The dynamin-related protein DRP-1 and the insulin signaling pathway cooperate to modulate Caenorhabditis elegans longevity

Here, we report that inactivation of the Caenorhabditis elegans dynamin-related protein DRP-1, a key component responsible for mitochondrial fission and conserved from yeast to humans, dramatically enhanced the effect of reduced insulin signaling (IIS) to extend lifespan. This represents the first report of a beneficial impact of manipulating mitochondrial dynamics on animal lifespan and suggests that mitochondrial morphology and IIS cooperate to modulate aging.     

小线虫,大发现:Caenorhabditis elegans在生命科学研究中的重要贡献

自20世纪60年代开始,秀丽线虫作为重要的模式生物在生命科学的发展过程中发挥着举足轻重的作用。线虫中的许多重大发现为人们理解复杂的细胞生命活动做出了极大的贡献。本文对秀丽线虫的研究历史、重要成果及研究前景作一简要综述。     

Conditions for Production of Microbial Cell Flocculant by Aspergillus sojae AJ7002

Cultivation is reported on Aspergillus sojae AJ7002 which synthesized an extracellular bio-flocculant. Growth studies in shaking flasks and fermentors were conducted to obtain higher flocculant production. The highest level of polymer accumulation was attained after 48–72 hr cultivation at 30–34°C. The favorable substrates for polymer formation were casein, yeast extract, polypepton and amino acids, such as glutamic acid and alanine. The addition of saccharides to the medium was found to reduce the pH of the culture broths, and hence inhibit the accumulation of flocculant in the culture broth. The finding that the product was a single substance from the early stage of fermentation suggested that the polymer was not a product of cell autolysis. The components of the polymer which were produced by Asp. sojae did not vary even if the medium composition or culture condition changed. The addition of 2-ketogluconic acid, which is one of the constituents of the polymer increased the flocculating activity of the culture medium.     

The DAF-2 insulin-like signaling pathway independently regulates aging and immunity in C. elegans

The Caenorhabditis elegans DAF-2 insulin-like signaling pathway, which regulates lifespan and stress resistance, has also been implicated in resistance to bacterial pathogens. Loss-of-function daf-2 and age-1 mutants have increased lifespans and are resistant to a variety of bacterial pathogens. This raises the possibility that the increased longevity and the pathogen resistance of insulin-like signaling pathway mutants are reflections of the same underlying mechanism. Here we report that regulation of lifespan and resistance to the bacterial pathogen Pseudomonas aeruginosa is mediated by both shared and genetically distinguishable mechanisms. We find that loss of germline proliferation enhances pathogen resistance and this effect requires daf-16, similar to the regulation of lifespan. In contrast, the regulation of pathogen resistance and lifespan is decoupled within the DAF-2 pathway. Long-lived mutants of genes downstream of daf-2, such as pdk-1 and sgk-1, show wildtype resistance to pathogens. However, mutants of akt-1 and akt-2, which we find to individually have modest effects on lifespan, show enhanced resistance to pathogens. We also demonstrate that pathogen resistance of daf-2, akt-1, and akt-2 mutants is associated with restricted bacterial colonization, and that daf-2 mutants are better able to clear an infection after challenge with P. aeruginosa. Moreover, we find that pathogen resistance among insulin-like signaling mutants is associated with increased expression of immunity genes during infection. Other processes that affect organismal longevity, including Jun kinase signaling and caloric restriction, do not affect resistance to bacterial pathogens, further establishing that aging and innate immunity are regulated by genetically distinct mechanisms.     

The two C. elegans ATG-16 homologs have partially redundant functions in the basal autophagy pathway

