Skubatz H, Orellana M V, Yablonka-Reuveni Z
Department of Botany, University of Washington, Seattle 98195, USA.
Histochem J. 2000 Aug;32(8):467-74. doi: 10.1023/a:1004140215519.
Immunoflorescence microscopy of sections of the voodoo lily Sauromatum guttatum appendix stained with monoclonal antibodies against alpha-smooth muscle actin and cytoplasmic actin revealed different staining intensity of different parts of the cell. The anti-cytoplasmic-actin recognized antigens present mainly in the cytoplasm, and the anti-alpha-smooth muscle-actin recognized more intensively antigens present in the nuclei. A positive staining of the nucleus was also obtained with FITC-phalloidin confirming the presence of actin in its filamenous form in the nucleus. The presence of a nuclear alpha-smooth muscle-actin-like protein was further confirmed by confocal laser microscopy. On Western blots, the two anti-actins labelled a protein band that comigrated with standard actin at the approximate molecular weight of 43 kDa. Several other proteins interacted with the two antibodies to a different degree. The monoclonal antibodies against beta-tubulin subunit stained only the periphery of the cytoplasm and anti-pan cytoplasmic myosin stained the cytoplasm weakly. On a Western blot, anti-beta-tubulin subunit primarily recognized a protein band at the appropriate molecular weight of 50 kDa. This is the first cytochemical evidence for the presence of alpha-smooth muscle-actin-like protein in the plant nucleus.
用抗α-平滑肌肌动蛋白和细胞质肌动蛋白的单克隆抗体对巫毒百合魔芋附属器切片进行免疫荧光显微镜检查,结果显示细胞不同部位的染色强度不同。抗细胞质肌动蛋白识别主要存在于细胞质中的抗原,而抗α-平滑肌肌动蛋白更强烈地识别存在于细胞核中的抗原。用异硫氰酸荧光素-鬼笔环肽也获得了细胞核的阳性染色,证实细胞核中存在丝状形式的肌动蛋白。共聚焦激光显微镜进一步证实了细胞核中存在α-平滑肌肌动蛋白样蛋白。在蛋白质免疫印迹分析中,两种抗肌动蛋白抗体标记了一条与标准肌动蛋白共迁移的蛋白带,其近似分子量为43 kDa。其他几种蛋白质与这两种抗体有不同程度的相互作用。抗β-微管蛋白亚基的单克隆抗体仅对细胞质周边进行染色,抗泛细胞质肌球蛋白对细胞质的染色较弱。在蛋白质免疫印迹分析中,抗β-微管蛋白亚基主要识别一条分子量为50 kDa的合适蛋白带。这是植物细胞核中存在α-平滑肌肌动蛋白样蛋白的首个细胞化学证据。