scholarly journals Rhodopsin-based voltage imaging tools for use in excitable cells of Caenorhabditis elegans

2019 ◽  
Author(s):  
Negin Azimi Hashemi ◽  
Amelie C. F. Bergs ◽  
Rebecca Scheiwe ◽  
Wagner Steuer Costa ◽  
Jana F. Liewald ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Body Wall ◽  
Ca 125 ◽  
Excitable Cells ◽  
Voltage Imaging ◽  
C Elegans ◽  
Non Invasive ◽  
All Optical ◽  
Body Wall Muscles ◽  
Voltage Dynamics

AbstractGenetically encoded voltage indicators (GEVIs) based on microbial rhodopsins utilize the voltage-sensitive fluorescence of the all-trans retinal (ATR) cofactor, while in electrochromic (eFRET) sensors, donor fluorescence drops when the rhodopsin acts as depolarization-sensitive acceptor. We systematically assessed Arch(D95N), Archon, and QuasAr, as well as the eFRET sensors MacQ-mCitrine and QuasAr-mOrange, in C. elegans. ATR-bearing rhodopsins reported on voltage changes in body wall muscles (BWMs) and the pharynx, the feeding organ, where Arch(D95N) showed ca. 125 % ΔF/F increase per 100 mV. The ATR fluorescence is very dim, however, using the retinal analog dimethylaminoretinal (DMAR), it was boosted 250-fold. eFRET sensors provided sensitivities of 45 % to 78 % ΔF/F per 100 mV, induced by BWM action potentials (APs). All sensors reported differences in muscle depolarization induced by a voltage-gated Ca2+-channel mutant. Optogenetically evoked de-or hyperpolarization of motor neurons increased or eliminated AP activity and caused a rise or drop in BWM sensor fluorescence. Last, we could analyze voltage dynamics across the entire pharynx, showing uniform depolarization but compartmentalized repolarization of anterior and posterior parts. Our work establishes all-optical, non-invasive electrophysiology in intact C. elegans.

scholarly journals Rhodopsin-based voltage imaging tools for use in muscles and neurons of Caenorhabditis elegans

2019 ◽  
Vol 116 (34) ◽  
pp. 17051-17060 ◽  
Author(s):  
Negin Azimi Hashemi ◽  
Amelie C. F. Bergs ◽  
Christina Schüler ◽  
Anna Rebecca Scheiwe ◽  
Wagner Steuer Costa ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Motor Neurons ◽  
Mammalian Cells ◽  
Voltage Imaging ◽  
C Elegans ◽  
All Optical ◽  
Body Wall Muscles ◽  
Potential Activity ◽  
Voltage Dynamics ◽  
Noninvasive Electrophysiology

Genetically encoded voltage indicators (GEVIs) based on microbial rhodopsins utilize the voltage-sensitive fluorescence of all-trans retinal (ATR), while in electrochromic FRET (eFRET) sensors, donor fluorescence drops when the rhodopsin acts as depolarization-sensitive acceptor. In recent years, such tools have become widely used in mammalian cells but are less commonly used in invertebrate systems, mostly due to low fluorescence yields. We systematically assessed Arch(D95N), Archon, QuasAr, and the eFRET sensors MacQ-mCitrine and QuasAr-mOrange, in the nematode Caenorhabditis elegans. ATR-bearing rhodopsins reported on voltage changes in body wall muscles (BWMs), in the pharynx, the feeding organ [where Arch(D95N) showed approximately 128% ΔF/F increase per 100 mV], and in neurons, integrating circuit activity. ATR fluorescence is very dim, yet, using the retinal analog dimethylaminoretinal, it was boosted 250-fold. eFRET sensors provided sensitivities of 45 to 78% ΔF/F per 100 mV, induced by BWM action potentials, and in pharyngeal muscle, measured in simultaneous optical and sharp electrode recordings, MacQ-mCitrine showed approximately 20% ΔF/F per 100 mV. All sensors reported differences in muscle depolarization induced by a voltage-gated Ca2+-channel mutant. Optogenetically evoked de- or hyperpolarization of motor neurons increased or eliminated action potential activity and caused a rise or drop in BWM sensor fluorescence. Finally, we analyzed voltage dynamics across the entire pharynx, showing uniform depolarization but compartmentalized repolarization of anterior and posterior parts. Our work establishes all-optical, noninvasive electrophysiology in live, intact 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 Inhibition underlies fast undulatory locomotion in C. elegans

2020 ◽  
Author(s):  
Lan Deng ◽  
Jack Denham ◽  
Charu Arya ◽  
Omer Yuval ◽  
Netta Cohen ◽  
...  
Keyword(s):  
Computational Models ◽  
Body Wall ◽  
Activity Patterns ◽  
Wild Type ◽  
C Elegans ◽  
Body Wall Muscles ◽  
Undulatory Locomotion ◽  
Gaba Transmission ◽  
Cross Inhibition

