Lin J et al. (2015) Front Cell Infect Microbiol "The icmF3 locus is involved in multiple adaptation- and virulence-related characteristics in Pseudomonas aeruginosa PAO1."

Shen X, Guo C, Lin J, Ma L, Ding W, Yang X, Chen K, Zhang W, Cheng J, Wang Y

[Front Cell Infect Microbiol, 2015]

The type VI secretion system (T6SS) is widely distributed in Gram-negative bacteria. Three separate T6SSs called H1-, H2-, and H3-T6SS have been discovered in Pseudomonas aeruginosa PAO1. Recent studies suggest that, in contrast to the H1-T6SS that targets prokaryotic cells, H2- and H3-T6SS are involved in interactions with both prokaryotic and eukaryotic cells. However, the detailed functions of T6SS components are still uncharacterized. The intracellular multiplication factor (IcmF) protein is conserved in type VI secretion systems (T6SS) of all different bacterial pathogens. Bioinformatic analysis revealed that IcmF3 in P. aeruginosa PAO1 is different from other IcmF homologs and may represent a new branch of these proteins with distinct functions. Herein, we have investigated the function of IcmF3 in this strain. We have shown that deletion of the icmF3 gene in P. aeruginosa PAO1 is associated with pleiotropic phenotypes. The icmF3 mutant has variant colony morphology and an hypergrowth phenotype in iron-limiting medium. Surprisingly, this mutant is also defective for the production of pyoverdine, as well as defects in swimming motility and virulence in a C. elegans worm model. The icmF3 mutant exhibits higher conjugation frequency than the wild type and increased biofilm formation on abiotic surfaces. Additionally, expression of two phenazine biosynthetic loci is increased in the icmF3 mutant, leading to the overproduction of pyocyanin. Finally, the mutant exhibits decreased susceptibility to aminoglycosides such as tobramycin and gentamicin. And the detected phenotypes can be restored completely or partially by trans complementation of wild type icmF3 gene. The pleiotropic effects observed upon icmF3 deletion demonstrate that icmF3 plays critical roles in both pathogenesis and environmental adaptation in P. aeruginosa PAO1.

Lin J et al. (2019) PLoS One "Human Marfan and Marfan-like Syndrome associated mutations lead to altered trafficking of the Type II TGF receptor in Caenorhabditis elegans."

Lin J, Kane NS, Vora M, Padgett RW, Gleason RJ

[PLoS One, 2019]

The transforming growth factor- (TGF) family plays an important role in many developmental processes and when mutated often contributes to various diseases. Marfan syndrome is a genetic disease with an occurrence of approximately 1 in 5,000. The disease is caused by mutations in fibrillin, which lead to an increase in TGF ligand activity, resulting in abnormalities of connective tissues which can be life-threatening. Mutations in other components of TGF signaling (receptors, Smads, Schnurri) lead to similar diseases with attenuated phenotypes relative to Marfan syndrome. In particular, mutations in TGF receptors, most of which are clustered at the C-terminal end, result in Marfan-like (MFS-like) syndromes. Even though it was assumed that many of these receptor mutations would reduce or eliminate signaling, in many cases signaling is active. From our previous studies on receptor trafficking in C. elegans, we noticed that many of these receptor mutations that lead to Marfan-like syndromes overlap with mutations that cause mis-trafficking of the receptor, suggesting a link between Marfan-like syndromes and TGF receptor trafficking. To test this hypothesis, we introduced three of these key MFS and MFS-like mutations into the C. elegans TGF receptor and asked if receptor trafficking is altered. We find that in every case studied, mutated receptors mislocalize to the apical surface rather than basolateral surface of the polarized intestinal cells. Further, we find that these mutations result in longer animals, a phenotype due to over-stimulation of the nematode TGF pathway and, importantly, indicating that function of the receptor is not abrogated in these mutants. Our nematode models of Marfan syndrome suggest that MFS and MFS-like mutations in the type II receptor lead to mis-trafficking of the receptor and possibly provides an explanation for the disease, a phenomenon which might also occur in some cancers that possess the same mutations within the type II receptor (e.g. colon cancer).

