The voltage-dependent anion channel 1 (VDAC1) is a crucial gatekeeper in the outer mitochondrial membrane, controlling metabolic and energy homeostasis. The available methodological approaches fell short of accurate visualization of VDAC1 in living cells. To permit precise VDAC1 imaging, we utilized the tetracysteine (TC)-tag and visualized VDAC1 dynamics in living cells. TC-tagged VDAC1 had a cluster-like distribution on mitochondria. The labeling of TC-tagged VDAC1 was validated with immunofluorescence. The majority of VDAC1-TC-clusters were localized at endoplasmic reticulum (ER)-mitochondria contact sites. Notably, VDAC1 colocalized with BCL-2 Antagonist/Killer (BAK)-clusters upon apoptotic stimulation. Using this new tool, we were able to observe VDAC1-TC at mitochondrial fission sites. These findings highlight the suitability of the TC-tag for live-cell imaging of VDAC1, shedding light on the roles of VDAC1 in cellular processes.
The endoplasmic reticulum (ER) is a complex, multifunctional organelle of eukaryotic cells and responsible for the trafficking and processing of nearly 30% of all human proteins. Any disturbance to these processes can cause ER stress, which initiates an adaptive mechanism called unfolded protein response (UPR) to restore ER functions and homeostasis. Mitochondrial ATP production is necessary to meet the high energy demand of the UPR, while the molecular mechanisms of ER to mitochondria crosstalk under such stress conditions remain mainly enigmatic. Thus, better understanding the regulation of mitochondrial bioenergetics during ER stress is essential to combat many pathologies involving ER stress, the UPR, and mitochondria. This article investigates the role of Sigma-1 Receptor (S1R), an ER chaperone, has in enhancing mitochondrial bioenergetics during early ER stress using human neuroblastoma cell lines. Our results show that inducing ER stress with tunicamycin, a known ER stressor, greatly enhances mitochondrial bioenergetics in a time- and S1R-dependent manner. This is achieved by enhanced ER Ca2+ leak directed towards mitochondria by S1R during the early phase of ER stress. Our data point to the importance of S1R in promoting mitochondrial bioenergetics and maintaining balanced H2O2 metabolism during early ER stress.
Abstract Background Aggression is an adaptive behaviour that animals use to protect offspring, defend themselves and obtain resources. Zebrafish, like many other animals, are not able to recognize themselves in the mirror and typically respond to their own reflection with aggression. However, mirror aggression is not an all-or-nothing phenomenon, with some individuals displaying high levels of aggression against their mirror image, while others show none at all. In the current work, we have investigated the genetic basis of mirror aggression by using a classic forward genetics approach - selective breeding for high and low mirror aggression zebrafish (HAZ and LAZ). Results We characterized AB wild-type zebrafish for their response to the mirror image. Both aggressive and non-aggressive fish were inbred over several generations. We found that HAZ were on average more aggressive than the corresponding LAZ across generations and that the most aggressive adult HAZ were less anxious than the least aggressive adult LAZ after prolonged selective breeding. RNAseq analysis of these fish revealed that hundreds of protein-encoding genes with important diverse biological functions such as arsenic metabolism ( as3mt ), cell migration ( arl4ab ), immune system activity ( ptgr1 ), actin cytoskeletal remodelling ( wdr1 ), corticogenesis ( dgcr2 ), protein dephosphorylation ( ublcp1 ), sialic acid metabolism ( st6galnac3 ) and ketone body metabolism ( aacs ) were differentially expressed between HAZ and LAZ, suggesting a strong genetic contribution to this phenotype. DAVID pathway analysis showed that a number of diverse pathways are enriched in HAZ over LAZ including pathways related to immune function, oxidation-reduction processes and cell signalling. In addition, weighted gene co-expression network analysis (WGCNA) identified 12 modules of highly correlated genes that were significantly associated with aggression duration and/or experimental group. Conclusions The current study shows that selective breeding based of the mirror aggression phenotype induces strong, heritable changes in behaviour and gene expression within the brain of zebrafish suggesting a strong genetic basis for this behaviour. Our transcriptomic analysis of fish selectively bred for high and low levels of mirror aggression revealed specific transcriptomic signatures induced by selective breeding and mirror aggression and thus provides a large and novel resource of candidate genes for future study.
Metabolic enzymes can adapt during energy stress, but the precise mechanisms and consequences of these adaptations remain understudied. Here, we discovered that hexokinase 1 (HK1), a key glycolytic enzyme, clusters into ring-like structures around mitochondria during energy stress. These HK1-rings constrict mitochondria at contact sites with the endoplasmic reticulum (ER) and prevent mitochondrial fission by displacing the dynamin-related protein 1 (Drp1) from mitochondrial constriction sites. Mechanistically, we identified that the lack of ATP and glucose-6-phosphate (G6P) promotes the clustering of HK1. Moreover, we found several mutations that are critical for the formation of HK1-rings. Utilizing these mutations, we could show that HK1-rings keep mitochondria connected and rewire cellular metabolism during energy stress. Our findings highlight that HK1 is a robust energy stress sensor that regulates the shape, connectivity and metabolic activity of mitochondria. Thus, the formation of HK1-rings may affect mitochondrial function in energy stress-related pathologies.
