De Henau S et al. (2016) Worm "Globin-based redox signaling."

De Henau S, Braeckman BP

[Worm, 2016]

In recent years, moderate levels of reactive oxygen species (ROS) have become recognized as signaling cues that participate at all levels of cellular organization. Globins, with their redox-active heme iron and ubiquitous presence, seem ideally suited to participate in ROS metabolism. Here we comment on our recent findings that show the participation of a globin, GLB-12, in a redox signaling pathway in Caenorhabditis elegans. We found that GLB-12 produces superoxide, a type of ROS, after which this is converted to what appears to be a hydrogen peroxide gradient over the plasma membrane by the activity of intracellular and extracellular superoxide dismutases. In the first part, we discuss in more detail the different regulatory mechanisms that increase the effectiveness of this redox signal. In the second part, we comment on how specific structural and biochemical properties allow this globin to perform redox reactions. Interestingly, these properties are also observed in 2 other C. elegans globins that appear to be involved in redox biology. We therefore hypothesize that globins involved in redox signaling display similar structural and biochemical characteristics and propose that a subgroup of globins can be added to the group of proteins that play a vital role in redox signaling.

De Henau, S. et al. (2017) International Worm Meeting "Mitochondrial redox signaling induces polarization in the C. elegans embryo."

De Henau, S., Dansen, T.B.

[International Worm Meeting, 2017]

Mitochondrial redox signaling plays a critical role in many biological processes and relies on the well-regulated production of H2O2 as signaling molecule. However, the regulatory mechanisms that lead to specificity in mitochondrial H2O2-signaling remain elusive, mainly because good model systems to address this question are not available. Cell polarization requires the dynamic regulation of signaling cascades in both time and space, making it an attractive model to study localized, subcellular signal transduction. We take advantage of this and use the C. elegans early embryo, one of the most-studied systems for cell polarization, to analyze the spatiotemporal regulation of redox signaling. We find that, coinciding with polarization, a subgroup of mitochondria relocates to the cell membrane at the site of symmetry breaking. After this, mitochondria become highly motile and localize closely to the posterior cortex of the embryo. An ultrasensitive H2O2-specific sensor shows that mitochondrial relocation to the cell membrane is accompanied by a striking increase in cortical H2O2-levels. Furthermore, mitochondrial H2O2 directly influences polarization, since compounds that alter mitochondrial H2O2-production affect symmetry breaking and maximal polarization. Key in polarization is the asymmetric rearrangement of cortical actomyosin-rich cables away from the newly formed posterior pole. We find that mitochondrial H2O2 inhibits cortical NMY-2 dynamics as well as activity of RHO-1, an actomyosin regulator. RHO-1 displays highly conserved and well described redox sensitivity, and we are currently mutating its redox-sensitive cysteines to determine if this protein is a direct target of mitochondrial H2O2 during polarization. In conclusion, we have developed a pioneering model based on cell polarization to study spatiotemporal redox signaling. Based on our results, we propose that 1) mitochondria induce symmetry-breaking by increasing H2O2-levels at the cell cortex and so inhibit actomyosin contractions, and 2) by moving anteriorly along the cortex during polarization, mitochondria generate a "H2O2 wave" that signals to collapse the actomyosin network in the anterior direction.

De Henau S et al. (2015) Nat Commun "A redox signalling globin is essential for reproduction in Caenorhabditis elegans."

Moens L, Pauwels M, Dewilde S, Tilleman L, De Henau S, Germani F, Vanfleteren JR, Vangheel M, Bolognesi M, Nardini M, Pesce A, Bert W, De Wael K, Vlaeminck C, Luyckx E, Trashin S, Braeckman BP

[Nat Commun, 2015]

Moderate levels of reactive oxygen species (ROS) are now recognized as redox signalling molecules. However, thus far, only mitochondria and NADPH oxidases have been identified as cellular sources of ROS in signalling. Here we identify a globin (GLB-12) that produces superoxide, a type of ROS, which serves as an essential signal for reproduction in C. elegans. We find that GLB-12 has an important role in the regulation of multiple aspects in germline development, including germ cell apoptosis. We further describe how GLB-12 displays specific molecular, biochemical and structural properties that allow this globin to act as a superoxide generator. In addition, both an intra- and extracellular superoxide dismutase act as key partners of GLB-12 to create a transmembrane redox signal. Our results show that a globin can function as a driving factor in redox signalling, and how this signal is regulated at the subcellular level by multiple control layers.

