TBC-2 Is Required for Embryonic Yolk Protein Storage and Larval Survival during L1 Diapause in Caenorhabditis elegans

C. elegans first stage (L1) larvae hatched in the absence of food, arrest development and enter an L1 diapause, whereby they can survive starvation for several weeks. The physiological and metabolic requirements for survival during L1 diapause are poorly understood. However, yolk, a cholesterol binding/transport protein, has been suggested to serve as an energy source. Here, we demonstrate that C. elegans TBC-2, a RAB-5 GTPase Activating Protein (GAP) involved in early-to-late endosome transition, is important for yolk protein storage during embryogenesis and for L1 survival during starvation. We found during embryogenesis, that a yolk::green fluorescent protein fusion (YP170::GFP), disappeared much more quickly in tbc-2 mutant embryos as compared with wild-type control embryos. The premature disappearance of YP170::GFP in tbc-2 mutants is likely due to premature degradation in the lysosomes as we found that YP170::GFP showed increased colocalization with Lysotracker Red, a marker for acidic compartments. Furthermore, YP170::GFP disappearance in tbc-2 mutants required RAB-7, a regulator of endosome to lysosome trafficking. Although tbc-2 is not essential in fed animals, we discovered that tbc-2 mutant L1 larvae have strongly reduced survival when hatched in the absence of food. We show that tbc-2 mutant larvae are not defective in maintaining L1 diapause and that mutants defective in yolk uptake, rme-1 and rme-6, also had strongly reduced L1 survival when hatched in the absence of food. Our findings demonstrate that TBC-2 is required for yolk protein storage during embryonic development and provide strong correlative data indicating that yolk constitutes an important energy source for larval survival during L1 diapause

Folliculin Regulates Ampk-Dependent Autophagy and Metabolic Stress Survival.

Dysregulation of AMPK signaling has been implicated in many human diseases, which emphasizes the importance of characterizing AMPK regulators. The tumor suppressor FLCN, responsible for the Birt-Hogg Dubé renal neoplasia syndrome (BHD), is an AMPK-binding partner but the genetic and functional links between FLCN and AMPK have not been established. Strikingly, the majority of naturally [...] which induces autophagy, inhibits apoptosis, improves cellular bioenergetics, and confers resistance to energy-depleting stresses including oxidative stress, heat, anoxia, and serum deprivation. We further show that AMPK activation conferred by FLCN loss is independent of the cellular energy state suggesting that FLCN controls the AMPK energy sensing ability. Together, our data suggest that FLCN is an evolutionarily conserved regulator of AMPK signaling that may act as a tumor suppressor by negatively regulating AMPK function

Premature depletion of YP170::GFP in <i>tbc-2(tm2241)</i> embryos.

<p>Differential Interference Contrast (DIC) (A, C, E, G, I, K, M, O, Q, S and U) and epifluorescence (B, D, F, H, J, L, N, P, R, T and V) images of wild-type (A, B, E, F, I, J, M, N, Q and R), <i>tbc-2(tm2241)</i> (C, D, G, H, K, L, O, P, S and T), and <i>tbc-2(tm2241) rab-7(ok511)</i> (U and V) oocytes (A–D) and different stage embryos (4-cell, E-H; bean stage at the beginning of morphogenesis, I-L; 1.5 fold stage of elongation, M-T; mid-stage of nonviable <i>tbc-2(tm2241) rab-7(ok511)</i> is late proliferative/early morphogenesis stage, U and V) carrying maternally deposited YP170::GFP from the integrated transgene <i>bIs1</i>. All images are of live animals except Q-T, which are fixed and immunostained with an anti-GFP antibody. Quantification of YP170::GFP fluorescence average intensity per unit area in wild-type and <i>tbc-2(tm2241)</i> proximal oocytes and bean stage embryos (<i>n</i> = 23 for both strains) (X and Y). A two-tailed unpaired Student t-test was used to determine statistical significance. n.s., not significant. ***, p<0.0001. Error bars represent standard deviations. Bars, 10 µm.</p

YP170::GFP localizes to lysosomes in <i>tbc-2(tm2241)</i> embryos.

<p>Confocal images of wild-type (A–C), <i>tbc-2(tm2241)</i> (D–F), and <i>tbc-2(tm2241) rab-7(ok511)</i> (G–I) embryos carrying YP170::GFP (green; A, D, and G) and stained with Lysotracker Red (B, E, and H) with the colocalization shown in the Merge images (C, F, and I). Arrows mark colocalization between YP170::GFP vesicles and Lysotracker Red. Quantification of the percentage of YP170::GFP fluorescence overlapping with Lysotracker Red compared to the total YP170::GFP fluorescence (J). <i>n = </i>25, wild-type; 17, <i>tbc-2(tm2241);</i> 10, <i>tbc-2(tm2241) rab-7(ok511)</i>. Statistical significance was determined using a two-tailed unpaired Student t-test. n.s., not significant. ***, p<0.0001. Error bars represent standard deviations. Bar, 5 µm.</p

<i>tbc-2(tm2241), rme-1(b1045),</i> and <i>rme-6(b1014)</i> L1 larvae have reduced survival during starvation.

<p>Survival curve of wild-type (blue), <i>tbc-2(tm2241)</i> (green), <i>rme-1(b1045)</i> (purple), and <i>rme-6(b1014)</i> (red) L1 larvae hatched in the absence of food. Graph represents the average of three independent experiments.</p

<i>tbc-2</i> is not required for maintaining L1 starvation-induced diapause.

<p>(A–D) Merged DIC and epifluorescence images of <i>hlh-8::GFP</i> expression in the M cell of wild-type (A and C) and <i>tbc-2(tm2241)</i> (B and D) L1 larvae at day 1 (A and B) and day 10 (C and D) of starvation. (E–H) Epifluorescence images of <i>cki-1::GFP</i> expression in wild-type (E and G) and <i>tbc-2(tm2241)</i> (F and H) in fed L1 larvae (E and F) and L1 larvae at day 10 of starvation (G and H). Bar, 10 µm.</p

Folliculin Regulates Ampk-Dependent Autophagy and Metabolic Stress Survival

<div><p>Dysregulation of AMPK signaling has been implicated in many human diseases, which emphasizes the importance of characterizing AMPK regulators. The tumor suppressor <i>FLCN</i>, responsible for the Birt-Hogg Dubé renal neoplasia syndrome (BHD), is an AMPK-binding partner but the genetic and functional links between FLCN and AMPK have not been established. Strikingly, the majority of naturally occurring <i>FLCN</i> mutations predisposing to BHD are predicted to produce truncated proteins unable to bind AMPK, pointing to the critical role of this interaction in the tumor suppression mechanism. Here, we demonstrate that FLCN is an evolutionarily conserved negative regulator of AMPK. Using <i>Caenorhabditis elegans</i> and mammalian cells, we show that loss of FLCN results in constitutive activation of AMPK which induces autophagy, inhibits apoptosis, improves cellular bioenergetics, and confers resistance to energy-depleting stresses including oxidative stress, heat, anoxia, and serum deprivation. We further show that AMPK activation conferred by FLCN loss is independent of the cellular energy state suggesting that FLCN controls the AMPK energy sensing ability. Together, our data suggest that FLCN is an evolutionarily conserved regulator of AMPK signaling that may act as a tumor suppressor by negatively regulating AMPK function.</p></div