Araujo Agarrayua de Souza, Danielle et al. (2021) International Worm Meeting "Safaty evaluation of nanoparticles prepared with differents polymers in Caenorhabditis elegans"

Araujo Agarrayua de Souza, Danielle, Elisa Haas, Sandra, Avila , Daiana, Carvalho, Felipe

[International Worm Meeting, 2021]

Nano-sized drug delivery systems have been the subject of intense research in recent years because polymeric materials allow the absorption and release of active substances in a controlled manner. The use of polymeric nanoparticles as a drug carrier has emerged as an alternative. Despite the benefits, the safety of nanoparticulate systems is an aspect to be understood, particularly in in vivo systems. Caenorhabditis elegans is a very useful alternative model for nanotoxicology and has been recently applied in this field. The aim of this study was to evaluate toxicological endpoints in worms exposed to nanomaterials prepared with different polymers: polyethylene glycol (PEG), chitosan (CH), eudragit (EU) and polysorbate 80 (P80) in C. elegans. First larval staged worms were obtained by a synchronization process and treated with nanomaterials at concentrations of 0.015, 0.225 and 0.45 mg/mL for 30 minutes in liquid medium (acute exposure). Soon after, they were washed to remove the treatments and transferred to Petri dishes containing NGM and E. coli OP50 for 48 h. Survival rate, brood size and worms length were determined. Data were expressed as mean ± standard error, and statistical analysis were done by one-way ANOVA followed by Tukey post-hoc test. We observed that the EU nanoparticles did not cause any significant change in toxicological endpoints when compared to control. On the other hand, exposure to CH, PEG and P80 nanoparticles decreased worms survival, reduced their progeny and significantly altered worms size. This work demonstrates the toxicological differences between polymers and a potential for EU to be used in new formulations for future drug vectoring and targeting systems.

Mastronardi, Karina et al. (2017) International Worm Meeting "Studying Ran-GTP regulation of cytokinesis."

Ozugergin, Imge, Williams, Brittany, Piekny, Alisa, Mastronardi, Karina, Beaudet, Daniel

[International Worm Meeting, 2017]

Cytokinesis physically divides a cell into two daughters at the end of mitosis and must be highly robust to avoid aneuploidy and cell fate changes. A RhoA-dependent actomyosin contractile ring ingresses to divide the appropriate genetic and cell fate determinants into each daughter. The prevailing dogma in the field is that the mitotic spindle regulates contractile ring assembly and ingression. However, microtubule-independent mechanisms also regulate cytokinesis, and may be crucial in polarized cells. During metaphase, a Ran-GTP gradient forms around chromatin where it mediates mitotic spindle assembly by releasing importins a/ beta complexes from microtubule-binding proteins and motors. Our data suggests that the Ran-GTP gradient persists in early anaphase where it affects the localization of contractile proteins for cytokinesis in human cells. Importantly, one of the Ran targets may be anillin, which is a scaffold that binds to and coordinates actin, myosin, RhoA and its upstream regulators, microtubules, and phospholipids. We found that human anillin contains a C-terminal nuclear localization signal (NLS) that binds to importin- beta . Point mutations in the NLS delay anillin's recruitment to the cortex, and the contractile ring oscillates and subsequently fails to ingress in a subset of cells rescued with this mutant. Interestingly, mutant anillin's localization is strongly demarcated by astral microtubules, suggesting that the mitotic spindle dominantly regulates cytokinesis in these cells. We hypothesize that the Ran-GTP regulation of cytokinesis is conserved among metazoans, and the strength of this pathway is proportional to ploidy, cell geometry and/or cell volume. To test this, we are determining if the Ran pathway regulates cytokinesis in early C. elegans embryos. Studies showed that there is a negative correlation between the rate of furrow ingression and cell volume as cells decrease in size through the first four rounds of divisions (Carvalho et al., 2009). We are determining how perturbing the Ran pathway alters the rate of furrow ingression and the localization of contractile proteins during these divisions. Also, we are determining if ANI-1 (C. elegans anillin), which is highly conserved with human anillin, contains an NLS that may function in the same way as human anillin. We also will determine if the Ran pathway regulates other well-known cytokinesis components. Carvalho et al. (2009) Cell 137: 926-37.

