman, A, Green, R.A.*, Oegema, K.F., Gomez-Cavasos, J.S.
[
International Worm Meeting,
2017]
In metazoa, germ cells undergo incomplete cytokinesis to form syncytia with cells connected by intercellular bridges. Centralspindlin, a complex containing two molecules of kinesin-6 and two of the Rho family GTPase-activating protein (GAP) CYK-4, is a conserved bridge component. Here, we analyze centralspindlin function in the C. elegans oogenic germline, which contains ~1000 cellular compartments connected by intercellular bridges to a cytoplasmic core. In contrast to cytokinesis, where both centralspindlin components are essential for spindle midzone and contractile ring assembly, CYK-4, but not kinesin-6, is required for a germline cytokinesis-like event where intracellular bridges close to bud oocytes off the syncytium. The CYK-4 C1 domain, predicted to interact with the plasma membrane, and GAP domain interface predicted to interact with Rho family GTPases were both required to recruit CYK-4 to intercellular bridges. Knockdown of RhoA, but not Cdc42 or Rac, disrupted germline structure, suggesting that the GAP domain recruits CYK-4 to intercellular bridges by binding RhoA.
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[
PLoS One,
2011]
Genetic and genome-wide RNAi approaches available in C. elegans, combined with tools for visualizing subcellular events with high-resolution, have led to increasing adoption of the early C. elegans embryo as a model for mechanistic and functional genomic analysis of cellular processes. However, a limitation of this system has been the impermeability of the embryo eggshell, which has prevented the routine use of small molecule inhibitors. Here, we present a method to permeabilize and immobilize embryos for acute inhibitor treatment in conjunction with live imaging. To identify a means to permeabilize the eggshell, we used a dye uptake assay to screen a set of 310 candidate genes defined by a combination of bioinformatic criteria. This screen identified 20 genes whose inhibition resulted in >75% eggshell permeability, and 3 that permeabilized embryos with minimal deleterious effects on embryo production and early embryonic development. To mount permeabilized embryos for acute drug addition in conjunction with live imaging, we combined optimized inhibition of one of these genes with the use of a microfabricated chamber that we designed. We demonstrate that these two developments enable the temporally controlled introduction of inhibitors for mechanistic studies. This method should also open new avenues of investigation by allowing profiling and specificity-testing of inhibitors through comparison with genome-wide phenotypic datasets.
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[
Elife,
2018]
Throughout metazoans, germ cells undergo incomplete cytokinesis to form syncytia connected by intercellular bridges. Gamete formation ultimately requires bridge closure, yet how bridges are reactivated to close is not known. The most conserved bridge component is centralspindlin, a complex of the Rho family GTPase-activating protein (GAP) CYK-4/MgcRacGAP and the microtubule motor ZEN-4/kinesin-6. Here, we show that oocyte production by the syncytial <i>C. elegans</i> germline requires CYK-4 but not ZEN-4, which contrasts with cytokinesis, where both are essential. Longitudinal imaging after conditional inactivation revealed that CYK-4 activity is important for oocyte cellularization, but not for the cytokinesis-like events that generate syncytial compartments. CYK-4's lipid-binding C1 domain and the GTPase-binding interface of its GAP domain were both required to target CYK-4 to intercellular bridges and to cellularize oocytes. These results suggest that the conserved C1-GAP region of CYK-4 constitutes a targeting module required for closure of intercellular bridges in germline syncytia.
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Mooney S, Vohra M, Jing B, Salazar DA, Soriano-Castell D, Burlingame AL, Kao AW, Dennissen FJA, Li B, Ashrafi K, Butler VJ, Alves-Ferreira M, Encalada SE, Gutierrez E, Oses-Prieto JA, Li KH, Groisman A, Mandelkow EM, Wang AL
[
Hum Mol Genet,
2018]
Mutations in the microtubule-associated protein tau (MAPT) underlie multiple neurodegenerative disorders, yet the pathophysiological mechanisms are unclear. A novel variant in MAPT resulting in an alanine to threonine substitution at position 152 (A152T tau) has recently been described as a significant risk factor for both frontotemporal lobar degeneration and Alzheimer's disease. Here we use complementary computational, biochemical, molecular, genetic and imaging approaches in Caenorhabditis elegans and mouse models to interrogate the effects of the A152T variant on tau function. In silico analysis suggests that a threonine at position 152 of tau confers a new phosphorylation site. This finding is borne out by mass spectrometric survey of A152T tau phosphorylation in C. elegans and mouse. Optical pulse-chase experiments of Dendra2-tau demonstrate that A152T tau and phosphomimetic A152E tau exhibit increased diffusion kinetics and the ability to traverse across the axon initial segment more efficiently than "wild-type (WT) tau. A C. elegans model of tauopathy reveals that A152T and A152E tau confer patterns of developmental toxicity distinct from WT tau, likely due to differential effects on retrograde axonal transport. These data support a role for phosphorylation of the variant threonine in A152T tau toxicity and suggest a mechanism involving impaired retrograde axonal transport contributing to human neurodegenerative disease.
