scholarly journals Epistatic, Synthetic, and Balancing Interactions among Tubulin Missense Mutations Affecting Neurite Growth in Caenorhabditis elegans

2020 ◽  
pp. mbc.E20-07-0492
Author(s):  
Ho Ming Terence Lee ◽  
Natalie Yvonne Sayegh ◽  
A. Sophia Gayek ◽  
Susan Laura Javier Jao ◽  
Martin Chalfie ◽  
...  
Keyword(s):  
Genetic Interaction ◽  
Neurite Growth ◽  
Phenotypic Characterization ◽  
Missense Mutations ◽  
Loss Of Function ◽  
C Elegans ◽  
Touch Receptor Neurons ◽  
Receptor Neurons ◽  
Tubulin Mutations

Mutations in tubulins affect microtubule (MT) dynamics and functions during neuronal differentiation and their genetic interaction provides insights into the regulation of MT functions. We previously used C. elegans touch receptor neurons to analyze the cellular impact of tubulin mutations and reported the phenotypes of 67 tubulin missense mutations, categorized into three classes: loss-of-function ( lf), antimorphic ( anti), and neomorphic ( neo) alleles. In this study, we isolated 54 additional tubulin alleles through suppressor screens in sensitized backgrounds that caused excessive neurite growth. These alleles included 32 missense mutations not analyzed before, bringing the total number of mutations in our collection to 99. Phenotypic characterization of these newly isolated mutations identified three new types of alleles: partial lf and weak neo alleles of mec-7/β-tubulin that had subtle effects and strong anti alleles of mec-12/α-tubulin. We also discovered complex genetic interactions among the tubulin mutations, including the suppression of neo mutations by intragenic lf and anti alleles, additive and synthetic effects between mec-7 neo alleles, and unexpected epistasis, in which weaker neo alleles masked the effects of stronger neo alleles in inducing ectopic neurite growth. We also observed balancing between neo and anti alleles, whose respective MT-hyperstablizing and -destabilizing effects neutralized each other.

scholarly journals Distinct effects of tubulin isotype mutations on neurite growth in Caenorhabditis elegans

2017 ◽  
Author(s):  
Chaogu Zheng ◽  
Margarete Diaz-Cuadros ◽  
Ken C.Q. Nguyen ◽  
David H. Hall ◽  
Martin Chalfie
Keyword(s):  
Function Analysis ◽  
Critical Role ◽  
Neurite Growth ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Post Translational Modification ◽  
Exterior Surface ◽  
Tubulin Isotypes ◽  
C Elegans ◽  
Tubulin Isotype

AbstractTubulins, the building block of microtubules (MTs), play a critical role in both supporting and regulating neurite growth. Eukaryotic genomes contain multiple tubulin isotypes, and their missense mutations cause a range of neurodevelopmental defects. Using the C. elegans touch receptor neurons, we analyzed the effects of 67 tubulin missense mutations on neurite growth. Three types of mutations emerged: 1) loss-of-function mutations, which cause mild defects in neurite growth; 2) antimorphic mutations, which map to the GTP binding site and intradimer and interdimer interfaces, significantly reduce MT stability, and cause severe neurite growth defects; and 3) neomorphic mutations, which map to the exterior surface, increase MT stability, and cause ectopic neurite growth. Structure-function analysis reveals a causal relationship between tubulin structure and MT stability. This stability affects neuronal morphogenesis. As part of this analysis, we engineered several disease-associated human tubulin mutations into C. elegans genes and examined their impact on neuronal development at the cellular level. We also discovered an α-tubulin (TBA-7) that appears to destabilize MTs. Loss of TBA-7 led to the formation of hyperstable MTs and the generation of ectopic neurites; the lack of potential sites for polyamination and polyglutamination on TBA-7 may be responsible for this destabilization.Table of Content (TOC) Highlight SummaryDifferent tubulin isotypes perform different functions in the regulation of MT structure and neurite growth, and missense mutations of tubulin genes have three types of distinct effects on MT stability and neurite growth. One α-tubulin isotype appears to induce relative instability due to the lack of potential post-translational modification sites.

scholarly journals Distinct effects of tubulin isotype mutations on neurite growth in Caenorhabditis elegans

