scholarly journals Identifying C. elegans Lifespan Mutants by Screening for Early-Onset Protein Aggregation

2021 ◽  
Author(s):  
Daniel F. Midkiff ◽  
Adriana San Miguel
Keyword(s):  
Protein Aggregation ◽  
High Throughput ◽  
Time Dependent ◽  
Biological Functions ◽  
Genetic Screens ◽  
High Throughput Analysis ◽  
Genetic Pathways ◽  
C Elegans ◽  
Forward Genetic Screens ◽  
Aggregate Growth

Genetic screens have been widely used to identify genetic pathways that control specific biological functions. In C. elegans, forward genetic screens rely on the isolation of reproductively active mutants that can self-propagate clonal populations. Since aged individuals are unable to generate clonal populations, screens that target post-reproductive phenotypes, such as longevity, are challenging. In this work, we developed an approach that combines microfluidic technologies and image processing to perform a high-throughput, automated screen for mutants with shortened lifespan using protein aggregation as a marker for aging. We take advantage of microfluidics for maintaining a reproductively-active adult mutagenized population and for performing serial high-throughput analysis and sorting of animals with increased protein aggregation, using fluorescently labeled PAB-1 as a readout. We identified five mutants with increased aggregation levels, of which two exhibited a reduced lifespan. We demonstrate that lifespan mutants can be identified by screening for accelerated protein aggregation through quantitative analysis of fluorescently-labeled aggregates in populations that do not require conditional sterilization or manual separation of parental and progeny populations. We further analyzed the morphology of protein aggregates and reveal that patterns of aggregation in naturally-aging animals differ from mutants with increased aggregation, suggesting aggregate growth is time-dependent. This screening approach can be customized to other non-developmental phenotypes that appear during adulthood, as well as to other aging markers to identify additional longevity-regulating genetic pathways.

scholarly journals Dendrites with specialized glial attachments develop by retrograde extension using SAX-7 and GRDN-1

2019 ◽  
Author(s):  
Elizabeth R. Cebul ◽  
Ian G. McLachlan ◽  
Maxwell G. Heiman
Keyword(s):  
Glial Cell ◽  
Molecular Mechanism ◽  
Mammalian Brain ◽  
Cytoplasmic Protein ◽  
Genetic Screens ◽  
Sense Organs ◽  
Adhesion Protein ◽  
C Elegans ◽  
Dendrite Development ◽  
Forward Genetic Screens

ABSTRACTDendrites develop elaborate morphologies in concert with surrounding glia, but the molecules that coordinate dendrite and glial morphogenesis are mostly unknown.C. elegansoffers a powerful model for identifying such factors. Previous work in this system examined dendrites and glia that develop within epithelia, similar to mammalian sense organs. Here, we focus on the neurons BAG and URX, which are not part of an epithelium but instead form membranous attachments to a single glial cell at the nose, reminiscent of dendrite-glia contacts in the mammalian brain. We show that these dendrites develop by retrograde extension, in which the nascent dendrite endings anchor to the presumptive nose and then extend by stretch during embryo elongation. Using forward genetic screens, we find that dendrite development requires the adhesion protein SAX-7/L1CAM and the cytoplasmic protein GRDN-1/CCDC88C to anchor dendrite endings at the nose. SAX-7 acts in neurons and glia, while GRDN-1 acts in glia to non-autonomously promote dendrite extension. Thus, this work shows how glial factors can help to shape dendrites, and identifies a novel molecular mechanism for dendrite growth by retrograde extension.

scholarly journals ADAMTS-family protease MIG-17 regulates synaptic allometry by modifying the extracellular matrix and modulating glia morphology during growth

2019 ◽  
Author(s):  
Tingting Ji ◽  
Kai Wang ◽  
Jiale Fan ◽  
Jichang Huang ◽  
Mengqing Wang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Extracellular Matrix ◽  
Collagen Type ◽  
Collagen Type Iv ◽  
Genetic Screens ◽  
Genetic Studies ◽  
C Elegans ◽  
Type Iv ◽  
Forward Genetic Screens ◽  
Signaling Axis

ABSTRACTSynapses are largely established during embryogenesis and maintained during growth. The mechanisms that regulate synaptic allometry—the maintenance of synaptic positions during growth—are largely unknown. We performed forward genetic screens inC. elegansfor synaptic allometry mutants and identifiedmig-17, a secreted metalloprotease of the conserved ADAMTS family. Through proteomic mass spectrometry analyses, cell biological and genetic studies we determined that MIG-17 is expressed by muscle cells to modulate glia location and morphology. Glia are proximal to synapses, and the glial location and morphology determine synaptic position during growth.Mig-17regulates synapse allometry by influencing epidermal-glia crosstalk through the regulation of basement membrane proteins, including collagen type IV, SPARC and fibulin. Our findings underscore the importance of glia location in the maintenance of synaptic allometry, and uncover a muscle-epidermal-glia signaling axis, mediated through the extracellular matrix, in the regulation of glia morphology and synaptic positions during growth.

