Novel elasticity measurements reveal C. elegans cuticle stiffens with age and in a long-lived mutant

Changes in biomechanical properties have profound impacts on human health. C. elegans might serve as a model for studying the molecular genetics of mammalian tissue decline. Previously, we found that collagens are required for insulin signaling mutants' long lifespan and that overexpression of specific collagens extends wild-type lifespan. However, whether these effects on lifespan are due to mechanical changes during aging has not yet been established. Here, we have developed two novel methods to study the cuticle: we measure mechanical properties of live animals using osmotic shock, and we directly perform the tensile test on isolated cuticles using microfluidic technology. Using these tools, we find that the cuticle, not the muscle, is responsible for changes in the “stretchiness” of C. elegans, and that cuticle stiffness is highly nonlinear and anisotropic. We also found that collagen mutations alter the integrity of the cuticle by significantly changing the elasticity. In addition, aging stiffens the cuticle under mechanical loads beyond the cuticle's healthy stretched state. Measurements of elasticity showed that long-lived daf-2 mutants were considerably better at preventing progressive mechanical changes with age. These tests of C. elegans biophysical properties suggest that the cuticle is responsible for their resilience.     

The Nicotinic Acetylcholine Receptors of the Parasitic Nematode Ascaris suum: Formation of Two Distinct Drug Targets by Varying the Relative Expression Levels of Two Subunits

Parasitic nematodes are of medical and veterinary importance, adversely affecting human health and animal welfare. Ascaris suum is a gastrointestinal parasite of pigs; in addition to its veterinary significance it is a good model of the human parasite Ascaris lumbricoides, estimated to infect ∼1.4 billion people globally. Anthelmintic drugs are essential to control nematode parasites, and nicotinic acetylcholine receptors (nAChRs) on nerve and muscle are the targets of cholinergic anthelmintics such as levamisole and pyrantel. Previous genetic analyses of nematode nAChRs have been confined to Caenorhabditis elegans, which is phylogenetically distinct from Ascaris spp. and many other important parasites. Here we report the cloning and expression of two nAChR subunit cDNAs from A. suum. The subunits are very similar in sequence to C. elegans UNC-29 and UNC-38, are expressed on muscle cells and can be expressed robustly in Xenopus oocytes to form acetylcholine-, nicotine-, levamisole- and pyrantel-sensitive channels. We also demonstrate that changing the stoichiometry of the receptor by injecting different ratios of the subunit cRNAs can reproduce two of the three pharmacological subtypes of nAChR present in A. suum muscle cells. When the ratio was 5∶1 (Asu-unc-38∶Asu-unc-29), nicotine was a full agonist and levamisole was a partial agonist, and oocytes responded to oxantel, but not pyrantel. At the reverse ratio (1∶5 Asu-unc-38∶Asu-unc-29), levamisole was a full agonist and nicotine was a partial agonist, and the oocytes responded to pyrantel, but not oxantel. These results represent the first in vitro expression of any parasitic nicotinic receptor and show that their properties are substantially different from those of C. elegans. The results also show that changing the expression level of a single receptor subunit dramatically altered the efficacy of some anthelmintic drugs. In vitro expression of these subunits may permit the development of parasite-specific screens for future anthelmintics.     

α-Actinin Is Required for the Proper Assembly of Z-Disk/Focal-Adhesion-Like Structures and for Efficient Locomotion in Caenorhabditis elegans

The actin binding protein α-actinin is a major component of focal adhesions found in vertebrate cells and of focal-adhesion-like structures found in the body wall muscle of the nematode Caenorhabditis elegans. To study its in vivo function in this genetic model system, we isolated a strain carrying a deletion of the single C. elegans α-actinin gene. We assessed the cytological organization of other C. elegans focal adhesion proteins and the ultrastructure of the mutant. The mutant does not have normal dense bodies, as observed by electron microscopy; however, these dense-body-like structures still contain the focal adhesion proteins integrin, talin, and vinculin, as observed by immunofluorescence microscopy. Actin is found in normal-appearing I-bands, but with abnormal accumulations near muscle cell membranes. Although swimming in water appeared grossly normal, use of automated methods for tracking the locomotion of individual worms revealed a defect in bending. We propose that the reduced motility of α-actinin null is due to abnormal dense bodies that are less able to transmit the forces generated by actin/myosin interactions.     

