Article
Reference
The ULK1 kinase, a necessary component of the pro-regenerative and anti-aging machinery in Hydra
SUKNOVIC, Nenad Slavko, et al.
Abstract
Hydra vulgaris (Hv) has a high regenerative potential and negligible senescence, as its stem cell populations divide continuously. In contrast, the cold-sensitive H. oligactis (Ho_CS) rapidly develop an aging phenotype under stress, with epithelial stem cells deficient for autophagy, unable to maintain their self-renewal. Here we tested in aging, non-aging and regenerating Hydra the activity and regulation of the ULK1 kinase involved in autophagosome formation. In vitro kinase assays show that human ULK1 activity is activated by Hv extracts but repressed by Ho_CS extracts, reflecting the ability or inability of their respective epithelial cells to initiate autophagosome formation. The factors that keep ULK1 inactive in Ho_CS remain uncharacterized. Hv_Basel1 animals exposed to the ULK1 inhibitor SBI-0206965 no longer regenerate their head, indicating that the sustained autophagy flux recorded in regenerating Hv_AEP2 transgenic animals expressing the DsRed-GFP-LC3A autophagy tandem sensor is necessary. The SBI-0206965 treatment also alters the contractility of intact Hv_Basel1 animals, and leads to a progressive reduction [...]
SUKNOVIC, Nenad Slavko,
et al. The ULK1 kinase, a necessary component of the pro-regenerative and anti-aging machinery in Hydra.
Mechanisms of Ageing and Development, 2021, vol. 194, no. 111414
DOI : 10.1016/j.mad.2020.111414 PMID : 33338499
Available at:
http://archive-ouverte.unige.ch/unige:148694
Disclaimer: layout of this document may differ from the published version.
The ULK1 kinase, a necessary component of
the pro-regenerative and anti-aging machinery in Hydra
Nenad SUKNOVIC, Szymon TOMCZYK, Delphine COLEVRET, Chrystelle PERRUCHOUD and Brigitte GALLIOT
Department of Genetics and Evolution, Institute of Genetics and Genomics in Geneva (iGE3), Faculty of Sciences, University of Geneva, 30 Quai Ernest Ansermet, CH-1211 Geneva 4, Switzerland
Supplemental methods ... 2
Quant-IM methods: quantification of fluorescence data ... 2
Supplemental Figures ... 3
Figure S1: Phylogenetic analysis of the ULK-related sequences from H. vulgaris and H. oligactis ... 3
Figure S2: Structure of ULK1 and alignment of Unc51/ULK1/ATG1 sequences ... 4
Figure S3: Phylogenetic analysis of the ATG6 / Beclin1 protein family ... 5
Figure S4: Conservation of the ATG6 / Beclin-1 sequences and regulatory sites across evolution. .... 6
Figure S5: Expression of the components of the Beclin-1 and ULK1 complexes in H. vulgaris. ... 7
Figure S6: Expression of the components of the Beclin-1 and ULK1 complexes in aging or non-aging H. oligactis ... 8
Figure S7: Transgenic Hv_AEP2 animals expressing the DsRed-GFP-LC3A tandem sensor ... 9
Figure S8: Chronic exposure of animals from the Hydra oligactis strains, Ho_CS and Ho_CR and Hydra vulgaris strain sf-1 to the ULK inhibitor SBI-0206965 ... 10
Figure S9: Efficiency of ULK1(RNAi) knock-down ... 10
Supplemental methods
Quant-IM methods: quantification of fluorescence data
After trypsin dissociation (see materials and methods), the cells spread on slides were imaged in a tile- scan fashion using the wide-field microscope Leica DM5500 with the 5x objective. Maximum projections of the tile-scan merge images were then analyzed with the Imaris software using the “surface” mode.
