The Aggregation-Prone Intracellular Serpin SRP-2 Fails to Transit the ER in Caenorhabditis elegans

Familial encephalopathy with neuroserpin inclusions bodies (FENIB) is a serpinopathy that induces a rare form of presenile dementia. Neuroserpin contains a classical signal peptide and like all extracellular serine proteinase inhibitors (serpins) is secreted via the endoplasmic reticulum (ER)–Golgi pathway. The disease phenotype is due to gain-of-function missense mutations that cause neuroserpin to misfold and aggregate within the ER. In a previous study, nematodes expressing a homologous mutation in the endogenous Caenorhabditis elegans serpin, srp-2, were reported to model the ER proteotoxicity induced by an allele of mutant neuroserpin. Our results suggest that SRP-2 lacks a classical N-terminal signal peptide and is a member of the intracellular serpin family. Using confocal imaging and an ER colocalization marker, we confirmed that GFP-tagged wild-type SRP-2 localized to the cytosol and not the ER. Similarly, the aggregation-prone SRP-2 mutant formed intracellular inclusions that localized to the cytosol. Interestingly, wild-type SRP-2, targeted to the ER by fusion to a cleavable N-terminal signal peptide, failed to be secreted and accumulated within the ER lumen. This ER retention phenotype is typical of other obligate intracellular serpins forced to translocate across the ER membrane. Neuroserpin is a secreted protein that inhibits trypsin-like proteinase. SRP-2 is a cytosolic serpin that inhibits lysosomal cysteine peptidases. We concluded that SRP-2 is neither an ortholog nor a functional homolog of neuroserpin. Furthermore, animals expressing an aggregation-prone mutation in SRP-2 do not model the ER proteotoxicity associated with FENIB.     

Mutants with altered muscle structure in Caenorhabditis elegans

In the small nematode, Caenorhabditis elegans, mutants with a disorganized myofilament lattice structure have been identified by polarized light and electron microscopy. Genetic analysis places the mutations in 12 complementation groups which are distributed over the six linkage groups of C. elegans. The phenotypes are described for the mutants from the 9 complementation groups not previously reported on in detail. Most are paralyzed, but some exhibit essentially normal movement; mutants of two loci show changes only in later larval stages and adulthood. Morphological studies show that, in general, all the members of a complementation group show similar changes in muscle structure and that these changes are distinctive for that group. In mutants of several genes, disorganization of the myofilament lattice is general with no one component of the lattice more obviously altered than others. In mutants of other genes specific structures are prominently altered. In one of the instances where thick filaments appear to be abnormal, double mutants combining mutations in this gene (unc-82 IV) with mutations in the gene for a myosin heavy chain (MacLeod et al., 1977a,b) or paramyosin (Waterston et al., 1977) were used to show that the unc-82 gene product probably affects thick filament assembly through its actions on paramyosin. Some possible implications of the morphological features of the mutants as well as the conclusions derived from the genetic studies are discussed.     

N-Glycans of Caenorhabditis elegans are specific to developmental stages

We have examined the N-glycans present during the developmental stages of Caenorhabditis elegans using two approaches, 1) a combination of permethylation followed by MALDI-TOF mass spectrometry (MS) and 2) derivatization with 2-aminobenzamide followed by separation by high-performance liquid chromatography and analyses by MALDI-TOF MS, post source decay (PSD) MS, and MALDI-QoTOF MS/MS. The N-glycan profile of each developmental stage (Larva 1, Larva 2, Larva 3, Larva 4, and Dauer and adult) appears to be unique. The pattern of complex N-glycans was stage-specific with the general trend of number and abundance of glycans being Dauer approximately = L1 > adult approximately = L4 > L3 approximately = L2. Dauer larvae contained complex N-glycans with higher molecular masses than those seen in other stages. MALDI-QoTOF MS/MS of Hex4HexNAc4 showed an N-acetyllac-tosamine substitution not previously observed in C. elegans. Phosphorylcholine (Pc)-substituted glycans were also found to be stage-specific. Higher molecular weight Pc-containing glycans, including fucose-containing ones such as difucosyl Pc-glycan (Pc1dHex2Hex5HexNAc6) seen in Dauer larvae, have not been observed in any organism. Pc2Hex4HexNAc3, from Dauer larvae, when subjected to PSD MS analyses, showed Pc may substitute both core and terminally linked GlcNAc; no such structure has previously been reported in any organism. C. elegans-specific fucosyl and native methylated glycans were found in all developmental stages. Taken together, the above results demonstrate that in-depth investigation of the role of the above N-glycans during C. elegans development should lead to a better understanding of their significance and the ways that they may govern interactions, both within the organism during development and between the mobile nematode and its pathogens.     

