Cytoplasmic dynein is a ubiquitous minus-end directed motor protein that is essential for a variety of cellular processes ranging from cargo transport to spindle and chromosome positioning. Specifically, in fission yeast during meiotic prophase, the fused nucleus follows the spindle pole body in oscillatory movements from one cell pole to the other. The three molecular players that are essential to this process are: (i) the motor protein dynein, which powers the movement of the nucleus, (ii) microtubules, which provide the tracts for the movement and (iii) Num1, the anchor protein of dynein at the cortex. Dyneins that are localized to the anchor protein at the cortex and simultaneously bound to the microtubule emanating from the spindle pole body, pull on that microtubule leading to the movement of the nucleus. The spindle pole body, by virtue of its movement establishes a leading and a trailing side. Previous work by Vogel et al. has elucidated the mechanism of these oscillations as that of asymmetric distribution of dynein between the leading and trailing sides. This differential distribution is a result of the load-dependent detachment of dynein preferentially from the trailing microtubules. This self-organization model for dynein, however, requires a continuous redistribution of dynein from the trailing to the leading side. In addition, dyneins need to be bound to the anchor protein to be able to produce force on the microtubules. Anchored dyneins are responsible for many other important processes in the cell such as spindle alignment and orientation, spindle separation and rotation. So we set out to elucidate the mechanism of redistribution of dynein as well as the targeting mechanism of dynein from the cytoplasm to cortical anchoring sites where they can produce pulling force on microtubules. By employing single-molecule observation using highly inclined laminated optical sheet (HILO) microscopy and tracking of fluorescently-tagged dyneins using a custom software, we were able to show that dyneins redistributed in the cytoplasm of fission yeast by simple diffusion. We also observed that dynein bound first to the microtubule and not directly to the anchor protein Num1. In addition, we were able to capture unbinding events of single dyneins from the microtubule to the cytoplasm. Surprisingly, dynein bound to the microtubule exhibited diffusive behaviour. The switch from diffusive to directed movement required to power nuclear oscillations occurred when dynein bound to its cortical anchor Num1. In summary, dynein employs a two-step targeting mechanism from the cytoplasm to the cortical anchoring sites, with the attachment to the microtubule acting as the intermediate step.

Cytoplasmic dynein is a ubiquitous minus-end directed motor protein that is essential for a variety of cellular processes ranging from cargo transport to spindle and chromosome positioning. Specifically, in fission yeast during meiotic prophase, the fused nucleus follows the spindle pole body in oscillatory movements from one cell pole to the other. The three molecular players that are essential to this process are: (i) the motor protein dynein, which powers the movement of the nucleus, (ii) microtubules, which provide the tracts for the movement and (iii) Num1, the anchor protein of dynein at the cortex. Dyneins that are localized to the anchor protein at the cortex and simultaneously bound to the microtubule emanating from the spindle pole body, pull on that microtubule leading to the movement of the nucleus. The spindle pole body, by virtue of its movement establishes a leading and a trailing side. Previous work by Vogel et al. has elucidated the mechanism of these oscillations as that of asymmetric distribution of dynein between the leading and trailing sides. This differential distribution is a result of the load-dependent detachment of dynein preferentially from the trailing microtubules. This self-organization model for dynein, however, requires a continuous redistribution of dynein from the trailing to the leading side. In addition, dyneins need to be bound to the anchor protein to be able to produce force on the microtubules. Anchored dyneins are responsible for many other important processes in the cell such as spindle alignment and orientation, spindle separation and rotation. So we set out to elucidate the mechanism of redistribution of dynein as well as the targeting mechanism of dynein from the cytoplasm to cortical anchoring sites where they can produce pulling force on microtubules. By employing single-molecule observation using highly inclined laminated optical sheet (HILO) microscopy and tracking of fluorescently-tagged dyneins using a custom software, we were able to show that dyneins redistributed in the cytoplasm of fission yeast by simple diffusion. We also observed that dynein bound first to the microtubule and not directly to the anchor protein Num1. In addition, we were able to capture unbinding events of single dyneins from the microtubule to the cytoplasm. Surprisingly, dynein bound to the microtubule exhibited diffusive behaviour. The switch from diffusive to directed movement required to power nuclear oscillations occurred when dynein bound to its cortical anchor Num1. In summary, dynein employs a two-step targeting mechanism from the cytoplasm to the cortical anchoring sites, with the attachment to the microtubule acting as the intermediate step.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19525号 / 理博第4185号 / 新制||理||1601(附属図書館) / 32561 / 京都大学大学院理学研究科化学専攻 / (主査)教授 杉山 弘, 教授 三木 邦夫, 教授 藤井 紀子 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM