Mizuta Yoko, Igarashi Masako, Shinagawa Tomomi, Kaneshiro Ikuma, Anan Haoto, Kurihara Daisuke
Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, Aichi, Japan.
Institute for Advanced Research (IAR), Nagoya University, Nagoya, Aichi, Japan.
Cytoskeleton (Hoboken). 2025 Jun 24. doi: 10.1002/cm.70003.
Pollen is a male gametophyte of angiosperms. Following meiosis, the microspore undergoes an asymmetric division called pollen mitosis I (PMI), which produces two cells of different sizes: a large vegetative cell and a small generative cell. Polarized nuclear migration and positioning during PMI are important for successful pollen development and cell differentiation. However, analyzing the pollen development process in real-time is challenging in many model plants with tricellular pollen, including Arabidopsis and rice. In this study, we established a method for live confocal imaging of microtubule and actin dynamics using suspension cultures with biolistic delivery of plasmid DNAs during PMI in Nicotiana benthamiana (Bentham's tobacco), containing bicellular pollen. Pharmacological studies have indicated that actin filaments are crucial for microspore nuclear positioning before PMI, cell plate expansion during cytokinesis, and chromatin dispersion in the vegetative cell nucleus after PMI. By contrast, the inhibition of microtubule assembly resulted in abnormal chromosome segregation and nuclear behavior after PMI, although nuclear positioning and asymmetric division were observed. Our in vitro live cell imaging system for PMI provides insights into the importance of cytoskeletal regulation in asymmetric division and differentiation during pollen development.
花粉是被子植物的雄配子体。减数分裂后，小孢子经历一次不对称分裂，称为花粉有丝分裂I（PMI），产生两个大小不同的细胞：一个大的营养细胞和一个小的生殖细胞。PMI过程中极化的核迁移和定位对于花粉的成功发育和细胞分化很重要。然而，在许多具有三细胞花粉的模式植物中，包括拟南芥和水稻，实时分析花粉发育过程具有挑战性。在本研究中，我们建立了一种方法，利用含有双细胞花粉的本氏烟草在PMI期间通过生物弹道递送质粒DNA的悬浮培养物，对微管和肌动蛋白动力学进行实时共聚焦成像。药理学研究表明，肌动蛋白丝对于PMI前小孢子核的定位、胞质分裂期间细胞板的扩展以及PMI后营养细胞核中的染色质分散至关重要。相比之下，微管组装的抑制导致PMI后染色体分离和核行为异常，尽管观察到了核定位和不对称分裂。我们用于PMI的体外活细胞成像系统揭示了细胞骨架调节在花粉发育过程中不对称分裂和分化中的重要性。
