a-rapid-protocol-for-integrating-extrachromosomal-arrays-with-high

A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the <em>C. elegans</em> Genome

Microinjecting DNA into the cytoplasm of the syncytial gonad of Caenorhabditis elegans is the main technique used to establish transgenic lines that ...

Université Claude Bernard Lyon

CNRS UMR 5534

Reversible acetylation of histone tails plays an important role in chromatin remodelling and regulation of gene activity. While modification by histone acetyltransferase (HAT) is usually linked to transcriptional activation, we provide here evidence for HAT function in several types of epigenetic repression. Chameau (Chm), a new Drosophila member of the MYST HAT family, dominantly suppresses position effect variegation (PEV), is required for the maintenance of Hox gene silencing by Polycomb group (PcG) proteins, and can partially substitute for the MYST Sas2 HAT in yeast telomeric position effect (TPE). Finally, we provide in vivo evidence that the acetyltransferase activity of Chm is required in these processes, since a variant protein mutated in the catalytic domain no longer rescues PEV modification, telomeric silencing of SAS2-deficient yeast cells, nor lethality of chm mutant flies. These findings emphasize the role of an acetyltransferase in gene silencing, which supports, according to the histone code hypothesis, that transcription at a particular locus is determined by a precise combination of histone tail modifications rather than by overall acetylation levels.

The MYST domain acetyltransferase Chameau functions in epigenetic mechanisms of transcriptional repression.

Cell motility is regulated by extracellular cues and by intracellular factors that accumulate at sites of contact between cells and the extracellular matrix. One of these factors, focal adhesion kinase (FAK), regulates the cycle of focal adhesion formation and disassembly that is required for cell movement to occur. Recently, Wnt signaling has also been implicated in the control of cell movement in vertebrates, but the mechanism through which Wnt proteins influence motility is unclear. We demonstrate that Drosphila Wnt4 is required for cell movement and FAK regulation during ovarian morphogenesis. Dfrizzled2, Disheveled, and protein kinase C are also required. The DWnt4 cell motility pathway is distinct from both the canonical Wnt pathway and the planar polarity pathway. Our data suggest that DWnt4 facilitates motility through regulation of focal adhesions.

DWnt4 regulates cell movement and focal adhesion kinase during Drosophila ovarian morphogenesis.

Duchenne muscular dystrophy is one of the most common neuromuscular diseases. It is caused by mutations in the dystrophin gene. Dystrobrevins are dystrophin-associated proteins potentially involved in signal transduction. The nematode Caenorhabditis elegans possesses one dystrophin-like (dys-1) and one dystrobrevin-like (dyb-1) gene. Mutations of dyb-1 and dys-1 lead to similar phenotypes, comprising hyperactivity and a tendency to hypercontract, which suggest that these proteins may participate in a common function. We show here that overexpression of the Dyb-1 protein delays the onset of the myopathy observed in the C. elegans double mutant (dys-1; hlh-1 mutations). This finding indicates that, in C. elegans, (1) the absence of dystrophin can be partly compensated for by extra doses of dystrobrevin, and (2) dystrobrevin is partly functional in absence of dystrophin.

Overexpression of dystrobrevin delays locomotion defects and muscle degeneration in a dystrophin-deficient Caenorhabditis elegans.

Dystrophin is the product of the gene mutated in Duchenne muscular dystrophy (DMD). Neither the function of dystrophin nor the physiopathology of the disease have been clearly established so far. In mammals, the dystrophin-glycoprotein complex (DGC) includes dystrophin, as well as transmembrane and cytoplasmic proteins. Since Caenorhabditis elegans possesses a dystrophin-like gene (dys-1), we investigated whether homologues of the DGC members could also be found in the C. elegans genome. Conserved homologues were found for dystroglycan, delta/gamma-sarcoglycan and syntrophin. Divergent but related proteins were found for alpha- and beta-sarcoglycans. No sarcospan counterpart was found. The expression of the conserved homologues was inactivated using the RNA interference technique. Phenotypes similar to that of dys-1 were obtained, both in the wild-type background and in combination with other mutations. These results strongly suggest that a protein complex comprising functional analogies with the DGC exists in C. elegans.

Genetic evidence for a dystrophin-glycoprotein complex (DGC) in Caenorhabditis elegans.

