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[
Dev Dyn,
2010]
Hemidesmosomes are evolutionarily conserved attachment complexes linked to intermediate filaments that connect epithelial cells to the extracellular matrix. They provide tissue integrity and resistance to mechanical forces. Alterations in hemidesmosome structures are responsible for skin blistering, carcinoma invasion, and wound-healing defects. Valuable information about hemidesmosome assembly and disassembly has been obtained from in vitro cell culture studies. However, how these processes take place in vivo still remains elusive. Here, we discuss recent data about the formation and reorganization of hemidesmosomes in several in vivo model systems, particularly zebrafish and Caenorhabditis elegans, focusing on various factors affecting their dynamics. Mechanisms found in different organisms reveal that hemidesmosome formation and maintenance in vivo are carefully controlled by ECM protein folding, ECM-receptor expression and trafficking, and by post-translational modification of hemidesmosome components. These findings validate and extend the in vitro studies, and shed light on our understanding about hemidesmosomes across species.
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Kovacs AL, Jia K, Sato K, Lu Q, Sato M, Lin L, Legouis R, Lapierre LR, O'Rourke EJ, Chang JT, Kumsta C, Guo B, Hansen M, Melendez A, Zhang H, Wu F, Wang X
[
Autophagy,
2015]
The cellular recycling process of autophagy has been extensively characterized with standard assays in yeast and mammalian cell lines. In multicellular organisms, numerous external and internal factors differentially affect autophagy activity in specific cell types throughout the stages of organismal ontogeny, adding complexity to the analysis of autophagy in these metazoans. Here we summarize currently available assays for monitoring the autophagic process in the nematode C. elegans. A combination of measuring levels of the lipidated Atg8 ortholog LGG-1, degradation of well-characterized autophagic substrates such as germline P granule components and the SQSTM1/p62 ortholog SQST-1, expression of autophagic genes and electron microscopy analysis of autophagic structures are presently the most informative, yet steady-state, approaches available to assess autophagy levels in C. elegans. We also review how altered autophagy activity affects a variety of biological processes in C. elegans such as L1 survival under starvation conditions, dauer formation, aging, and cell death, as well as neuronal cell specification. Taken together, C. elegans is emerging as a powerful model organism to monitor autophagy while evaluating important physiological roles for autophagy in key developmental events as well as during adulthood.
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[
Nature,
2011]
Mechanotransduction refers to the transformation of physical forces into chemical signals. It generally involves stretch-sensitive channels or conformational change of cytoskeleton-associated proteins. Mechanotransduction is crucial for the physiology of several organs and for cell migration. The extent to which mechanical inputs contribute to development, and how they do this, remains poorly defined. Here we show that a mechanotransduction pathway operates between the body-wall muscles of Caenorhabditis elegans and the epidermis. This pathway involves, in addition to a Rac GTPase, three signalling proteins found at the hemidesmosome:
p21-activated kinase (PAK-1), the adaptor GIT-1 and its partner PIX-1. The phosphorylation of intermediate filaments is one output of this pathway. Tension exerted by adjacent muscles or externally exerted mechanical pressure maintains GIT-1 at hemidesmosomes and stimulates PAK-1 activity through PIX-1 and Rac. This pathway promotes the maturation of a hemidesmosome into a junction that can resist mechanical stress and contributes to coordinating the morphogenesis of epidermal and muscle tissues. Our findings suggest that the C. elegans hemidesmosome is not only an attachment structure, but also a mechanosensor that responds to tension by triggering signalling processes. We suggest that similar pathways could promote epithelial morphogenesis or wound healing in other organisms in which epithelial cells adhere to tension-generating contractile cells.