Identification of time-dependent biomarkers and effects of exposure to volatile organic compounds using high-throughput analysis

2015 ◽  
Vol 31 (11) ◽  
pp. 1563-1570 ◽  
Author(s):  
Ji Young Hong ◽  
So Yeon Yu ◽  
Gi Won Kim ◽  
Jeong Jin Ahn ◽  
Youngjoo Kim ◽  
...  
Keyword(s):  
Volatile Organic Compounds ◽  
Organic Compounds ◽  
High Throughput ◽  
Time Dependent ◽  
High Throughput Analysis ◽  
Throughput Analysis ◽  
Volatile Organic

scholarly journals A sensitized genetic screen to identify regulators of C. elegans germline stem cells

2021 ◽  
Author(s):  
Sarah Robinson-Thiewes ◽  
Aaron M Kershner ◽  
Heaji Shin ◽  
Kimberly A Haupt ◽  
Peggy Kroll-Connor ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dna Polymerase ◽  
Transcriptional Activation ◽  
Regulatory Network ◽  
Genetic Screen ◽  
Germline Stem Cells ◽  
Meiotic Cell ◽  
Genetic Screens ◽  
C Elegans ◽  
Forward Genetic Screens

Abstract GLP-1/Notch signaling and a downstream RNA regulatory network maintain germline stem cells (GSCs) in Caenorhabditis elegans. In mutants lacking the GLP-1 receptor, all GSCs enter the meiotic cell cycle precociously and differentiate into sperm. This dramatic GSC defect is called the “Glp” phenotype. The lst-1 and sygl-1 genes are direct targets of Notch transcriptional activation and functionally redundant. Whereas single lst-1 and sygl-1 mutants are fertile, lst-1 sygl-1 double mutants are sterile with a Glp phenotype. We set out to identify genes that function redundantly with either lst-1 or sygl-1 to maintain GSCs. To this end, we conducted forward genetic screens for mutants with a Glp phenotype in genetic backgrounds lacking functional copies of either lst-1 or sygl-1. The screens generated nine glp-1 alleles, two lst-1 alleles, and one allele of pole-1, which encodes the catalytic subunit of DNA polymerase ε. Three glp-1 alleles reside in Ankyrin (ANK) repeats not previously mutated. pole-1 single mutants have a low penetrance Glp phenotype that is enhanced by loss of sygl-1. Thus, the screen uncovered one locus that interacts genetically with sygl-1 and generated useful mutations for further studies of GSC regulation.

scholarly journals Retrograde Transport and ATG-4.2-Mediated Maturation Cooperate to Remove Autophagosomes from the Synapse

2018 ◽  
Author(s):  
Sarah E. Hill ◽  
Daniel A. Colón-Ramos
Keyword(s):  
Cell Body ◽  
Retrograde Transport ◽  
List Type ◽  
Single Neurons ◽  
Genetic Screens ◽  
Autophagosome Formation ◽  
C Elegans ◽  
Forward Genetic Screens ◽  
Autophagosome Maturation

SUMMARYAutophagy is spatially compartmentalized in neurons, with autophagosome biogenesis occurring in the axon and degradation in the cell body. The mechanisms that coordinate autophagosome formation, trafficking and degradation across the polarized structure of the neuron are not well understood. Here we use genetic screens and in vivo imaging in single neurons of C. elegans to demonstrate that specific steps of autophagy are differentially required in distinct subcellular compartments of the neuron. We demonstrate that completion of autophagosome biogenesis and closure at the synapse are necessary for dynein-mediated retrograde transport. We uncover a role for UNC-16/JIP3/Sunday Driver in facilitating autophagosome retrograde transport. Through forward genetic screens we then determine that autophagosome maturation and degradation in the cell body depend on removal of LGG-1/Atg8/GABARAP from autophagosomes by the protease ATG-4.2. Our studies reveal that regulation of distinct ATG4 proteases contributes to the coordination of autophagy across subcellular regions of the neuron.HIGHLIGHTS and eTOC BlurbAutophagosome closure, but not maturation, occurs locally at presynaptic sitesRetrograde transport of autophagosomes requires the motor adaptor UNC-16/JIP3The autophagy protease ATG-4.2, but not the related ATG-4.1, is required for autophagosome maturation and degradationDefects in retrograde transport and maturation genetically interact and enhance accumulation of autophagosomes in presynaptic regions

Computer-enhanced high-throughput genetic screens of C. elegans in a microfluidic system