The properties of K(+) channels in body wall muscle cells acutely dissected from the nematode Caenorhabditis elegans were investigated at the macroscopic and unitary level using an in situ patch clamp technique. In the whole-cell configuration, depolarizations to potentials positive to -40 mV gave rise to outward currents resulting from the activation of two kinetically distinct voltage-dependent K(+) currents: a fast activating and inactivating 4-aminopyridine-sensitive component and a slowly activating and maintained tetraethylammonium-sensitive component. In cell-attached patches, voltage-dependent K(+) channels, with unitary conductances of 34 and 80 pS in the presence of 5 and 140 mM external K(+), respectively, activated at membrane potentials positive to -40 mV. Excision revealed that these channels corresponded to Ca(2+)-activated K(+) channels exhibiting an unusual sensitivity to internal Cl(-) and whose activity progressively decreased in inside-out conditions. After complete run-down of these channels, one third of inside-out patches displayed activity of another Ca(2+)-activated K(+) channel of smaller unitary conductance (6 pS at 0 mV in the presence of 5 mM external K(+)). In providing a detailed description of native K(+) currents in body wall muscle cells of C. elegans, this work lays the basis for further comparisons with mutants to assess the function of K(+) channels in this model organism that is highly amenable to molecular and classical genetics.

Characterization of K(+) currents using an in situ patch clamp technique in body wall muscle cells from Caenorhabditis elegans.

Caenorhabditis elegans is a powerful model system widely used to investigate the relationships between genes and complex behaviors like locomotion. However, physiological studies at the cellular level have been restricted by the difficulty to dissect this microscopic animal. Thus, little is known about the properties of body wall muscle cells used for locomotion. Using in situ patch clamp technique, we show that body wall muscle cells generate spontaneous spike potentials and develop graded action potentials in response to injection of positive current of increasing amplitude. In the presence of K+ channel blockers, membrane depolarization elicited Ca2+ currents inhibited by nifedipine and exhibiting Ca2+-dependent inactivation. Our results give evidence that the Ca2+ channel involved belongs to the L-type class and corresponds to EGL-19, a putative Ca2+ channel originally thought to be a member of this class on the basis of genomic data. Using Ca2+ fluorescence imaging on patch-clamped muscle cells, we demonstrate that the Ca2+ transients elicited by membrane depolarization are under the control of Ca2+ entry through L-type Ca2+ channels. In reduction of function egl-19 mutant muscle cells, Ca2+ currents displayed slower activation kinetics and provided a significantly smaller Ca2+ entry, whereas the threshold for Ca2+ transients was shifted toward positive membrane potentials.

The L-type voltage-dependent Ca2+ channel EGL-19 controls body wall muscle function in Caenorhabditis elegans.

Syntrophins are a family of PDZ domain-containing adaptor proteins required for receptor localization. Syntrophins are also associated with the dystrophin complex in muscles. We report here the molecular and functional characterization of the Caenorhabditis elegans gene stn-1 (F30A10.8), which encodes a syntrophin with homology to vertebrate alpha and beta-syntrophins. stn-1 is expressed in neurons and in muscles of C.elegans. stn-1 mutants resemble dystrophin (dys-1) and dystrobrevin (dyb-1) mutants: they are hyperactive, bend their heads when they move forward, tend to hypercontract, and are hypersensitive to the acetylcholinesterase inhibitor aldicarb. These phenotypes are suppressed when stn-1 is expressed under the control of a muscular promoter, indicating that they are caused by the absence of stn-1 in muscles. These results suggest that the role of syntrophin is linked to dystrophin function in C.elegans.

The stn-1 syntrophin gene of C.elegans is functionally related to dystrophin and dystrobrevin.

Degenerins have emerged from genetic studies in Caenorhabditis elegans as candidate mechanically gated amiloride-sensitive ion channels for transducing mechanical stimuli into cellular responses. In C. elegans muscle, the existence of a genetic interaction between the unc-105 degenerin gene and let-2, a gene encoding an alpha2(IV) collagen, raised the possibility that UNC-105 may function as a mechanically gated channel in a stretch receptor complex. However, to date, ion channel activity of UNC-105 has only been recorded in a gain-of-function mutant form in heterologous expression systems. In this study we investigated the in situ properties of UNC-105 using the whole cell configuration of the patch clamp technique on body wall muscle cells from acutely dissected C. elegans. Amiloride was found to be without effect on membrane potential of wild-type muscle cells, suggesting that the UNC-105 degenerin is electrically silent in resting muscle. Hypo-osmotic shocks induced a reversible depolarization of muscle cells but which was not affected by amiloride. Deformation of the cells by applying tension to the filamentous complex on which muscle cells remained attached or by ejecting external solution under pressure failed to induce any change of membrane potential. In gain-of-function unc-105(n506) mutant cells, an amiloride-sensitive inward Na(+) current was found to be constitutively active, leading to maintained muscle depolarization. An associated mutation in the alpha2(IV) collagen LET-2 led to the closure of the mutant UNC-105(n506) channel while a collagenase treatment of these double mutant cells caused it to re-open, giving evidence for a functional interaction between LET-2 collagen and mutant UNC-105 channel.