The kinetochores of Caenorhabditis elegans

Light microscopy of the mitotic chromosomes of Caenorhabditis elegans suggests that non-localized kinetochores are present, since the chromosomes appear as stiff rods 1 to 2 m in length and lack any visible constriction. The holokinetic structure was confirmed by reconstructions of electron micrographs of dividing nuclei in serially sectioned embryos. In prophase the kinetochore appears as an amorphous projection approximately 0.18–0.2 m in diameter in cross section and in longitudinal section it appears to be continuous along the chromatin. At prometaphase and metaphase the kinetochore is a convex plaque covering the poleward face of the chromosome and extending the length of the chromosome. In longitudinal section the kinetochore is a trilaminar structure with electron dense inner and outer layers of 0.02 m, and an electron lucent middle layer of 0.03 m. The inner layer is adjacent to a more electron dense region of chromatin. The kinetochore was also seen as a band extending the length of the chromosome in whole mount preparations of chromosomes stained with ethanolic phosphotungstic acid. Most gamma ray induced chromosome fragments segregate normally in embryonic mitoses, but some fragments display aberrant behavior. Similar behavior was seen in embryos carrying a genetically characterized free duplication. It is suggested that mitotic segregation of small fragments may be inefficient because the probability of attachment of microtubules to the kinetochore is proportional to kinetochore length.     

The microRNAs of Caenorhabditis elegans

The soil nematode, Caenorhabditis elegans, occupies a central place in the short history of microRNA (miRNA) research. The converse is also true: miRNAs have emerged as key regulatory components in the life cycle of the worm, as well as numerous other organisms. Since the landmark discovery in 1993 of the first miRNA gene, lin-4, several other miRNAs have been characterized in detail in C. elegans and shown to participate in diverse biological processes. Moreover, the worm has provided, by virtue of its ease of genetic manipulation and amenability to high-throughput methods, an ideal platform for elucidating many general and conserved aspects of miRNA biology, namely mechanisms of biogenesis, target recognition, gene silencing, and regulation thereof. In this review, we summarize both the contribution of miRNAs to C. elegans physiology and development, as well as the contribution of C. elegans research to our understanding of general features of miRNA biology.     

FGF negatively regulates muscle membrane extension in Caenorhabditis elegans

Striated muscles from Drosophila and several vertebrates extend plasma membrane to facilitate the formation of the neuromuscular junction (NMJ) during development. However, the regulation of these membrane extensions is poorly understood. In C. elegans, the body wall muscles (BWMs) also have plasma membrane extensions called muscle arms that are guided to the motor axons where they form the postsynaptic element of the NMJ. To investigate the regulation of muscle membrane extension, we screened 871 genes by RNAi for ectopic muscle membrane extensions (EMEs) in C. elegans. We discovered that an FGF pathway, including let-756(FGF), egl-15(FGF receptor), sem-5(GRB2) and other genes negatively regulates plasma membrane extension from muscles. Although compromised FGF pathway activity results in EMEs, hyperactivity of the pathway disrupts larval muscle arm extension, a phenotype we call muscle arm extension defective or MAD. We show that expression of egl-15 and sem-5 in the BWMs are each necessary and sufficient to prevent EMEs. Furthermore, we demonstrate that let-756 expression from any one of several tissues can rescue the EMEs of let-756 mutants, suggesting that LET-756 does not guide muscle membrane extensions. Our screen also revealed that loss-of-function in laminin and integrin components results in both MADs and EMEs, the latter of which are suppressed by hyperactive FGF signaling. Our data are consistent with a model in which integrins and laminins are needed for directed muscle arm extension to the nerve cords, while FGF signaling provides a general mechanism to regulate muscle membrane extension.     

