Anhydrobiotic organisms have the remarkable ability to lose extensive amounts of body water and survive in an ametabolic, suspended animation state. Distributed to various taxa of life, these organisms have evolved strategies to efficiently protect their cell membranes and proteins against extreme water loss. At the molecular level, a variety of mutually non-exclusive mechanisms have been proposed to account particularly for preserving the integrity of the cell membranes in the desiccated state. Recently, it has been shown that the dauer larva of the nematode Caenorhabditis elegans is anhydrobiotic and accumulates high amounts of trehalose during preparation for harsh desiccation (preconditioning), thereby allowing for a reversible desiccation / rehydration cycle. Here, we have used this genetic model to study the biophysical manifestations of anhydrobiosis and show that, in addition to trehalose accumulation, the dauer larvae exhibit a systemic chemical response upon preconditioning by dramatically reducing their phosphatidylcholine (PC) content. The C. elegans strain daf-2 was chosen for these studies, because it forms a constitutive dauer state under appropriate growth conditions. Using complementary approaches such as chemical analysis, time-resolved FTIR-spectroscopy, Langmuir-Blodgett monolayers, and fluorescence spectroscopy, it is shown that this chemical adaptation of the phospholipid (PL) composition has key consequences for their interaction with trehalose. Infrared-spectroscopic experiments were designed and automated to particularly address structural changes during fast hydration transients. Importantly, the coupling of headgroup hydration to acyl chain order at low humidity was found to be altered on the environmentally relevant time scale of seconds. PLs from preconditioned larvae with reduced PC content exhibit a higher trehalose affinity, a stronger hydration-induced gain in acyl chain free volume, and a wider spread of structural relaxation rates during lyotropic transitions and sub- headgroup H-bond interactions as compared to PLs from non-preconditioned larvae. The effects are related to the intrinsically different hydration properties of PC and phosphatidylethanolamine (PE) headgroups, and lead to a larger hydration-dependent rearrangement of trehalose-mediated H-bond network in PLs from preconditioned larvae. This results in a lipid compressibility modulus of ∼0.5 mN/m and 1.2 mN/m for PLs derived from preconditioned and non-preconditioned larvae, respectively. The ensemble of these changes evidences a genetically controlled chemical tuning of the native lipid composition of a true anhydrobiote to functionally interact with a ubiquitous protective disaccharide. The biological relevance of this adaptation is the preservation of plasma membrane integrity by relieving mechanical strain from desiccated trehalose- containing cells during fast rehydration. Finally, the thermo-tropic lipid phase behavior was studied by temperature-dependent ATR-FTIR and fluorescence spectroscopy of LAURDAN-labeled PLs. The results show that the adaptation to drought, which is accomplished to a significant part by the reduction of the PC content, relies on reducing thermo-tropic and enhancing lyotropic phase transitions. The data are interpreted on a molecular level emphasizing the influence of trehalose on the lipid phase transition under biologically relevant conditions by a detailed analysis of the lipid C=O H-bond environment. The salient feature of the deduced model is a dynamic interaction of trehalose at the PL headgroup region. It is proposed here that the location of trehalose is changed from a more peripheral to a more sub-headgroup-associated position. This appears to be particularly pronounced in PLs from preconditioned worms. The sugar slides deeper into the inter-headgroup space during hydration and thereby supports a quick lateral expansion such that membranes can more readily adapt to the volume changes in the swelling biological material at reduced humidity. The data show that the nature of the headgroup is crucial for its interaction with trehalose and there is no general mechanism by which the sugar affects lipidic phase transitions. The intercalation into a phosphatidylethanolamine-rich membrane appears to be unique. In this case, neither the phase transition temperature nor its width is affected by the protective sugar, whereas strong effects on these parameters were observed with other model lipids. With respect to membrane preservation, desiccation tolerance may be largely dependent on reducing phosphatidylcholine and increasing the phsophatidylethanolamine content in order to optimize trehalose headgroup interactions. As a consequence, fast mechanical adaptation of cell membranes to hydration-induced strain can be realized.

