Homann Ulrike, Meckel Tobias, Hewing Jennifer, Hütt Marc-Thorsten, Hurst Annette C
Institute of Botany, University of Technology Darmstadt, Schnittspahnstrasse 3-5, 64287 Darmstadt, Germany.
Eur J Cell Biol. 2007 Aug;86(8):489-500. doi: 10.1016/j.ejcb.2007.05.003. Epub 2007 Jun 28.
The organisation of membrane proteins into certain domains of the plasma membrane (PM) has been proposed to be important for signalling in yeast and animal cells. Here we describe the formation of a very distinct pattern of the K(+) channel KAT1 fused to the green fluorescent protein (KAT1::GFP) when transiently expressed in guard cells of Vicia faba. Using confocal laser scanning microscopy we observed a radially striped pattern of KAT1::GFP fluorescence in the PM in about 70% of all transfected guard cells. This characteristic pattern was found to be cell type and protein specific and independent of the stomatal aperture and the cytoskeleton. Staining of the cell wall of guard cells with Calcofluor White revealed a great similarity between the arrangement of cellulose microfibrils and the KAT1::GFP pattern. Furthermore, the radial pattern of KAT1::GFP immediately disappeared when turgor pressure was strongly decreased by changing from hypotonic to hypertonic conditions. The pattern reappeared within 15 min upon reestablishment of high turgor pressure in hypotonic solution. Evaluation of the staining pattern by a mathematical algorithm further confirmed this reversible abolishment of the radial pattern during hypertonic treatment. We therefore conclude that the radial organisation of KAT1::GFP depends on the close contact between the PM and cell wall in turgid guard cells. These results offer the first indication for a role of the cell wall in the localisation of ion channels. We propose a model in which KAT1 is located in the cellulose fibrils intermediate areas of the PM and discuss the physiological role of this phenomenon.
膜蛋白在质膜（PM）特定区域的组织被认为对酵母和动物细胞中的信号传导很重要。在此，我们描述了融合绿色荧光蛋白的钾离子通道KAT1（KAT1::GFP）在蚕豆保卫细胞中瞬时表达时形成的一种非常独特的模式。使用共聚焦激光扫描显微镜，我们在约70%的所有转染保卫细胞中观察到KAT1::GFP荧光在质膜上呈径向条纹模式。发现这种特征模式具有细胞类型和蛋白质特异性，且与气孔孔径和细胞骨架无关。用荧光增白剂对保卫细胞壁进行染色显示，纤维素微纤丝的排列与KAT1::GFP模式之间有很大的相似性。此外，当通过从低渗条件转变为高渗条件使膨压大幅降低时，KAT1::GFP的径向模式立即消失。在低渗溶液中重新建立高膨压后，该模式在15分钟内重新出现。通过数学算法对染色模式进行评估进一步证实了在高渗处理期间径向模式的这种可逆消失。因此，我们得出结论，KAT1::GFP的径向组织取决于膨压状态下保卫细胞质膜与细胞壁之间的紧密接触。这些结果首次表明细胞壁在离子通道定位中发挥作用。我们提出了一个模型，其中KAT1位于质膜的纤维素微纤丝中间区域，并讨论了这一现象的生理作用。