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[
Metallomics,
2010]
With their widespread applications in industry, agriculture and many other fields, more and more rare earth elements (REEs) are getting into the environment, especially the aquatic systems. Therefore, understanding the aquatic ecotoxicity of REEs has become more and more important. In the present work, Caenorhabditis elegans (C. elegans) was used as a test organism and life-cycle endpoints were chosen along with elemental assay to evaluate the aquatic toxicity of lanthanum (La), a representative of REEs. The results show La+ had significant adverse effects on the growth and reproduction of worms above a concentration of 10 mol L. The elemental mapping by microbeam synchrotron radiation X-ray fluorescence (-SRXRF) illustrated how La treatment disturbed the metals distribution in the whole body of a single tiny nematode at lower levels. Our results suggested that the high-level REEs in some polluted water bodies would lead to an aquatic ecological crisis. The assessment we performed in the present work could be developed as a standardized test design for aquatic toxicological research.
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[
Genes Dev,
2011]
The discovery of microRNAs (miRNAs)
lin-4 and
let-7 as temporal regulators in Caenorhabditis elegans led to broader searches for novel miRNAs and their biological roles. Unlike protein-coding genes and some long noncoding RNAs, canonical metazoan miRNAs are not known to contain introns within their genomic precursor sequences. Because the short length of miRNAs complicates a statistically definitive assignment of split genes in RNA sequencing data sets, we took an experimental approach toward testing the compatibility of splicing and functional miRNA biogenesis. To definitively evaluate the possibility that miRNAs could derive from interrupted genes, we constructed intron-interrupted variants of C. elegans
lin-4 and assayed for their miRNA-encoding capability and biological activity in the developing organism. Our studies indicate that (1) intron-containing miRNAs (inc-miRs) can be efficiently spliced and processed to produce miRNAs with normal termini, and (2) these miRNAs can be functional in full rescue of developmental phenotypes in null mutants lacking endogenous
lin-4. This study provides the first evidence to support the ability of intron-interrupted miRNA precursors to produce functional regulators and identifies an additional modality available for metazoan miRNA production.
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Piatkevich KD, Yao L, Sun X, Wu L, Namikawa K, Wang X, Zhang H, Rehbock M, Papadaki S, Drobizhev M, Kö\;ster RW
[
Nat Methods,
2023]
Recent progress in fluorescent protein development has generated a large diversity of near-infrared fluorescent proteins (NIR FPs), which are rapidly becoming popular probes for a variety of imaging applications. However, the diversity of NIR FPs poses a challenge for end-users in choosing the optimal one for a given application. Here we conducted a systematic and quantitative assessment of intracellular brightness, photostability, oligomeric state, chemical stability and cytotoxicity of 22 NIR FPs in cultured mammalian cells and primary mouse neurons and identified a set of top-performing FPs including emiRFP670, miRFP680, miRFP713 and miRFP720, which can cover a majority of imaging applications. The top-performing proteins were further validated for in vivo imaging of neurons in Caenorhabditis elegans, zebrafish, and mice as well as in mice liver. We also assessed the applicability of the selected NIR FPs for multicolor imaging of fusions, expansion microscopy and two-photon imaging.
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[
RNA,
1996]
Caenorhabditis elegans introns typically lack both branch point and polypyrimidine tract consensus sequences utilized in other organisms for intron recognition. However, most introns have an unusually long, highly conserved consensus, UUUCAG/R, at the 3' splice site. This site can be recognized even when the -1G is changed to A. To determine how the 3' splice site is defined, we tested mutations in the sequence UUUCAA/A at the 3' splice site of the first intron of a test gene in vivo. Replacement of individual U's with A's or C's compromised splicing. When the sequence UUUCAA/AAG was tested, splicing occurred following both the -1A and the +3G, indicating that both UUUC and the AG contain 3' splice site information. When the sequence UUUCAA/AAA was tested, all splicing occurred following the -1A, suggesting that the UUUC contains sufficient information in the absence of an AG to specify the location of the splice site. In support of this idea, when point mutations were introduced into the UUUC, unspliced RNAs accumulated. Surprisingly, RNAs containing the mutant intron often contained the second, nonmutated intron as well, suggesting that interference with splicing of one intron can interfere with splicing of a second intron in the same pre-mRNA. The majority of these unspliced RNAs were degraded by the system responsible for degradation of transcripts containing nonsense mutations (smg), even though the intron contained no nonsense codon.