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Pennington PR, Heistad RM, Nyarko JNK, Barnes JR, Bolanos MAC, Parsons MP, Knudsen KJ, De Carvalho CE, Leary SC, Mousseau DD, Buttigieg J, Maley JM, Quartey MO
[
Sci Rep,
2021]
The pool of -Amyloid (A) length variants detected in preclinical and clinical Alzheimer disease (AD) samples suggests a diversity of roles for A peptides. We examined how a naturally occurring variant, e.g. A(1-38), interacts with the AD-related variant, A(1-42), and the predominant physiological variant, A(1-40). Atomic force microscopy, Thioflavin T fluorescence, circular dichroism, dynamic light scattering, and surface plasmon resonance reveal that A(1-38) interacts differently with A(1-40) and A(1-42) and, in general, A(1-38) interferes with the conversion of A(1-42) to a -sheet-rich aggregate. Functionally, A(1-38) reverses the negative impact of A(1-42) on long-term potentiation in acute hippocampal slices and on membrane conductance in primary neurons, and mitigates an A(1-42) phenotype in Caenorhabditis elegans. A(1-38) also reverses any loss of MTT conversion induced by A(1-40) and A(1-42) in HT-22 hippocampal neurons and APOE 4-positive human fibroblasts, although the combination of A(1-38) and A(1-42) inhibits MTT conversion in APOE 4-negative fibroblasts. A greater ratio of soluble A(1-42)/A(1-38) [and A(1-42)/A(1-40)] in autopsied brain extracts correlates with an earlier age-at-death in males (but not females) with a diagnosis of AD. These results suggest that A(1-38) is capable of physically counteracting, potentially in a sex-dependent manner, the neuropathological effects of the AD-relevant A(1-42).
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[
Worm Breeder's Gazette,
2003]
Wormgenes is a new resource for C.elegans offering a detailed summary about each gene and a powerful query system.
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[
Front Pharmacol,
2020]
Oligomeric assembly of Amyloid- (A) is the main toxic species that contribute to early cognitive impairment in Alzheimer's patients. Therefore, drugs that reduce the formation of A oligomers could halt the disease progression. In this study, by using transgenic <i>Caenorhabditis elegans</i> model of Alzheimer's disease, we investigated the effects of frondoside A, a well-known sea cucumber <i>Cucumaria frondosa</i> saponin with anti-cancer activity, on A aggregation and proteotoxicity. The results showed that frondoside A at a low concentration of 1 M significantly delayed the worm paralysis caused by A aggregation as compared with control group. In addition, the number of A plaque deposits in transgenic worm tissues was significantly decreased. Frondoside A was more effective in these activities than ginsenoside-Rg3, a comparable ginseng saponin. Immunoblot analysis revealed that the level of small oligomers as well as various high molecular weights of A species in the transgenic <i>C. elegans</i> were significantly reduced upon treatment with frondoside A, whereas the level of A monomers was not altered. This suggested that frondoside A may primarily reduce the level of small oligomeric forms, the most toxic species of A. Frondoside A also protected the worms from oxidative stress and rescued chemotaxis dysfunction in a transgenic strain whose neurons express A. Taken together, these data suggested that low dose of frondoside A could protect against A-induced toxicity by primarily suppressing the formation of A oligomers. Thus, the molecular mechanism of how frondoside A exerts its anti-A aggregation should be studied and elucidated in the future.
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[
International Journal of Developmental Biology,
1998]
Pleiotropy , a situation in which a single gene influences multiple phenotypic tra its, can arise in a variety of ways. This paper discusses possible underlying mechanisms and proposes a classification of the various phenomena involved.
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[
Curr Biol,
2011]
Recent work on a Caenorhabditis elegans transmembrane ATPase reveals a central role for the aminophospholipid phosphatidylethanolamine in the production of a class of extracellular vesicles.