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. 2024 Mar:214:19-27.
doi: 10.1016/j.freeradbiomed.2024.01.050. Epub 2024 Jan 30.

ULP-2 SUMO protease regulates UPRmt and mitochondrial homeostasis in Caenorhabditis elegans

Lirin Michaeli  1 , Eyal Spector  1 , Simon Haeussler  2 , Cátia A Carvalho  1 , Hanna Grobe  1 , Ulrike Bening Abu-Shach  1 , Hen Zinger  1 , Barbara Conradt  3 , Limor Broday  4
Affiliations

ULP-2 SUMO protease regulates UPRmt and mitochondrial homeostasis in Caenorhabditis elegans

Lirin Michaeli et al. Free Radic Biol Med. 2024 Mar.

Abstract

Mitochondria are the powerhouses of cells, responsible for energy production and regulation of cellular homeostasis. When mitochondrial function is impaired, a stress response termed mitochondrial unfolded protein response (UPRmt) is initiated to restore mitochondrial function. Since mitochondria and UPRmt are implicated in many diseases, it is important to understand UPRmt regulation. In this study, we show that the SUMO protease ULP-2 has a key role in regulating mitochondrial function and UPRmt. Specifically, down-regulation of ulp-2 suppresses UPRmt and reduces mitochondrial membrane potential without significantly affecting cellular ROS. Mitochondrial networks are expanded in ulp-2 null mutants with larger mitochondrial area and increased branching. Moreover, the amount of mitochondrial DNA is increased in ulp-2 mutants. Downregulation of ULP-2 also leads to alterations in expression levels of mitochondrial genes involved in protein import and mtDNA replication, however, mitophagy remains unaltered. In summary, this study demonstrates that ULP-2 is required for mitochondrial homeostasis and the UPRmt.

Keywords: Mitochondrial unfolded protein response; SENP; SUMO; SUMO protease; Smo-1; ULP-2; UPRmt.

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Conflict of interest statement

Declaration of competing interest None.

