Reprogramming of the transcriptome after heat stress mediates heat hormesis in Caenorhabditis elegans.

Transient stress experiences not only trigger acute stress responses, but can also have long-lasting effects on cellular functions. In Caenorhabditis elegans, a brief exposure to heat shock during early adulthood extends lifespan and improves stress resistance, a phenomenon known as heat hormesis. Here, we investigated the prolonged effect of hormetic heat stress on the transcriptome of worms and found that the canonical heat shock response is followed by a profound transcriptional reprogramming in the post-stress period. This reprogramming relies on the endoribonuclease ENDU-2 but not the heat shock factor 1. ENDU-2 co-localizes with chromatin and interacts with RNA polymerase II, enabling specific regulation of transcription after the stress period. Failure to activate the post-stress response does not affect the resistance of animals to heat shock but eliminates the beneficial effects of hormetic heat stress. In summary, our work discovers that the RNA-binding protein ENDU-2 mediates the long-term impacts of transient heat stress via reprogramming transcriptome after stress exposure.

Mitochondrial Perturbations Couple mTORC2 to Autophagy in C. elegans.

Autophagy is stimulated by stress conditions and needs to be precisely tuned to ensure cellular homeostasis and organismal development and health. The kinase mechanistic target of rapamycin (mTOR) forms the enzymatic core of the highly conserved mTOR complexes mTORC1 and mTORC2. mTORC1 is a key inhibitor of autophagy, yet the function of mTORC2 in autophagy is controversial. We here show that inactivation of mTORC2 and its direct target serum- and glucocorticoid-inducible kinase 1 (SGK-1) potently induces autophagy and the autophagic degradation of mitochondria in C. elegans. Enhanced autophagy in mTORC2- or SGK-1-deficient animals contributes to their developmental and reproductive defects and is independent of the canonical SGK-1 effector DAF-16/FOXO. Importantly, we find that inactivation of mTORC2-SGK-1 signaling impairs mitochondrial homeostasis and triggers an increased release of mitochondria-derived reactive oxygen species (mtROS) to induce autophagy. Thus, mitochondrial stress couples reduced mTORC2 activity to enhanced autophagic turnover.

Exploring the electron transfer pathways in photosystem I by high-time-resolution electron paramagnetic resonance: observation of the B-side radical pair P700(+)A1B(-) in whole cells of the deuterated green alga Chlamydomonas reinhardtii at cryogenic temperatures.

Crystallographic models of photosystem I (PS I) highlight a symmetrical arrangement of the electron transfer cofactors which are organized in two parallel branches (A, B) relative to a pseudo-C2 symmetry axis that is perpendicular to the membrane plane. Here, we explore the electron transfer pathways of PS I in whole cells of the deuterated green alga Chlamydomonas reinhardtii using high-time-resolution electron paramagnetic resonance (EPR) at cryogenic temperatures. Particular emphasis is given to quantum oscillations detectable in the tertiary radical pairs P700(+)A1A(-) and P700(+)A1B(-) of the electron transfer chain. Results are presented first for the deuterated site-directed mutant PsaA-M684H in which electron transfer beyond the primary electron acceptor A0A on the PsaA branch of electron transfer is impaired. Analysis of the quantum oscillations, observed in a two-dimensional Q-band (34 GHz) EPR experiment, provides the geometry of the B-side radical pair. The orientation of the g tensor of P700(+) in an external reference system is adapted from a time-resolved multifrequency EPR study of deuterated and 15N-substituted cyanobacteria (Link, G.; Berthold, T.; Bechtold, M.; Weidner, J.-U.; Ohmes, E.; Tang, J.; Poluektov, O.; Utschig, L.; Schlesselman, S. L.; Thurnauer, M. C.; Kothe, G. J. Am. Chem. Soc. 2001, 123, 4211-4222). Thus, we obtain the three-dimensional structure of the B-side radical pair following photoexcitation of PS I in its native membrane. The new structure describes the position and orientation of the reduced B-side quinone A1B(-) on a nanosecond time scale after light-induced charge separation. Furthermore, we present results for deuterated wild-type cells of C. reinhardtii demonstrating that both radical pairs P700(+)A1A(-) and P700(+)A1B(-) participate in the electron transfer process according to a mole ratio of 0.71/0.29 in favor of P700(+)A1A(-). A detailed comparison reveals different orientations of A1A(-) and A1B(-) in their respective binding sites such that formation of a strong hydrogen bond from A1(-) to the protein backbone is possible only in the case of A1A(-). We suggest that this is relevant to the rates of forward electron transfer from A1A(-) or A1B(-) to the iron-sulfur center F(X), which differ by a factor of 10. Thus, the present study sheds new light on the orientation of the phylloquinone acceptors in their binding pockets in PS I and the effect this has on function.