Chez la majorité des espèces, les mitochondries et leur ADN sont hérités de manière uniparentale maternelle. Au moment de la fécondation, le spermatozoïde entre dans l'ovocyte avec ses mitochondries et leur ADN menant à se demander pourquoi et comment les mitochondries paternelles ne sont plus détectées chez le nouvel individu. Chez le ver C. elegans, les mitochondries d’origine spermatique sont activement dégradées par autophagie dans l’embryon une cellule. Les marqueurs de l’autophagie chez le ver, les protéines LGG-1 et LGG-2, sont observés autour des organites d’origine spermatique après la fécondation et l’interférence avec l’autophagie bloque l’élimination de ces organites. Néanmoins, il n’est toujours pas clair comment ce ciblage s’effectue ni le rôle des différentes protéines de l’autophagies impliquées dans le processus. La première partie des résultats montre que LGG-2 permet le transport des autophagosomes et de leur contenu vers la zone pericentrosomale afin de faciliter leur fusion avec les lysosomes qui se concentrent dans cette zone. En parallèle, j’ai testé plusieurs hypothèses afin d’identifier les mécanismes de ciblage des mitochondries d’origine spermatique. J’ai montré que l’ubiquitine joue un rôle dans le recrutement de la protéine LGG-1 autour des organites spermatiques. J’ai également décrit plusieurs propriétés des mitochondries spermatiques et ovocytaires qui semblent jouer un rôle dans le recrutement de la machinerie de l’autophagie. Ainsi, la dégradation des mitochondries d’origine spermatique représente une forme originale et physiologique de mitophagie. / In most animal species, mitochondria and their DNA are maternally inherited. Upon fertilization, the spermatozoid and its mitochondria enter into the oocyte leading to the questions why and how are those mitochondria not detected in the new born. The sperm derived mitochondria are selectively degraded by autophagy in C. elegans. The autophagy proteins, LGG-1 and LGG-2, are recruited around sperm-derived organelles upon fertilization in the early embryo of C. elegans and the interference with the autophagy blocks the degradation of those organelles. The mechanism permitting this specific targeting of the paternal mitochondria and the role of the different autophagy proteins are still unclear. First, we showed that LGG-2 plays an important role in the clearance of sperm-derived organelles by targeting them to the pericentrosomal area to facilitate their fusion with lysosomes. In parallel, I tested several hypotheses to identify the mechanism permitting the specific targeting of sperm-derived mitochondria. I showed that the ubiquitin plays a role in the recruitment of LGG-1 around sperm-derived organelles and described several properties of the sperm and oocyte-derived mitochondria that are likely to play an important role for the recruitment of the autophagy machinery. This led us to conclude that sperm derived mitochondria degradation represent an original physiologic mitophagy.

In order to survive, cells must be able to cope with a variety of environmental stressors. The heat shock response (HSR) is a pro-survival mechanism employed by cells in response to protein denaturing stress, such as heat. Since its discovery in 1960, the heat shock response has been found to be regulated by the transcription factor heat shock factor 1 (HSF1). During periods of increased stress, HSF1 undergoes a multi-step process of activation that involves homotrimerization, DNA-binding, and post-translational regulatory modifications, all of which ultimately function to control the transcription of chaperone genes. These chaperone genes encode molecular chaperone proteins which function to promote survival during stress by restoring protein homeostasis to the cell. Although HSF1 is classically studied for its role in regulating the HSR, HSF1 also has roles in regulating metabolism, development, and longevity. Studies in the nematode Caenorhabditis elegans demonstrate the HSF1 homolog, HSF-1, as a global regulator of gene expression that has both stress-dependent and -independent functions. Modulating HSF1 activity therefore has implications beyond stress-induced processes, and has been suggested as a promising therapeutic target for diseases of aging and protein dysfunction. We were interested in determining regulators of the HSR using C. elegans as a model to test for effects on proteostasis and longevity. In these studies, we observed the effects of compound treatment (Chapters 1 and 2), genetic manipulation (Chapters 3 and 4), and environmental stimuli (Chapters 5 and 6), on the HSR in C. elegans. In Chapters 1 and 2, we describe our findings that treatment with the DNA synthesis inhibitor Fluorodeoxyuridine, and treatment with coffee and caffeine, enhance the heat shock response and improve proteostasis in aging worms in an HSF-1-dependent manner. In Chapters 3 and 4, we uncovered that negative regulation of the HSR by the cell cycle and apoptosis regulator CCAR2 is conserved in C. elegans, and is mediated by the CCAR2 ortholog, LST-3. We also uncovered that negative regulation of the HSR by LST-3 requires the SIRT1 homolog Sir-2.1, and knockdown of LST-3 via lst-3 RNAi works through Sir-2.1 to enhance stress-resistance, fitness, proteostasis and longevity. In Chapters 5 and 6, we describe the global impact of HSF-1 in regulating transcriptional processes during a heat stress. The profiling of global HSF-1 mRNA and miRNA targets has allowed us to uncover a heat-dependent and -independent role for HSF-1 in regulating gene expression to impact stress-resistance, proteostasis, and longevity. Altogether, these studies demonstrate the impact of compound treatment, genetic manipulation, and environmental stimuli on the heat shock response, while also uncovering global stress-dependent and -independent roles for HSF-1. This work therefore provides insight into various methods of activating the HSR by modulating HSF-1 activity, and uncovering global HSF-1 target genes, which may be useful for designing therapeutic treatment strategies for diseases of protein dysfunction.