De Henau, S. et al. (2013) International Worm Meeting "A Redox Signaling Globin Regulates Germ Cell Apoptosis in Caenorhabditis elegans."

Pesce, A., Tilleman, L., Nardini, M., Braeckman, B.P., Bolognesi, M., De Wael, K., De Henau, S., Moens, L., Pauwels, M., Dewilde, S.

[International Worm Meeting, 2013]

It has become clear that reactive oxygen species (ROS) can modulate signal transduction pathways, thereby influencing cellular functioning. A striking aspect hereby is that ROS can be purposely generated by enzymes. However, in the majority of reported redox-sensitive pathways, it is unclear what the enzymatic source is of ROS. We show that a globin of C. elegans, globin-12 (GLB-12), regulates multiple aspects in reproduction by redox signaling. glb-12 RNAi causes severely reduced fecundity and multiple defects during oocyte development, including increased germline apoptosis levels. By focusing on the increase in germline apoptosis, we found that GLB-12 signals through the JNK and P38 MAPK pathways: abolishing these pathways eliminates the increase in germline apoptosis. Biochemical analysis of GLB-12 showed that, unlike the well studied hemo- and myoglobin, this globin cannot bind oxygen. Instead, we see that GLB-12 has a reduction potential sufficiently low to favor electron transfer from its heme iron to oxygen. In addition, the crystal structure of GLB-12 shows that this globin possesses unique properties that support a role in electron transfer. In vitro, GLB-12 can indeed actively convert oxygen to superoxide by electron transfer, thus creating a redox signal. In vivo, the unstable superoxide produced by GLB-12 could potentially be converted by superoxide dismutases (SODs) to the more stable hydrogen peroxide, which in turn may act as a biological messenger. Applying glb-12 RNAi in mutants for the five C. elegans sod genes showed that fecundity is further reduced in the intracellular SOD-1 mutant and restored to almost normal levels in the extracellular SOD-4 mutant. We also observed such opposite effects of SOD-1 and SOD-4 on the antiapoptotic role of GLB-12, and on the inhibitory role of GLB-12 on the P38 pathway. SOD-1 and SOD-4 thus modulate the signal created by GLB-12, creating a redox signaling pathway that influences germline apoptosis levels.

De Henau, S. et al. (2011) International Worm Meeting "Caenorhabditis elegans' GLB-12 regulates germline apoptosis levels and vulval development."

De Henau, S., Tilleman, L., Hoogewijs, D., Moens, L., Dewilde, S., Braeckman, B.P., Vanfleteren, J.R.

[International Worm Meeting, 2011]

