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

了解分裂面定向的机理是植物形态建成的重要问题。在细胞进入有丝分裂之前，微管（Mts）聚集形成前期带（PPB）。由于PPB位于胞质分裂末期细胞板融合的部位，因此了解Mts是如何组织成PPB的，对于理解分裂面取向的调控机制是至关重要的。肌动蛋白和cdk被报道存在于PPBs中，但它们的作用尚不清楚。为了了解PPB中这些分子与Mts之间的关系，电子显微镜（EM）研究是必要的。然而，我们不能得到可靠的结果，通过传统的EM，因为Mts是非常动态的结构。为了解决这一问题，需要引入高压冷冻法。在本研究中，我们用HPF检测洋葱根尖，以了解HPF是否对我们的目的有用。结果表明，用HPF固定Spurr树脂薄片，能获得比化学固定法更好的PPB图像。为了检验电磁水平三维分析的可能性，对HPF样品进行了冷冻断裂和深加工。我们的方法似乎适用于HPF样品的断裂和深加工方法。由于我们的方法似乎是有前途的，我们得出结论，这个项目是值得继续在未来。

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.