Lab on a Chip ◽  
2009 ◽  
Vol 9 (1) ◽  
pp. 38-40 ◽  
Author(s):  
Matthew M. Crane ◽  
Kwanghun Chung ◽  
Hang Lu
Keyword(s):  
High Throughput ◽  
Microfluidic System ◽  
Genetic Screens ◽  
C Elegans

scholarly journals Clarinet (CLA-1), a novel active zone protein required for synaptic vesicle clustering and release

2017 ◽  
Author(s):  
Zhao Xuan ◽  
Laura Manning ◽  
Jessica Nelson ◽  
Janet E. Richmond ◽  
Daniel Colón-Ramos ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Active Zone ◽  
Neurotransmitter Release ◽  
Synaptic Vesicles ◽  
Synaptic Depression ◽  
Genetic Screens ◽  
C2 Domains ◽  
Specific Loss ◽  
C Elegans ◽  
Forward Genetic Screens

AbstractActive zone proteins cluster synaptic vesicles at presynaptic terminals and coordinate their release. In forward genetic screens we isolated a novel C. elegans active zone gene, clarinet (cla-1). cla-1 mutants exhibit defects in synaptic vesicle clustering, reduced spontaneous neurotransmitter release, increased synaptic depression and reduced synapse number. Ultrastructurally, cla-1 mutants have fewer synaptic vesicles adjacent to the dense projection and an increased number of docked vesicles. Cla-1 encodes 3 isoforms containing common C-terminal PDZ and C2 domains with homology to vertebrate active zone proteins Piccolo and RIM. The short isoform localizes exclusively to the active zone while a longer ~9000 amino acid isoform colocalizes with synaptic vesicles. Specific loss of CLA-1L results in synaptic vesicle clustering defects and increased synaptic depression, but not in reduced synapse number or mini frequency. Together our data indicate that specific isoforms of clarinet serve distinct functions, regulating synapse development, synaptic vesicle clustering and release.

scholarly journals Necessity and contingency in developmental genetic screens: LIN-3, Wnt and semaphorin pathways in vulval induction of the nematode Oscheius tipulae

2018 ◽  
Author(s):  
Amhed M. Vargas-Velazquez ◽  
Fabrice Besnard ◽  
Marie-Anne Félix
Keyword(s):  
Caenorhabditis Elegans ◽  
Cell Fate ◽  
Wnt Pathway ◽  
Precursor Cell ◽  
Genetic Screens ◽  
C Elegans ◽  
Forward Genetic Screens ◽  
Anchor Cell ◽  
Notch Pathways ◽  
Vulval Precursor Cell

AbstractGenetic screens in the nematode Caenorhabditis elegans identified the EGF/Ras and Notch pathways as central for vulval precursor cell fate patterning. Schematically, the anchor cell secretes EGF, inducing the P6.p cell to a 1° vulval fate; P6.p in turn induces its neighbors to a 2° fate through Delta-Notch signaling and represses Ras signaling. In the nematode Oscheius tipulae, the anchor cell successively induces 2° then 1° vulval fates. Here we report on the molecular identification of mutations affecting vulval induction in O. tipulae. A single Induction Vulvaless mutation was found, which we identify as a cis-regulatory deletion in a tissue-specific enhancer of the O. tipulae lin-3 homolog, confirmed by CRISPR/Cas9 mutation. In contrast to this predictable Vulvaless mutation, mutations resulting in an excess of 2° fates unexpectedly correspond to the plexin/semaphorin pathway, which was not implicated in vulval fate induction in C. elegans. Hyperinduction of P4.p and P8.p in these mutants likely results from mispositioning of these cells due to a lack of contact inhibition. The third signaling pathway found by forward genetics in O. tipulae is the Wnt pathway: decrease in Wnt pathway activity results in loss of vulval precursor competence and induction, and 1° fate miscentering on P5.p. Our results suggest that the EGF and Wnt pathways have qualitatively similar activities in vulval induction in C. elegans and O. tipulae, albeit with quantitative differences in the effects of mutation. This study highlights both necessity and contingency in forward genetic screens.100-word summaryGenetic screens in the nematode Caenorhabditis elegans identified EGF and Notch pathways as key for vulval precursor cell fate patterning. Here we report on the molecular identification of mutations affecting vulval induction in another nematode, Oscheius tipulae. The single mutation with reduced induction is identified as a cis-regulatory deletion in the O. tipulae lin-3 homolog, confirmed by CRISPR/Cas9 mutation. In contrast to this predictable Vulvaless mutation, mutations resulting in an excess of 2° vulval fates unexpectedly correspond to the plexin/semaphorin pathway, not implicated in vulval induction in C. elegans. This study highlights both necessity and contingency in forward genetic screens.

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