Caenorhabditis elegans has the surprisingly high number of 33 globins. These globins are diverse in both gene and protein structure and are localized in a variety of cells, mostly neurons. Here, for the first time, we show that a globin, GLB-12, is actively involved in the regulation of developmental processes, namely in the regulation of germline apoptosis levels and vulval formation. This is demonstrated by the following results. Firstly, RNAi against glb-12 in RNAi-hypersensitive strains, such as nre-1(hd20) lin-15B(hd126), causes severely reduced fecundity and, in accordance with this observation, increased levels of apoptosis in the germline. This indicates that GLB-12 is an active inhibitor of germline apoptosis. Furthermore, glb-12(RNAi) also induces a protruding vulva (58% of adult worms) and developmental abnormalities (15% of adult worms). Secondly, using a translational reporter, we observed expression of GLB-12 in several head and tail neurons as well as in the nerve cord. Interestingly, this globin is also expressed in the developing vulva and in the gonadal sheath cells of the adult hermaphrodite, which may be related to the protruding vulva phenotype and the increased levels of apoptosis of the glb-12(RNAi) worms respectively. Thirdly, both major glb-12(RNAi) phenotypes, the reduced fecundity and the protruding vulva, seem to be regulated through a Ras/MAPK signaling pathway, as worms defective in this pathway do not show these phenotypes. Lastly, we have found that applying glb-12(RNAi) to the N2 bristol strain - in which neurons are less sensitive to RNAi - causes neither a protruding vulva nor developmental abnormalities. However, the RNAi does cause a reduction in the fecundity of the worm, though not to the same extent as seen in the RNAi-hypersensitive strain. In addition, applying glb-12(RNAi) to the glb-12 translational reporter, which can be considered a wild type, showed that GLB-12 is only absent in the gonadal sheath; it is still present in the nerve cells and in the developing vulva. These results indicate that GLB-12 is active in both neuronal and non-neuronal tissues, and that its effect in both tissue types seems to be cumulative. GLB-12, thus, functions as a repressor of germline apoptosis and a regulator of vulval development and seems to act through the Ras/MAPK pathway.

De Henau, S et al. (2009) International Worm Meeting "Wide diversity in structure and expression profiles among members of the Caenorhabditis elegans globin protein family."

Vanfleteren, J R, De Henau, S, Hoogewijs, D, Braeckman, B P

[International Worm Meeting, 2009]

Globins are small globular proteins, with a characteristic 3-over-3 a-helical sandwich structure that encloses an iron-containing heme group. Organisms can express multiple globin molecules with variant properties and functions (Vinogradov and Moens, 2008). In silico analysis of the genome of Caenorhabditis elegans revealed an unexpectedly high number of globin genes featuring a remarkable diversity in gene structure, amino acid sequence and expression profiles (Hoogewijs et al., 2004). The availability of full genomic sequences and EST data from several other nematode species presents a unique opportunity to explore the evolutionary globin dynamics of these organisms. Caenorhabditis species contain a very large number of globin genes, and even distantly related nematodes harbor orthologs to many of them. Bayesian phylogenetic analysis resolves all nematode globins into two distinct globin classes. Analysis of the globin intron-exon structures suggests extensive loss of ancestral introns and gain of new positions in deep nematode ancestors, and mainly loss in the Caenorhabditis lineage. Also, our analysis provides some evidence for a number of gene duplication events giving rise to a class of globin genes that is likely unique to the nematode phylum (Hoogewijs et al., 2008). Secondly, to reveal tissue-specific expression profiles of this protein family, we constructed gene fusions of the promoter regions for all 33 globin genes to the coding region of GFP. The majority of these fusion products is expressed in neuronal cells in the head and tail portions of the body. The other globin genes seem to be expressed in non-neuronal tissues, including body wall muscle, vulval muscle and the pharynx. Based on these results, most globin genes are likely to have a cell-specific function (Hoogewijs et al., 2008). At this moment, we are extending this expression analysis by including the complete coding region of each globin gene. By doing this we will be able to determine whether the globin gene introns and 3''UTR contain additional cis-regulatory information. Lastly, we used real-time PCR (qPCR) to analyze expression patterns of this globin family under hypoxia stress condition. We found that oxygen shortage (0,5% O2) modulates the expression of several globin-like genes. This seems to indicate that these globins are involved in the protection of tissues from hypoxia. Strikingly, there is little overlap in genes responsive to hypoxia and genes responsive to anoxia (12h <0,001% O2) (Hoogewijs et al., 2007), which might illustrate functional diversity in this protein family.