Little is known about the molecular components and interactions of the planar cell polarity pathway that regulate neuronal polarity. This study uses a prkl-1 induced backwards locomotion defect as an array to perform a prkl-1 suppressor screen in C. elegans looking for new components of the planar cell polarity pathway involved in the neuronal polarization of VC4 and VC5. The screen discovered twelve new alleles of vang-1, one new allele of fntb-1 and five new mutations in unknown polarity genes. fntb-1 encodes for the worm ortholog of Farnesyltransferase β-subunit and is important for neuronal polarization. Acting cell and non-cell autonomously, fntb-1 regulates the function and localization of prkl-1 through the recognition of a CAAX motif. Therefore, fntb-1 modifies prkl-1 to regulate the neuronal polarity of VC4 and VC5.

We sought to investigate the role of the Planar Cell Polarity (PCP) pathway in neuronal positioning along the Anterior-Posterior (AP) axis of C. elegans, and chose the worm’s DD-type motor neurons as a model. The six DD neurons (DD1-DD6) are evenly spaced in the ventral nerve cord of wild type animals. Here we showed that mutations in core PCP genes caused DD neuron spacing and positioning defects. prkl-1 double mutant combinations with vang-1 and fmi-1 showed a suppression of the more severe prkl-1 single mutant defects, which was evidence of genetic interactions between these PCP components. We also conducted a candidate screen of Frizzled, Dishevelled, Wnt, and ROCK genes, and found that dsh-1/Dishevelled, mom-2/Wnt and let-502/ROCK also played roles in DD neuronal positioning. Both vang-1 and prkl-1 were found to function within the nervous system to guide DD neuronal positioning, and prkl-1 was further identified as playing a cell autonomous role. The origins of observed DD neuron anterior positioning defects were investigated during embryogenesis, in which 1.5 fold stage prkl-1(ok3182) embryos displayed delayed intercalation of the DD neurons. This represents a novel role for the PCP pathway in mediating DD neuronal intercalation.

Oxygen deprivation can be pathological for many organisms, including humans. Consequently, there are several biologically and economically relevant negative impacts associated with oxygen deprivation. Developing an understanding of which genes can influence survival of oxygen deprivation will enable the formulation of more effective policies and practices. In this dissertation, genes that influence adult anoxia survival in the model metazoan system, C. elegans, are identified and characterized. Insulin-like signaling, gonad function and gender have been shown to influence longevity and stress resistance in the soil nematode, C. elegans. Thus, either of these two processes or gender may influence anoxia survival. The hypothesis that insulin-like signaling alters anoxia survival in C. elegans is tested in Aim I. The hypotheses that gonad function or gender modulates anoxia survival are tested in Aim II. Insulin-like signaling affects anoxia survival in C. elegans. Reduction of insulin-like signaling through mutation of the insulin-like receptor, DAF-2, increases anoxia survival rates in a gpd-2/3 dependent manner. The glycolytic genes gpd-2/3 are necessary for wild-type response to anoxia, and sufficient for increasing anoxia survival through overexpression. Gonad function and gender both affect anoxia survival in C. elegans. A reduction of ovulation and oocyte maturation, as measured by oocyte flux, is associated with enhanced anoxia survival in all cases examined to date. Reduction of function of several genes involved in germline development and RTK/Ras/MAPK signaling reduce ovulation and oocyte maturation while concurrently increasing anoxia survival. The act of mating does not influence anoxia survival, but altering ovulation through breeding or chemical treatment does. The male phenotype also increases anoxia survival rates independent of genotype. These studies have identified and characterized over ten different genotypes that affect adult survival of anoxia in C. elegans. Before these studies were conducted, there were no genes known to influence adult anoxia survival in C. elegans. Furthermore, these studies have begun to uncouple mechanisms of longevity and stress resistance.