The presence of multiple homologs of the same yeast ATG genes endows an extra layer of complexity on the autophagic machinery in higher eukaryotes. The physiological function of individual homologs in the autophagy pathway remains poorly understood. Here we characterized the function of the two atg16 homologs, atg-16.1 and atg-16.2, in the autophagy pathway in C. elegans. We showed that atg-16.2 mutants exhibit a stronger autophagic defect than atg-16.1 mutants. atg-16.2; atg-16.1 double mutants display a much more severe defect than either single mutant. ATG-16.1 and ATG-16.2 interact with themselves and each other and also directly associate with ATG-5. atg-16.1 mutant embryos exhibit a wild-type expression and distribution pattern of LGG-1/Atg8, while LGG-1 puncta are markedly fewer in number and weaker in intensity in atg-16.2 mutants. In atg-16.2; atg-16.1 double mutants, the lipidated form of LGG-1 accumulates, but LGG-1 puncta are completely absent. ATG-16.2 ectopically expressed on the plasma membrane provides novel sites of LGG-1 puncta formation. We also demonstrated that the C-terminal WD repeats are dispensable for the role of atg-16.2 in aggrephagy (the degradation of protein aggregates by autophagy). Genetic epistasis analysis placed atg-16.2 upstream of atg-2, epg-6, and atg-18. Our study indicated that C. elegans ATG-16s are involved in specifying LGG-1 puncta formation and the two ATG-16 homologs have partially redundant yet distinct functions in the aggrephagy pathway.     

The secreted Alzheimer-related amyloid precursor protein fragment has an essential role in C. elegans

Mutations in the gene encoding the amyloid precursor protein (APP) or the enzymes that process APP are correlated with familial Alzheimer disease. Alzheimer disease is also associated with insulin resistance (type 2 diabetes). In our recently published study,¹ Ewald CY, Raps DA, Li C. APL-1, the Alzheimer’s Amyloid precursor protein in Caenorhabditis elegans, modulates multiple metabolic pathways throughout development. Genetics 2012; 191:493 - 507; http://dx.doi.org/10.1534/genetics.112.138768; PMID: 22466039 [Crossref], [PubMed], [Web of Science ®] , [Google Scholar] we obtained genetic evidence that the extracellular fragment of APL-1, the C. elegans ortholog of human APP, may act as a signaling molecule to modulate insulin and nuclear hormone pathways in C. elegans development. In addition, independent of insulin and nuclear hormone signaling, high levels of the extracellular fragment of APL-1 (sAPL-1) leads to a temperature-sensitive embryonic lethality, which is dependent on activity of a predicted receptor protein tyrosine phosphatase (MOA-1/R155.2). Furthermore, this embryonic lethality is enhanced by knockdown of a predicted prion-like protein (pqn-29). The precise molecular mechanisms underlying these processes remain to be determined. Here, we present hypothetical models as to how sAPL-1 signaling influences metabolic and developmental pathways. Together, with previous findings in mammals that the extracellular domain of mammalian APP (sAPP) binds to a death-receptor,² Nikolaev A, McLaughlin T, O’Leary DD, Tessier-Lavigne M. APP binds DR6 to trigger axon pruning and neuron death via distinct caspases. Nature 2009; 457:981 - 9; http://dx.doi.org/10.1038/nature07767; PMID: 19225519 [Crossref], [PubMed], [Web of Science ®] , [Google Scholar] our findings support the model that sAPP signaling affects critical biological processes.     

细菌介导的RNA干扰对C.elegans中par-3基因的作用

设计并构建了针对par-3基因的发夹RNA载体,将构建好的质粒转入大肠杆菌HT115,25℃喂食Caenorhabditis elegans（C.elegans）野生型虫体,24h后观察par-3（RNA干扰）celegans的胚胎发育情况。结果显示通过喂食形成发夹结构dsRNA的细菌可以对celegans中par-3基因进行RNA干扰,干扰率可以达到60％以上。干扰后的早期胚胎发育丧失第一次卵裂的不对称性,第二次卵裂的纺锤体方向发生改变,与par-3突变体的观察结果一致,为进一步在mex-3转基因虫体中通过RNA干扰研究基因表达打下了基础。     

Evidence for the contribution of LTR retrotransposons to C. elegans gene evolution

LTR retrotransposons may be important contributors to host gene evolution because they contain regulatory and coding signals. In an effort to assess the possible contribution of LTR retrotransposons to C. elegans gene evolution, we searched upstream and downstream of LTR retrotransposon sequences for the presence of predicted genes. Sixty-three percent of LTR retrotransposon sequences (79/124) are located within 1 kb of a gene or within gene boundaries. Most gene-retrotransposon associations were located along the chromosome arms. Our results are consistent with the hypothesis that LTR retrotransposons have contributed to the structural and/or regulatory evolution of genes in C. elegans.     

