张丁, 余雯, 刘梦楠, 青昕, 丁祥, 侯怡铃. 线粒体分裂基因dnm1缺失对粟酒裂殖酵母细胞有丝分裂及能量代谢的影响[J]. 北京师范大学学报(自然科学版), 2024, 60(3): 331-343. DOI: 10.12202/j.0476-0301.2023229
引用本文: 张丁, 余雯, 刘梦楠, 青昕, 丁祥, 侯怡铃. 线粒体分裂基因dnm1缺失对粟酒裂殖酵母细胞有丝分裂及能量代谢的影响[J]. 北京师范大学学报(自然科学版), 2024, 60(3): 331-343. DOI: 10.12202/j.0476-0301.2023229
ZHANG Ding, YU Wen, LIU Mengnan, QING Xin, DING Xiang, HOU Yiling. Deletion of dnm1(a mitochondrial division gene) affects mitosis and energy metabolism in Schizosaccharomyces pombe[J]. Journal of Beijing Normal University(Natural Science), 2024, 60(3): 331-343. DOI: 10.12202/j.0476-0301.2023229
Citation: ZHANG Ding, YU Wen, LIU Mengnan, QING Xin, DING Xiang, HOU Yiling. Deletion of dnm1(a mitochondrial division gene) affects mitosis and energy metabolism in Schizosaccharomyces pombe[J]. Journal of Beijing Normal University(Natural Science), 2024, 60(3): 331-343. DOI: 10.12202/j.0476-0301.2023229

线粒体分裂基因dnm1缺失对粟酒裂殖酵母细胞有丝分裂及能量代谢的影响

Deletion of dnm1(a mitochondrial division gene) affects mitosis and energy metabolism in Schizosaccharomyces pombe

  • 摘要: 在粟酒裂殖酵母细胞中,线粒体分裂蛋白Dnm1是调控线粒体分裂和融合动态过程的关键蛋白.为研究酵母细胞dnm1基因缺失后对有丝分裂和能量代谢的影响,采用活细胞成像技术分析其细胞有丝分裂动力学的变化、RT-qPCR技术分析cdc2cdc13基因的转录水平、高效液相色谱质谱联用技术检测其能量代谢物的变化并验证.结果表明dnm1基因缺失后抑制裂殖酵母生长.活细胞成像显示dnm1Δ菌株在有丝分裂间期微管束长度较野生型增长了(0.83±0.70) μm(P<0.01),产生5根微管束的菌株增加、3根微管束的菌株减少.有丝分裂前期dnm1Δ菌株中纺锤体伸长时间延长(0.85±0.02) min (P<0.05),后期时间延长(5.80±1.62) min (P<0.01),后期纺锤体伸长速度减慢0.06 μm·min−1(P<0.05),且dnm1Δ菌株出现纺锤体延迟断裂和滞后的染色体分离.高效液相色谱质谱联用技术检测结果表明,dnm1Δ菌株存在辅酶合成缺陷,NADPH含量显著降低(P<0.05),中间代谢产物6-磷酸葡萄糖、6-磷酸果糖、柠檬酸、顺式乌头酸、丙酮酸、异柠檬酸和L-苹果酸相对含量显著降低 (P<0.05),出现ATP产生障碍.验证结果显示代谢物分析结果可靠,且dnm1Δ菌株在对数生长期cdc2基因的表达量显著低于野生型.本研究为进一步探寻Dnm1蛋白在细胞有丝分裂中的功能和相关分子机制提供了一定的科学依据.

     

    Abstract: In Schizosaccharomyces pombe, the mitochondrial dynamin-related protein Dnm1 regulates dynamic mitochondrial fission/fusion. To examine the effect of dnm1 gene deletion on mitosis and energy metabolism in the fission yeast, live cells were imaged to analyze cell mitosis kinetics, RT-qPCR was performed to analyze transcription level of cdc2 and cdc13, high performance liquid chromatography-mass spectrometry was done to measure energy metabolites. The deletion of dnm1 gene led to slowed growth of fission yeast, microtubule bundles in dnm1Δ strain increased by (0.83 ± 0.70) μm (P < 0.01) when compared to the wild-type in interphase, the number of strains producing 5 microtubule bundles increased, but the number of strains producing 3 microtubule bundles decreased. The spindle elongation time in dnm1Δ prolonged by (0.85 ± 0.02) min (P < 0.05) in prophase, by (5.80 ± 1.62) min (P<0.01) in anaphase. Spindle elongation slowed down by 0.06 μm·min−1 (P < 0.05) in anaphase. The dnm1Δ strain exhibited delayed spindle breakage and chromosome segregation. High-performance liquid chromatography-mass spectrometry revealed that dnm1Δ strain was deficient in coenzyme synthesis, resulting in a significant decrease in NADPH content (P < 0.05). The relative content of intermediate metabolites (glucose 6-phosphate, fructose 6-phosphate, citric acid, cis aconitic acid, pyruvate, isocitrate, and L-malic acid) were significantly reduced (P < 0.05), with major defect in ATP production. The cdc2 gene expression in logarithmic growth phase in dnm1Δ strain was significantly lower than the wild-type.

     

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