Quantitative fluorescence imaging of mitochondria in body wall muscles of Caenorhabditis elegans under hyperglycemic conditions using a microfluidic chip

2020 ◽  
Vol 12 (6) ◽  
pp. 150-160 ◽  
Author(s):  
Samuel Sofela ◽  
Sarah Sahloul ◽  
Sukanta Bhattacharjee ◽  
Ambar Bose ◽  
Ushna Usman ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Fluorescence Imaging ◽  
Microfluidic Device ◽  
Microfluidic Chip ◽  
Body Wall ◽  
The Body ◽  
C Elegans ◽  
Technological Advantage ◽  
Body Wall Muscles ◽  
Quantitative Fluorescence

Abstract Type 2 diabetes is the most common metabolic disease, and insulin resistance plays a role in the pathogenesis of the disease. Because completely functional mitochondria are necessary to obtain glucose-stimulated insulin from pancreatic beta cells, dysfunction of mitochondrial oxidative pathway could be involved in the development of diabetes. As a simple animal model, Caenorhabditis elegans renders itself to investigate such metabolic mechanisms because it possesses insulin/insulin-like growth factor-1 signaling pathway similar to that in humans. Currently, the widely spread agarose pad-based immobilization technique for fluorescence imaging of the mitochondria in C. elegans is laborious, batchwise, and does not allow for facile handling of the worm. To overcome these technical challenges, we have developed a single-channel microfluidic device that can trap a C. elegans and allow to image the mitochondria in body wall muscles accurately and in higher throughput than the traditional approach. In specific, our microfluidic device took advantage of the proprioception of the worm to rotate its body in a microfluidic channel with an aspect ratio above one to gain more space for its undulation motion that was favorable for quantitative fluorescence imaging of mitochondria in the body wall muscles. Exploiting this unique feature of the microfluidic chip-based immobilization and fluorescence imaging, we observed a significant decrease in the mitochondrial fluorescence intensity under hyperglycemic conditions, whereas the agarose pad-based approach did not show any significant change under the same conditions. A machine learning model trained with these fluorescence images from the microfluidic device could classify healthy and hyperglycemic worms at high accuracy. Given this significant technological advantage, its easiness of use and low cost, our microfluidic imaging chip could become a useful immobilization tool for quantitative fluorescence imaging of the body wall muscles in C. elegans.

scholarly journals Mutations Affecting Nerve Attachment of Caenorhabditis elegans

Genetics ◽  
2001 ◽  
Vol 157 (4) ◽  
pp. 1611-1622 ◽  
Author(s):  
Go Shioi ◽  
Michinari Shoji ◽  
Masashi Nakamura ◽  
Takeshi Ishihara ◽  
Isao Katsura ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Nerve Cord ◽  
Ventral Nerve Cord ◽  
Body Wall ◽  
The Body ◽  
Genetic Loci ◽  
Muscle Attachment ◽  
C Elegans ◽  
Ventral Cord ◽  
Excretory Canal

Abstract Using a pan-neuronal GFP marker, a morphological screen was performed to detect Caenorhabditis elegans larval lethal mutants with severely disorganized major nerve cords. We recovered and characterized 21 mutants that displayed displacement or detachment of the ventral nerve cord from the body wall (Ven: ventral cord abnormal). Six mutations defined three novel genetic loci: ven-1, ven-2, and ven-3. Fifteen mutations proved to be alleles of previously identified muscle attachment/positioning genes, mup-4, mua-1, mua-5, and mua-6. All the mutants also displayed muscle attachment/positioning defects characteristic of mua/mup mutants. The pan-neuronal GFP marker also revealed that mutants of other mua/mup loci, such as mup-1, mup-2, and mua-2, exhibited the Ven defect. The hypodermis, the excretory canal, and the gonad were morphologically abnormal in some of the mutants. The pleiotropic nature of the defects indicates that ven and mua/mup genes are required generally for the maintenance of attachment of tissues to the body wall in C. elegans.