The ALP-Enigma Protein ALP-1 Functions in Actin Filament Organization to Promote Muscle Structural Integrity in Caenorhabditis elegans

Mutations that affect the Z-disk–associated ALP-Enigma proteins have been linked to human muscular and cardiac diseases. Despite their clear physiological significance for human health, the mechanism of action of ALP-Enigma proteins is largely unknown. In Caenorhabditis elegans, the ALP-Enigma protein family is encoded by a single gene, alp-1; thus C. elegans provides an excellent model to study ALP-Enigma function. Here we present a molecular and genetic analysis of ALP-Enigma function in C. elegans. We show that ALP-1 and α-actinin colocalize at dense bodies where actin filaments are anchored and that the proper localization of ALP-1 at dense bodies is dependent on α-actinin. Our analysis of alp-1 mutants demonstrates that ALP-1 functions to maintain actin filament organization and participates in muscle stabilization during contraction. Reducing α-actinin activity enhances the actin filament phenotype of the alp-1 mutants, suggesting that ALP-1 and α-actinin function in the same cellular process. Like α-actinin, alp-1 also interacts genetically with a connectin/titin family member, ketn-1, to provide mechanical stability for supporting body wall muscle contraction. Taken together, our data demonstrate that ALP-1 and α-actinin function together to stabilize actin filaments and promote muscle structural integrity.     

Scaling behaviors of weighted food webs as energy transportation networks

Food webs can be regarded as energy transporting networks in which the weight of each edge denotes the energy flux between two species. By investigating 21 empirical weighted food webs as energy flow networks, we found several ubiquitous scaling behaviors. Two random variables A_i and C_i defined for each vertex i, representing the total flux (also called vertex intensity) and total indirect effect or energy store of i, were found to follow power law distributions with the exponents α≈1.32 and β≈1.33, respectively. Another scaling behavior is the power law relationship, , where η≈1.02. This is known as the allometric scaling power law relationship because A_i can be treated as metabolism and C_i as the body mass of the sub-network rooted from the vertex i, according to the algorithm presented in this paper. Finally, a simple relationship among these power law exponents, η=(α−1)/(β−1), was mathematically derived and tested by the empirical food webs.     

Intrinsic viscosities of polyelectrolytes in the absence and in the presence of extra salt: Consequences of the stepwise conversion of dextran into a polycation

Viscosities of dilute polymer solutions were measured in capillary viscometers for samples varying in their fraction f of charged units from 0.00 to 0.90. The dependence of the logarithm of the relative viscosity on polymer concentration c is in all cases reproduced quantitatively by three characteristic parameters: [η], the intrinsic viscosity; B, a viscometric interaction parameter (related to the Huggins constant); [η], a parameter required only for polyelectrolytes at low concentrations of extra salt. In pure water [η] increases more than 80 times as the fraction f rises from zero to 0.90 and [η] starts from zero and goes up to ≈71 mL/g. Upon the addition of NaCl [η] decreases by at least one order of magnitude (depending on the value of f). The observed dependences of log [η] on log c_salt can be modeled quantitatively by Boltzmann sigmoids.     

The DH-PH region of the giant protein UNC-89 activates RHO-1 GTPase in Caenorhabditis elegans body wall muscle

Mutation of the Caenorhabditis elegans gene unc-89 results in disorganization of muscle A-bands. unc-89 encodes a giant polypeptide (900 kDa) containing a DH domain followed by a PH domain at its N terminus, which is characteristic of guanine nucleotide exchange factor proteins for Rho GTPases. To obtain evidence that the DH-PH region has activity toward specific Rho family small GTPases, we conducted an experiment using the yeast three-hybrid system. The DH-PH region of UNC-89 has exchange activity for RHO-1 (C. elegans RhoA), but not for CED-10 (C. elegans Rac), MIG-2 (C. elegans RhoG), or CDC-42 (C. elegans Cdc42). The DH domain alone has similar activity for RHO-1. An in vitro binding assay demonstrates interaction between the DH-PH region of UNC-89 and each of the C. elegans Rho GTPases. Partial knockdown of rho-1 in C. elegans adults showed a pattern of disorganization of myosin thick filaments similar to the phenotype caused by unc-89 (su75), a mutant allele in which all of the isoforms containing the DH-PH region are missing. Taken together, we propose a model in which the DH-PH region of UNC-89 activates RHO-1 GTPase for organization of myosin filaments in C. elegans muscle cells.     

