STEPHANE ROLLAND et al. (2007) International Worm Meeting "How do the Bcl-2-like protein CED-9 and the mitofusin-like GTPase FZO-1 control mitochondria morphology in C. elegans?"

BARBARA CONRADT, STEPHANE ROLLAND

[International Worm Meeting, 2007]

In mammals, mitochondria play a central role in apoptosis by releasing several pro-apoptotic factors. In Caenorhabditis elegans, mitochondria have so far only been proposed to serve as docks for CED-9/CED-4 and CED-9/EGL-1 complexes in healthy cells and apoptotic cells, respectively. Recent work has demonstrated that DRP-1-mediated mitochondrial fragmentation is required for efficient apoptosis in C. elegans. Furthermore, DRP-1-mediated mitochondrial fragmentation in apoptotic cells has been shown to be dependant on CED-9 (1). However, how CED-9 participates in the control of mitochondrial morphology is still unknown. To address this question, we have systematically screened the ability of CED-9 to interact with components of the mitochondrial fission and fusion machineries using a yeast two-hybrid system. This screen revealed that CED-9 interacts with the mitofusin-like protein FZO-1 and that this interaction is enhanced by the presence of EGL-1 and inhibited by the presence of CED-4. We also confirmed this interaction in vitro by GST pull-down. Mitofusin-like proteins are large GTPases localized to the mitochondrial outer membrane and required for mitochondrial fusion. Mitofusin-like proteins have been proposed to form trans-heterodimers allowing the tethering of adjacent mitochondria and to participate in outer membrane fusion through a GTP-dependent mechanism (2). We have shown that the mitofusin-like protein FZO-1 is also required for mitochondrial fusion in C. elegans. Indeed, whereas mitochondria of wild-type animals form an elongated network, mitochondria of animals homozygous for the fzo-1 loss-of-function mutation tm1133 are fragmented. The same phenotype is observed when the fzo-1 gene is knocked-down by RNAi. These results are consistent with a previous report of Kanazawa et al (3). In addition, we have shown that the over-expression of fzo-1 induces the aggregation of mitochondria in grape-like structures, due to either hyper-tethering of mitochondria and/or fusion specifically of their outer membranes. Contrary to fzo-1 over-expression, the over-expression of ced-9 seems to induce complete fusion of mitochondria. Furthermore, CED-9-induced fusion is dependent on a functional fzo-1 gene, indicating that CED-9 regulates the function of FZO-1. However, in contrast to findings reported by Delivani et al (4), animals lacking ced-9 function do not have an obvious defect in mitochondrial morphology. Therefore, the physiological significance of the fusogenic activity of CED-9 remains to be elucidated. (1. Jagasia, R et al (2005) Nature 433:754-60. 2. Zhang, Y et al (2007) FEBS Lett. 3. Kanazawa, T et al (2004) West Coast Worm Meeting. 4. Delivani, P et al (2006) Mol. Cell 21:761-773).

Koehler, Fabian et al. (2015) International Worm Meeting "The loss of mma-1 LRPPRC function induces the mitochondrial unfolded protein response."

Koehler, Fabian, Rolland, Stephane, Mueller-Rischart, Kathrin, Conradt, Barbara

[International Worm Meeting, 2015]

In a genetic screen for Caenorhabditis elegans mutants with abnormal mitochondrial morphology, we identified mma-1 (mitochondrial morphology abnormal-1), a C. elegans homolog of the French Canadian Leigh Syndrome gene LRPPRC (leucine-rich pentatricopeptide repeat containing). Inactivation of mma-1 in C. elegans or LRPPRC in mammalian cells results in a decrease in the production of mitochondria-encoded subunits of cytochrome c oxidase (COX) and consequently a reduction in COX activity. We have previously shown that in this context, mitochondria form a hyperfused network that transiently maintains cellular ATP levels (Rolland et al, 2013). We now demonstrate that the inactivation of mma-1 LRPPRC also leads to an imbalance between mitochondria- and nuclear-encoded COX subunits, which triggers a conserved mitochondrial unfolded protein response (UPRmt). We also show that the activation of UPRmt leads to the restoration of mitochondrial proteostasis. Finally, we present evidence that UPRmt and the mitochondrial hyperfusion response are coordinated but mediated by genetically distinct pathways. We propose that in the context of mma-1 LRPPRC knock-down, mitochondrial hyperfusion helps to transiently maintain cellular ATP levels enabling UPRmt to restore mitochondrial proteostasis.Rolland, S.G., Motori, E., Memar, N., Hench, J., Frank, S., Winklhofer, K.F. , and Conradt, B. (2013). Impaired complex IV activity in response to loss of LRPPRC function can be compensated by mitochondrial hyperfusion. PNAS 110, E2967-2976.Barbara Conradt and Stephane Rolland are co-corresponding authors.