Developmental decisions: Balancing genetics and the environment by C. elegans

The small nematode C. elegans is characterized by developing through a highly coordinated, reproducible cell lineage that serves as the basis of many studies focusing on the development of multi-lineage organisms. Indeed, the reproducible cell lineage enables discovery of developmental defects that occur in even a single cell. Only recently has attention been focused on how these animals modify their genetically programmed cell lineages to adapt to altered environments. Here, we summarize the current understanding of how C. elegans responds to food deprivation by adapting their developmental program in order to conserve energy. In particular, we highlight the AMPK-mediated and insulin-like growth factor signaling pathways that are the principal regulators of induced cell cycle quiescence.     

MAU-8 is a Phosducin-like Protein required for G protein signaling in C. elegans

The mau-8(qm57) mutation inhibits the function of GPB-2, a heterotrimeric G protein beta subunit, and profoundly affects behavior through the Galphaq/Galphao signaling network in C. elegans. mau-8 encodes a nematode Phosducin-like Protein (PhLP), and the qm57 mutation leads to the loss of a predicted phosphorylation site in the C-terminal domain of PhLP that binds the Gbetagamma surface implicated in membrane interactions. In developing embryos, MAU-8/PhLP localizes to the cortical region, concentrates at the centrosomes of mitotic cells and remains associated with the germline blastomere. In adult animals, MAU-8/PhLP is ubiquitously expressed in somatic tissues and germline cells. MAU-8/PhLP interacts with the PAR-5/14.3.3 protein and with the Gbeta subunit GPB-1. In mau-8 mutants, the disruption of MAU-8/PhLP stabilizes the association of GPB-1 with the microtubules of centrosomes. Our results indicate that MAU-8/PhLP modulates G protein signaling, stability and subcellular location to regulate various physiological functions, and they suggest that MAU-8 might not be limited to the Galphaq/Galphao network.     

Immuno-MALDI (iMALDI) mass spectrometry for the analysis of proteins in signaling pathways

Introduction: Biomarkers are commonly used to stratify cancer patients and guide targeted therapies, but most biomarkers are of a genomic nature. Discrepancies between the genome and proteome and the high rates of drug resistance indicate that proteomic analyses may provide additional critically important information. Here we present immuno-Matrix-Assisted Laser Desorption/Ionization (iMALDI), the combination of immuno-affinity enrichment of peptides followed by direct MALDI-mass spectrometry analysis. iMALDI is a highly sensitive, targeted protein-quantitation technique with the potential to measure clinically relevant signaling-pathway proteins using minimal sample amounts, thus improving upon existing methodologies.

Areas covered: We provide a brief overview of the current state of biomarker analysis technologies for modern cancer treatment. We also show the advantages of iMALDI for translating potential new biomarkers into the clinic, factors to consider for iMALDI assay development, and the utility of iMALDI for the quantitation of cell-signaling proteins.

Expert commentary: We see targeted mass spectrometry approaches such as iMALDI as an important part of improving patient responses to targeted therapies by providing highly sensitive, accurate, precise, and specific measurements of signaling-pathway proteins, both in tumor cells and in cells from the tumor microenvironment. iMALDI results can be integrated with other -omics data to aid in tumor-targeting therapies and immuno-oncology.     

Two types of chloride transporters are required for GABA(A) receptor-mediated inhibition in C. elegans

Chloride influx through GABA-gated Cl(-) channels, the principal mechanism for inhibiting neural activity in the brain, requires a Cl(-) gradient established in part by K(+)-Cl(-) cotransporters (KCCs). We screened for Caenorhabditis elegans mutants defective for inhibitory neurotransmission and identified mutations in ABTS-1, a Na(+)-driven Cl(-)-HCO(3)(-) exchanger that extrudes chloride from cells, like KCC-2, but also alkalinizes them. While animals lacking ABTS-1 or the K(+)-Cl(-) cotransporter KCC-2 display only mild behavioural defects, animals lacking both Cl(-) extruders are paralyzed. This is apparently due to severe disruption of the cellular Cl(-) gradient such that Cl(-) flow through GABA-gated channels is reversed and excites rather than inhibits cells. Neuronal expression of both transporters is upregulated during synapse development, and ABTS-1 expression further increases in KCC-2 mutants, suggesting regulation of these transporters is coordinated to control the cellular Cl(-) gradient. Our results show that Na(+)-driven Cl(-)-HCO(3)(-) exchangers function with KCCs in generating the cellular chloride gradient and suggest a mechanism for the close tie between pH and excitability in the brain.     