Lin J et al. (2020) Am J Transl Res "Fisetin regulates gut microbiota to decrease CCR9+/CXCR3+/CD4+ T-lymphocyte count and IL-12 secretion to alleviate premature ovarian failure in mice."

Chen J, Huang Y, Xiong Y, Nie X, Gong Z, Lin J, Chen C, Liu T

[Am J Transl Res, 2020]

Currently, there are no studies reporting the efficacy of fisetin in premature ovarian failure (POF). In this study, using mouse and <i>Caenorhabditis elegans</i> models, we found that fisetin not only significantly reversed ovarian damage in POF mice, but also effectively increased <i>C. elegans</i> lifespan and fertility. Subsequently, we carried out 16S rRNA v3+v4 sequencing using fresh feces samples from each group of mice. Results showed that although there was no significant difference in the number of gut microbiomes between the different groups of mice, fisetin affected the diversity and distribution of gut microbiota in POF mice. Alpha and beta diversity analyses showed that in the gut of POF mice in the fisetin group, the bacterial count of uncultured_bacterium_f_Lachnospiraceae was significantly increased, while that of <i>Akkermansia</i> was significantly decreased. Finally, flow cytometry analysis showed that the numbers of CCR9⁺/CXCR3⁺/CD4⁺ T lymphocytes in the peripheral blood of POF mice in the fisetin group were significantly reduced, along with the number of CD4⁺/interleukin (IL)-12⁺ cells. Therefore, our data suggested that fisetin regulates the distribution and bacterial counts of <i>Akkermansia</i> and uncultured_bacterium_f_Lachnospiracea in POF mice, and reduces peripheral blood CCR9⁺/CXCR3⁺/CD4⁺ T-lymphocyte count and IL-12 secretion to regulate the ovarian microenvironment and reduce inflammation, thus exerting therapeutic effects against POF.

Lins J et al. (2023) MicroPubl Biol "Generation of a C. elegans tdp-1 null allele and humanized TARDBP containing human disease-variants."

Hart AC, Lins J, Brock TJ, Hopkins CE

[MicroPubl Biol, 2023]

Clinical variants of <i>TARDBP</i> are associated with frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS) and other degenerative diseases. The predicted <i>C. elegans</i> ortholog of <i>TARDBP</i> is encoded by <i>tdp-1</i> , but functional orthology has not been demonstrated <i>in vivo.</i> We undertook CRISPR/Cas9-based genome editing of the <i>tdp-1</i> locus to create a complete loss of function allele; all <i>tdp-1</i> exons and introns were deleted, creating <i>tdp-1(tgx58)</i> , which resulted in neurodegeneration after oxidative stress. Next, we undertook CRISPR-based genome editing to replace <i>tdp-1</i> exons with human TARDBP coding sequences, creating humanized ( <i>hTARDBP</i> ) <i>C. elegans</i> expressing TDP-43 <i>.</i> Based on the efficiency of this genome editing, we suggest that iterative genome editing of the <i>tdp-1</i> target locus using linked coCRISPR markers, like <i>dpy-10</i> , would be a more efficient strategy for sequential assembly of the large engineered transgenes. <i>hTARDBP</i> decreased the neurodegeneration defect of <i>tdp-1(tgx58)</i> , demonstrating functional cross-species orthology. To develop <i>C. elegans</i> models of FTD and ALS, we inserted five different patient <i>TARDBP</i> variants in the <i>C. elegans</i> <i>hTARDBP</i> locus. Only one clinical variant increased stress-induced neurodegeneration; other variants caused inconsistent or negligible defects under these conditions. Combined, this work yielded an unambiguous null allele for <i>tdp-1</i> , a validated, humanized <i>hTARDBP,</i> and multiple ALS/FTD patient-associated variant models that can be used for future studies.

Lin H et al. (2001) J Cell Biol "Polyploids require Bik1 for kinetochore-microtubule attachment."

