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[
J Cell Biol,
1982]
Two distinct types of surface membrane rearrangement occur during the differentiation of Caenorhabditis elegans spermatids into amoeboid spermatozoa. The first, detected by the behavior of latex beads attached to the surface, is a nondirected, intermittent movement of discrete portions of the membrane. This movement starts when spermatids are stimulated to differentiate and stops when a pseudopod is formed. The second type of movement is a directed, continual flow of membrane components from the tip of the pseudopod to its base. Both membrane glycoproteins and fluorescent phospholipids inserted in the membrane flow backward at the same rate, approximately 4 micrometers/min, although their lateral diffusion coefficients in the membrane differ by at least a factor of 5. These observations suggest that pseudopodial membrane movement is due to bulk flow of membrane components away from the tip
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[
J Mol Biol,
1987]
The core histone genes (H2A, H2B, H3 and H4) of Caenorhabditis elegans are arranged in approximately 11 dispersed clusters and are not tandemly arrayed in the genome. Three well-characterized genomic clones, which contain histone genes, have one copy of each core histone gene per cluster. One of the clones (lambda Ceh-1) carries one histone cluster surrounded by several thousand base-pairs of non- histone DNA, and another clone (lambda Ceh-3) contains a histone cluster duplication surrounded by non-histone DNA. A third clone (lambda Ceh-2) carries a cluster of core histone genes flanked on one side (12,000 base-pairs away) by a single H2B gene and on the other by non-histone DNA. A fourth cluster (clone BE9) has one copy each of H3 and H4 and two copies each of H2A and H2B. This cluster is also flanked by non-histone DNA. Analysis of cosmid clones which overlap three of the clusters shows that no other histone clusters are closer than 8000 to 60,000 base-pairs, although unidentified non-histone transcription units are present on the flanking regions. Gene order within the histone clusters varies, and histone mRNAs are transcribed from both DNA strands. No H1 sequences are found on these core histone clones. Restriction fragment length polymorphisms between two related nematode strains (Bristol and Bergerac) were used as phenotypic markers in genetic crosses to map one histone cluster to linkage group V and another to linkage group IV. Hybridization of gene-specific probes from sea urchin to C. elegans RNA identifies C. elegans core histone messenger RNAs of sizes similar to sea urchin early stage histone mRNAs (H2A, H2B, H3 and H4). The organization of histone genes in C. elegans resembles the clustering found in most vertebrate organisms and does not resemble the tandem patterns of the early stage histone gene family of sea urchins or the major histone locus of Drosophila.
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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,
1982]
Latex beads and wheat germ agglutinin (WGA) were used to examine the movement of membrane components on amoeboid spermatozoa of Caenorhabditis elegans. The behavior of beads attached to the cell revealed continuous, directed movement from the tip of the pseudopod to its base, but no movement on the cell body. Lectin receptors are also cleared from the pseudopod (4). Blocking preexisting lectin receptors with unlabeled WGA followed by pulse-labeling wih fluorescent WGA showed that new lectin receptors are continuously inserted at the tip of the pseudopod. Like latex beads, these new lectin receptors move continuously over the pseudopod surface to the cell body-pseudopod junction where they are probably internalized. Mutants altering the rate of membrane flow, and eliminating its topographical asymmetry, have been identified. Together with the observation that fluorescent phospholipids are cleared from the pseudopod of developing spermatozoa at the same rate as lectin receptors (25), these results show that there is bulk membrane flow over the pseudopod with assembly at the tip and apparent disassembly at the base. There are no vesicles visible at either the pseudopodial tip or base, so these spermatozoa must have a novel mechanism for insertion and uptake of membrane components. This membrane flow could provide the forward propulsion of spermatozoa attached to a substrate by their pseudopods.
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[
J Cell Sci,
1984]
Caenorhabditis elegans spermatozoa use a single, persistent pseudopod to crawl at about 20 micrometers/min but, unlike other types of crawling cells, sperm lack both filamentous actin and myosin. Interference reflection microscopy has revealed that sperm form broad grey areas of contact, analogous to the close contacts that have been described underneath other crawling eukaryotic cells, between their pseudopods and their substrate. Individual sperm change the size, shape and pattern of their substrate attachments as they crawl but we found no correlation between the extent of underside of the cell in contact with the substrate and the velocity of locomotion. Two predominant attachment patterns were observed: (1) a single broad contact extending from the front of the pseudopod nearly to the rear of the cell; and (2) two separate contact sites, one under the front of the pseudopod and one under the cell body. Occasionally, under cells exhibiting the second type of attachment pattern, portions of the anterior contact separated and remained stationary relative to the substrate while the cell moved forward. This observation, as well as the continuous change in shape of the contact areas, suggests that sperm continually form new contacts near the tip of the pseudopod and release these contacts backwards. In extreme cases, sperm were able to crawl with only the front of the pseudopod in contact with the substrate. Therefore, we propose that sperm locomotion depends on the interaction of several key events (traction, propulsion, membrane insertion) occurring at the leading edge of the
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[
J Mol Biol,
2002]
The amoeboid locomotion of nematode sperm is mediated by the assembly dynamics of the major sperm protein (MSP). MSP forms fibrous networks based on a hierarchy of macromolecular assemblies: helical subfilaments are built from MSP dimers; filaments are formed from two subfilaments coiling round one another; and filaments themselves super-coil to produce bundles. To provide a structural context for understanding the role of these macromolecular assemblies in cell locomotion, we have determined the 2.6 Angstrom resolution structure of crystals of Caenorhabditis elegans MSP that are constructed from helices of MSP chains that are analogous to the subfilaments from which filaments are constructed. Comparison with the crystal structures of dimers and helical assemblies of Ascaris suum MSP has identified five conserved interaction interfaces that suggest how subfilaments interact in filaments and how filaments can form bundles. The interfaces frequently involve the loop containing residues 78-85, which is divergent between MSP homologues, and the loop containing residues 98-103, which is highly conserved.