Unique C. elegans telomeric overhang structures reveal the evolutionarily conserved properties of telomeric DNA

There are two basic mechanisms that are associated with the maintenance of the telomere length, which endows cancer cells with unlimited proliferative potential. One mechanism, referred to as alternative lengthening of telomeres (ALT), accounts for approximately 10–15% of all human cancers. Tumours engaged in the ALT pathway are characterised by the presence of the single stranded 5′-C-rich telomeric overhang (C-overhang). This recently identified hallmark of ALT cancers distinguishes them from healthy tissues and renders the C-overhang as a clear target for anticancer therapy. We analysed structures of the 5′-C-rich and 3′-G-rich telomeric overhangs from human and Caenorhabditis elegans, the recently established multicellular in vivo model of ALT tumours. We show that the telomeric DNA from C. elegans and humans forms fundamentally different secondary structures. The unique structural characteristics of C. elegans telomeric DNA that are distinct not only from those of humans but also from those of other multicellular eukaryotes allowed us to identify evolutionarily conserved properties of telomeric DNA. Differences in structural organisation of the telomeric DNA between the C. elegans and human impose limitations on the use of the C. elegans as an ALT tumour model.     

A novel fluorescent timer based on bicistronic expression strategy in Caenorhabditis elegans

Fluorescent timers are useful tools for studying the spatial and temporal cellular or molecular events. Based on the trans-splicing mechanism in Caenorhabditis elegans, we constructed a “fluorescent timer” through bicistronic expression of two fluorescent proteins with different maturation times. When used in vivo, this “timer” changes its color over time and therefore can be used to monitor the activity of the targeted promoters in C. elegans. Using this “timer”, we have successfully traced the time-dependent activity of myo-3 promoter which drives expression in body wall muscle and vulval muscle. We found that the myo-3 promoter started to be active about 7 h after egg-laying and sustained its activity in the following hatching process. We have also determined the myo-3 promoter activity during larval development by this “timer”. We anticipate that more new “fluorescent timers” with variable time-resolution could be designed by bicistronic expression of different fluorescent protein pairs.     

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm’s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.     

Study on properties and regulation peculiarities of glycogenolysis enzymes in fish skeletal muscles depending on peculiarities of the species motor activity

The activity values, properties and peculiarities of activation of glycogen phosphorylase (GP, EC 2.4.1.1) and glycogen phosphorylase kinase (GPK, EC 2.7.138) were studied in the white skeletal muscle of fishes differing in motor behavior. No differences in the GP and GPK activity levels were revealed in porgy Diplodus annularis (L.), horse mackerel Trachurus mediterraneus ponticus, trout Salmo trutta morphario, scorpionfish Scorpaena porcus, flounder Scophtalnus maeoticus maeoticus, and carp Cyprinus carpio; however, properties of the isolated enzymes and peculiarities of formation of their activated forms during swimming in a hydrodynamic tube are determined by functional peculiarities of the muscle tissue and are associated with the motor activity character of the species. The more rapid ion regulation prevails in fishes capable for the burst swimming type (scorpionfish, trout). The glycogenolysis hormonal regulation leading to a change of the GPK activity index has been found in other species.     

The influences of PRG-1 on the expression of small RNAs and mRNAs

Background

In metazoans, Piwi-related Argonaute proteins play important roles in maintaining germline integrity and fertility and have been linked to a class of germline-enriched small RNAs termed piRNAs. Caenorhabditis elegans encodes two Piwi family proteins called PRG-1 and PRG-2, and PRG-1 interacts with the C. elegans piRNAs (21U-RNAs). Previous studies found that mutation of prg-1 causes a marked reduction in the expression of 21U-RNAs, temperature-sensitive defects in fertility and other phenotypic defects.

Results

In this study, we wanted to systematically demonstrate the function of PRG-1 in the regulation of small RNAs and their targets. By analyzing small RNAs and mRNAs with and without a mutation in prg-1 during C. elegans development, we demonstrated that (1) mutation of prg-1 leads to a decrease in the expression of 21U-RNAs, and causes 35 ~ 40% of miRNAs to be down-regulated; (2) in C. elegans, approximately 3% (6% in L4) of protein-coding genes are differentially expressed after mutating prg-1, and 60 ~ 70% of these substantially altered protein-coding genes are up-regulated; (3) the target genes of the down-regulated miRNAs and the candidate target genes of the down-regulated 21U-RNAs are enriched in the up-regulated protein-coding genes; and (4) PRG-1 regulates protein-coding genes by down-regulating small RNAs (miRNAs and 21U-RNAs) that target genes that participate in the development of C. elegans.