Danielle F Kelleher et al. (2005) International Worm Meeting "C. briggsae tra mutants"

Eric S Haag, David B Pilgrim, John Salogiannis, Carlos Carvalho, Marnie R Layton, Danielle F Kelleher

[International Worm Meeting, 2005]

To explore how sex determination evolves over short, tractable time scales, we are developing Caenorhabditis briggsae, a close relative of the model nematode C. elegans, as a true genetic system. The function of C. briggsae sex determination genes has previously been assessed by RNA interference (RNAi). RNAi against the C. briggsae and C. remanei fem genes suggests differences in their germline function between species (1-3). However, RNAi rarely eliminates all target expression, and it is not suitable for the genetic approaches essential for pathway reconstruction (1,4). Here, we describe the isolation and characterization of Cb-tra mutant alleles. The best-characterized tra alleles fall into three linkage groups tightly linked to Cb-tra-1, Cb-tra-2, and Cb-tra-3. Sequencing of reference alleles from the Cb-tra-1 and Cb-tra-2 classes revealed missense and nonsense mutations, suggesting these are Cb-tra-1 and Cb-tra-2 mutants. Sequencing of a likely Cb-tra-3 reference allele is in progress. Intra-species suppression of the Cb-tra(ed24ts) Tra phenotype by genomic variants in the wild-type strain HK104 suggests that molecular interactions underlying sex determination are changing between two C. briggsae populations. Epistasis analysis of Cb-tra-2 with Cb-fem-2 and other fem-like Cb-tra-2 suppressors (see posters by Carvalho et al. and Hill et al.) indicates that the order of sex determination gene interactions in the soma is similar between the two species.1 Haag, ES, Wang, S, and Kimble, J (2002). Curr. Biol. 12: 2035-41. 2 Stothard, P, Hansen, D, and Pilgrim, D (2002). J. Mol. Evol. 54: 267-282. 3 Stothard, P, and Pilgrim, D (2003). BioEssays 25: 221-31. 4 Kuwabara, PE (1996). Genetics 144: 597-607.

Eric S Haag et al. (2003) International Worm Meeting "Sex determination mutants in C. briggsae"

Eric S Haag, Marnie R Layton, Danielle F Kelleher

[International Worm Meeting, 2003]

Sex determination is accomplished via highly disparate mechanisms in different phyla, and the homologous sex determination genes of closely related species often undergo rapid molecular evolution. To systematically explore how sex determination evolves over short, tractable time scales, we are developing Caenorhabditis briggsae as a true genetic system. Until now, the function of C. briggsae sex determination genes has been assessed using RNAi. However, RNAi rarely eliminates all target expression and it is not suitable for suppressor screens, epistasis and mosaic analyses, etc. Such genetic approaches are essential for accurate reconstruction of an entire pathway. Further, RNAi targeting of the C. briggsae and C. remanei fem genes suggests there may be large differences in germline fem function between these species1-3, a result that, if verified, would have major implications for the evolution of hermaphroditism in the genus. These results may in fact be due to divergence in the tissue-specificity of fem function, but could also merely reflect limited efficacy of RNAi. This can be resolved by the isolation of true mutations, done most readily in the hermaphrodite C. briggsae.Here, we describe the isolation of loss-of-funtion Cb-tra mutant alleles, work being carried out in conjunction with the Pilgrim Lab at the Univ. of Alberta (see poster by de Carvalho and Pilgrim). Using standard C. elegans methods, we have obtained six such alleles. 4 are completely penetrant at all temperatures, whereas 2 are partially rescued at 15 degrees . The alleles appear to fall into two complementation groups, one resembling Ce-tra-1 and the other Ce-tra-2 in phenotype. Mapping with visible and SNP markers is consistent with the tra-2-like complementation group (nm1 and nm10 alleles) being actual Cb-tra-2 mutations. Sequencing of these alleles is underway. If they are indeed Cb-tra-2 mutants, then the penetrance of Cb-tra-2(RNAi)), about 10%1,4, is much lower than that of true mutants. This suggests that the qualitative species differences seen in fem gene RNAi phenotypes may also be the result of incomplete target inactivation.1Haag, ES, Wang, S, and Kimble, J (2002). Current Biol. 12: 2035-41; 2Stodhard, P, Hansen, D, and Pilgrim, D (2002). J. Mol. Evol. 54: 267-282; 3Stodhard, P, and Pilgrim, D (2003). BioEssays 25: 221-31; 4Kuwabara, PE (1996). Genetics 144: 597-607.