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[
BMC Bioinformatics,
2023]
BACKGROUND: Locomotion behaviors of Caenorhabditis elegans play an important role in drug activity screening, anti-aging research, and toxicological assessment. Previous studies have provided important insights into drug activity screening, anti-aging, and toxicological research by manually counting the number of body bends. However, manual counting is often low-throughput and takes a lot of time and manpower. And it is easy to cause artificial bias and error in counting results. RESULTS: In this paper, an algorithm is proposed for automatic counting and analysis of the body bending behavior of nematodes. First of all, the numerical coordinate regression method with convolutional neural network is used to obtain the head and tail coordinates. Next, curvature-based feature point extraction algorithm is used to calculate the feature points of the nematode centerline. Then the maximum distance between the peak point and the straight line between the pharynx and the tail is calculated. The number of body bends is counted according to the change in the maximum distance per frame. CONCLUSION: Experiments are performed to prove the effectiveness of the proposed algorithm. The accuracy of head coordinate prediction is 0.993, and the accuracy of tail coordinate prediction is 0.990. The Pearson correlation coefficient between the results of the automatic count and manual count of the number of body bends is 0.998 and the mean absolute error is 1.931. Different strains of nematodes are selected to analyze differences in body bending behavior, demonstrating a relationship between nematode vitality and lifespan. The code is freely available at https://github.com/hthana/Body-Bend-Count .
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[
BMC Bioinformatics,
2022]
BACKGROUND: Locomotive behaviors are a rapid evaluation indicator reflecting whether the nervous system of worms is damaged, and has been proved to be sensitive to chemical toxicity. In many toxicological studies, C. elegans head thrashes is a key indicator of locomotive behaviors to measure the vitality of worms. In previous studies, the number of head thrashes was manually counted, which is time-consuming and labor-intensive. RESULTS: This paper presents an automatic recognition and counting method for head thrashes behavior of worms from experimental videos. First, the image processing algorithm is designed for worm morphology features calculation, mean gray values of head and tail are used to locate the head of worm accurately. Next, the worm skeleton is extracted and divided into equal parts. The angle formulas are used to calculate the bending angle of the head of worm. Finally, the number of head thrashes is counted according to the bending angle of the head in each frame. The robustness of the proposed algorithm is evaluated by comparing the counting results of the manual counting. It is proved that the proposed algorithm can recognize the occurrence of head thrashes of C. elegans of different strains. In addition, the difference of the head thrashes behavior of different worm strains is analyzed, it is proved that the relationship between worm head thrashes behavior and lifespan. CONCLUSIONS: A new method is proposed to automatically count the number of head thrashes of worms. This algorithm makes it possible to count the number of head thrashes from the worm videos collected by the automatic tracking system. The proposed algorithm will play an important role in toxicological research and worm vitality research. The code is freely available at https://github.com/hthana/HTC .
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[
FASEB J,
2015]
The retinitis pigmentosa 2 polypeptide (RP2) functions as a GTPase-activating protein (GAP) for ARL3 (Arf-like protein 3), a small GTPase. ARL3 is an effector of phosphodiesterase 6 (PDE6D), a prenyl-binding protein and chaperone of prenylated protein in photoreceptors. Mutations in the human RP2 gene cause X-linked retinitis pigmentosa (XLRP) and cone-rod dystrophy (XL-CORD). To study mechanisms causing XLRP, we generated an RP2 knockout mouse. The Rp2h(-/-) mice exhibited a slowly progressing rod-cone dystrophy simulating the human disease. Rp2h(-/-) scotopic a-wave and photopic b-wave amplitudes declined at 1 mo of age and continued to decline over the next 6 mo. Prenylated PDE6 subunits and G-protein coupled receptor kinase 1 (GRK1) were unable to traffic effectively to the Rp2h(-/-) outer segments. Mechanistically, absence of RP2 GAP activity increases ARL3-GTP levels, forcing PDE6D to assume a predominantly "closed" conformation that impedes binding of lipids. Lack of interaction disrupts trafficking of PDE6 and GRK1 to their destination, the photoreceptor outer segments. We propose that hyperactivity of ARL3-GTP in RP2 knockout mice and human patients with RP2 null alleles leads to XLRP resembling recessive rod-cone dystrophy.