A study on MTs in the cell cortex by HP freezing

间期皮层微管（Interphase cortical microtubules，MTs）和前期带（preprophase band，PPB）是高等植物皮层微管的两种排列方式，分别在细胞伸长方向的调控和分裂位点的确定中起着重要作用。然而，很少有人知道的机制，调节这些皮层MT组织和营业额。我们采用了高压冷冻和冷冻置换或冷冻蚀刻技术的组合，以保存和可视化形成的MT在洋葱和烟草幼苗的超微结构分析。这些研究发现了一些以前没有报道过的新结构特征。(1)聚合/解聚MT的体外研究表明，它们的末端显示出扩口和非扩口构型。我们的显微照片表明，皮质MT也表现出扩口和非扩口的结束，一致的想法，他们正在翻转。卷曲末端构型（预期在体内解聚MT末端）的数量通过用MT解聚药物安磺灵治疗而增加。这表明卷曲末端的频率反映了活细胞中收缩MT的频率。(2)影响F-肌动蛋白稳定性的药物已被证明会扰乱PPB形态。为了更多地了解这些药物如何引起这些变化，我们正在定量分析它们对MT-MT和MT-质膜间距的影响。由于高压冷冻细胞中质膜的光滑、非波状构象，冷冻固定样品比化学固定样品更容易进行这种类型的分析。(3)先前的研究表明，PPB区域含有类似于细胞壁染色的囊泡，这导致了它们可能参与将特殊类型的细胞壁分子递送到PPB区域的建议。然而，我们的显微照片表明，PPB MT之间形成的包被内吞囊泡数量增加。

Interphase cortical microtubules (MTs) and the preprophase band (PPB) are two cortical arrays of MTs in higher plants, and are known to play ezzentrrial role for the regulation of the direction of cell elongation and for the determination of the division site, respectively. However, little is known about the mechanism that regulates these cortical MT organization and turnover. We have employed a combination of high pressure freezing and freeze-substitution or freeze-etch techniques to preserve and visualize forming MTs in onion and tobacco seedlings for ultrastructural analysis. These studies have led to the detection of several novel structural features that have not been reported previously.(1) In vitro studies of polymerizing/depolymerizing MTs have shown that their ends display both flared and non-flared configurations. Our micrographs demonstrate that cortical MTs also exhibit flared and non-flared ends, consistent with the idea that they are turning over. The number of coiled end configurations (expected depolymerizing MT end in vivo) increased by the treatment with a MT-depolymerizing drug, oryzalin. This suggests that the the frequency of coiled ends reflects that of shrinklng MTs in living cells. (2) Drugs that affect F-actin stability have been shown to perturb PPB morphology. To learn more about how such drugs may cause these changes, we are analyzing quantitatively their effects on both MT-MT and MT-plasma membrane spacings. This type of analysis is more easily done with cryofixed than with chemically fixed samples due to the smooth, non-wavy conformation of the plasma membrane in high pressure frozen cells. (3) Previous studies have shown that PPB regions contain vesicles that stain similar to cell walls, which has led to the suggestion that they could be Involved in delivering special types of cell wall molecules to PPB regions. Our micrographs demonstrate, however, that increased numbers of coated, endocytic vesicles are formed between the PPB MTs.