Lin H, Fink GR, Pellman D, de Carvalho P, Kho D, Pierre P, Tai CY

[J Cell Biol, 2001]

The attachment of kinetochores to spindle microtubules (MTs) is essential for maintaining constant ploidy in eukaryotic cells. Here, biochemical and imaging data is presented demonstrating that the budding yeast CLIP-170 orthologue Bik1is a component of the kinetochore-MT binding interface. Strikingly, Bik1 is not required for viability in haploid cells, but becomes essential in polyploids. The ploidy-specific requirement for BIK1 enabled us to characterize BIK1 without eliminating nonhomologous genes, providing a new approach to circumventing the overlapping function that is a common feature of the cytoskeleton. In polyploid cells, Bik1 is required before anaphase to maintain kinetochore separation and therefore contributes to the force that opposes the elastic recoil of attached sister chromatids. The role of Bik1 in kinetochore separation appears to be independent of the role of Bik1 in regulating MT dynamics. The finding that a protein involved in kinetochore-MT attachment is required for the viability of polyploids has potential implications for cancer therapeutics.

Lin XH et al. (1998) J Biol Chem "Caenorhabditis elegans contains two distinct acid sphingomyelinases."

Lin XH, Hengartner MO, Kolesnick RN

[J Biol Chem, 1998]

Mounting evidence supports a role for acid sphingomyelinase (ASM) in cellular stress signaling. Only murine and human sphingomyelinases have been defined at the molecular level. These enzymes are the products of a conserved gene and at the amino acid level share 82% identity. In this study, we show that the nematode Caenorhabditis elegans possesses two ASMs, termed ASM-1 and ASM-2 encoded by two distinct genes, but lacks detectable neutral sphingomyelinase activity. The C. elegans ASMs are about 30% identical with each other and with the human and murine enzymes. The conserved regions include a saposin-like domain, proline-rich domain, and a putative signal peptide. In addition, 16 cysteines distributed throughout the molecules, and selected glycosylation sites, are conserved. The expression of these genes in C. elegans is regulated during development. Asm-1 is preferentially expressed in the embryo, whereas asm-2 is predominantly expressed in postembryonic stages. When transfected as Flag-tagged proteins into COS-7 cells, ASM-1 is found almost entirely in a secreted form whereas only 20% of ASM-2 is secreted. Only the secreted forms display enzymatic activity. Furthermore, ASM-2 requires addition of Zn2+ to be fully active, whereas ASM-1 is active in the absence of cation. C. elegans is the first organism to display two ASMs. This finding suggests the existence of an ASM gene family.

Lin CH et al. (2010) J Neurosci "Insulin signaling plays a dual role in Caenorhabditis elegans memory acquisition and memory retrieval."

Sellings L, Pereira S, Lin CH, Tomioka M, Iino Y, van der Kooy D

[J Neurosci, 2010]

Insulin signaling plays a prominent role in regulation of dauer formation and longevity in Caenorhabditis elegans. Here, we show that insulin signaling also is required in benzaldehyde-starvation associative plasticity, in which worms pre-exposed to the odor attractant benzaldehyde in the absence of food subsequently demonstrate a conditioned aversion response toward the odorant. Animals with mutations in insulin-related 1 (ins-1), abnormal dauer formation 2 (daf-2), and aging alteration 1 (age-1), which encode the homolog of human insulin, insulin/IGF-1 receptor, and PIP3 kinase, respectively, demonstrated significant deficits in benzaldehyde-starvation associative plasticity. Using a conditional allele, we show that the behavioral roles of DAF-2 signaling in associative plasticity can be dissociated, with DAF-2 signaling playing a more significant role in the memory retrieval than in memory acquisition. We propose DAF-2 signaling acts as a learning-specific starvation signal in the memory acquisition phase of benzaldehyde-starvation associative plasticity but functions to switch benzaldehyde-sensing amphid wing C neurons into an avoidance signaling mode during memory retrieval.

Lin L et al. (2013) J Cell Biol "The scaffold protein EPG-7 links cargo-receptor complexes with the autophagic assembly machinery."

