Nanoscale Structure and Mechanics of Skin in a C. elegans Model of Touch Sensation

Abstract Background and Aims NPHS2 is the most frequently mutated gene in steroid-resistant nephrotic syndrome. NPHS2 encodes podocin, a key component of the glomerular filtration barrier. Podocin is a 42 kDa integral membrane protein, which accumulates in lipid raft microdomains at the podocyte slit diaphragm and is known to form oligomers. The c.686G>A, p.R229Q is the most common non-silent variant of NPHS2. We formerly described that R229Q is pathogenic only when trans-associated to specific 3’ missense mutations on the other parental allele. These C-terminal podocin mutants exert a dominant negative effect on R229Q podocin and retain it in intracellular compartments. As such, R229Q is the first variant in human genetics with a mutation-dependent pathogenicity. Based on FRET analysis and structural modeling, we formerly showed that the dominant negative effect is mediated by an altered oligomerization. However, the pathogenicity of several [mutation];[R229Q] associations is still in question and the identification of other rare NPHS2 variants with a mutation-dependent pathogenicity require an in vivo model. We therefore aimed to generate a Caenorhabditis elegans model, deficient for the homologous gene of NPHS2 (mec-2) and coexpressing differently (GFP- and mCherry-) tagged human podocin pairs. MEC-2 shares 45% identity and 83% similarity over 275aa (72%) of podocin (383aa). Expressed in six neurons, it is responsible for gentle-touch mechanosensation. The mec-2 mutants are insensitive to gentle touch in the center part of the body. Method Vectors encoding C-terminal GFP- or mCherry-tagged MEC-2 or human podocin with C. elegans codon optimization under mec-2 promoter and a selection marker (unc-119) were generated. Double (mec-2 and unc-119) mutant strains were established. Mutant strains were transformed by microparticle bombardment. The gentle-touch mechanosensation was examined by cat’s whiskers in a blinded experiment. Results The expression pattern of GFP and mCherry under mec-2 promoter corresponded to the six neurons responsible for gentle-touch sensation, indicating the proper functioning of the promoter. Strains with extrachromosomal MEC-2 or podocin coding vectors were successfully established (GFP-tagged MEC-2: n= 55 strains, GFP-tagged podocin: n= 81, mCherry-tagged podocin: n= 18). However, we found no rescue of the gentle touch sensation in any of them in blinded experiments. We hypothesized that either the fluorescent tag or the lack of chromosomal integration prevents the rescue effect of MEC-2. We therefore aimed to achieve self-cleaving of MEC-2 and the fluorescent tag, and inserted a T2A self-cleaving peptide-encoding sequence between them. To achieve chromosomal integration, we are implementing the MosSCI (Mos1-mediated Single Copy Insertion) technique. Conclusion Once the rescue with MEC-2 is achieved, the rescue effect of wild type and next different human podocin variant(s) will be aimed to analyze. The generation of the first animal model to study human interallelic interactions is challenging.

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The Tactile Receptive Fields of Freely Moving Caenorhabditis elegans Nematodes

10.1101/259937 ◽

2018 ◽

Author(s):

E. A. Mazzochette ◽

A. L. Nekimken ◽

F. Loizeau ◽

J. Whitworth ◽

B. Huynh ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Receptive Fields ◽

Behavioral Responses ◽

The Body ◽

Stimulus Strength ◽

Wild Type ◽

C Elegans ◽

Speed Up ◽

Touch Sensation ◽

Freely Moving

AbstractSensory neurons embedded in skin are responsible for the sense of touch. In humans and other mammals, touch sensation depends on thousands of diverse somatosensory neurons. By contrast, Caenorhabditis elegans nematodes have six gentle touch receptor neurons linked to simple behaviors. The classical touch assay uses an eyebrow hair to stimulate freely moving C. elegans, evoking evasive behavioral responses. While this assay has led to the discovery of genes required for touch sensation, it does not provide control over stimulus strength or position. Here, we present an integrated system for performing automated, quantitative touch assays that circumvents these limitations and incorporates automated measurements of behavioral responses. Highly Automated Worm Kicker (HAWK) unites microfabricated silicon force sensors and video analysis with real-time force and position control. Using this system, we stimulated animals along the anterior-posterior axis and compared responses in wild-type and spc-1(dn) transgenic animals, which have a touch defect due to expression of a dominant-negative α spectrin protein fragment. As expected from prior studies, delivering large stimuli anterior to the mid-point of the body evoked a reversal, but such a stimulus applied posterior to the mid-point evoked a speed-up. The probability of evoking a response of either kind depended on stimulus strength and location; once initiated, the magnitude and quality of both reversal and speed-up behavioral responses were uncorrelated with stimulus location, strength, or the absence or presence of the spc-1(dn) transgene. Wild-type animals failed to respond when the stimulus was applied near the mid-point. These results establish that stimulus strength and location govern the activation of a stereotyped motor program and that the C. elegans body surface consists of two receptive fields separated by a gap.

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In Vivo Imaging of C. elegans Mechanosensory Neurons Demonstrates a Specific Role for the MEC-4 Channel in the Process of Gentle Touch Sensation

Neuron ◽

10.1016/j.neuron.2003.08.015 ◽

2003 ◽

Vol 39 (6) ◽

pp. 1005-1017 ◽

Cited By ~ 186

Author(s):

Hiroshi Suzuki ◽

Rex Kerr ◽

Laura Bianchi ◽

Christian Frøkjær-Jensen ◽

Dan Slone ◽

...

Keyword(s):

In Vivo Imaging ◽

Specific Role ◽

C Elegans ◽

Mechanosensory Neurons ◽

Touch Sensation

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Faculty Opinions recommendation of In vivo imaging of C. elegans mechanosensory neurons demonstrates a specific role for the MEC-4 channel in the process of gentle touch sensation.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1000043.201164 ◽

2003 ◽

Author(s):

Ellen A Lumpkin

Keyword(s):

In Vivo Imaging ◽

Specific Role ◽

C Elegans ◽

Mechanosensory Neurons ◽

Touch Sensation

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Body Mechanics Regulate the Force Threshold for Gentle Touch Sensation in the Nematode C. Elegans

Biophysical Journal ◽

10.1016/j.bpj.2011.11.185 ◽

2012 ◽

Vol 102 (3) ◽

pp. 29a

Author(s):

Bryan C. Petzold ◽

Sung-Jin Park ◽

Miriam B. Goodman ◽

Beth L. Pruitt

Keyword(s):

C Elegans ◽

Touch Sensation

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Spatial Asymmetry in the Mechanosensory Phenotypes of the C. elegans DEG/ENaC Gene mec-10

Journal of Neurophysiology ◽

10.1152/jn.00330.2010 ◽

2010 ◽

Vol 104 (6) ◽

pp. 3334-3344 ◽

Cited By ~ 20

Author(s):

Marios Chatzigeorgiou ◽

Laura Grundy ◽

Katie S. Kindt ◽

Wei-Hsiang Lee ◽

Monica Driscoll ◽

...

Keyword(s):

Cell Body ◽

Neuronal Cell ◽

The Body ◽

Neuronal Cell Body ◽

Mechanical Stimuli ◽

C Elegans ◽

Mechanosensory Transduction ◽

Proximal Dendrite ◽

Touch Sensation ◽

Asymmetrically Distributed

DEG/ENaC channels have been broadly implicated in mechanosensory transduction, yet many questions remain about how these proteins contribute to complexes that sense mechanical stimuli. In C. elegans, two DEG/ENaC channel subunits are thought to contribute to a gentle touch transduction complex: MEC-4, which is essential for gentle touch sensation, and MEC-10, whose importance is less well defined. By characterizing a mec-10 deletion mutant, we have found that MEC-10 is important, but not essential, for gentle touch responses in the body touch neurons ALM, PLM, and PVM. Surprisingly, the requirement for MEC-10 in ALM and PLM is spatially asymmetric; mec-10 animals show significant behavioral and physiological responses to stimulation at the distal end of touch neuron dendrites, but respond poorly to stimuli applied near the neuronal cell body. The subcellular distribution of a rescuing MEC-10::GFP translational fusion was found to be restricted to the neuronal cell body and proximal dendrite, consistent with the hypothesis that MEC-10 protein is asymmetrically distributed within the touch neuron process. These results suggest that MEC-10 may contribute to only a subset of gentle touch mechanosensory complexes found preferentially at the proximal dendrite.

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The glycomes of Caenorhabditis elegans and other model organisms

Biochemical Society Symposium ◽

10.1042/bss0690117 ◽

2002 ◽

Vol 69 ◽

pp. 117-134 ◽

Cited By ~ 47

Author(s):

Stuart M. Haslam ◽

David Gems ◽

Howard R. Morris ◽

Anne Dell

Keyword(s):

Caenorhabditis Elegans ◽

Current Knowledge ◽

Structural Data ◽

Model Organisms ◽

Entire Genome ◽

C Elegans ◽

The Face ◽

Area Of Concern ◽

Nematode Worm

There is no doubt that the immense amount of information that is being generated by the initial sequencing and secondary interrogation of various genomes will change the face of glycobiological research. However, a major area of concern is that detailed structural knowledge of the ultimate products of genes that are identified as being involved in glycoconjugate biosynthesis is still limited. This is illustrated clearly by the nematode worm Caenorhabditis elegans, which was the first multicellular organism to have its entire genome sequenced. To date, only limited structural data on the glycosylated molecules of this organism have been reported. Our laboratory is addressing this problem by performing detailed MS structural characterization of the N-linked glycans of C. elegans; high-mannose structures dominate, with only minor amounts of complex-type structures. Novel, highly fucosylated truncated structures are also present which are difucosylated on the proximal N-acetylglucosamine of the chitobiose core as well as containing unusual Fucα1–2Gal1–2Man as peripheral structures. The implications of these results in terms of the identification of ligands for genomically predicted lectins and potential glycosyltransferases are discussed in this chapter. Current knowledge on the glycomes of other model organisms such as Dictyostelium discoideum, Saccharomyces cerevisiae and Drosophila melanogaster is also discussed briefly.

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How do histone modifications contribute to transgenerational epigenetic inheritance in C. elegans?

Biochemical Society Transactions ◽

10.1042/bst20190944 ◽

2020 ◽

Vol 48 (3) ◽

pp. 1019-1034 ◽

Cited By ~ 1

Author(s):

Rachel M. Woodhouse ◽

Alyson Ashe

Keyword(s):

Histone Modifications ◽

Molecular Mechanisms ◽

Epigenetic Inheritance ◽

Nematode Caenorhabditis Elegans ◽

Histone Methyltransferases ◽

Transgenerational Epigenetic Inheritance ◽

C Elegans ◽

Recent Developments ◽

Gene Regulatory

Gene regulatory information can be inherited between generations in a phenomenon termed transgenerational epigenetic inheritance (TEI). While examples of TEI in many animals accumulate, the nematode Caenorhabditis elegans has proven particularly useful in investigating the underlying molecular mechanisms of this phenomenon. In C. elegans and other animals, the modification of histone proteins has emerged as a potential carrier and effector of transgenerational epigenetic information. In this review, we explore the contribution of histone modifications to TEI in C. elegans. We describe the role of repressive histone marks, histone methyltransferases, and associated chromatin factors in heritable gene silencing, and discuss recent developments and unanswered questions in how these factors integrate with other known TEI mechanisms. We also review the transgenerational effects of the manipulation of histone modifications on germline health and longevity.

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