Conclusions

In prg-1-mutated C. elegans, the expression of miRNAs and 21U-RNAs was reduced, and the protein-coding targets, which were associated with the development of C. elegans, were up-regulated. This may be the mechanism underlying PRG-1 function.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-321) contains supplementary material, which is available to authorized users.     

Quantitative Analysis of Cytokinesis In Situ during C. elegans Postembryonic Development

The physical separation of a cell into two daughter cells during cytokinesis requires cell-intrinsic shape changes driven by a contractile ring. However, in vivo, cells interact with their environment, which includes other cells. How cytokinesis occurs in tissues is not well understood. Here, we studied cytokinesis in an intact animal during tissue biogenesis. We used high-resolution microscopy and quantitative analysis to study the three rounds of division of the C. elegans vulval precursor cells (VPCs). The VPCs are cut in half longitudinally with each division. Contractile ring breadth, but not the speed of ring closure, scales with cell length. Furrowing speed instead scales with division plane dimensions, and scaling is consistent between the VPCs and C. elegans blastomeres. We compared our VPC cytokinesis kinetics data with measurements from the C. elegans zygote and HeLa and Drosophila S2 cells. Both the speed dynamics and asymmetry of ring closure are qualitatively conserved among cell types. Unlike in the C. elegans zygote but similar to other epithelial cells, Anillin is required for proper ring closure speed but not asymmetry in the VPCs. We present evidence that tissue organization impacts the dynamics of cytokinesis by comparing our results on the VPCs with the cells of the somatic gonad. In sum, this work establishes somatic lineages in post-embryonic C. elegans development as cell biological models for the study of cytokinesis in situ.     

CeleST: Computer Vision Software for Quantitative Analysis of C. elegans Swim Behavior Reveals Novel Features of Locomotion

In the effort to define genes and specific neuronal circuits that control behavior and plasticity, the capacity for high-precision automated analysis of behavior is essential. We report on comprehensive computer vision software for analysis of swimming locomotion of C. elegans, a simple animal model initially developed to facilitate elaboration of genetic influences on behavior. C. elegans swim test software CeleST tracks swimming of multiple animals, measures 10 novel parameters of swim behavior that can fully report dynamic changes in posture and speed, and generates data in several analysis formats, complete with statistics. Our measures of swim locomotion utilize a deformable model approach and a novel mathematical analysis of curvature maps that enable even irregular patterns and dynamic changes to be scored without need for thresholding or dropping outlier swimmers from study. Operation of CeleST is mostly automated and only requires minimal investigator interventions, such as the selection of videotaped swim trials and choice of data output format. Data can be analyzed from the level of the single animal to populations of thousands. We document how the CeleST program reveals unexpected preferences for specific swim “gaits” in wild-type C. elegans, uncovers previously unknown mutant phenotypes, efficiently tracks changes in aging populations, and distinguishes “graceful” from poor aging. The sensitivity, dynamic range, and comprehensive nature of CeleST measures elevate swim locomotion analysis to a new level of ease, economy, and detail that enables behavioral plasticity resulting from genetic, cellular, or experience manipulation to be analyzed in ways not previously possible.

This is a PLOS Computational Biology Software Article

     

Effects of accumulations of sediments and drift algae on recruitment of sessile organisms associated with oyster reefs