2017 ◽  
Vol 28 (21) ◽  
pp. 2786-2801 ◽  
Author(s):  
Chaogu Zheng ◽  
Margarete Diaz-Cuadros ◽  
Ken C. Q. Nguyen ◽  
David H. Hall ◽  
Martin Chalfie
Keyword(s):  
Caenorhabditis Elegans ◽  
Neuronal Development ◽  
Function Analysis ◽  
Critical Role ◽  
Neurite Growth ◽  
Cellular Level ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Exterior Surface ◽  
C Elegans

Tubulins, the building block of microtubules (MTs), play a critical role in both supporting and regulating neurite growth. Eukaryotic genomes contain multiple tubulin isotypes, and their missense mutations cause a range of neurodevelopmental defects. Using the Caenorhabditis elegans touch receptor neurons, we analyzed the effects of 67 tubulin missense mutations on neurite growth. Three types of mutations emerged: 1) loss-of-function mutations, which cause mild defects in neurite growth; 2) antimorphic mutations, which map to the GTP binding site and intradimer and interdimer interfaces, significantly reduce MT stability, and cause severe neurite growth defects; and 3) neomorphic mutations, which map to the exterior surface, increase MT stability, and cause ectopic neurite growth. Structure-function analysis reveals a causal relationship between tubulin structure and MT stability. This stability affects neuronal morphogenesis. As part of this analysis, we engineered several disease-associated human tubulin mutations into C. elegans genes and examined their impact on neuronal development at the cellular level. We also discovered an α-tubulin (TBA-7) that appears to destabilize MTs. Loss of TBA-7 led to the formation of hyperstable MTs and the generation of ectopic neurites; the lack of potential sites for polyamination and polyglutamination on TBA-7 may be responsible for this destabilization.

scholarly journals Functional Characterization of the Adenylyl Cyclase Genesgs-1by Analysis of a Mutational Spectrum inCaenorhabditis elegans

Genetics ◽  
2002 ◽  
Vol 161 (1) ◽  
pp. 133-142 ◽  
Author(s):  
Celine Moorman ◽  
Ronald H A Plasterk
Keyword(s):  
Life Span ◽  
Adenylyl Cyclase ◽  
Neuronal Degeneration ◽  
Functional Characterization ◽  
Adenylyl Cyclases ◽  
Loss Of Function ◽  
C Elegans ◽  
Larval Stages ◽  
Pharyngeal Pumping

AbstractThe sgs-1 (suppressor of activated Gαs) gene encodes one of the four adenylyl cyclases in the nematode C. elegans and is most similar to mammalian adenylyl cyclase type IX. We isolated a complete loss-of-function mutation in sgs-1 and found it to result in animals with retarded development that arrest in variable larval stages. sgs-1 mutant animals exhibit lethargic movement and pharyngeal pumping and (while not reaching adulthood) have a mean life span that is >50% extended compared to wild type. An extensive set of reduction-of-function mutations in sgs-1 was isolated in a screen for suppressors of a neuronal degeneration phenotype induced by the expression of a constitutively active version of the heterotrimeric Gαs subunit of C. elegans. Although most of these mutations change conserved residues within the catalytic domains of sgs-1, mutations in the less-conserved transmembrane domains are also found. The sgs-1 reduction-of-function mutants are viable and have reduced locomotion rates, but do not show defects in pharyngeal pumping or life span.

scholarly journals glp-3 Is Required for Mitosis and Meiosis in the Caenorhabditis elegans Germ Line

Genetics ◽  
1997 ◽  
Vol 145 (1) ◽  
pp. 111-121 ◽  
Author(s):  
Lisa C Kadyk ◽  
Eric J Lambie ◽  
Judith Kimble
Keyword(s):  
Caenorhabditis Elegans ◽  
Germ Cells ◽  
Germ Line ◽  
Stem Cell Population ◽  
Phenotypic Characterization ◽  
Loss Of Function ◽  
Dapi Staining ◽  
Cell Cycles ◽  
C Elegans ◽  
Mitosis And Meiosis

