A study on MTs in the cell cortex by HP freezing

间期皮质微管（Interphase cortical microtubules, MTs）和前期带（preprophase band， PPB）是高等植物中两个皮层微管阵列，分别在调控细胞伸长方向和确定细胞分裂位点方面起着中心作用。然而，关于调节这些皮层MT组织和转换的机制知之甚少。我们采用高压冷冻和冷冻替代或冷冻蚀刻技术的组合来保存和可视化洋葱和烟草幼苗中形成的mt，用于超微结构分析。这些研究发现了一些以前没有报道过的新的结构特征。(1)在体外对聚合/解聚MTs的研究表明，它们的末端呈现出喇叭形和非喇叭形构型。我们的显微照片显示，皮质mt也表现出喇叭和非喇叭末端，与他们翻转的想法一致。使用MT解聚药物oryzalin治疗后，卷曲末端构型（体内预期的解聚MT端）的数量增加。这表明，卷曲末端的频率反映了活细胞中mtts的收缩频率。(2)影响f -肌动蛋白稳定性的药物已被证明会扰乱PPB的形态。为了进一步了解这些药物是如何引起这些变化的，我们正在定量分析它们对MT-MT和mt -质膜间隔的影响。由于高压冷冻细胞的质膜光滑、无波浪形，冷冻固定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区域含有与细胞壁相似的染色囊泡，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。然而，我们的显微照片显示，在PPB mt之间形成了数量增加的包被内吞囊泡。

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.