A study on PPB-associated molecules by the HP freezing.

了解分裂面方向的形成机制是植物形态发生的一个重要问题。在细胞进入有丝分裂前，微管聚集形成前期带（PPB）。由于PPB位于细胞质分裂结束时细胞板融合的位置，因此了解Mts如何作为PPB组织对于理解分裂面方向的调节机制至关重要。据报道，Actins和cdks存在于PPBs中，但它们的作用尚不清楚。为了了解这些分子与PPB中Mts之间的关系，有必要进行电镜研究。然而，由于Mts是一个非常动态的结构，我们无法通过传统的EM得到可靠的结果。为了解决这一问题，我们需要引入高压冷冻（HPF）方法。在本研究中，我们通过HPF检查洋葱根尖，以了解HPF是否对我们的目的有用。结果表明，与化学固定法相比，高功率光镜法可以获得更好的树脂薄片PPB图像。为了检验在EM水平上进行三维分析的可能性，对HPF样品进行了冻结断裂和深加工。我们的方法似乎是断裂和深度教学方法应用于HPF样本。由于我们的方法似乎很有希望，我们得出结论，这个项目值得在未来继续下去。

To know the mechanism of division plane oriention is an important problem for plant morphogenesis. Just befor cells enter the mitosis, Microtubules (Mts) gather to form a preprophase band (PPB). As the PPB locates at the site where cell plate will be fused at the end of cytokinesis, to know how Mts are organized as a PPB is essential for understanding regulatory nmechanisms of the division plane orientation. Actins and cdks are reported to be in PPBs but their role have not yet clear. In oreder to know the relationship among these molecules and Mts in a PPB,electron microscopic (EM) study is necessary. However, we could not get reliable results by the conventional EM because Mts are very dynamic structure. In order to solve this problem, we need to introduce high pressure freeezing (HPF) method. In the present study, we examine onion root tips by HPF to know whether HPF is useful for our purpose. Results showed that we could get better PPB images of the thin section of Spurr's resin by HPF than by the chemical fixation. In order to examine the possibility of the three dimentional analysis at the EM level, freeze fracture and deep teching mehtods are applied to the HPF samples. As our approach seems fracture and deep teching mehtods are applied to the HPF samples. As our approach seems to be promissing, we concluded that this project is worth to continue in future.