Abstract
Plant cells exhibit highly dynamic protein and membrane trafficking pathways. Many key protein and membrane sorting events occur in very small compartments or even in subdomains within an organelle, which makes their visualization by light microscopy very challenging. To analyze the endosomal sorting of plasma membrane proteins for degradation and the vacuolar transport of storage proteins in seeds, we perform dual-axis electron tomography of high-pressure frozen/freeze substituted plant material. We obtain 3D reconstructions of large areas of the cells containing organelles of interest and subject them to segmentation, model calculation, and quantitative analysis. Electron tomography in combination with structural pattern recognition of known macromolecular complexes and/or immunogold labeling allow for the correlation between structure and biochemical composition. Several examples on how these imaging approaches have been applied to the understanding of plant trafficking pathways will be discussed.