Positioning and maintenance of embryonic body wall muscle attachments in C. elegans requires the mup-1 gene

Development ◽  
1991 ◽  
Vol 111 (3) ◽  
pp. 667-681 ◽  
Author(s):  
P.Y. Goh ◽  
T. Bogaert
Keyword(s):  
Extracellular Matrix ◽  
Body Wall ◽  
Early Stage ◽  
Muscle Growth ◽  
The Body ◽  
Body Wall Muscle ◽  
C Elegans ◽  
Extracellular Matrix Molecule ◽  
Extracellular Matrix Components ◽  
Body Wall Muscles

As part of a general study of genes specifying a pattern of muscle attachments, we identified and genetically characterised mutants in the mup-1 gene. The body wall muscles of early stage mup-1 embryos have a wild-type myofilament pattern but may extend ectopic processes. Later in embryogenesis, some body wall muscles detach from the hypodermis. Genetic analysis suggests that mup-1 has both a maternal and a zygotic component and is not required for postembryonic muscle growth and attachment. mup-1 mutants are suppressed by mutations in several genes that encode extracellular matrix components. We propose that mup-1 may encode a cell surface/extracellular matrix molecule required both for the positioning of body wall muscle attachments in early embryogenesis and the subsequent maintenance of these attachments to the hypodermis until after cuticle synthesis.

scholarly journals Three-dimensional simulation of the Caenorhabditis elegans body and muscle cells in liquid and gel environments for behavioural analysis

2018 ◽  
Vol 373 (1758) ◽  
pp. 20170376 ◽  
Author(s):  
Andrey Palyanov ◽  
Sergey Khayrulin ◽  
Stephen D. Larson
Keyword(s):  
Caenorhabditis Elegans ◽  
Muscle Activation ◽  
Body Wall ◽  
Particle System ◽  
Three Dimensional ◽  
Muscle Cells ◽  
The Body ◽  
C Elegans ◽  
Muscle Activation Patterns ◽  
Simulation Engine

To better understand how a nervous system controls the movements of an organism, we have created a three-dimensional computational biomechanical model of the Caenorhabditis elegans body based on real anatomical structure. The body model is created with a particle system–based simulation engine known as Sibernetic, which implements the smoothed particle–hydrodynamics algorithm. The model includes an elastic body-wall cuticle subject to hydrostatic pressure. This cuticle is then driven by body-wall muscle cells that contract and relax, whose positions and shape are mapped from C. elegans anatomy, and determined from light microscopy and electron micrograph data. We show that by using different muscle activation patterns, this model is capable of producing C. elegans -like behaviours, including crawling and swimming locomotion in environments with different viscosities, while fitting multiple additional known biomechanical properties of the animal.  This article is part of a discussion meeting issue ‘Connectome to behaviour: modelling C. elegans at cellular resolution’.

Calsequestrin, a calcium sequestering protein localized at the sarcoplasmic reticulum, is not essential for body-wall muscle function in Caenorhabditis elegans

2000 ◽  
Vol 113 (22) ◽  
pp. 3947-3958 ◽  
Author(s):  
J.H. Cho ◽  
Y.S. Oh ◽  
K.W. Park ◽  
J. Yu ◽  
K.Y. Choi ◽  
...  
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Calcium Binding ◽  
Body Wall ◽  
Functional Characterization ◽  
The Body ◽  
Body Wall Muscle ◽  
C Elegans ◽  
Muscle Formation ◽  
Body Wall Muscles

Calsequestrin is the major calcium-binding protein of cardiac and skeletal muscles whose function is to sequester Ca(2+)in the lumen of the sarcoplasmic reticulum (SR). Here we describe the identification and functional characterization of a C. elegans calsequestrin gene (csq-1). CSQ-1 shows moderate similarity (50% similarity, 30% identity) to rabbit skeletal calsequestrin. Unlike mammals, which have two different genes encoding cardiac and fast-twitch skeletal muscle isoforms, csq-1 is the only calsequestrin gene in the C. elegans genome. We show that csq-1 is highly expressed in the body-wall muscles, beginning in mid-embryogenesis and maintained through the adult stage. In body-wall muscle cells, CSQ-1 is localized to sarcoplasmic membranes surrounding sarcomeric structures, in the regions where ryanodine receptors (UNC-68) are located. Mutation in UNC-68 affects CSQ-1 localization, suggesting that the two possibly interact in vivo. Genetic analyses of chromosomal deficiency mutants deleting csq-1 show that CSQ-1 is not essential for initiation of embryonic muscle formation and contraction. Furthermore, double-stranded RNA injection resulted in animals completely lacking CSQ-1 in body-wall muscles with no observable defects in locomotion. These findings suggest that although CSQ-1 is one of the major calcium-binding proteins in the body-wall muscles of C. elegans, it is not essential for body-wall muscle formation and contraction.

scholarly journals Purified thick filaments from the nematode Caenorhabditis elegans: evidence for multiple proteins associated with core structures.

1988 ◽  
Vol 106 (6) ◽  
pp. 1985-1995 ◽  
Author(s):  
H F Epstein ◽  
G C Berliner ◽  
D L Casey ◽  
I Ortiz
Keyword(s):  
Caenorhabditis Elegans ◽  
Body Wall ◽  
Gradient Centrifugation ◽  
Blue Staining ◽  
Nematode Caenorhabditis Elegans ◽  
C Elegans ◽  
Thick Filaments ◽  
Cell Biol ◽  
Associated Proteins ◽  
Body Wall Muscles

The thick filaments of the nematode, Caenorhabditis elegans, arising predominantly from the body-wall muscles, contain two myosin isoforms and paramyosin as their major proteins. The two myosins are located in distinct regions of the surfaces, while paramyosin is located within the backbones of the filaments. Tubular structures constitute the cores of the polar regions, and electron-dense material is present in the cores of the central regions (Epstein, H.F., D.M. Miller, I. Ortiz, and G.C. Berliner. 1985. J. Cell Biol. 100:904-915). Biochemical, genetic, and immunological experiments indicate that the two myosins and paramyosin are not necessary core components (Epstein, H.F., I. Ortiz, and L.A. Traeger Mackinnon. 1986. J. Cell Biol. 103:985-993). The existence of the core structures suggests, therefore, that additional proteins may be associated with thick filaments in C. elegans. To biochemically detect minor associated proteins, a new procedure for the isolation of thick filaments of high purity and structural preservation has been developed. The final step, glycerol gradient centrifugation, yielded fractions that are contaminated by, at most, 1-2% with actin, tropomyosin, or ribosome-associated proteins on the basis of Coomassie Blue staining and electron microscopy. Silver staining and radioautography of gel electrophoretograms of unlabeled and 35S-labeled proteins, respectively, revealed at least 10 additional bands that cosedimented with thick filaments in glycerol gradients. Core structures prepared from wild-type thick filaments contained at least six of these thick filament-associated protein bands. The six proteins also cosedimented with thick filaments purified by gradient centrifugation from CB190 mutants lacking myosin heavy chain B and from CB1214 mutants lacking paramyosin. For these reasons, we propose that the six associated proteins are potential candidates for putative components of core structures in the thick filaments of body-wall muscles of C. elegans.

scholarly journals Muscle cell attachment in Caenorhabditis elegans.