Fischer, Christian et al. (2021) International Worm Meeting "MitoSegNet: Easy-to-use Deep Learning Segmentation for Analysing Mitochondrial Morphology"

Fischer, Christian, Haeussler, Simon, Marr, Carsten, Duchen, Michael, Conradt, Barbara, Rolland, Stephane, Singh, Kritarth, Besora-Casals, Laura

[International Worm Meeting, 2021]

While the analysis of mitochondrial morphology has emerged as an important tool in the study of mitochondrial function, efficient quantification of mitochondrial microscopy images presents a difficult task and bottleneck for statistically robust conclusions. Here, we present the Mitochondrial Segmentation Network (MitoSegNet), a pretrained deep learning segmentation model that enables researchers to easily exploit the power of deep learning for the quantification of mitochondrial morphology (Fischer, Besora-Casals et al. 2020). The MitoSegNet was generated by training a modified fully convolutional neural network with fluorescent microscopy, maximum-intensity projection images, depicting mitochondria in body wall muscle cells of adult C. elegans worms. We tested the performance of MitoSegNet against three feature-based segmentation algorithms and the machine-learning segmentation tool Ilastik. MitoSegNet outperformed all other methods in both pixelwise and morphological segmentation accuracy. We successfully applied MitoSegNet to unseen fluorescence microscopy images of mitoGFP expressing mitochondria in wild-type and catp-6ATP13A2 mutant C. elegans adults. Additionally, MitoSegNet was capable of accurately segmenting mitochondria in HeLa cells treated with fragmentation inducing reagents. We provide MitoSegNet for all operating systems as an easy-to-use graphical user interface tool that combines segmentation with morphological analysis. Reference Fischer, C. A., L. Besora-Casals, S. G. Rolland, S. Haeussler, K. Singh, M. Duchen, B. Conradt and C. Marr (2020). "MitoSegNet: Easy-to-use Deep Learning Segmentation for Analyzing Mitochondrial Morphology." iScience 23(10).

Rackles, Elisabeth et al. (2021) International Worm Meeting "Impaired peroxisomal import triggers a peroxisomal retrograde signaling"

Ewbank, Jonathan, Forne , Ignasi, Imhof, Axel, Rolland, Stephane, Fischer, Christian, Zhang, Xing, Zacherl, Judith, Osman, Christof, Rackles, Elisabeth, Witting, Michael, Schrott, Simon

[International Worm Meeting, 2021]

Retrograde signaling pathways between different cellular organelles (such as mitochondria or ER) and the nucleus have been identified over the years. These retrograde signaling pathways have been shown to activate the expression of organelle-specific quality control proteins, in order to maintain the organelles' function in response to organelle-specific stress. Until now, the existence of a retrograde signaling pathway for other organelles such as peroxisomes had remained elusive. Using the nematode Caenorhabditis elegans, we identified for the first time such signaling for peroxisomes. Specifically, we showed that peroxisomal import stress caused by the knock-down of prx-5, the homolog of the human peroxisomal matrix import receptor PEX5, triggers a peroxisomal retrograde signaling (PRS). We show that the PRS is dependent on the transcription factor NHR-49, the homolog of human PPARalpha and its co-factor MDT-15, the homolog of human Mediator MED15. Lipidomic analysis revealed that prx-5 knock-down causes increased levels of triacylglycerols with longer acyl chains, which are normally broken-down by peroxisomal beta-oxidation. The presence of excess long chain fatty acids could represent the signal that activates the PRS. Consistent with this hypothesis, we found that directly perturbing peroxisomal beta-oxidation is sufficient to activate the PRS. Using transcriptomic/proteomic approaches we found that peroxisomal import stress also induces the up-regulation of peroxisomal lipid metabolism, which could represent a mechanism to cope with the reduced beta-oxidation. Additionally, proteins involved in the immune response are up-regulated, and we found that the PRS is activated upon infection with Pseudomonas aeruginosa. The PRS thus may fulfill two functions, maintaining the homeostasis of lipid metabolism and acts as a potential surveillance mechanism to protect against pathogens