Optogenetically engineered calcium oscillations promote autophagy-mediated cell death via AMPK activation.

Autophagy is a double-edged sword for cells; it can lead to both cell survival and death. Calcium (Ca2+) signalling plays a crucial role in regulating various cellular behaviours, including cell migration, proliferation and death. In this study, we investigated the effects of modulating cytosolic Ca2+ levels on autophagy using chemical and optogenetic methods. Our findings revealed that ionomycin and thapsigargin induce Ca2+ influx to promote autophagy, whereas the Ca2+ chelator BAPTA-AM induces Ca2+ depletion and inhibits autophagy. Furthermore, the optogenetic platform allows the manipulation of illumination parameters, including density, frequency, duty cycle and duration, to create different patterns of Ca2+ oscillations. We used the optogenetic tool Ca2+-translocating channelrhodopsin, which is activated and opened by 470 nm blue light to induce Ca2+ influx. These results demonstrated that high-frequency Ca2+ oscillations induce autophagy. In addition, autophagy induction may involve Ca2+-activated adenosine monophosphate (AMP)-activated protein kinases. In conclusion, high-frequency optogenetic Ca2+ oscillations led to cell death mediated by AMP-activated protein kinase-induced autophagy.

Store-operated calcium entry inhibits primary ciliogenesis via the activation of Aurora A.

The primary cilium is an antenna-like organelle protruding from the cell surface that can detect physical and chemical stimuli in the extracellular space to activate specific signaling pathways and downstream gene expressions. Calcium ion (Ca2+ ) signaling regulates a wide spectrum of cellular processes, including fertilization, proliferation, differentiation, muscle contraction, migration, and death. This study investigated the effects of the regulation of cytosolic Ca2+ levels on ciliogenesis using chemical, genetic, and optogenetic approaches. We found that ionomycin-induced Ca2+ influx inhibited ciliogenesis and Ca2+ chelator BATPA-AM-induced Ca2+ depletion promoted ciliogenesis. In addition, store-operated Ca2+ entry and the endoplasmic reticulum Ca2+ sensor stromal interaction molecule 1 (STIM1) negatively regulated ciliogenesis. Moreover, an optogenetic platform was used to create different Ca2+ oscillation patterns by manipulating lighting parameters, including density, frequency, exposure time, and duration. Light-activated Ca2+ -translocating channelrhodopsin (CatCh) is activated by 470-nm blue light to induce Ca2+ influx. Our results show that high-frequency Ca2+ oscillations decrease ciliogenesis. Furthermore, the inhibition of cilia formation induced by Ca2+ may occur via the activation of Aurora kinase A. Cilia not only induce Ca2+ signaling but also regulate cilia formation by Ca2+ signaling.

YAP nuclear translocation induced by HIF-1α prevents DNA damage under hypoxic conditions.

Maladaptive repair of acute kidney injury (AKI) is associated with a high risk of developing chronic kidney disease deemed irremediable even in present days. When AKI arises from ischemia-reperfusion injury, hypoxia usually plays a major role. Although both hypoxia-inducible factor-1α (HIF-1α) and yes-associated protein (YAP) have been proven to promote renal cell survival under hypoxia, there is a lack of research that studies the crosstalk of the two and its effect on kidney repair. In studying the crosstalk, CoCl2 was used to create a mimetic hypoxic environment. Immunoprecipitation and proximity ligation assays were performed to verify protein interactions. The results show that HIF-1α interacts with YAP and promotes nuclear translocation of YAP at a high cell density under hypoxic conditions, suggesting HIF-1α serves as a direct carrier that enables YAP nuclear translocation. This is the first study to identify HIF-1α as a crucial pathway for YAP nuclear translocation under hypoxic conditions. Once translocated into a nucleus, YAP protects cells from DNA damage and apoptosis under hypoxic conditions. Since it is unlikely for YAP to translocate into a nucleus without HIF-1α, any treatment that fosters the crosstalk between the two holds the potential to improve cell recovery from hypoxic insults.

Depletion of intracellular Ca2+ induces FOXM1 SUMOylation and accumulation on the inner nuclear membrane and accelerates G2/M cell cycle transition.

Intracellular calcium (Ca2+) has been reported to regulate transcription factor activity and cancer development, but how it affects the function of Forkhead box protein M1 (FOXM1), a crucial transcription factor and key oncogene participating in tumorigenesis, remains unclear. Here, we investigated the regulatory role of Ca2+ on FOXM1 and found that Ca2+ depletion caused the distribution of FOXM1 to aggregate on the nuclear envelope, which was also observed in many cell lines. Further experiments revealed that sequestrated FOXM1 colocalized with lamin B in the inner nuclear membrane (INM) and was affected by the activity of nuclear export protein exportin 1 (XPO1). To investigate how intracellular Ca2+ affects FOXM1, we found that among the posttranscriptional modifications, only SUMOylation of FOXM1 showed a pronounced increase under reduced Ca2+, and suppressed SUMOylation rescued FOXM1 sequestration. In addition, Ca2+-dependent SUMOylated FOXM1 appeared to enhance the G2/M transition of the cell cycle and decrease cell apoptosis. In conclusion, our findings provide a molecular basis for the relationship between Ca2+ signaling and FOXM1 regulation, and we look to elucidate Ca2+-dependent FOXM1 SUMOylation-related biological functions in the future.

Optogenetically engineered Ca2+ oscillation-mediated DRP1 activation promotes mitochondrial fission and cell death.

Mitochondrial dynamics regulate the quality and morphology of mitochondria. Calcium (Ca2+) plays an important role in regulating mitochondrial function. Here, we investigated the effects of optogenetically engineered Ca2+ signaling on mitochondrial dynamics. More specifically, customized illumination conditions could trigger unique Ca2+ oscillation waves to trigger specific signaling pathways. In this study, we found that modulating Ca2+ oscillations by increasing the light frequency, intensity and exposure time could drive mitochondria toward the fission state, mitochondrial dysfunction, autophagy and cell death. Moreover, illumination triggered phosphorylation at the Ser616 residue but not the Ser637 residue of the mitochondrial fission protein, dynamin-related protein 1 (DRP1, encoded by DNM1L), via the activation of Ca2+-dependent kinases CaMKII, ERK and CDK1. However, optogenetically engineered Ca2+ signaling did not activate calcineurin phosphatase to dephosphorylate DRP1 at Ser637. In addition, light illumination had no effect on the expression levels of the mitochondrial fusion proteins mitofusin 1 (MFN1) and 2 (MFN2). Overall, this study provides an effective and innovative approach to altering Ca2+ signaling for controlling mitochondrial fission with a more precise resolution than pharmacological approaches in the temporal dimension.

Hypoxia-induced YAP activation and focal adhesion turnover to promote cell migration in mesenchymal TNBC cells.

BACKGROUND: Hypoxia is commonly characterized by malignant tumors that promote the aggressiveness and metastatic potential of cancer. Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, with approximately 46% capacity related to distant metastasis. Transcriptional factor yes-associated protein (YAP), a core component of the Hippo pathway, is associated with poor prognosis and outcome in cancer metastasis. Here, we explored the effect of hypoxia-mediated YAP activation and focal adhesions (FAs) turnover in mesenchymal TNBC cell migration. METHODS: We characterized the effect of hypoxia on YAP in different breast cancer cell lines using a hypoxia chamber and CoCl2 . RESULTS: Hypoxia-induced YAP nuclear translocation is significantly observed in normal breast epithelial cells, non-TNBC cells, mesenchymal TNBC cells, but not in basal-like TNBC cells. Functionally, we demonstrated that YAP activation was required for hypoxia to promote mesenchymal TNBC cell migration. Furthermore, hypoxia induced the localization of FAs at the leading edge of mesenchymal TNBC cells. In contrast, verteporfin (VP), a YAP inhibitor, significantly reduced the migration and the recruitment of nascent FAs at the cell periphery under hypoxia conditions, which only showed in mesenchymal TNBC cells. CONCLUSIONS: Our data support the hypothesis that YAP is novel factor and positively responsible for hypoxia-promoting mesenchymal TNBC cell migration. Our findings provide further evidence and outcomes to help prevent the progression of TNBC.