tcl-2 encodes a novel protein that acts synergistically with Wnt signaling pathways in C. elegans

Mutations in tcl-2 cause defects in the specification of the fates of the descendants of the TL and TR blast cells, whose polarity is regulated by lin-44/Wnt and lin-17/frizzled, during Caenorhabditis elegans development. In wild-type animals, POP-1/TCF/LEF, is asymmetrically distributed to the T cell daughters, resulting in a higher level of POP-1 in the nucleus of the anterior daughter. The POP-1 asymmetric distribution is controlled by lin-44 and lin-17. However, in tcl-2 mutants, POP-1 is equally distributed to T cell daughters as is observed in lin-17 mutants, indicating that, like lin-17, tcl-2 functions upstream of pop-1. In addition, tcl-2 mutations cause defects in the development of the gonad and the specification of fate of the posterior daughter of the P12 cell, both of which are controlled by the Wnt pathway. Double mutant analyses indicate that tcl-2 can act synergistically with the Wnt pathway to control gonad development as well as P12 descendant cell fate specification. tcl-2 encodes a novel protein. A functional tcl-2::gfp construct was weakly expressed in the nuclei of the T cell and its descendants. Our results suggest that tcl-2 functions with Wnt pathways to control T cell fate specification, gonad development, and P12 cell fate specification.     

Four KH domains of the C. elegans Bicaudal-C ortholog GLD-3 form a globular structural platform

Caenorhabditis elegans GLD-3 is a five K homology (KH) domain-containing protein involved in the translational control of germline-specific mRNAs during embryogenesis. GLD-3 interacts with the cytoplasmic poly(A)-polymerase GLD-2. The two proteins cooperate to recognize target mRNAs and convert them into a polyadenylated, translationally active state. We report the 2.8-Å-resolution crystal structure of a proteolytically stable fragment encompassing the KH2, KH3, KH4, and KH5 domains of C. elegans GLD-3. The structure reveals that the four tandem KH domains are organized into a globular structural unit. The domains are involved in extensive side-by-side interactions, similar to those observed in previous structures of dimeric KH domains, as well as head-to-toe interactions. Small-angle X-ray scattering reconstructions show that the N-terminal KH domain (KH1) forms a thumb-like protrusion on the KH2–KH5 unit. Although KH domains are putative RNA-binding modules, the KH region of GLD-3 is unable in isolation to cross-link RNA. Instead, the KH1 domain mediates the direct interaction with the poly(A)-polymerase GLD-2, pointing to a function of the KH region as a protein–protein interaction platform.     

Diverse regulation of sensory signaling by C. elegans nPKC-epsilon/eta TTX-4

Molecular and pharmacological studies in vitro suggest that protein kinase C (PKC) family members play important roles in intracellular signal transduction. Nevertheless, the in vivo roles of PKC are poorly understood. We show here that nPKC-epsilon/eta TTX-4 in the nematode Caenorhabditis elegans is required for the regulation of signal transduction in various sensory neurons for temperature, odor, taste, and high osmolality. Interestingly, the requirement for TTX-4 differs in different sensory neurons. In AFD thermosensory neurons, gain or loss of TTX-4 function inactivates or hyperactivates the neural activity, respectively, suggesting negative regulation of temperature sensation by TTX-4. In contrast, TTX-4 positively regulates the signal sensation of ASH nociceptive neurons. Moreover, in AWA and AWC olfactory neurons, TTX-4 plays a partially redundant role with another nPKC, TPA-1, to regulate olfactory signaling. These results suggest that C. elegans nPKCs regulate different sensory signaling in various sensory neurons. Thus, C. elegans provides an ideal model to reveal genetically novel components of nPKC-mediated molecular pathways in sensory signaling.