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J Cell Biochem,
1989]
We have examined the mechanism of membrane protein insertion in the ameboid spermatozoa of Caenorhabditis elegans using two monoclonal antibodies which recognize the same set of eight sperm-specific polypeptides. Previous electron microscopic studies demonstrated that these antibodies label surface and cytoplasmic populations of antigen. Cells whose surface antigen had been removed by proteolysis were able to localize new membrane protein insertion at the tips of pseudopodial projections. C. elegans sperm do not contain the protein synthesizing machinery needed for delivery of new membrane to the cell surface. It has, therefore, been of interest to determine how localized membrane assembly occurs. Here we have determined the subcellular location of each of these eight polypeptides. A closely positioned doublet of bands around 97 kD (comprising 40% of the total antigen in sperm) represents surface (larger member of doublet) and cytoplasmic (lower member) forms of protein. Proteolysis of live cells eliminated this surface form from immunoblots but did not affect the cytoplasmic protein. When cells were allowed to reinsert new protein following removal of the enzyme, this surface form was regenerated. Since sperm are unable to synthesize new protein, this higher molecular weight species may arise from a posttranslational modification of proteins in the cytoplasmic pool. We present evidence suggesting that the surface protein is generated from this cytoplasmic pool by addition of fatty acid. Fatty acid acylation would account for both the observed decrease in electrophoretic mobility of the surface form and provide increased hydrophobicity to the protein which may allow for its insertion into the lipid bilayer.
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Cell Motil Cytoskeleton,
1988]
Caenorhabditis elegans sperm are nonflagellated cells that lack actin and myosin yet can form pseudopods to propel themselves over solid substrates. Surface-attached probes such as latex beads, lectins, and antimembrane protein monoclonal antibodies move rearward over the dorsal pseudopod surface of sessile cells. Using monoclonal antibodies against membrane proteins of C. elegans sperm to examine the role of localized membrane assembly and rearward flow in crawling movement, we determined that substrates prepared by coating glass with antimembrane protein antibodies, but not naked glass or other nonmembrane-binding proteins, promote sperm motility. Sperm locomotion is inhibited in a concentration-dependent fashion when cells are bathed with soluble antimembrane protein monoclonal antibodies but not with antimouse Ig antibodies or a monoclonal antibody against a sperm cytoplasmic protein. Our results suggest that C. elegans sperm crawl by gaining traction with substrate- attached ligands via their surface proteins and by using the motor that moves those proteins rearward on unattached cells to pull the entire cell forward. Continuous insertion of new proteins at the front of the cell and their subsequent adhesion to the substrate allows this process to continue.
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J Cell Biol,
1986]
During the development of pseudopodial spermatozoa of the nematode, Caenorhabditis elegans, protein synthesis stops before differentiation is completed. Colloidal gold conjugates of monoclonal antibody SP56, which binds to the surface of spermatozoa, and TR20, which recognizes the major sperm cytoplasmic protein (MSP), were used to label thin sections of testes embedded in Lowicryl K4M in order to follow polypeptides from their synthesis early in spermatogenesis to their segregation to specific compartments of the mature cell. Both antigens are synthesized in primary spermatocytes and are assembled into a unique double organelle, the fibrous body-membranous organelle (FB-MO) complex. However, the antigens are localized in different regions of this FB-MO complex. As described in detail, the assembly of proteins into the FB-MO complex allows both membrane and cytoplamsic components to be concentrated in the spermatids after meiosis. Then, the stepwise disassembly of this transient structure ensures delivery of each component to its final destination in the mature spermatozoan: MSP filaments in the fibrous body depolymerize, releasing MSP into the cytoplasm and the membranous organelles fuse with the plasma membrane, delivering SP56 antigen to the surface.
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J Cell Biol,
1986]
Four monoclonal antibodies that are directed against antigens present in sperm and absent from other worm tissues were characterized. Antibody TR20 is directed against the major sperm proteins, a family of small, abundant, cytoplasmic proteins that have been previously described (Klass, M. R., and D. Hirsh, 1981, Dev. Biol., 84:299-312; Burke, D. J., and S. Ward, 1983, J. Mol. Biol., 171:1-29). Three other antibodies, SP56, SP150, and TR11, are all directed against the same set of minor sperm polypeptides that range in size from 29 to 215 kD. More than eight different sperm polypeptides are antigenic by both immunotransfer and immunoprecipitation assays. The three antibodies are different immunoglobulin subclasses, yet they compete with each other for antigen binding so they are directed against the same antigenic determinant on the multiple sperm proteins. This antigenic determinant is sensitive to any of six different proteases, is insensitive to periodate oxidation or N-glycanase digestion, and is detectable on a polypeptide synthesized in vitro. Therefore, the antigenic determinant resides in the polypeptide chain. However, peptide fragments of the proteins are not antigenic, thus the determinant is likely to be dependent on polypeptide conformation. The antigenic determinant shared by these proteins could represent a common structural feature of importance to the localization or cellular specificity of these proteins.
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BMC Bioinformatics,
2017]
BACKGROUND: High-throughput sequencing offers higher throughput and lower cost for sequencing a genome. However, sequencing errors, including mismatches and indels, may be produced during sequencing. Because, errors may reduce the accuracy of subsequent de novo assembly, error correction is necessary prior to assembly. However, existing correction methods still face trade-offs among correction power, accuracy, and speed. RESULTS: We develop a novel overlap-based error correction algorithm using FM-index (called FMOE). FMOE first identifies overlapping reads by aligning a query read simultaneously against multiple reads compressed by FM-index. Subsequently, sequencing errors are corrected by k-mer voting from overlapping reads only. The experimental results indicate that FMOE has highest correction power with comparable accuracy and speed. Our algorithm performs better in long-read than short-read datasets when compared with others. The assembly results indicated different algorithms has its own strength and weakness, whereas FMOE is good for long or good-quality reads. CONCLUSIONS: FMOE is freely available at https://github.com/ythuang0522/FMOC .
