Genetic characterization of AMPK-interacting genes in germline stem cell quiescence and integrity in «Caernorhabditis elegans»

In response to adverse conditions, C. elegans can enter an alternative developmental stage called dauer. In this state the larva undergoes several changes in order to conserve energy and withstand environmental stress. One such change occurs in the germline, where the germline stem cells (GSC) arrest their mitotic divisions and remain quiescent for the duration of this stage. Previous studies have identified the catalytic subunit of AMP-activated protein kinase (AMPK), aak-2, to be necessary in establishing this cell cycle quiescence. The aak-2 mutants exhibit pronounced germline hyperplasia compared to the normal dauer, however the molecular mechanism through which AMPK controls the GSC proliferation remains unclear. To address this question, we identified potential AMPK targets involved in the GSC quiescence by conducting a genome-wide RNAi suppressor screen for candidates that rescue the aak-2-induced hyperplasia. After screening 18,432 genes, we identified 59 candidates, which were further categorized into gene ontology groups. Of particular interest are genes necessary for miRNA-dependent gene silencing and components of the extracellular matrix (ECM), important constituents of stem cell niches. In addition we also discovered that animals exhibiting the aak-2-dependent hyperplasia in dauer are sterile and have severe morphological germline defects following the recovery from dauer. This demonstrates the importance of AMPK-dependent dauer germline quiescence on post-dauer reproduction. Candidate suppressors of the dauer germline hyperplasia failed to rescue the sterility phenotype, suggesting that although AMPK-dependent, the processes that control the GSC quiescence in dauer and the germline recovery post-dauer are independent.En réponse à des conditions défavorables, les vers C. elegans peuvent entrer dans un état de développement alternatif appelé dauer. Dans cet état, la larve subit plusieurs changements afin d'économiser son énergie et résister aux contraintes de l'environnement. Un tel changement se produit dans la lignée germinale; les cellules souches de lignée germinale (CGC) stoppent leurs divisions mitotiques et restent au repos durant toute la durée de cette étape. Des études précédentes ont identifié la sous-unité catalytique de la protéine kinase AMP-dépendante (AMPK), aak-2, nécessaire à l'établissement de ce repos du cycle cellulaire. Les mutants aak-2 présentent une hyperplasie de la lignée germinale prononcée par rapport à un dauer normal, mais le mécanisme moléculaire par lequel l'AMPK contrôle la prolifération des CGC reste incertain. Pour répondre à cette question, nous avons identifié des cibles potentielles de l'AMPK impliquées dans la quiescence des CGC, en effectuant un crible ARNi à l'échelle du genome entier pour trouver les candidats qui reduisent l'hyperplasie induite par aak-2. Après avoir testé 18 432 gènes, nous avons identifié 59 candidats, qui ont ensuite été classés en groupe en function de leur ontologie respective. D'un intérêt particulier sont les gènes nécessaires pour l'extinction de gènes grace au mechanisme de miARN ainsi que les composants de la matrice extracellulaire (ECM), constituants importants des niches de cellules souches. En outre, nous avons également découvert que les animaux présentant une hyperplasie durant dauer dependante de aak-2 sont stériles et conservent de graves anomalies morphologiques de la lignée germinale après la reprise du development normal. Cela demontre l'importance de la quiescence de la lignee germinale,d'une maniere dependante de AMPK, pour la reproduction post-dauer. Nourrir les animaux en dauer avec des gènes candidats qui suppriment l'hyperplasie dauer de la lignée germinale a échoué à sauver le phénotype de stérilité, ce qui suggère que, bien que dependant de l'AMPK, les processus qui contrôlent la mise au repos de la CGC dans dauer et la récupération post-dauer de la lignée germinales

Genome-wide surveys reveal polarity and cytoskeletal regulators mediate LKB1-associated germline stem cell quiescence

Abstract Background Caenorhabditis elegans can endure long periods of environmental stress by altering their development to execute a quiescent state called “dauer”. Previous work has implicated LKB1 - the causative gene in the autosomal dominant, cancer pre-disposing disease called Peutz-Jeghers Syndrome (PJS), and its downstream target AMPK, in the establishment of germline stem cell (GSC) quiescence during the dauer stage. Loss of function mutations in both LKB1/par-4 and AMPK/aak(0) result in untimely GSC proliferation during the onset of the dauer stage, although the molecular mechanism through which these factors regulate quiescence remains unclear. Curiously, the hyperplasia observed in par-4 mutants is more severe than AMPK-compromised dauer larvae, suggesting that par-4 has alternative downstream targets in addition to AMPK to regulate germline quiescence. Results We conducted three genome-wide RNAi screens to identify potential downstream targets of the protein kinases PAR-4 and AMPK that mediate dauer-dependent GSC quiescence. First, we screened to identify genes that phenocopy the par-4-dependent hyperplasia when compromised by RNAi. Two additional RNAi screens were performed to identify genes that suppressed the germline hyperplasia in par-4 and aak(0) dauer larvae, respectively. Interestingly, a subset of the candidates we identified are involved in the regulation of cell polarity and cytoskeletal function downstream of par-4, in an AMPK-independent manner. Moreover, we show that par-4 temporally regulates actin cytoskeletal organization within the dauer germ line at the rachis-adjacent membrane, in an AMPK-independent manner. Conclusion Our data suggest that the regulation of the cytoskeleton and cell polarity may contribute significantly to the tumour suppressor function of LKB1/par-4