Mitotic phosphorylation of Drp1, a dynamin-related GTPase, is essential for mitochondrial fission during mitosis. In this study, researchers analyzed mitochondrial dynamics in mitotic mammalian cells, finding that mitochondria in interphase HeLa cells are long tubular structures that become fragmented in early mitosis and are subsequently reformed in daughter cells. In contrast, Drp1 knockdown cells maintain tubular mitochondrial structures during mitosis, indicating that Drp1 is required for mitochondrial fission. Drp1 was specifically phosphorylated by Cdk1/cyclin B on Ser-585 during mitosis. Exogenous expression of an unphosphorylated Drp1 mutant (Drp1^585A) reduced mitotic mitochondrial fragmentation, suggesting that phosphorylation of Drp1 on Ser-585 promotes mitochondrial fission in mitotic cells. Mitochondria are dynamic organelles that undergo constant fusion and fission, maintaining their shape and function. Fusion proteins such as mitofusin (Mfn) and Fzo1 are essential for mitochondrial fusion, while dynamin-related proteins like Drp1 and Dnm1 are involved in mitochondrial fission. Mitochondrial fragmentation is induced by factors such as dissipation of the mitochondrial membrane potential and apoptotic stimuli. The molecular mechanisms regulating mitochondrial fusion and fission are not well understood. In this study, researchers found that Drp1 is mitotically phosphorylated by Cdk1/cyclin B, and this phosphorylation is essential for mitochondrial fission during mitosis. Phosphorylation of Drp1 at Ser-585 was confirmed using specific antibodies and in vitro phosphorylation assays. The phosphorylation of Drp1 at Ser-585 was shown to stimulate mitochondrial fission in mitotic cells. Drp1 knockdown cells showed reduced mitochondrial fragmentation, while expression of wild-type Drp1 restored mitochondrial fragmentation. These results suggest that Drp1-dependent mitochondrial fission is crucial for mitochondrial inheritance during mitosis. The study provides the first evidence that Drp1-dependent mitochondrial fragmentation occurs in the early mitotic phase.Mitotic phosphorylation of Drp1, a dynamin-related GTPase, is essential for mitochondrial fission during mitosis. In this study, researchers analyzed mitochondrial dynamics in mitotic mammalian cells, finding that mitochondria in interphase HeLa cells are long tubular structures that become fragmented in early mitosis and are subsequently reformed in daughter cells. In contrast, Drp1 knockdown cells maintain tubular mitochondrial structures during mitosis, indicating that Drp1 is required for mitochondrial fission. Drp1 was specifically phosphorylated by Cdk1/cyclin B on Ser-585 during mitosis. Exogenous expression of an unphosphorylated Drp1 mutant (Drp1^585A) reduced mitotic mitochondrial fragmentation, suggesting that phosphorylation of Drp1 on Ser-585 promotes mitochondrial fission in mitotic cells. Mitochondria are dynamic organelles that undergo constant fusion and fission, maintaining their shape and function. Fusion proteins such as mitofusin (Mfn) and Fzo1 are essential for mitochondrial fusion, while dynamin-related proteins like Drp1 and Dnm1 are involved in mitochondrial fission. Mitochondrial fragmentation is induced by factors such as dissipation of the mitochondrial membrane potential and apoptotic stimuli. The molecular mechanisms regulating mitochondrial fusion and fission are not well understood. In this study, researchers found that Drp1 is mitotically phosphorylated by Cdk1/cyclin B, and this phosphorylation is essential for mitochondrial fission during mitosis. Phosphorylation of Drp1 at Ser-585 was confirmed using specific antibodies and in vitro phosphorylation assays. The phosphorylation of Drp1 at Ser-585 was shown to stimulate mitochondrial fission in mitotic cells. Drp1 knockdown cells showed reduced mitochondrial fragmentation, while expression of wild-type Drp1 restored mitochondrial fragmentation. These results suggest that Drp1-dependent mitochondrial fission is crucial for mitochondrial inheritance during mitosis. The study provides the first evidence that Drp1-dependent mitochondrial fragmentation occurs in the early mitotic phase.