No description available.

Maternal mRNA regulation is essential to germline and embryo development in metazoans. Over the past few decades, it has become clear that many RNA-binding proteins (RBPs) containing highly conserved RNA-binding domains orchestrate spatiotemporal expression pattern of germline and embryonic genes to control gametogenesis and embryogenesis in the nematode Caenorhabditis elegans. These RBPs bind regulatory elements situated primarily in the UTRs of their target mRNAs to regulate expression by influencing transcript stability or translational efficiency. The 3´UTR is the main determinant of patterned expression in the germline of C. elegans. MEX-3 is a KH-domain RBP that is required for anterior cell fate specification and maintenance of germ cell totipotency. MEX-3 is expressed in mitotic germ cells, maturing oocytes, and early embryos. MEX-3 is absent in the meiotic pachytene region as well as the diplotene loop region. The 3´UTR of mex-3 is sufficient to confer MEX-3’s expression to a transgenic reporter. Here, I assessed the importance of the endogenous 3´UTR of mex-3 to MEX-3’s expression pattern and function using CRISPR/Cas9 mutagenesis followed by molecular and phenotypic analysis. 3´UTR deletion allelic series demonstrated that the endogenous 3´UTR of mex-3 is indeed required for MEX-3’s pattern in the germline in vivo. I identified regions of the 3´UTR that contribute to repression of MEX-3 in different regions of the germline. Surprisingly, the 3´UTR was dispensable for viability. However, several 3´UTR deletions exhibited reduced fertility. Analysis of the transcriptome of these mutants revealed that the 3´UTR deletions altered expression of soma-specific genes, consistent with MEX-3’s role in repressing somatic gene programs. These data sets also showed that mex-3 mRNA levels do not correlate with MEX-3 protein levels. In order to determine which germline RBPs regulate expression of mex-3 through its 3´UTR, I used RNAi to knock down several candidate RBPs including three that were previously shown to regulate expression of MEX-3. My RNAi studies showed that GLD-1, LIN-41, and OMA-1/2 repress expression of mex-3 through its 3´UTR in the meiotic pachytene region, diplotene loop region, and oocytes in the proximal end, respectively. Furthermore, I have identified DAZ-1, an RRM-containing RBP, as a novel repressor of MEX-3 expression in the distal mitotic germ cells. Using RNAi, I demonstrated that poly(A) tail length control and the translation initiation factor IFE-3 contribute to MEX-3’s expression in the germline. Poly(A) polyadenylation and deadenylation cycles govern expression of mex-3 in the distal mitotic germ cells, while IFE-3 contributes to repression of mex-3 in the meiotic pachytene region, presumably by control of translation initiation. Using high throughput sequencing-based poly(A) tail assay, I have shown that the poly(A) tail length distribution of mex-3 mRNA shifts towards shorter tails in the mex-3 3´UTR deletion mutants with reduced fertility phenotypes. Our study is the first as far as we know to address the importance of an endogenous 3´UTR to in vivo expression and function in C. elegans germline. It will be interesting to determine how different RBPs and cis-regulatory elements orchestrate the spatiotemporal expression pattern of a single germline gene. It will also be interesting to assess whether other germline 3´UTRs are similarly dispensable for viability, and if so, what role do 3´UTRs play in enhancing reproductive success.

I used the C. elegans genetic model to examine the role of ceramide biosynthesis (sphingolipid pathway) and iron regulation and found that each process impacts germline development and function. Using a sphingolipid specific antibody mAb15B4, I found that sphingolipids are associated with germ granules (P granules) within C. elegans and zebrafish; thus, suggesting conservation of macromolecules associated with germ granules. Phenotype analysis of ceramide biosynthesis mutants in C. elegans revealed that this pathway is essential for normal germline function in the aging adult hermaphrodite; specifically, precocious germline senescence was observed. Furthermore, I found that disruption of ceramide biosynthesis, via the hyl-2 deletion mutation, negatively impacts mAb15B4 localization at the P granules. Through genetic suppression analysis, I determined that insulin signaling and lipid biosynthesis can modulate the mAb15B4 localization to P granules. Additional, phenotype analysis showed that ceramide biosynthesis dysfunction decreased fecundity, and led to germline structure defects and uterine tumors. Through suppression analysis, I determined that modulation of the insulin signaling pathway suppressed the precocious germline senescence due to ceramide biosynthesis dysfunction. Since the presence of uterine tumors is associated with reproductive senescence I concluded that ceramide biosynthesis has a role in germline maintenance in the aging of the germline (germline senescence). The other important fate of a germ cell is programmed cell death. Apoptosis, which occurs through a highly conserved molecular pathway, is a normal component of growth and homeostatic processes. I used C. elegans to gain a greater understanding of the cisd gene function. The C. elegans genome has three previously uncharacterized cisd genes which code for CISD-1 (homology to vertebrate mitoNEET/CISD1 and NAF-1/CISD2) and CISD-3.1 and CISD-3.2 (homology to vertebrate Miner2/CISD3). I determined that independent disruption of the cisd genes resulted in a significant increase in the number of cell corpses within the adult hermaphrodite germline. Genetic analysis was used to examine the dysfunction of cisd-1 relative to the cell death canonical pathway genes. The increased gamete cell death in the cisd-1 hermaphrodite is suppressed by the ced-9 (Bcl-2 homolog) gain-of-function and requires functional CED-3 (caspase) and CED-4 (APAF). Additionally, the increased germ cell programmed cell death is facilitated by the pro-apoptotic, CED-9-binding protein, CED-13. Further analysis of the cisd gene family members show that cisd-3.2 dysfunction leads to germline defects and reproductive dysfunction, suggesting defects in germline stem cell proliferation. Expression analysis using the cisd promoters to drive fluorescent protein reporters showed that the cisd gene family is expressed in various tissues including the germline; fusion protein analysis showed that CISD-3 is mitochondrial localized. I propose that cisd-3.2 germline defects are a result of abnormal mitochondrial function. Combined, this work is significant because it identifies sphingolipids as a new component of embryonic P granules, a role for ceramide biosynthesis in reproductive senescence, and places the cisd gene family members as regulators of physiological germline programmed cell death acting through CED-13 and the core apoptotic machinery. Furthermore, it is the first study to show that a CISD3 protein family member is required for normal germline function. These findings support the idea that ceramide biosynthesis and iron regulation are core components in germline development and function.

NEET proteins belong to a unique class of [2Fe-2S] cluster proteins that have been shown to participate in various biological processes such as regulating iron, reactive oxygen species and apoptosis within the cell and are localized to the mitochondria. Disruption of the mitochondrial NEET proteins are associated with different human diseases such as obesity, neurodegeneration, cancer and diabetes. In humans, a missense mutation in the CISD2 gene results in a heritable multisystem disorder termed Wolfram syndrome 2 (WFS2), a disease which displays an early onset of juvenile diabetes and various neuropsychiatric disorders. The C. elegans genome contains three previously uncharacterized cisd genes: cisd-1, which has homology to the human CISD1 and CISD2, and cisd-3.1 and cisd-3.2, both of which have homology to the human CISD3. Disruption of the cisd-3 gene(s) function results in mis-regulation of proteostasis in the mitochondria, whereas cisd-1 and cisd-3.1 disruption impacts proteostasis in the endoplasmic reticulum. Reduction of cisd-3.2 gene function also leads to a developmental delay in C. elegans. A knockout mutation of the cisd-3.2(pn68) gene function results in various germline defects including delayed development progression and morphological defects. Furthermore, I show the cisd gene(s) and protein expression profiles is present relative to sex, tissue type and developmental stages. This work is significant because it provides further insight of the essential role of CISD-3 relative to C. elegans. Furthermore, my studies can contribute to new genetic discoveries that will widen the scientific research relative to NEET protein family studies.