The germ line is the only tissue in Caenorhabditis elegans in which a stem cell population continues to divide mitotically throughout life; hence the cell cycles of the germ line and the soma are regulated differently. Here we report the genetic and phenotypic characterization of the glp-3 gene. In animals homozygous for each of five recessive loss-of-function alleles, germ cells in both hermaphrodites and males fail to progress through mitosis and meiosis, but somatic cells appear to divide normally. Germ cells in animals grown at 15° appear by DAPI staining to be uniformly arrested at the G2/M transition with <20 germ cells per gonad on average, suggesting a checkpoint-mediated arrest. In contrast, germ cells in mutant animals grown at 25° frequently proliferate slowly during adulthood, eventually forming small germ lines with several hundred germ cells. Nevertheless, cells in these small germ lines never undergo meiosis. Double mutant analysis with mutations in other genes affecting germ cell proliferation supports the idea that glp-3 may encode a gene product that is required for the mitotic and meiotic cell cycles in the C. elegans germ line.

scholarly journals Molecular Characterization of eutF Mutants ofSalmonella typhimurium LT2 Identifies eutFLesions as Partial-Loss-of-Function tonB Alleles

1999 ◽  
Vol 181 (2) ◽  
pp. 368-374 ◽  
Author(s):  
Michael G. Thomas ◽  
George A. O’Toole ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Amino Acid ◽  
Molecular Characterization ◽  
Immunoblot Analysis ◽  
Phenotypic Characterization ◽  
Loss Of Function ◽  
Wild Type ◽  
Partial Loss ◽  
Acid Addition ◽  
Fusion Site

ABSTRACT The eutF locus of Salmonella typhimuriumLT2 was identified as a locus necessary for the utilization of ethanolamine as a sole carbon source. Initial models suggested that EutF was involved in either ethanolamine transport or was a transcriptional regulator of an ethanolamine transporter. Phenotypic characterization of eutF mutants suggested EutF was somehow involved in 1,2-propanediol, propionate, and succinate utilization. Here we provide evidence that two alleles defining the eutFlocus, Δ903 and eutF1115, are partial-loss-of-function tonB alleles. Both mutations were complemented by plasmids containing a wild-type allele of theEscherichia coli tonB gene. Immunoblot analysis using TonB monoclonal antibodies detected a TonB fusion protein in strains carrying eutF alleles. Molecular analysis of the Δ903 allele identified a deletion that resulted in the fusion of the 3′ end of tonB with the 3′ end oftrpA. In-frame translation of the tonB-trpAfusion resulted in the final 9 amino acids of TonB being replaced by a 45-amino-acid addition. We isolated a derivative of a strain carrying allele Δ903 that regained the ability to grow on ethanolamine as a carbon and energy source. The molecular characterization of the mutation that corrected the Eut−phenotype caused by allele Δ903 showed that the new mutation was a deletion of two nucleotides at the tonB-trpAfusion site. This deletion resulted in a frameshift that replaced the 45-amino-acid addition with a 5-amino-acid addition. This change resulted in a TonB protein with sufficient activity to restore growth on ethanolamine and eut operon expression to nearly wild-type levels. It was concluded that the observed EutF phenotypes were due to the partial loss of TonB function, which is proposed to result in reduced cobalamin and ferric siderophore transport in an aerobic environment; thus, the eutF locus does not exist.

scholarly journals Neuronal KGB-1 JNK MAPK signaling regulates the dauer developmental decision in response to environmental stress in C. elegans

2021 ◽  
Author(s):  
Deepshikha Dogra ◽  
Warakorn Kulalert ◽  
Frank Schroeder ◽  
Dennis H Kim
Keyword(s):  
Elevated Temperature ◽  
Signaling Pathways ◽  
Mapk Pathway ◽  
Negative Regulator ◽  
Loss Of Function ◽  
C Elegans ◽  
Metabolic Remodeling ◽  
Dauer Formation ◽  
Identification And Characterization

In response to stressful growth conditions of high population density, food scarcity and elevated temperature, young larvae of nematode Caenorhabditis elegans can enter a developmentally arrested stage called dauer that is characterized by dramatic anatomic and metabolic remodeling. Genetic analysis of dauer formation of C. elegans has served as an experimental paradigm for the identification and characterization of conserved neuroendocrine signaling pathways. Here, we report the identification and characterization of a conserved JNK-like mitogen-activated protein kinase (MAPK) pathway that is required for dauer formation in response to environmental stressors. We observed that loss-of-function mutations in the MLK-1-MEK-1-KGB-1 MAPK pathway suppress dauer entry. Loss-of-function mutation in the VHP-1 MAPK phosphatase, a known negative regulator of KGB-1 signaling, results in constitutive dauer formation which is dependent on the presence of dauer pheromone but independent of diminished food levels or elevated temperatures. Our data suggest that KGB-1 pathway acts in the sensory neurons, in parallel to established insulin and TGF-β signaling pathways, to transduce the dauer-inducing environmental cues of diminished food levels and elevated temperature.