1991 ◽  
Vol 114 (3) ◽  
pp. 465-479 ◽  
Author(s):  
R Francis ◽  
R H Waterston
Keyword(s):  
Caenorhabditis Elegans ◽  
Basement Membrane ◽  
Muscle Cell ◽  
Body Wall ◽  
Cell Attachment ◽  
Muscle Cells ◽  
The Body ◽  
Nematode Caenorhabditis Elegans ◽  
Hypodermal Cell ◽  
Body Wall Muscles

In the nematode Caenorhabditis elegans, the body wall muscles exert their force on the cuticle to generate locomotion. Interposed between the muscle cells and the cuticle are a basement membrane and a thin hypodermal cell. The latter contains bundles of filaments attached to dense plaques in the hypodermal cell membranes, which together we have called a fibrous organelle. In an effort to define the chain of molecules that anchor the muscle cells to the cuticle we have isolated five mAbs using preparations enriched in these components. Two antibodies define a 200-kD muscle antigen likely to be part of the basement membrane at the muscle/hypodermal interface. Three other antibodies probably identify elements of the fibrous organelles in the adjacent hypodermis. The mAb IFA, which reacts with mammalian intermediate filaments, also recognizes these structures. We suggest that the components recognized by these antibodies are likely to be involved in the transmission of tension from the muscle cell to the cuticle.

scholarly journals Comparing Caenorhabditis elegans gentle and harsh touch response behavior using a multiplexed hydraulic microfluidic device

2017 ◽  
Author(s):  
Patrick D. McClanahan ◽  
Joyce H. Xu ◽  
Christopher Fang-Yen
Keyword(s):  
Caenorhabditis Elegans ◽  
Microfluidic Device ◽  
Receptive Fields ◽  
The Body ◽  
Mechanical Stimuli ◽  
C Elegans ◽  
Response Characteristics ◽  
Touch Receptor Neurons ◽  
Touch Response ◽  
Receptor Neurons

AbstractThe roundworm Caenorhabditis elegans is an important model system for understanding the genetics and physiology of touch. Classical assays for C. elegans touch, which involve manually touching the animal with a probe and observing its response, are limited by their low throughput and qualitative nature. We developed a microfluidic device in which several dozen animals are subject to spatially localized mechanical stimuli with variable amplitude. The device contains 64 sinusoidal channels through which worms crawl, and hydraulic valves that deliver touch stimuli to the worms. We used this assay to characterize the behavioral responses to gentle touch stimuli and the less well studied harsh (nociceptive) touch stimuli. First, we measured the relative response thresholds of gentle and harsh touch. Next, we quantified differences in the receptive fields between wild type worms and a mutant with non-functioning posterior touch receptor neurons. We showed that under gentle touch the receptive field of the anterior touch receptor neurons extends into the posterior half of the body. Finally, we found that the behavioral response to gentle touch does not depend on the locomotion of the animal immediately prior to the stimulus, but does depend on the location of the previous touch. Responses to harsh touch, on the other hand, did not depend on either previous velocity or stimulus location. Differences in gentle and harsh touch response characteristics may reflect the different innervation of the respective mechanosensory cells. Our assay will facilitate studies of mechanosensation, sensory adaptation, and nociception.

Genetic defects in acetylcholine signalling promote protein degradation in muscle cells of Caenorhabditis elegans

2000 ◽  
Vol 113 (11) ◽  
pp. 2003-2010 ◽  
Author(s):  
N.J. Szewczyk ◽  
J.J. Hartman ◽  
S.J. Barmada ◽  
L.A. Jacobson
Keyword(s):  
Caenorhabditis Elegans ◽  
Body Wall ◽  
Muscle Protein ◽  
Muscle Cells ◽  
Negative Control ◽  
The Body ◽  
Reporter Protein ◽  
Release Of Acetylcholine ◽  
Body Wall Muscles ◽  
Ach Receptors

A myosin-lacZ fusion, expressed in 103 muscle cells of Caenorhabditis elegans, reports on how proteolysis in muscle is controlled by neural and intramuscular signals. Upon acute starvation, the fusion protein is degraded in the posterior 63 cells of the body-wall muscle, but remains stable in 32 anterior body-wall muscles and 8 vulval muscle cells. This distinction correlates with differences in the innervation of these cells. Reporter protein in the head and vulval muscles becomes labile upon genetic ‘denervation’ in mutants that have blocks in pre-synaptic synthesis or release of acetylcholine (ACh) or post-synaptic reception at nicotinic ACh receptors (nAChR), whereas protein in all 103 muscles is stabilized by the nicotinic agonist levamisole in the absence of ACh production. Levamisole does not stabilize muscle protein in nAChR mutants that are behaviorally resistant to levamisole. Neural inputs thus exert negative control over the proteolytic process in muscle by stimulating muscle nicotinic ACh receptors.

scholarly journals The Caenorhabditis elegans vab-10 spectraplakin isoforms protect the epidermis against internal and external forces

2003 ◽  
Vol 161 (4) ◽  
pp. 757-768 ◽  
Author(s):  
Julia M. Bosher ◽  
Bum-Soo Hahn ◽  
Renaud Legouis ◽  
Satis Sookhareea ◽  
Robby M. Weimer ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Body Wall ◽  
Epidermal Thickness ◽  
C Elegans ◽  
Attachment Structures ◽  
Body Wall Muscles ◽  
Embryonic Morphogenesis ◽  
External Forces ◽  
New Mutations

Morphogenesis of the Caenorhabditis elegans embryo is driven by actin microfilaments in the epidermis and by sarcomeres in body wall muscles. Both tissues are mechanically coupled, most likely through specialized attachment structures called fibrous organelles (FOs) that connect muscles to the cuticle across the epidermis. Here, we report the identification of new mutations in a gene known as vab-10, which lead to severe morphogenesis defects, and show that vab-10 corresponds to the C. elegans spectraplakin locus. Our analysis of vab-10 reveals novel insights into the role of this plakin subfamily. vab-10 generates isoforms related either to plectin (termed VAB-10A) or to microtubule actin cross-linking factor plakins (termed VAB-10B). Using specific antibodies and mutations, we show that VAB-10A and VAB-10B have distinct distributions and functions in the epidermis. Loss of VAB-10A impairs the integrity of FOs, leading to epidermal detachment from the cuticle and muscles, hence demonstrating that FOs are functionally and molecularly related to hemidesmosomes. We suggest that this isoform protects against forces external to the epidermis. In contrast, lack of VAB-10B leads to increased epidermal thickness during embryonic morphogenesis when epidermal cells change shape. We suggest that this isoform protects cells against tension that builds up within the epidermis.

scholarly journals Caenorhabditis elegans Unc-45 Is a Component of Muscle Thick Filaments and Colocalizes with Myosin Heavy Chain B, but Not Myosin Heavy Chain a

2000 ◽  
Vol 148 (2) ◽  
pp. 375-384 ◽  
Author(s):  
Wanyuan Ao ◽  
Dave Pilgrim
Keyword(s):  
Caenorhabditis Elegans ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Body Wall ◽  
Thick Filament ◽  
The Body ◽  
Temperature Sensitive ◽  
Filament Assembly ◽  
Thick Filaments ◽  
Body Wall Muscles

In the nematode Caenorhabditis elegans, animals mutant in the gene encoding the protein product of the unc-45 gene (UNC-45) have disorganized muscle thick filaments in body wall muscles. Although UNC-45 contains tetratricopeptide repeats (TPR) as well as limited similarity to fungal proteins, no biochemical role has yet been found. UNC-45 reporters are expressed exclusively in muscle cells, and a functional reporter fusion is localized in the body wall muscles in a pattern identical to thick filament A-bands. UNC-45 colocalizes with myosin heavy chain (MHC) B in wild-type worms as well as in temperature-sensitive (ts) unc-45 mutants, but not in a mutant in which MHC B is absent. Surprisingly, UNC-45 localization is also not seen in MHC B mutants, in which the level of MHC A is increased, resulting in near-normal muscle thick filament structure. Thus, filament assembly can be independent of UNC-45. UNC-45 shows a localization pattern identical to and dependent on MHC B and a function that appears to be MHC B–dependent. We propose that UNC-45 is a peripheral component of muscle thick filaments due to its localization with MHC B. The role of UNC-45 in thick filament assembly seems restricted to a cofactor for assembly or stabilization of MHC B.

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