As well as the classic bean-shaped structures observed in electron micrographs, mitochondria are frequently found as long, snake-shaped tubules, and extended reticular networks whose overall morphology depends on the balance of fission and fusion of mitochondrial tubules in growing cells. [1][2][3][4] Such mitochondrial morphology, size, distribution, and copy number were demonstrated to change in living cells during cellular differentiation, development, or under pathological conditions, including liver disease, muscle dystrophy, cardiomyopathy, and cancer. [5][6][7] In differentiated cells, mitochondria are often localized to specific cytoplasmic regions rather than randomly distributed. 8) Additionally, mitochondrial morphology and distribution changed by the metabolic state of the cell or during cell growth. [9][10][11][12] Although the role of mitochondria in metabolism, ATP production, and apoptosis is more widely recognized, alterations in mitochondrial morphology and abundance are also important for cellular functions. In fact, the distribution of mitochondria in dendrites of living hippocampal neurons, which are regulated by mitochondrial fission/fusion, has been shown to be an essential and limiting factor for synapse density and plasticity. 13) Detail, however, on the mitochondrial behaviour during cellular differentiation and cell cycle progression in higher eukaryotes has little been revealed.It was shown that at S-phase, there is a substantial increase in the amount of phosphorylated histone H1 and that the phosphorylated histone H1 is diffusely distributed throughout the cell nucleus.14) On the other hand, at the end of Sphase, phosphorylation of histone H3 begins immediately following the replication of the centromeric heterochromatin.15) This phosphorylation is diffusely distributed throughout the nucleus, but, initiating at the centromers, spreads throughout the genome as the cells progress to prophase. Thus, when used in combination, staining with antibodies to phosphorylated histone H1 and H3 can be used to clearly and easily resolve mixed cell populations into each of the major stages of the cell cycle (UPSTATE, 2002 Catalog, Technical Appendix pp. 365).In the present study, we investigated mitochondrial dynamics during the cell cycle progression using synchronized HeLa cells in which the cells were transfected with mitoDsRed1 to visualize mitochondria in the cells and the major stages of the cell cycle of the observed cells were resolved by staining the cells with phosphorylated histone H1 and H3 antibodies. We found that mitochondria exist as filamentous network structures throughout the cell cycle progression, changing their morphology, distribution, and abundance. MATERIALS AND METHODS Double-Thymidine Block and Cell Cycle AnalysisHeLa cells were maintained in Dulbecco's modified Eagle's medium (DMEM; GIBCO Invitrogen Corp., Carlsbad, CA, U.S.A.) plus 10% fetal calf serum at 37°C under an atmosphere of 5% CO 2 . Cells were synchronized for the G1/S boundary by a double-thymidine block p...

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