Each surface represented one cell from the image analyzed for the mean fluorescence intensity in both green and red channel. Quantification data containing both green and the red mean fluorescence intensities for each cell were imported in R-studio and plotted using the custom written R-script:
g <- read.csv("Data_green.csv") r <- read.csv("Data_red.csv")
g_sel <- select(g, ID, Intensity_Mean)
g_sel <- rename(g_sel, Green_fluorescence = Intensity_Mean) r_sel <- select(r, ID, Intensity_Mean)
r_sel <- rename(r_sel, Red_fluorescence = Intensity_Mean) Data_full <- left_join(g_sel, r_sel, by = "ID")
Data_full <- mutate(Data_full, total_fluo = Green_fluorescence + Red_fluorescence) Data_full <- mutate(Data_full, Green_perc = (Green_fluorescence/total_fluo)*100) Data_full <- mutate(Data_full, Red_perc = (Red_fluorescence/total_fluo)*100) Data_full %>%
pivot_longer(c(Green_perc, Red_perc), names_to = "Channel_perc", values_to = "Fluo_perc")
%>%
ggplot(aes(fill = Channel_perc, y=Fluo_perc, x=ID)) + geom_bar(position="stack", stat="identity") + scale_fill_manual(values = c("Green","red")) + labs(y="Fluo %", x = "Cell' ID")
Data_full_with_means <- rbind(colMeans(Data_full), Data_full) View (Data_full_with_means)
Graphs can be represented as stacked percentage bars showing the green/red fluorescence on the y-axis and cell ID on x-axis.
Supplemental Figures
Figure S1: Phylogenetic analysis of the ULK-related sequences from H. vulgaris and H. oligactis
Phylogenetic tree of ULK protein sequences aligned with MUSCLE and built with PhyML 3.0, tested with 100 bootstraps. Hydra sequences are written dark red. Species code is as follows: Arath: Arabidopsis thaliana;
Caeel: Caenorhabditis elegans; Cragi: Crassostrea gigas (oyster); Dicdi: Dictyostelium discoideum (slime mold);
Galga: Gallus gallus (chick); Human, Hydvu: Hydra vulgaris; Hydol: Hydra oligactis; Lotgi: Lottia gigantean (owl limpet, mollusk); Nemve: Nematostella vectensis (sea anemone); Neucr: Neurospora crassa (yeast); Sacko:
Saccoglossus kowalesvskii (acorn worm); Schpo: Schizosaccharomyces pombe (yeast); Xentr: Xenopus tropicalis (Western clawed frog). The ULK1/ATG1 family includes sequences from yeast, plants and metazoans, whereas the ULK3 and ULK4 families include sequences from deuterostomes, protostomes, cnidarians. The ULK1 sequence from Dicdi appears divergent.
Figure S2: Structure of ULK1 and alignment of Unc51/ULK1/ATG1 sequences
(A) Scheme of the human and Hydra ULK1 (Unc-51 like kinase) proteins after (Papinski and Kraft, 2016). LIR:
LC3-Interacting Region; EAT: Early Autophagy Targetting/Tethering domain; MIT: Microtubule Interacting and Transport domain. (B) Alignment of the ULK1-related sequences from human (O75385, Q8IYT8), H. vulgaris (Hydvu, seq40808_loc15218), H. oligactis cold resistant (HolCR, R034566c0g1_i03), Crassostrea gigas (Cragi,
K1PNL8), C. elegans (Caeel, Q23023) and Drosophila melanogaster (Drome, Q9VU14). Sequences can be retrieved at Uniprot.org and HydrAtlas.unige.ch. Note the conservation of the kinase domain at the N-terminus (red arrowheads) and the C-terminal EAT / MIT domain that binds ATG13 and FIP200 (purple background). Key residues for ULK1 regulation in mammals appear conserved in Hydra: Thr180 for ULK1 autophosphorylation, the LIR motif, Ser317 and Ser775 for AMPK phosphorylation in response to starvation, Ser758 for TOR phosphorylation in response to nutrient sufficiency, which inhibits ULK1 activity (Kim et al., 2011).
Figure S3: Phylogenetic analysis of the ATG6 / Beclin1 protein family
Phylogenetic tree of Beclin1 (BECN1) protein sequences aligned with MUSCLE and built with PhyML 3.0, tested with the aLRT software at the LIRMM (http://www.phylogeny.fr/index.cgi). The Beclin1 family includes sequences from yeast, plants and metazoans (deuterostomes, protostomes, cnidarians, porifers). The BECN1 sequence from Caenorhabditis elegans (Caeel) appears highly divergent among the metazoan sequences.
Phallusia mammillata (tunicate); Sacce: Saccharomyces cerevisiae (yeast); Stija: Stichopus japonicus (sea cucumber); Strpu: Strongylocentrotus purpuratus (sea urchin); Stypi: Stylophora pistillata (hood coral); Triad:
Trichoplax adherens (placozoan).
Figure S4: Conservation of the ATG6 / Beclin-1 sequences and regulatory sites across evolution.
