McFarlane Heather E, Lee Eun Kyoung, van Bezouwen Laura S, Ross Bradford, Rosado Abel, Samuels A Lacey
Department of Botany, University of British Columbia, University Blvd., Vancouver, Canada.
School of Biosciences, University of Melbourne, Australia.
Plant Cell Physiol. 2017 Mar 1;58(3):478-484. doi: 10.1093/pcp/pcw224.
Membrane contact sites are recognized across eukaryotic systems as important nanostructures. Endoplasmic reticulum (ER)-plasma membrane (PM) contact sites (EPCS) are involved in excitation-contraction coupling, signaling, and plant responses to stress. In this report, we perform a multiscale structural analysis of Arabidopsis EPCS that combines live cell imaging, quantitative transmission electron microscopy (TEM) and electron tomography over a developmental gradient. To place EPCS in the context of the entire cortical ER, we examined green fluorescent protein (GFP)-HDEL in living cells over a developmental gradient, then Synaptotagmin1 (SYT1)-GFP was used as a specific marker of EPCS. In all tissues examined, young, rapidly elongating cells showed lamellar cortical ER and higher density of SYT1-GFP puncta, while in mature cells the cortical ER network was tubular, highly dynamic and had fewer SYT1-labeled puncta. The higher density of EPCS in young cells was verified by quantitative TEM of cryo-fixed tissues. For all cell types, the size of each EPCS had a consistent range in length along the PM from 50 to 300 nm, with microtubules and ribosomes excluded from the EPCS. The structural characterization of EPCS in different plant tissues, and the correlation of EPCS densities over developmental gradients illustrate how ER-PM communication evolves in response to cellular expansion.
膜接触位点在真核生物系统中被公认为重要的纳米结构。内质网（ER）-质膜（PM）接触位点（EPCS）参与兴奋-收缩偶联、信号传导以及植物对胁迫的反应。在本报告中，我们对拟南芥EPCS进行了多尺度结构分析，该分析结合了活细胞成像、定量透射电子显微镜（TEM）以及在发育梯度上的电子断层扫描。为了将EPCS置于整个皮质内质网的背景下，我们在发育梯度上检查了活细胞中的绿色荧光蛋白（GFP）-HDEL，然后使用突触结合蛋白1（SYT1）-GFP作为EPCS的特异性标记。在所有检查的组织中，年轻的、快速伸长的细胞显示出层状皮质内质网和更高密度的SYT1-GFP斑点，而在成熟细胞中，皮质内质网网络是管状的，高度动态且SYT1标记的斑点较少。通过对冷冻固定组织的定量TEM验证了年轻细胞中EPCS的较高密度。对于所有细胞类型，每个EPCS沿质膜的长度大小在50至300nm范围内一致，微管和核糖体被排除在EPCS之外。不同植物组织中EPCS的结构特征以及发育梯度上EPCS密度的相关性说明了内质网-质膜通讯如何随着细胞扩张而演变。