Patch clamp study of the UNC-105 degenerin and its interaction with the LET-2 collagen in Caenorhabditis elegans muscle.

The Caenorhabditis elegans SLO-1 channel belongs to the family of calcium-activated large conductance BK potassium channels. SLO-1 has been shown to be involved in neurotransmitter release and ethanol response. Here, we report that SLO-1 also has a critical role in muscles. Inactivation of the slo-1 gene in muscles leads to phenotypes similar to those caused by mutations of the dystrophin homologue dys-1. Notably, slo-1 mutations result in a progressive muscle degeneration when put into a sensitized genetic background. slo-1 localization was observed by gfp reporter gene in both the M-line and the dense bodies (Z line) of the C.elegans body-wall muscles. Using the inside-out configuration of the patch clamp technique on body-wall muscle cells of acutely dissected wild-type worms, we characterized a Ca2+-activated K+ channel that was identified unambiguously as SLO-1. Since neither the abundance nor the conductance of SLO-1 was changed significantly in dys-1 mutants compared to wild-type animals, it is likely that the inactivation of dys-1 causes a misregulation of SLO-1. All in all, these results indicate that SLO-1 function in C.elegans muscles is related to the dystrophin homologue DYS-1.

The SLO-1 BK channel of Caenorhabditis elegans is critical for muscle function and is involved in dystrophin-dependent muscle dystrophy.

Prevention of muscle fiber degeneration is a key issue in the treatment of muscular dystrophies such as Duchenne Muscular Dystrophy (DMD). It is widely postulated that existing pharmaceutical compounds might potentially be beneficial to DMD patients, but tools to identify them are lacking. Here, by using a Caenorhabditis elegans model of dystrophin-dependent muscular dystrophy, we show that the neurohormone serotonin and some of its agonists are potent suppressors of muscle degeneration. Inhibitors of serotonin reuptake transporters, which prolong the action of endogenous serotonin, have a similar effect. Moreover, reduction of serotonin levels leads to degeneration of non-dystrophic muscles. Our results demonstrate that serotonin is critical to C. elegans striated muscles. These findings reveal a new function of serotonin in striated muscles.

Blocking of striated muscle degeneration by serotonin in C. elegans.

In mammals, the lack of dystrophin leads to a degeneration of skeletal muscles. It has been known for many years that this pathology can be blocked by denervation or immobilization of muscles. It is not yet clear, however, whether this suppressing effect is due to the absence of fiber contraction per se, or to other mechanisms which may be induced by such treatments. We took advantage of the genetic tools available in the animal model Caenorhabditis elegans to address this question. Using RNA interference and existing mutants, we genetically impaired the excitation-contraction cascade at specific points in a dystrophin-deficient C. elegans strain which normally undergoes extensive muscle degeneration. Our data show that reducing sarcomere contraction by slightly impairing the contraction machinery is sufficient to dramatically suppress muscle degeneration. Thus, it is the physical tension exerted on the muscle fibers which is the key deleterious event in the absence of dystrophin.

Dystrophin-dependent muscle degeneration requires a fully functional contractile machinery to occur in C. elegans.