AbstractInhibition plays important roles in modulating the neural activities of sensory and motor systems at different levels from synapses to brain regions. To achieve coordinated movement, motor systems produce alternating contraction of antagonist muscles, whether along the body axis or within and among limbs. In the nematode C. elegans, a small network involving excitatory cholinergic and inhibitory GABAergic motoneurons generates the dorsoventral alternation of body-wall muscles that supports undulatory locomotion. Inhibition has been suggested to be necessary for backward undulation because mutants that are defective in GABA transmission exhibit a shrinking phenotype in response to a harsh touch to the head, whereas wild-type animals produce a backward escape response. Here, we demonstrate that the shrinking phenotype is exhibited by wild-type as well as mutant animals in response to harsh touch to the head or tail, but only GABA transmission mutants show slow locomotion after stimulation. Impairment of GABA transmission, either genetically or optogenetically, induces lower undulation frequency and lower translocation speed during crawling and swimming in both directions. The activity patterns of GABAergic motoneurons are different during low and high undulation frequencies. During low undulation frequency, GABAergic VD and DD motoneurons show similar activity patterns, while during high undulation frequency, their activity alternates. The experimental results suggest at least three non-mutually exclusive roles for inhibition that could underlie fast undulatory locomotion in C. elegans, which we tested with computational models: cross-inhibition or disinhibition of body-wall muscles, or inhibitory reset.Significance StatementInhibition serves multiple roles in the generation, maintenance, and modulation of the locomotive program and supports the alternating activation of antagonistic muscles. When the locomotor frequency increases, more inhibition is required. To better understand the role of inhibition in locomotion, we used C. elegans as an animal model, and challenged a prevalent hypothesis that cross-inhibition supports the dorsoventral alternation. We find that inhibition is related to the speed rather than the direction of locomotion and demonstrate that inhibition is unnecessary for muscle alternation during slow undulation in either direction but crucial to sustain rapid dorsoventral alternation. We combined calcium imaging of motoneurons and muscle with computational models to test hypotheses for the role of inhibition in locomotion.

scholarly journals A conserved neuropeptide system links head and body motor circuits to enable adaptive behavior

2020 ◽  
Author(s):  
Shankar Ramachandran ◽  
Navonil Banerjee ◽  
Raja Bhattacharya ◽  
Denis Touroutine ◽  
Christopher M. Lambert ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Adaptive Behavior ◽  
Environmental Conditions ◽  
Body Wall ◽  
Behavioral Responses ◽  
Physiological Changes ◽  
C Elegans ◽  
Motor Circuits ◽  
Concerted Activity ◽  
Stimulation Of

SUMMARYNeuromodulators promote adaptive behaviors in response to either environmental or internal physiological changes. These responses are often complex and may involve concerted activity changes across circuits that are not physically connected. It is not well understood how neuromodulatory systems act across circuits to elicit complex behavioral responses. Here we show that the C. elegans NLP-12 neuropeptide system shapes responses to food availability by selectively modulating the activity of head and body wall motor neurons. NLP-12 modulation of the head and body wall motor circuits is generated through conditional involvement of alternate GPCR targets. The CKR-1 GPCR is highly expressed in the head motor circuit, and functions to enhance head bending and increase trajectory reorientations during local food searching, primarily through stimulatory actions on SMD head motor neurons. In contrast, NLP-12 activation of CKR-1 and CKR-2 GPCRs regulates body bending under basal conditions, primarily through actions on body wall motor neurons. Thus, locomotor responses to changing environmental conditions emerge from conditional NLP-12 stimulation of head or body wall motor neuron targets.

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.

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

10.3791/59175 ◽  
2019 ◽  
Author(s):  
Ashley A. Martin ◽  
Simon Alford ◽  
Janet E. Richmond
Keyword(s):  
Calcium Imaging ◽  
Body Wall ◽  
C Elegans ◽  
Body Wall Muscles ◽  
In Vivo Calcium Imaging

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 Non-canonical apical constriction shapes emergent matrices in C. elegans

2018 ◽  
Author(s):  
Sophie S. Katz ◽  
Chloe Maybrun ◽  
Hannah M. Maul-Newby ◽  
Alison R. Frand
Keyword(s):  
Actin Filament ◽  
Larval Stage ◽  
Body Wall ◽  
Matrix Remodeling ◽  
Apical Constriction ◽  
Actin Bundles ◽  
C Elegans ◽  
Filament Bundles ◽  
Body Wall Muscles

ABSTRACTSpecialized epithelia produce apical matrices with distinctive topographies by enigmatic mechanisms. Here, we describe a holistic mechanism that integrates cortical actomyosin dynamics with apical matrix remodeling to pattern C. elegans cuticles. Therein, axial AFBs appear near the surface of lateral epidermal syncytia during an interval of transverse apical constriction (AC). AC gives rise to three temporary semi-circular cellular protrusions beneath a provisional matrix (sheath). In turn, sheath components pattern durable ridges along the midline of adult cuticles (alae). We propose that forces generated by AC are relayed via the sheath to sculpt the the acellular adult cuticle manifest several hours later. Furthermore, we provide evidence that circumferential actin filament bundles (CFBs) near the surface of the adjacent syncytia (hyp7) are largely dispensable for the propagation of annular cuticle structures from one larval stage to the next. Rather, the temporary CFBs extend from actin bundles overlying body wall muscles, which are situated between Ce. hemidesmosomes. Similar mechanisms may contribute to the morphogenesis of integumentary organs in higher metazoans.

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 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.

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