CLUSTAL multiple sequence alignment of the Human (Q14457), Hydra (Hydvu, T2MDF4), C. elegans (Caeel, Q22592), Drosophila (Drome, Q9VCE1) and Arabidopsis thaliana (Arath, Q9M367) Beclin-1/ATG6 protein sequences obtained with MUSCLE (3.8) (www.ebi.ac.uk/Tools/msa/muscle). Note the conservation of the three overlapping domains: the BH3 motif required for the BCL2/BCL-XL binding, the Coiled-Coiled Domain
(CCD) and the Evolutionary-Conserved Domain (ECD). Sites written black bold correspond to phosphorylation or acetylation sites characterized experimentally (Sun et al., 2015; Denon and Dhamijia, 2018; Phosphonet);
Ser and Tyr sites written red indicate predicted phosphorylation sites identified by Kinexus P-site prediction algorithm: http://www.phosphonet.ca/?search=Q14457.
isolated by FACS (see Wenger et al. 2016). Abbreviations: bc: body column, eESC: epidermal Epithelial Stem Cell; gESC: gastrodermal ESC; i-cells: Interstitial Stem Cell. All RNA-seq sequences and profiles are available on HydrAtlas server (https://hydratlas.unige.ch) (Wenger et al., 2019). In yeast and plants, PIK3C3 is named Vps34 and PIK3R4 corresponds toVps15.
Figure S6: Expression of the components of the Beclin-1 and ULK1 complexes in aging or non-aging H.
oligactis
RNA-seq analysis was performed at several time-points on aging Cold Sensitive (Ho_CS) and non-aging Cold Resistant (Ho_CR) animals either maintained at 18°C or transferred to 10°C to induce aging (see details in Tomczyk et al., 2020) of the Beclin1 complex (A), the ULK1 complex (B) and the gene encoding UVRAG that interacts with Beclin1 (C). In the Beclin1 complex, the expression of PIK3R4 is not shown as expressed at too low levels. The accession numbers of the Ho_CS sequences (upper rows in A and B) starts with S0---c0g-_i0-, those of the Ho_CR ones (lower row in A and B) with R0---c0g-_i0-. Sequences and RNA_seq expression profiles are available on the Hydratlas server (https://hydratlas.unige.ch). Note the different expression profiles of ULK1 and ATG13 between Ho_CS and Ho_CR maintained at 10°C.
Figure S7: Transgenic Hv_AEP2 animals expressing the DsRed-GFP-LC3A tandem sensor
red cells, indicating that the autophagy flux is not modified. This negative result reflects the low sensitivity of Hv_AEP2 animals to the MG132 drug when compared to Hv_Basel1 ones (see Figure 3). (B) Quant-IM quantification of the green fluorescence in ga-LC3A cells located in the basal-regenerating tips (region R1), apical-regenerating tips (region R2) and subjacent body column (region R3), dissected 1, 24 and 48 hours after bisection at mid-gastric position. The results obtained in three independent experiments are shown here. Each dot corresponds to the value of a single cell. Individual and median values are depicted in box plots. The p values were determined with the Welch’s unpaired t-test. **** p < 0.001.
Figure S8: Chronic exposure of animals from the Hydra oligactis strains, Ho_CS and Ho_CR and Hydra vulgaris strain sf-1 to the ULK inhibitor SBI-0206965
(A) Effect of the ULK inhibitor SBI-0206965 (1 µM) on the survival of Hv_sf1 and Ho_CS animals maintained starved at 18°C. (B) Effect of the TORC inhibitor rapamycin (0.8 µM) and the ULK inhibitor SBI-0206965 (1 µM) on survival of Ho_CS animals maintained at 10°C. In A and B, the survival rate was measured on 6x 10 animals continuously exposed to SBI-0206965. (C) Effect of the ULK inhibitor SBI-0206965 (0.5 or 1 µM) on head regeneration of Ho_CR and Ho_CS animals. Regeneration was tested on 10-20 animals per condition, maintained at 18°C, starved for 4 days and exposed to SBI-0206965 for 3 days prior to mid- gastric bisection and continuously after bisection.
Figure S9: Efficiency of ULK1(RNAi) knock-down Q-PCR analysis of ULK1 expression after three EPs in Hv_AEP2 (A) and Hv_Basel1 (B) animals. The ULK1-A siRNA does not lead to ULK1 silencing whereas the ULK1- B and ULK1-C siRNAs lead to a similar 45% reduction of ULK1 expression in Hv_AEP2 and Hv_Basel1 animals.