Figures

Image 1
Graphical abstract
Fig. 1
Fig. 1
Downregulation of ulp-2 suppresses markers of the UPRmt. (A) hsp-6p::gfp (bcSi9) fluorescence in WT(+/+), drp-1(tm1108) and fzo-1(tm1133) genetic backgrounds, each following control(L4440), ulp-2 or atfs-1 RNAi treatments as indicated [atfs-1(RNAi) was used as a positive control]. (B) Quantification of hsp-6p::gfp (bcSi9) fluorescence presented in A (One-way ANOVA, ****P < 0.0001, n is indicated for each group). (C) Real-time qPCR quantification of ulp-2 or atfs-1 mRNA in the different genetic backgrounds following the RNAi treatments. (t-test was used to analyze all data except for hsp-6p::gfp following atfs-1(RNAi) and hsp-6p::gfp;drp-1(tm1108) and hsp-6p::gfp;fzo-1(tm1133) both following ulp-2(RNAi) where U test was used, ****P < 0.0001). (D) hsp-6p::gfp (bcSi9) fluorescence in WT(+/+) and ulp-2(tv380) genetic backgrounds following control(L4440) or cco-1 RNAi treatment. (E) Quantification of hsp-6p::gfp (bcSi9) fluorescence presented in D (One-way ANOVA, **P < 0.01; ****P < 0.0001, n is indicated for each group). (F) Real-time qPCR quantification of hsp-6 (U test, ****P < 0.0001) or hsp-60 (t-test, ****P < 0.0001) mRNA in WT(+/+) or ulp-2(tv380) genetic backgrounds. Data is from three biological repeats shown as mean ± SEM. Scale bar 100 μM.
Fig. 2
Fig. 2
Loss of ULP-2 leads to reduced mitochondrial membrane potential without affecting cellular redox potential. (A) TMRE fluorescence of representative worms of the indicated genotypes. (B) Quantification of TMRE fluorescence shown in A. Fluorescent values were normalized to WT(+/+) (Kruskal-Wallis test with Dunn's post hoc test, **P < 0.01; ***P < 0.001; ****P < 0.0001, n is indicated for each group). (C) TMRE fluorescence of representative WT(+/+) or ulp-2(tv380) worms following cco-1(RNAi) or L4440 empty vector control(RNAi). (D) Quantification of TMRE fluorescence shown in C. Values were normalized to WT(+/+) Control(RNAi). (Kruskal-Wallis test with Dunn's post hoc test, ****P < 0.0001. ns-not significant, n is indicated for each group). (E) Quantification of Grx1-roGFP2 fluorescence ratios of its oxidative to reduced state (λex405, λem580)/(λex490, λem580) in WT(+/+) and ulp-2(tv380) worms (U test, ns-not significant, n is indicated for each condition). Data is from at least three biological repeats, shown as mean ± SEM. Scale bar = 10 μM.
Fig. 3
Fig. 3
Mitochondrial networks are expanded in the absence of ULP-2. (A–B) Upper panel: Confocal images of the hypodermis and muscle tissue respectively of WT(+/+) or ulp-2(tv380) worms stained with MitoView™ Green. Middle and Lower panel: Segmented and skeletonized images of the original images shown in the upper panel. (C) Insets of the white rectangles in the upper panel of A-B as indicated. (D–E) Quantifications of mitochondrial morphology parameters of the images presented in A-B respectively. Each dot on the graph represents the average measurements from a single worm. Definitions of morphology parameters are described in ‘Materials and Methods’ (all morphology parameters were analyzed using U test except for ‘Total Mitochondrial Area Normalized to Worm Area’ and muscle junctions and branches that were analyzed using t-test; n = 21,18 for WT and ulp-2(tv380) respectively, **P < 0.01; ***P < 0.001; ****P < 0.0001). Data is from at least three biological repeats shown as mean ± SEM. Scale bar = 10 μM.
Fig. 4
Fig. 4
Ulp-2 deficiency increases relative mtDNA and modifies mitochondrial biogenesis gene expression without altering basal mitophagy. (A) RT-qPCR of relative mitochondrial DNA (mtDNA) normalized to nuclear DNA (nDNA) of ulp-2(tv380) compared to WT(+/+) (U test, ****P < 0.0001). (B) RT-qPCR measurement for expression level of mitochondrial genes in WT(+/+) or ulp-2(tv380) worms. C. elegans genes are indicated followed by their mammalian orthologues. Genes are grouped according to their known function as indicated in the upper part of the graph but may have additional functions (U test, *P < 0.05, **P < 0.01, ***P < 0.001 ns-not significant). (C) Worms expressing the mitophagy biosensor myo-3p::tomm-20::Rosella in muscle tissue following control L4440(RNAi) or ulp-2(RNAi) as indicated. White rectangles are presented on the right as insets. (E) Positive control to C where worms expressing myo-3p::tomm-20::Rosella were treated with NaN3 or M9 buffer. White rectangles are presented on the right as insets. (D,F) Related to C and E respectively: Quantification of the GFPSEP/DsRed fluorescence ratio in the indicated experimental conditions (t-test, ****P < 0.0001, ns-not significant, n is indicated for each group). For visualization purposes, images presented as GFP or DsRed were pseudo colored in Green or Magenta respectively. Data is from at least three biological repeats, shown as mean ± SEM. Scale bar = 10 μM.
figs1
figs1
ulp-2(RNAi) suppress UPRmt induced by disruption to mitochondrial fusion. (A) hsp-6::gfp(zcIs13) UPRmt reporter fluorescence in fzo-1(tm1133) genetic background following Control (L4440 empty vector), ulp-2 or atfs-1 RNAi treatments as indicated. (B) Quantification of hsp-6::gfp(zcIs13) fluorescence presented in A. Data is normalized to the control(RNAi) condition (One-way ANOVA, ****P<0.0001, n≥20 in each group). Data is one of three biological repeats, shown as mean ± SEM. Scale bar=100µM.
figs2
figs2
A schematic representation of TMRE quantification. A raw image is converted to 8-bit (A), followed by segmentation of mitochondria using Ilastik (B). An additional segmentation is performed on the worm’s body (C) to avoid measuring signals outside of the worm. The ImageJ plugin integrates the 8-bit image with the segmented images to generate Regions Of Interest (ROI) only around the mitochondria that are found within the worm body while additional noise is removed by filtering out particles below a defined size. The plugin fills holes within the worm body segmentation and considers only the largest area while ignoring smaller isolated areas. The resulting ROIs (D) are measured for intensity. Scale bar: 10 μm.
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