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[
J Mol Biol,
1989]
We have determined the nucleotide sequence of core histone genes and flanking regions from two of approximately 11 different genomic histone clusters of the nematode Caenorhabditis elegans. Four histone genes from one cluster (H3, H4, H2B, H2A) and two histone genes from another (H4 and H2A) were analyzed. The predicted amino acid sequences of the two H4 and H2A proteins from the two clusters are identical, whereas the nucleotide sequences of the genes have diverged 9% (H2A) and 12% (H4). Flanking sequences, which are mostly not similar, were compared to identify putative regulatory elements. A conserved sequence of 34 base-pairs is present 19 to 42 nucleotides 3' of the termination codon of all the genes. Within the conserved sequence is a 16-base dyad sequence homologous to the one typically found at the 3' end of histone genes from higher eukaryotes. The C. elegans core histone genes are organized as divergently transcribed pairs of H3-H4 and H2A-H2B and contain 5' conserved sequence elements in the shared spacer regions. One of the sequence elements, 5' CTCCNCCTNCCCACCNCANA 3', is located immediately upstream from the canonical TATA homology of each gene. Another sequence element, 5' CTGCGGGGACACATNT 3', is present in the spacer of each heterotypic pair. These two 5' conserved sequences are not present in the promoter region of histone genes from other organisms, where 5' conserved sequences are usually different for each histone class. They are also not found in non-histone genes of C. elegans. These putative regulatory sequences of C. elegans core histone genes are similar to the regulatory elements of both higher and lower eukaryotes. The coding regions of the genes and the 3' regulatory sequences are similar to those of higher eukaryotes, whereas the presence of common 5' sequence elements upstream from genes of different histone classes is similar to histone promoter elements in yeast.
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[
Biochemistry,
1987]
The major intestinal esterase from the nematode Caenorhabditis elegans has been purified to essential homogeneity. Starting from whole worms, the overall purification is 9000-fold with a 10% recovery of activity. The esterase is a single polypeptide chain of Mr 60,000 and is stoichiometrically inhibited by organophosphates. Substrate preferences and inhibition patterns classify the enzyme as a carboxylesterase (EC 3.1.1.1), but the physiological function is unknown. The sequence of 13 amino acid residues at the esterase N- terminus has been determined. This partial sequence shows a surprisingly high degree of similarity to the N-terminal sequence of two carboxylesterases recently isolated from Drosophila mojavensis [Pen, J., van Beeumen, J., & Beintema, J. J. (1986) Biochem. J. 238, 691-699].
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[
EMBO J,
2007]
The Drosophila element Mos1 is a class II transposon, which moves by a ''cut-and-paste'' mechanism and can be experimentally mobilized in the Caenorhabditis elegans germ line. Here, we triggered the excision of identified Mos1 insertions to create chromosomal breaks at given sites and further manipulate the broken loci. Double-strand break (DSB) repair could be achieved by gene conversion using a transgene containing sequences homologous to the broken chromosomal region as a repair template. Consequently, mutations engineered in the transgene could be copied to a specific locus at high frequency. This pathway was further characterized to develop an efficient tool-called MosTIC-to manipulate the C. elegans genome. Analysis of DSB repair during MosTIC experiments demonstrated that DSBs could also be sealed by end-joining in the germ line, independently from the evolutionarily conserved Ku80 and ligase IV factors. In conjunction with a publicly available Mos1 insertion library currently being generated, MosTIC will provide a general tool to customize the C. elegans genome.
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Berynskyy M, Morimoto RI, Bukau B, Stengel F, Kirstein J, Szlachcic A, Arnsburg K, Stank A, Scior A, Nillegoda NB, Gao X, Guilbride DL, Aebersold R, Wade RC, Mayer MP
[
Nature,
2015]
Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states. Healthy metazoan cells effectively eliminate intracellular protein aggregates, indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro. This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are flexible with respect to subunit composition. Complex formation allows J-proteins to initiate transient higher order chaperone structures involving HSP70 and interacting nucleotide exchange factors. A network of cooperative class A and B J-protein interactions therefore provides the metazoan HSP70 machinery with powerful, flexible, and finely regulatable disaggregase activity and a further level of regulation crucial for cellular protein quality control.