In Caenorhabditis elegans, mutations of the dystrophin homologue, dys-1, produce a peculiar behavioral phenotype (hyperactivity and a tendency to hypercontract). In a sensitized genetic background, dys-1 mutations also lead to muscle necrosis. The dyc-1 gene was previously identified in a genetic screen because its mutation leads to the same phenotype as dys-1, suggesting that the two genes are functionally linked. Here, we report the detailed characterization of the dyc-1 gene. dyc-1 encodes two isoforms, which are expressed in neurons and muscles. Isoform-specific RNAi experiments show that the absence of the muscle isoform, and not that of the neuronal isoform, is responsible for the dyc-1 mutant phenotype. In the sarcomere, the DYC-1 protein is localized at the edges of the dense body, the nematode muscle adhesion structure where actin filaments are anchored and linked to the sarcolemma. In yeast two-hybrid assays, DYC-1 interacts with ZYX-1, the homologue of the vertebrate focal adhesion LIM domain protein zyxin. ZYX-1 localizes at dense bodies and M-lines as well as in the nucleus of C. elegans striated muscles. The DYC-1 protein possesses a highly conserved 19 amino acid sequence, which is involved in the interaction with ZYX-1 and which is sufficient for addressing DYC-1 to the dense body. Altogether our findings indicate that DYC-1 may be involved in dense body function and stability. This, taken together with the functional link between the C. elegans DYC-1 and DYS-1 proteins, furthermore suggests a requirement of dystrophin function at this structure. As the dense body shares functional similarity with both the vertebrate Z-disk and the costamere, we therefore postulate that disruption of muscle cell adhesion structures might be the primary event of muscle degeneration occurring in the absence of dystrophin, in C. elegans as well as vertebrates.

DYC-1, a protein functionally linked to dystrophin in Caenorhabditis elegans is associated with the dense body, where it interacts with the muscle LIM domain protein ZYX-1.

In vertebrates, zyxin is a LIM-domain protein belonging to a family composed of seven members. We show that the nematode Caenorhabditis elegans has a unique zyxin-like protein, ZYX-1, which is the orthologue of the vertebrate zyxin subfamily composed of zyxin, migfilin, TRIP6, and LPP. The ZYX-1 protein is expressed in the striated body-wall muscles and localizes at dense bodies/Z-discs and M-lines, as well as in the nucleus. In yeast two-hybrid assays ZYX-1 interacts with several known dense body and M-line proteins, including DEB-1 (vinculin) and ATN-1 (α-actinin). ZYX-1 is mainly localized in the middle region of the dense body/Z-disk, overlapping the apical and basal regions containing, respectively, ATN-1 and DEB-1. The localization and dynamics of ZYX-1 at dense bodies depend on the presence of ATN-1. Fluorescence recovery after photobleaching experiments revealed a high mobility of the ZYX-1 protein within muscle cells, in particular at dense bodies and M-lines, indicating a peripheral and dynamic association of ZYX-1 at these muscle adhesion structures. A portion of the ZYX-1 protein shuttles from the cytoplasm into the nucleus, suggesting a role for ZYX-1 in signal transduction. We provide evidence that the zyx-1 gene encodes two different isoforms, ZYX-1a and ZYX-1b, which exhibit different roles in dystrophin-dependent muscle degeneration occurring in a C. elegans model of Duchenne muscular dystrophy.

ZYX-1, the unique zyxin protein of Caenorhabditis elegans, is involved in dystrophin-dependent muscle degeneration.

Duchenne muscular dystrophy (DMD) is a neuromuscular disease caused by mutations in the dystrophin gene. The subcellular mechanisms of DMD remain poorly understood and there is currently no curative treatment available. Using a Caenorhabditis elegans model for DMD as a pharmacologic and genetic tool, we found that cyclosporine A (CsA) reduces muscle degeneration at low dose and acts, at least in part, through a mitochondrial cyclophilin D, CYN-1. We thus hypothesized that CsA acts on mitochondrial permeability modulation through cyclophilin D inhibition. Mitochondrial patterns and dynamics were analyzed, which revealed dramatic mitochondrial fragmentation not only in dystrophic nematodes, but also in a zebrafish model for DMD. This abnormal mitochondrial fragmentation occurs before any obvious sign of degeneration can be detected. Moreover, we demonstrate that blocking/delaying mitochondrial fragmentation by knocking down the fission-promoting gene drp-1 reduces muscle degeneration and improves locomotion abilities of dystrophic nematodes. Further experiments revealed that cytochrome c is involved in muscle degeneration in C. elegans and seems to act, at least in part, through an interaction with the inositol trisphosphate receptor calcium channel, ITR-1. Altogether, our findings reveal that mitochondria play a key role in the early process of muscle degeneration and may be a target of choice for the design of novel therapeutics for DMD. In addition, our results provide the first indication in the nematode that (i) mitochondrial permeability transition can occur and (ii) cytochrome c can act in cell death.

Marie-Christine Mariol

Université Claude Bernard Lyon

A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome