[
Genesis,
2013]
In Caenorhabditis elegans, physiological germ cell apoptosis eliminates more than half of the cells in the hermaphrodite gonad to support gamete quality and germline homeostasis by a still unidentified mechanism. External factors can also affect germ cell apoptosis. The BH3-only protein EGL-1 induces germ cell apoptosis when animals are exposed to pathogens or agents that produce DNA damage. DNA damage-induced apoptosis also requires the nematode
p53 homolog CEP-1. Previously, we found that heat shock, oxidative, and osmotic stresses induce germ cell apoptosis through an EGL-1 and CEP-1 independent mechanism that requires the MAPKK pathway. However, we observed that starvation increases germ cell apoptosis by an unknown pathway. Searching for proteins that participate in stress-induced apoptosis, we found the RNA-binding protein TIAR-1 (a homolog of the mammalian TIA-1/TIAR family of proteins). Here, we show that TIAR-1 in C. elegans is required to induce apoptosis in the germline under several conditions. We also show that TIAR-1 acts downstream of CED-9 (a BCL2 homolog) to induce apoptosis under stress conditions, and apparently does not seem to regulate
ced-4 or
ced-3 mRNAs accumulation directly. TIAR-1 is expressed ubiquitously in the cytoplasm of the soma as well as the germline, where it sometimes associates with P granules. We show that animals lacking TIAR-1 expression are temperature sensitive sterile due to oogenesis and spermatogenesis defects. Our work shows that TIAR-1 is required for proper germline function and demonstrates that this protein is important to induce germ cell apoptosis under several conditions.
[
International Worm Meeting,
2017]
Extracellular vesicles are emerging as an important aspect of intercellular communication by delivering a parcel of proteins, lipids even nucleic acids to specific target cells over short or long distances (Maas 2017). A subset of C. elegans ciliated neurons release EVs to the environment and elicit changes in male behaviors in a cargo-dependent manner (Wang 2014, Silva 2017). Our studies raise many questions regarding these social communicating EV devices. Why is the cilium the donor site? What mechanisms control ciliary EV biogenesis? How are bioactive functions encoded within EVs? EV detection is a challenge and obstacle because of their small size (100nm). However, we possess the first and only system to visualize and monitor GFP-tagged EVs in living animals in real time. We are using several approaches to define the properties of an EV-releasing neuron (EVN) and to decipher the biology of ciliary-released EVs. To identify mechanisms regulating biogenesis, release, and function of ciliary EVs we took an unbiased transcriptome approach by isolating EVNs from adult worms and performing RNA-seq. We identified 335 significantly upregulated genes, of which 61 were validated by GFP reporters as expressed in EVNs (Wang 2015). By characterizing components of this EVN parts list, we discovered new components and pathways controlling EV biogenesis, EV shedding and retention in the cephalic lumen, and EV environmental release. We also identified cell-specific regulators of EVN ciliogenesis and are currently exploring mechanisms regulating EV cargo sorting. Our genetically tractable model can make inroads where other systems have not, and advance frontiers of EV knowledge where little is known. Maas, S. L. N., Breakefield, X. O., & Weaver, A. M. (2017). Trends in Cell Biology. Silva, M., Morsci, N., Nguyen, K. C. Q., Rizvi, A., Rongo, C., Hall, D. H., & Barr, M. M. (2017). Current Biology. Wang, J., Kaletsky, R., Silva, M., Williams, A., Haas, L. A., Androwski, R. J., Landis JN, Patrick C, Rashid A, Santiago-Martinez D, Gravato-Nobre M, Hodgkin J, Hall DH, Murphy CT, Barr, M. M. (2015).Current Biology. Wang, J., Silva, M., Haas, L. A., Morsci, N. S., Nguyen, K. C. Q., Hall, D. H., & Barr, M. M. (2014). Current Biology.
[
International Worm Meeting,
2019]
Dietary restriction (DR) has been shown to increase lifespan and promote healthy aging in a wide range of organisms, but the mechanisms through which this occurs remain poorly defined. AMP-activated protein kinase (AMPK) is activated in response to DR, is pro-longevity, and is thought to mediate the effect of DR on lifespan extension. Previously we have shown that AMPK promotes longevity via a cell nonautonomous signaling cascade involving the cyclic AMP-responsive element binding protein (CREB)-regulated transcriptional coactivator (CRTC)-1. However, the role and requirement of this signaling cascade in DR remains unclear. We hypothesize that AMPK is only required in specific tissues for DR-mediated longevity. To begin to test this hypothesis, we have used CRISPR to generate a tool set which will allow us to sensitively and specifically probe the requirement for AMPK in various tissues. Using a newly developed SKILODGE safe harbor system from our lab, we have knocked in either wildtype or truncated constitutively
aak-2 driven by either ubiquitous (
eft-3) or neuronal-specific (
rab-3) promoters. We can use these strains for overexpression experiments or we can cross them into an
aak-2-deficient mutant to perform tissue-specific rescue experiments. Here we will discuss the spatial requirement for AMPK in DR. Since the implementation of DR in humans is both impractical and unpleasant, specific characterization of the required components will be essential in designing therapies that can mimic DR's beneficial effects on lifespan and healthspan.