scholarly journals Touch-induced Mechanical Strain in Somatosensory Neurons is Independent of Extracellular Matrix Mutations in C. elegans

2019 ◽  
Author(s):  
Adam L. Nekimken ◽  
Beth L. Pruitt ◽  
Miriam B. Goodman
Keyword(s):  
Extracellular Matrix ◽  
Fluorescent Protein ◽  
Mechanical Strain ◽  
C Elegans ◽  
Mechanosensory Neurons ◽  
State Touch ◽  
Touch Receptor Neurons ◽  
Somatosensory Neurons ◽  
Measured Strain ◽  
Receptor Neurons

AbstractCutaneous mechanosensory neurons are activated by mechanical loads applied to the skin, and these stimuli are proposed to generate mechanical strain within sensory neurons. Using a microfluidic device to deliver controlled stimuli to intact animals and large, immobile, and fluorescent protein-tagged mitochondria as fiducial markers in the touch receptor neurons (TRNs), we visualized and measured touch-induced mechanical strain in C. elegans worms. At steady-state, touch stimuli sufficient to activate TRNs induce an average strain of 3.1% at the center of the actuator and this strain decays to near zero at the edges of the actuator. We also measured strain in animals carrying mutations affecting links between the extracellular matrix (ECM) and the TRNs but could not detect any differences in touch-induced mechanical strain between wild-type and mutant animals. Collectively, these results demonstrate that touching the skin induces local mechanical strain in intact animals and suggest that a fully intact ECM is not essential for transmitting mechanical strain from the skin to cutaneous mechanosensory neurons.

scholarly journals Nine Novel PAX9 Mutations and a Distinct Tooth Agenesis Genotype-Phenotype

2017 ◽  
Vol 97 (2) ◽  
pp. 155-162 ◽  
Author(s):  
S.-W. Wong ◽  
D. Han ◽  
H. Zhang ◽  
Y. Liu ◽  
X. Zhang ◽  
...  
Keyword(s):  
Dna Analysis ◽  
Direct Sequencing ◽  
Taste Perception ◽  
Phenotypic Characterization ◽  
Tooth Agenesis ◽  
Reaction Products ◽  
Loss Of Function ◽  
Second Molar ◽  
Functional Studies

Tooth agenesis is one of the most common developmental anomalies affecting function and esthetics. The paired-domain transcription factor, Pax9, is critical for patterning and morphogenesis of tooth and taste buds. Mutations of PAX9 have been identified in patients with tooth agenesis. Despite significant progress in the genetics of tooth agenesis, many gaps in knowledge exist in refining the genotype-phenotype correlation between PAX9 and tooth agenesis. In the present study, we complete genetic and phenotypic characterization of multiplex Chinese families with nonsyndromic (NS) tooth agenesis. Direct sequencing of polymerase chain reaction products revealed 9 novel (c.140G>C, c.167T>A, c.332G>C, c.194C>A, c.271A>T, c.146delC, c.185_189dup, c.256_262dup, and c.592delG) and 2 known heterozygous mutations in the PAX9 gene among 120 probands. Subsequently, pedigrees were extended, and we confirmed that the mutations co-segregated with the tooth agenesis phenotype (with exception of families in which DNA analysis was not available). In 1 family ( n = 6), 2 individuals harbored both the PAX9 c.592delG mutation and a heterozygous missense mutation (c.739C>T) in the MSX1 gene. Clinical characterization of families segregating a PAX9 mutation reveal that all affected individuals were missing the mandibular second molar and their maxillary central incisors are most susceptible to microdontia. A significant reduction of bitter taste perception was documented in individuals harboring PAX9 mutations ( n = 3). Functional studies revealed that PAX9 haploinsufficiency or a loss of function of the PAX9 protein underlies tooth agenesis.

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