The Study On The Role And Mechanism Of Mitochondrial Ca²⁺transients In Mitotic Progression

Cells constantly have to adapt their bioenergetic state to respond to the environmental fluctuations.As such,it is important to evolve mechanisms to match energy supply with energetic demand.The cell cycle is a finely-tuned cellular process with high energy demand for DNA synthesis and chromosome segregation.Growing evidence indicates that metabolic signals are integrated and coupled to cell cycle progression.However,it remains unclear how cells sense the energy demand,signal to the energy generation machinery,and maintain energy homeostasis for proper cell cycle progression.Calcium signaling triggers numerous physiological processes in the intracellular environment and plays an important role in cellular energy homeostasis.It is well established that a rise in the cytosolic Ca²⁺levels leads to a rapid Ca²⁺uptake by mitochondria.The subsequent increase in mitochondrial matrix Ca²⁺can activate a number of enzymes of the mitochondrial electron transport chain and the tricarboxylic acid?TCA?cycle,thereby increasing mitochondrial respiration and ATP production.Several early cell cycle studies have shown a rapid rise in Ca²⁺levels in cytosol,termed Ca²⁺transients,in metaphase cells during mitosis.However,whether mitochondrial Ca²⁺fluctuations occur and play a role in mitosis have long been neglected.Moreover,the upstream events that trigger these Ca²⁺transients remain unclear.To visualize the mitochondrial calcium dynamics in living cells during the cell cycle progression,we generated a HeLa cell line expressing a mitochondrial localized,genetically encoded Ca²⁺indicator,4mt-GCaMP6,whose fluorescence intensity reflects the free Ca²⁺level in mitochondria.Mitochondrial Ca²⁺signals were then monitored by time-lapse imaging.We observed a rapid Ca²⁺burst in mitochondria,termed mitochondrial Ca²⁺transient,occurs in a majority of cells that entered mitosis.This mitochondrial Ca²⁺transient is mediated by the mitochondrial calcium uniporter?MCU?.SiRNA-mediated MCU silencing dramatically decreases the mitochondrial respiration and causes an energy shortage in mitotic cells,thus leading to a defect of microtubule depolymerization and tension establishment,hyperactivation of the spindle checkpoint and delayed mitotic progression.Interestingly,the observed transients predominantly occur in mitotic cells,indicating an unexpected role of the MCU-mediated mitochondrial Ca²⁺transients in acute energy supply during mitosis.We next explored the relationship of the mitochondrial Ca²⁺transients and cellular energy status in mitotic cells.By simultaneously recording the dynamics of mitochondrial Ca²⁺and cytosolic ATP levels during mitosis,we demonstrate that the mitotic mitochondrial Ca²⁺transients is likely a direct reponse to the cellular energy status.We observed a decline in cytosolic ATP level after mitotic entry thus leading to a low cellular status and consequently activation of the energy sensor AMP-activated protein kinase?AMPK?.The activated AMPK,on the one hand,promotes cytosolic Ca²⁺release.On the other hand,it translocates into the mitochondria,phosphorylates and activates MCU,allowing Ca²⁺entry into mitochondria to boost mitochondrial respiration.In this study,we found an unexpected role for MCU-mediated mitochondrial Ca²⁺transient in proper mitotic progression.This energy production mechanism acts as an adaptive response to the low ATP level in mitosis.As a result,our findings establish a critical role for AMPK-and MCU-dependent mitochondrial Ca²⁺signaling in mitosis and reveal a mechanism of mitochondrial adaptation to acute cellular energy stress..