Caenorhabditits elegans LRK-1 and PINK-1 act antagonistically in stress response and neurite outgrowth.

Mutations in two genes encoding the putative kinases LRRK2 and PINK1 have been associated with inherited variants of Parkinson disease. The physiological role of both proteins is not known at present, but studies in model organisms have linked their mutants to distinct aspects of mitochondrial dysfunction, increased vulnerability to oxidative and endoplasmic reticulum stress, and intracellular protein sorting. Here, we show that a mutation in the Caenorhabditits elegans homologue of the PTEN-induced kinase pink-1 gene resulted in reduced mitochondrial cristae length and increased paraquat sensitivity of the nematode. Moreover, the mutants also displayed defects in axonal outgrowth of a pair of canal-associated neurons. We demonstrate that in the absence of lrk-1, the C. elegans homologue of human LRRK2, all phenotypic aspects of pink-1 loss-of-function mutants were suppressed. Conversely, the hypersensitivity of lrk-1 mutant animals to the endoplasmic reticulum stressor tunicamycin was reduced in a pink-1 mutant background. These results provide the first evidence of an antagonistic role of PINK-1 and LRK-1. Due to the similarity of the C. elegans proteins to human LRRK2 and PINK1, we suggest a common role of both factors in cellular functions including stress response and regulation of neurite outgrowth. This study might help to link pink-1/PINK1 and lrk-1/LRRK2 function to the pathological processes resulting from Parkinson disease-related mutants in both genes, the first manifestations of which are cytoskeletal defects in affected neurons.

Heme, a plastid-derived regulator of nuclear gene expression in Chlamydomonas.

To gain insight into the chloroplast-to-nucleus signaling role of tetrapyrroles, Chlamydomonas reinhardtii mutants in the Mg-chelatase that catalyzes the insertion of magnesium into protoporphyrin IX were isolated and characterized. The four mutants lack chlorophyll and show reduced levels of Mg-tetrapyrroles but increased levels of soluble heme. In the mutants, light induction of HSP70A was preserved, although Mg-protoporphyrin IX has been implicated in this induction. In wild-type cells, a shift from dark to light resulted in a transient reduction in heme levels, while the levels of Mg-protoporphyrin IX, its methyl ester, and protoporphyrin IX increased. Hemin feeding to cultures in the dark activated HSP70A. This induction was mediated by the same plastid response element (PRE) in the HSP70A promoter that has been shown to mediate induction by Mg-protoporphyrin IX and light. Other nuclear genes that harbor a PRE in their promoters also were inducible by hemin feeding. Extended incubation with hemin abrogated the competence to induce HSP70A by light or Mg-protoporphyrin IX, indicating that these signals converge on the same pathway. We propose that Mg-protoporphyrin IX and heme may serve as plastid signals that regulate the expression of nuclear genes.

Identification of a plastid response element that acts as an enhancer within the Chlamydomonas HSP70A promoter.

Chloroplast-derived signals control a subset of nuclear genes in higher plants and eukaryotic algae. Among the types of signals identified are intermediates of chlorophyll biosynthesis such as Mg-protoporphyrin IX (MgProto). In Chlamydomonas reinhardtii, it was suggested that this tetrapyrrole mediates the light induction of chaperone gene HSP70A. Here we have analyzed cis elements involved in the regulation of HSP70A by MgProto and light. We identified two promoters and between their transcription start sites two regulatory regions that each may confer inducibility by MgProto and light to both HSP70A promoters. These regulatory regions, when cloned in front of basal non-light inducible heterologous promoters, conferred inducibility by MgProto and light. The orientation and distance independent function of these cis-regulatory sequences qualifies them as enhancers that mediate the response of nuclear genes to a chloroplast signal. Mutational analysis of one of these regulatory regions and an alignment with promoters of other MgProto-inducible genes revealed the sequence motif (G/C)CGA(C/T)N(A/G)N15 (T/C/A)(A/T/G) which, as shown for HSP70A, may confer MgProto responsiveness. This cis-acting sequence element is employed for induction of HSP70A by both MgProto and light, lending support to the model that light induction of this gene is mediated via MgProto.