Huang X, Lin L, Lu Q, Zhang H, Yang P

[J Cell Biol, 2013]

The mechanism by which protein aggregates are selectively degraded by autophagy is poorly understood. Previous studies show that a family of Atg8-interacting proteins function as receptors linking specific cargoes to the autophagic machinery. Here we demonstrate that during Caenorhabditis elegans embryogenesis, epg-7 functions as a scaffold protein mediating autophagic degradation of several protein aggregates, including aggregates of the p62 homologue SQST-1, but has little effect on other autophagy-regulated processes. EPG-7 self-oligomerizes and is degraded by autophagy independently of SQST-1. SQST-1 directly interacts with EPG-7 and colocalizes with EPG-7 aggregates in autophagy mutants. Mutations in epg-7 impair association of SQST-1 aggregates with LGG-1/Atg8 puncta. EPG-7 interacts with multiple ATG proteins and colocalizes with ATG-9 puncta in various autophagy mutants. Unlike core autophagy genes, epg-7 is dispensable for starvation-induced autophagic degradation of substrate aggregates. Our results indicate that under physiological conditions a scaffold protein endows cargo specificity and also elevates degradation efficiency by linking the cargo-receptor complex with the autophagic machinery.

Lin JL et al. (2012) J Biol Chem "Structural insights into apoptotic DNA degradation by CED-3 protease suppressor-6 (CPS-6) from Caenorhabditis elegans."

Doudeva LG, Nakagawa A, Lin CL, Wang YT, Hsiao YY, Skeen-Gaar RR, Yang WZ, Xue D, Yuan HS, Lin JL

[J Biol Chem, 2012]

Endonuclease G (EndoG) is a mitochondrial protein that traverses to the nucleus and participates in chromosomal DNA degradation during apoptosis in yeast, worms, flies, and mammals. However, it remains unclear how EndoG binds and digests DNA. Here we show that the Caenorhabditis elegans CPS-6, a homolog of EndoG, is a homodimeric Mg(2+)-dependent nuclease, binding preferentially to G-tract DNA in the optimum low salt buffer at pH 7. The crystal structure of CPS-6 was determined at 1.8 resolution, revealing a mixed topology with the two -metal finger nuclease motifs located distantly at the two sides of the dimeric enzyme. A structural model of the CPS-6-DNA complex suggested a positively charged DNA-binding groove near the Mg(2+)-bound active site. Mutations of four aromatic and basic residues: Phe(122), Arg(146), Arg(156), and Phe(166), in the protein-DNA interface significantly reduced the DNA binding and cleavage activity of CPS-6, confirming that these residues are critical for CPS-6-DNA interactions. In vivo transformation rescue experiments further showed that the reduced DNase activity of CPS-6 mutants was positively correlated with its diminished cell killing activity in C. elegans. Taken together, these biochemical, structural, mutagenesis, and in vivo data reveal a molecular basis of how CPS-6 binds and hydrolyzes DNA to promote cell death.

Lin MY et al. (2019) Chin J Nat Med "Effect of loureirin A against Candida albicans biofilms."

Wang Y, Zhong H, Zhang RL, Hu GH, Yan L, Hu DD, Lin MY, Su J, Jiang YY, Yuan ZL

[Chin J Nat Med, 2019]

Loureirin A is a major active component of Draconis sanguis, a traditional Chinese medicine. This work aimed to investigate the activity of loureirin A against Candida albicans biofilms. 2, 3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT)reduction assay and scanning electron microscopy were used to investigate the anti-biofilm effect. Minimal inhibitory concentration testing and time-kill curve assay were used to evaluate fungicidal activity. Cell surface hydrophobicity (CSH) assay and hyphal formation experiment were respectively carried out to investigate adhesion and morphological transition, two virulence traits of C. albicans. Real-time RT-PCR was used to investigate gene expression. Galleria mellonella-C. albicans and Caenorhabditis elegans-C. albicans infection models were used to evaluate the in-vivo antifungal effect. Human umbilical vein endothelial cells and C. elegans nematodes were used to evaluate the toxicity ofloureirin A. Our data indicated that loureirin A had a significant effect on inhibiting C. albicans biofilms, decreasing CSH, and suppressing hyphal formation. Consistently, loureirin A down-regulated the expression of some adhesion-related genes and hypha/biofilm-related genes. Moreover, loureirin A prolonged the survival of Galleria mellonella and Caenorhabditis elegans in C. albicans infection models and exhibited low toxicity. Collectively, loureirin A inhibits fungal biofilms, and this effect may be associated with the suppression of pathogenic traits, adhesion and hyphal formation.