De Henau S et al. (2009) European Worm Neurobiology Meeting "Wide diversity in structure and expression profiles among members of the Caenorhabditis elegans globin protein family"

Hoogewijs D, De Henau S, Vanfleteren JR, Braeckman BP

[European Worm Neurobiology Meeting, 2009]

Globins are small globular proteins, with a characteristic 3-over-3 alpha-helical sandwich structure that encloses an iron-containing heme group. Organisms can express multiple globin molecules that have variant properties and functions (Weber and Vinogradov 2001; Vinogradov and Moens, 2008). In silico analysis of the genome of Caenorhabditis elegans revealed an unexpectedly high number of globin genes featuring a remarkable diversity in gene structure, amino acid sequence and expression profiles (Hoogewijs et al., 2004). To reveal tissue-specific expression profiles of this protein family, we constructed gene fusions of the promoter and enhancer regions for all 33 globin genes to the coding region of GFP. The majority of these fusion products is expressed in neuronal cells in the head and tail portions of the body, as well as in the nerve cord. The other globin genes seem to be expressed in non-neuronal tissues, including body wall muscle, vulval muscle and the pharynx. Based on these results, most globin genes are likely to have a cell-specific function (Hoogewijs et al., 2008). At this moment, we are extending this expression analysis by including the complete coding region of each globin gene. By doing this we will be able to determine whether the globin gene introns and 3.UTR contain additional cis-regulatory information. Secondly, we used real-time PCR (qPCR) to analyze expression patterns of this globin family under hypoxia stress condition. C. elegans, as a soil nematode, frequently encounters severe hypoxic conditions in its natural environment. We found that oxygen shortage (0,1% O2) modulates the expression of several globin-like genes. This seems to indicate that these globins are involved in the protection of tissues from hypoxia. Strikingly, there is little overlap in genes responsive to hypoxia and genes responsive to anoxia (12h <0,001% O2) (Hoogewijs et al., 2007), which might illustrate functional diversity in this protein family. In conclusion, the strong variation in expression localization and stress responses of this protein family might indicate that the majority of these globins are not simple oxygen carriers. Rather, they could be involved in a variation of signaling and stress responses.

De Henau S et al. (2020) Dev Cell "Mitochondria-Derived H2O2 Promotes Symmetry Breaking of the C.elegans Zygote."

De Henau S, Pages-Gallego M, Dansen TB, Pannekoek WJ

[Dev Cell, 2020]

Symmetry breaking is an essential step in cell differentiation and early embryonic development. However, the molecular cues that trigger symmetry breaking remain largely unknown. Here, we show that mitochondrial H₂O₂ acts as a symmetry-breaking cue in the C.elegans zygote. We find that symmetry breaking is marked by a local H₂O₂ increase and coincides with a relocation of mitochondria to the cell cortex. Lowering endogenous H₂O₂ levels delays the onset of symmetry breaking, while artificially targeting mitochondria to the cellular cortex using a light-induced heterodimerization technique is sufficient to initiate symmetry breaking in a H₂O₂-dependent manner. In wild-type development, both sperm and maternal mitochondria contribute to symmetry breaking. Our findings reveal that mitochondrial H₂O₂-signaling promotes the onset of polarization, a fundamental process in development and cell differentiation, and this is achieved by both mitochondrial redistribution and differential H₂O₂-production.

Dansen TB et al. (2021) STAR Protoc "Modulating organelle distribution using light-inducible heterodimerization in C.elegans."

De Henau S, Dansen TB

[STAR Protoc, 2021]

The relative positioning of organelles underlies fundamental cellular processes, including signaling, polarization, and cellular growth. Here, we describe the usage of a light-dependent heterodimerization system, LOVpep-ePDZ, to alter organelle positioning locally and reversibly in order to study the functional consequences of organelle positioning. The protocol gives details on how to accomplish expression of fusion proteins encoding this system, describes the imaging parameters to achieve subcellular activation in <i>C.elegans</i>, and may be adapted for use in other model systems. For complete details on the use and execution of this protocol, please refer to De Henau etal. (2020).

Schwartz AZA et al. (2020) Dev Cell "Stimulating Embryo Polarization with Mitochondrial Peroxide."

Nance J, Schwartz AZA

[Dev Cell, 2020]

Centrosomes break symmetry in the C.elegans one-cell embryo, triggering its anterior-posterior polarization and initiating segregation of somatic and germline cell lineages. In this issue of Developmental Cell, De Henau etal. show that mitochondria also contribute to symmetry breaking by producing hydrogen peroxide at the egg's future posterior pole.