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Elife,
2022]
Analyses across imaging modalities allow the integration of complementary spatiotemporal information about brain development, structure and function. However, systematic atlasing across modalities is limited by challenges to effective image alignment. We combine highly spatially resolved electron microscopy (EM) and highly temporally resolved time-lapse fluorescence microscopy (FM) to examine the emergence of a complex nervous system in C. elegans embryogenesis. We generate an EM time series at four classic developmental stages and create a landmark-based co-optimization algorithm for cross-modality image alignment, which handles developmental heterochrony among datasets to achieve accurate single-cell level alignment. Synthesis based on the EM series and time-lapse FM series carrying different cell-specific markers reveals critical dynamic behaviors across scales of identifiable individual cells in the emergence of the primary neuropil, the nerve ring, as well as a major sensory organ, the amphid. Our study paves the way for systematic cross-modality data synthesis in C. elegans and demonstrates a powerful approach that may be applied broadly.
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Opt Express,
1998]
Multiphoton laser scanning microscopy (MPLSM) enables the production of long timelapse recordings from live fluorescent specimens. 1047- and 900-nm excitation were used to image both a vital fluorescent membrane probe, FM 4-64, and a modified green fluorescent protein (GFP) in live Caenorhabditis elegans embryos. Automated four-dimensional (4D) data collection yielded individual recordings comprising thousands of images, each allowing analysis of all of the cell divisions, contacts, migrations, and fusions that occur during a span of several hours of embryogenesis.
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Sci Rep,
2020]
A bioelectronic nose device based on micelle-stabilized olfactory receptors is developed for the selective discrimination of a butter flavor substance in commercial fermented alcoholic beverages. In this work, we have successfully overexpressed ODR-10, a type of olfactory receptor, from Caenorhabditis elegans using a bacterial expression system at a low cost and high productivity. The highly-purified ODR-10 was stabilized in micelle structures, and it was immobilized on a carbon nanotube field-effect transistor to build a bioelectronic nose for the detection of diacetyl, a butter flavor substance, via the specific interaction between diacetyl and ODR-10. The bioelectronic nose device can sensitively detect diacetyl down to 10 fM, and selectively discriminate it from other substances. In addition, this sensor could directly evaluate diacetyl levels in a variety of real fermented alcoholic beverages such as beer, wine, and makgeolli (fermented Korean wine), while the sensor did not respond to soju (Korean style liquor without diacetyl). In this respect, our sensor should be a powerful tool for versatile food industrial applications such as the quality control of alcoholic beverages and foods.
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Proc Natl Acad Sci U S A,
2011]
Chemical synapses contain substantial numbers of neurotransmitter-filled synaptic vesicles, ranging from approximately 100 to many thousands. The vesicles fuse with the plasma membrane to release neurotransmitter and are subsequently reformed and recycled. Stimulation of synapses in vitro generally causes the majority of the synaptic vesicles to release neurotransmitter, leading to the assumption that synapses contain numerous vesicles to sustain transmission during high activity. We tested this assumption by an approach we termed cellular ethology, monitoring vesicle function in behaving animals (10 animal models, nematodes to mammals). Using FM dye photooxidation, pHluorin imaging, and HRP uptake we found that only approximately 1-5% of the vesicles recycled over several hours, in both CNS synapses and neuromuscular junctions. These vesicles recycle repeatedly, intermixing slowly (over hours) with the reserve vesicles. The latter can eventually release when recycling is inhibited in vivo but do not seem to participate under normal activity. Vesicle recycling increased only to 5% in animals subjected to an extreme stress situation (frog predation on locusts). Synapsin, a molecule binding both vesicles and the cytoskeleton, may be a marker for the reserve vesicles: the proportion of vesicles recycling in vivo increased to 30% in synapsin-null Drosophila. We conclude that synapses do not require numerous reserve vesicles to sustain neurotransmitter release and thus may use them for other purposes, examined in the accompanying paper.
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Biotechniques,
1998]
We present a novel approach to the viewing and analysis of 4-dimensional (4-D) data sets recorded from live fluorescent samples. With stereo-4-D reconstructions, the observer manipulates a rotatable projection of the full 3-dimensional (3-D) specimen while simultaneously controlling animation of the recording forward or backward in time. The result is a unique lifelike perspective on the development of an entire living subject. Here, we apply this technique to the observation of the cell membranes of developing Caenorhabditis elegans. Embryos labeled with the vital plasma membrane probe FM 4-64 were imaged by multiphoton laser scanning fluorescence microscopy, yielding 4-D data sets of entire embryos over several hours of development. Stereo 4-D and standard focal-plane 4-D viewing of these novel time-lapse recordings provide the observer with detail at both the subcellular and whole-animal level from a single data set and produce a unique record of the lineage, cell shape changes, cell contacts and morphogenetic dynamics that make up embryogenesis. The procedures by which stereo-4-D reconstructions are created and viewed rely on public domain software running on a personal computer and should therefore be accessible by a general audience. Data output utilizes the versatile and well-supported QuickTime animation format. Additional features allow for stereo-4-D reconstruction of isolated 3-D volumes of interest from within the larger specimen.