FLR-4, a novel serine/threonine protein kinase, regulates defecation rhythm in Caenorhabditis elegans

The defecation behavior of the nematode Caenorhabditis elegans is controlled by a 45-s ultradian rhythm. An essential component of the clock that regulates the rhythm is the inositol trisphosphate receptor in the intestine, but other components remain to be discovered. Here, we show that the flr-4 gene, whose mutants exhibit very short defecation cycle periods, encodes a novel serine/threonine protein kinase with a carboxyl terminal hydrophobic region. The expression of functional flr-4::GFP was detected in the intestine, part of pharyngeal muscles and a pair of neurons, but expression of flr-4 in the intestine was sufficient for the wild-type phenotype. Furthermore, laser killing of the flr-4-expressing neurons did not change the defecation phenotypes of wild-type and flr-4 mutant animals. Temperature-shift experiments with a temperature-sensitive flr-4 mutant suggested that FLR-4 acts in a cell-functional rather than developmental aspect in the regulation of defecation rhythms. The function of FLR-4 was impaired by missense mutations in the kinase domain and near the hydrophobic region, where the latter allele seemed to be a weak antimorph. Thus, a novel protein kinase with a unique structural feature acts in the intestine to increase the length of defecation cycle periods.     

The SH3 domain of UNC-89 (obscurin) interacts with paramyosin,a coiled-coil protein,in Caenorhabditis elegans muscle

UNC-89 is a giant polypeptide located at the sarcomeric M-line of Caenorhabditis elegans muscle. The human homologue is obscurin. To understand how UNC-89 is localized and functions, we have been identifying its binding partners. Screening a yeast two-hybrid library revealed that UNC-89 interacts with paramyosin. Paramyosin is an invertebrate-specific coiled-coil dimer protein that is homologous to the rod portion of myosin heavy chains and resides in thick filament cores. Minimally, this interaction requires UNC-89’s SH3 domain and residues 294–376 of paramyosin and has a K_D of ∼1.1 μM. In unc-89 loss-of-function mutants that lack the SH3 domain, paramyosin is found in accumulations. When the SH3 domain is overexpressed, paramyosin is mislocalized. SH3 domains usually interact with a proline-rich consensus sequence, but the region of paramyosin that interacts with UNC-89’s SH3 is α-helical and lacks prolines. Homology modeling of UNC-89’s SH3 suggests structural features that might be responsible for this interaction. The SH3-binding region of paramyosin contains a “skip residue,” which is likely to locally unwind the coiled-coil and perhaps contributes to the binding specificity.     

SEL-5, a serine/threonine kinase that facilitates lin-12 activity in Caenorhabditis elegans

Ligands present on neighboring cells activate receptors of the LIN-12/Notch family by inducing a proteolytic cleavage event that releases the intracellular domain. Mutations that appear to eliminate sel-5 activity are able to suppress constitutive activity of lin-12(d) mutations that are point mutations in the extracellular domain of LIN-12, but cannot suppress lin-12(intra), the untethered intracellular domain. These results suggest that sel-5 acts prior to or during ligand-dependent release of the intracellular domain. In addition, sel-5 suppression of lin-12(d) mutations is tissue specific: loss of sel-5 activity can suppress defects in the anchor cell/ventral uterine precursor cell fate decision and a sex myoblast/coelomocyte decision, but cannot suppress defects in two different ventral hypodermal cell fate decisions in hermaphrodites and males. sel-5 encodes at least two proteins, from alternatively spliced mRNAs, that share an amino-terminal region and differ in the carboxy-terminal region. The amino-terminal region contains the hallmarks of a serine/threonine kinase domain, which is most similar to mammalian GAK1 and yeast Pak1p.     

The neurexin superfamily of Caenorhabditis elegans

The neurexin superfamily is a group of transmembrane molecules mediating cell-cell contacts and generating specialized membranous domains in polarized epithelial and nerves cells. We describe here the domain organization and expression of the entire, core neurexin superfamily in the nematode Caenorhabditis elegans, which is composed of three family members. One of the superfamily members, nrx-1, is an ortholog of vertebrate neurexin, the other two, itx-1 and nlr-1, are orthologs of the Caspr subfamily of neurexin-like genes. Based on reporter gene analysis, we find that nrx-1 is exclusively expressed in most if not all cells of the nervous system and localizes to presynaptic specializations. itx-1 and nrx-1 reporter genes are expressed in non-overlapping patterns within and outside the nervous system. ITX-1 protein co-localizes with β-G-spectrin to a subapical domain within intestinal cells. These studies provide a starting point for further functional analysis of this family of proteins.     

