Fluorescence microscopic localization of actin in pollen tubes: comparison of actin antibody and phalloidin staining.
Fluorescence microscopic localization of actin in pollen tubes: comparison of actin antibody and phalloidin staining.
复制标题
DOI：
10.1002/cm.970120404
复制
发表时间：
1989
影响因子：
--
通讯作者：
Xiaojing Tang;S. Lancelle;P. K. Hepler
中科院分区：
文献类型：
--
作者：
Xiaojing Tang;S. Lancelle;P. K. Hepler
用肌动蛋白单克隆抗体和罗丹明鬼笔环肽（RP）对烟草（Nicotiana）花粉管中肌动蛋白的定位进行了比较。单克隆抗肌动蛋白基于对花粉管提取物的蛋白质印迹，标记了一种与肌肉肌动蛋白共迁移的45 kD多肽。一个51 kD的未知蛋白和三个小于45 kD的条带，推测是肌动蛋白的蛋白水解片段，也观察到。使用该抗体的结构观察揭示了微丝（MF）的轴向取向链的网络。微纤维分布在整个长度的花粉管，除了在最顶端，在那里通常观察到弥漫染色。RP染色后，MF的类似模式很明显。当用细胞松弛素（CB或CD）处理花粉管时，细胞质流被抑制，管伸长也被抑制。显微镜分析表明，微丝（MF）模式显着改变，然而，抗体和RP产生不同的染色模式。抗体显示许多MF链分布在整个管长度上并延伸到最尖端。相反，RP主要表现为弥漫性染色模式，只有少数丝状物质的短团块。通过快速冷冻固定和冷冻置换制备的花粉管切片的免疫金标记显示，细胞松弛素处理后确实存在肌动蛋白MF束。因此，我们的研究结果质疑文献中的报告，鬼笔环肽染色的基础上，断言细胞松弛素片段或破坏肌动蛋白MFs。
A comparison of actin localization in pollen tubes of Nicotiana has been made using a monoclonal actin antibody and rhodamine-phalloidin (RP). The monoclonal antiactin, based on Western blotting of pollen tube extract, labels a polypeptide at 45 kD that comigrates with muscle actin. A 51-kD unknown protein and three bands less than 45 kD, presumed to be proteolytic fragments of actin, are also observed. Structural observations using this antibody reveal a network of axially oriented strands of microfilaments (MFs). The MFs are distributed throughout the length of the pollen tube except at the very tip, where diffuse staining is usually observed. A similar pattern of MFs is evident after RP staining. When pollen tubes are treated with cytochalasins (CB or CD) cytoplasmic streaming is inhibited, as is tube elongation. Microscopic analysis reveals that the microfilament (MF) pattern is markedly altered; however, the antibody and RP produce different staining patterns. The antibody reveals many MF strands that distribute throughout the tube length and extend into the very tip. In contrast, RP shows mostly a diffuse staining pattern with only a few short clumps of filamentous material. Immunogold labelling of sections of pollen tubes prepared by rapid-freeze fixation and freeze substitution reveals that actin MF bundles are indeed present after cytochalasin treatment. Our results thus question reports in the literature, based on phalloidin staining, asserting that cytochalasin fragments or destroys actin MFs.