Increases in sediment and drift algae accumulations have caused degradation of coastal lagoons worldwide. It is well known that these factors are stressors of seagrass beds, sediment fauna and coral reefs. However, little is known about the impacts on temperate hard-bottom assemblages within soft-bottom lagoons. To test if accumulations of sediment and drift algae (stress) affected recruitment of sessile oyster reef organisms, we constructed cages in Hog Island Bay, Virginia that trapped drifting macroalgae (≈ 2.7 kg WW m²) and facilitated sedimentation (≈ 7 mm per 2-3 month). The stress treatments and unstressed controls were placed in front, between, and behind reefs (position) to represent wave exposed (≈ 0.3 m, windy conditions), current exposed (≈ 0.2 m s^− 1, peak tide) and protected (≈ 0 m, 0.0 m s^− 1) habitats. The percentage cover of recruited taxa onto bricks was mapped 4 times during a 1-year period. There were strong significant effects of stress on the total assemblage, plant (but not animal) richness, total plant and animal cover, and cover of the most common taxa. Unstressed bricks had high plant richness, high animal and plant cover, and high cover of the oyster Crassostrea virginica, the alien algae Gracilaria vermiculophylla and Codium fragile, the alga Agardhiella subulata, and high to medium cover of the opportunistic algae Ulva curvata and Enteromorpha spp. In comparison, sediment-stressed bricks had low plant richness, low animal and plant cover, and low cover of C. virginica, G. vermiculophylla, C. fragile, A. subulata, U. curvata and Enteromorpha spp. Similarly, algae-stressed bricks also had low cover of animals, C. virginica, G. vermiculophylla, C. fragile, and A. subulata, but intermediate plant richness and plant cover and high cover of U. curvata and Enteromorpha spp. Although reef position caused significant multivariate results, this factor was clearly less important than the stress factor. Our study shows that accumulations of sediments and drift algae have an adverse impact on sessile temperate reef organisms, reducing richness and abundance, but favoring a few small opportunistic taxa. As the reef-generating oysters themselves performed poorly under these stressors, the long-term impact of the causes of these stressors, eutrophication and urbanization, is likely to be diminished reefs with cascading adverse effects on sessile reef organisms.     

Germline Expression Influences Operon Organization in the Caenorhabditis elegans Genome

Operons are found across multiple kingdoms and phyla, from prokaryotes to chordates. In the nematode Caenorhabditis elegans, the genome contains >1000 operons that compose ~15% of the protein-coding genes. However, determination of the force(s) promoting the origin and maintenance of operons in C. elegans has proved elusive. Compared to bacterial operons, genes within a C. elegans operon often show poor coexpression and only sometimes encode proteins with related functions. Using analysis of microarray and large-scale in situ hybridization data, we demonstrate that almost all operon-encoded genes are expressed in germline tissue. However, genes expressed during spermatogenesis are excluded from operons. Operons group together along chromosomes in local clusters that also contain monocistronic germline-expressed genes. Additionally, germline expression of genes in operons is largely independent of the molecular function of the encoded proteins. These analyses demonstrate that mechanisms governing germline gene expression influence operon origination and/or maintenance. Thus, gene expression in a specific tissue can have profound effects on the evolution of genome organization.     

C. elegans Germ Cells Show Temperature and Age-Dependent Expression of Cer1, a Gypsy/Ty3-Related Retrotransposon

Virus-like particles (VLPs) have not been observed in Caenorhabditis germ cells, although nematode genomes contain low numbers of retrotransposon and retroviral sequences. We used electron microscopy to search for VLPs in various wild strains of Caenorhabditis, and observed very rare candidate VLPs in some strains, including the standard laboratory strain of C. elegans, N2. We identified the N2 VLPs as capsids produced by Cer1, a retrotransposon in the Gypsy/Ty3 family of retroviruses/retrotransposons. Cer1 expression is age and temperature dependent, with abundant expression at 15°C and no detectable expression at 25°C, explaining how VLPs escaped detection in previous studies. Similar age and temperature-dependent expression of Cer1 retrotransposons was observed for several other wild strains, indicating that these properties are common, if not integral, features of this retroelement. Retrotransposons, in contrast to DNA transposons, have a cytoplasmic stage in replication, and those that infect non-dividing cells must pass their genomic material through nuclear pores. In most C. elegans germ cells, nuclear pores are largely covered by germline-specific organelles called P granules. Our results suggest that Cer1 capsids target meiotic germ cells exiting pachytene, when free nuclear pores are added to the nuclear envelope and existing P granules begin to be removed. In pachytene germ cells, Cer1 capsids concentrate away from nuclei on a subset of microtubules that are exceptionally resistant to microtubule inhibitors; the capsids can aggregate these stable microtubules in older adults, which exhibit a temperature-dependent decrease in egg viability. When germ cells exit pachytene, the stable microtubules disappear and capsids redistribute close to nuclei that have P granule-free nuclear pores. This redistribution is microtubule dependent, suggesting that capsids that are released from stable microtubules transfer onto new, dynamic microtubules to track toward nuclei. These studies introduce C. elegans as a model to study the interplay between retroelements and germ cell biology.