Comparative analysis of various gene finders specific to Caenorhabditis elegans genome

Computational gene prediction and identifying alternatively spliced isoforms have always been a challenging task. In this paper, we describe the performance of three gene/exon finding programmes namely Fex, Gen view2 and Gene builder capable of predicting open reading frames or exons for a given set of sequences from C. elegans genome. The predicted exons were compared with the 'sequencing consortium' identified exons and degree of consensus among them is discussed. We found that exon prediction by Fex was similar to the consortium prediction as compared to Gen view2 and Gene builder results. Interestingly, some exons (six exons in five genes) predicted positive only by Fex and not by the 'sequencing consortium' are found at the C. elegans EST database. This data is critical for further debate and discussion on gene finding in C. elegans.     

Identification of specific let-7 microRNA binding complexes in Caenorhabditis elegans

Little is known about the protein complexes required for microRNA formation and function. Here we used native gel electrophoresis to identify miRNA ribonucleoprotein complexes (miRNPs) in Caenorhabditis elegans. Our data reveal multiple distinct miRNPs that assemble on the let-7 miRNA in vitro. The formation of these complexes is affected but not abolished by alg-1 or alg-2 null mutations. The largest complex (M*) with an estimated molecular mass of >669 kDa cofractionates with the known RISC factors ALG-1, VIG-1, and TSN-1. The M* complex and two complexes, M3 and M4, with similar molecular weights of ~500 kDa, also assemble on all other miRNAs used in our experiments. Two smaller complexes, M1 (~160 kDa) and M2 (~250 kDa), assemble on the members of the let-7 miRNAs family but not lin-4 or mir-234, and their formation is highly dependent on specific sequences in the 5′ seed region of let-7. Moreover, an unidentified protein, p40, which only appears in the M1 and M2 complexes, was detected by UV triggered cross-linking to let-7 but not to lin-4. The cross-linking of p40 to let-7 is also dependent on the let-7 sequence. Another unidentified protein, p13, is detected in all let-7 binding complexes and lin-4 cross-linked products. Our data suggest that besides being present in certain large miRNPs with sizes similar to reported RISC, the let-7 miRNA also assembles with specific binding proteins and forms distinct small complexes.     

Location of specific messenger RNAs in Caenorhabditis elegans by cytological hybridization

We have developed an autoradiographic technique for detecting specific Caenorhabditis elegans messenger RNA molecules in situ by hybridization of labeled, cloned DNA probes to fixed tissue sections and squashes of embryos and adults. We report analyses with probes of actin and collagen gene sequences from a C. elegans genomic clone library. Hybridization is RNase sensitive and tissue specific. In adults the actin probe, which recognizes cytoplasmic as well as muscle actin mRNA, hybridizes strongly to muscle and distal gonad (ovary), somewhat less strongly to maturing oocytes, and weakly to intestine. The collagen probe hybridizes weakly to distal gonad and intestine and very strongly to subcuticular tissues, in particular to the hypodermal cells and syncytial cytoplasm of the lateral hypodermal ridges, which are the sites of cuticle synthesis. In embryos, hybridization to squashes indicates that actin message is present at fertilization, decreases during early cleavage, and then increases again during morphogenesis. By contrast, collagen message is absent until the 100-cell stage and then increases rapidly during morphogenesis. The number of cells labeled is consistent with the view that the collagen probe hybridizes to hypodermal precursor cells. We estimate that our present methods can detect messages representing about 0.2% or more of the total mRNA population, and increases in this sensitivity should be possible. Therefore, the cytological hybridization technique should be useful for determining temporal and spatial patterns of specific mRNA distributions during development, at least for abundant and moderately abundant messages.     

The structural puzzle of how serpin serine proteinase inhibitors work

Serine proteinase cleavage of proteins is essential to a wide variety of biological processes and is primarily regulated by protein inhibitors. Many inhibitors are conformationally rigid simulations of optimal serine proteinase substrates, which makes them highly efficient competitive inhibitors of target proteinases. In contrast, members of the serpin family of serine proteinase inhibitors display extensive flexibility and polymorphism, particularly in their reactive site segments and in β-sheet secondary structure, which can take up and expel strands. Reactive site and β-sheet polymorphism appear to be coupled in the serpins and may account for the extreme stability of serpinproteinase complexes through the insertion of the reactive site strand into a β-sheet. These unusual properties may have opened an adaptive pathway of proteinase regulation that was unavailable to the conformationally rigid proteinase inhibitors.