Unicellular cyanobacteria Synechocystis 6803 were fixed using high-pressure freezing （HPF） and freeze substitution without any chemical cross-linkers. Immunoelectron microscopy of these cells showed that five sequen...Unicellular cyanobacteria Synechocystis 6803 were fixed using high-pressure freezing （HPF） and freeze substitution without any chemical cross-linkers. Immunoelectron microscopy of these cells showed that five sequential enzymes of the Calvin cycle （phosphoriboisomerase, phosphoribulokinase, ribulose-1,5-bisphosphate carboxylase/oxygenase （RuBisCO）, 3-phosphoglyceratekinase and glyceraldehyde-3-phosphate dehydrogenase） and the catalytic portion of the chloroplast H^＋-ATP synthase （CF1） are located adjacent to the thylakoid membranes. Cell-free extracts of Synechocystis were processed by ultracentrifugation to isolate thylakoid fractions sedimenting at 40 000, 90 000, and 150 000 g. Among these, the 150 000-g fraction showed the highest linked activity of the above five sequential Calvin cycle enzymes and also the highest coordinated activity of light and dark reactions as assessed by ribose-5-phosphate （R-5-P） ＋ADP dependent CO2 fixation. Immunogold labeling of this membrane fraction confirmed the presence of the above five enzymes as well as the catalytic portion of the CF1 ATP synthase. Notably, the protein A-gold labeling of the thylakoids was observed without use of chemical cross-linkers and in spite of the normal washing steps used during standard immunolabeling. The results showed that soluble Calvin cycle enzymes might be organized along the thylakoid membranes.展开更多
This review focuses on the contribution of electron tomography-based techniques to our understanding of cellular processes in plant cells. Electron microscopy techniques have evolved to provide better three-dimensiona...This review focuses on the contribution of electron tomography-based techniques to our understanding of cellular processes in plant cells. Electron microscopy techniques have evolved to provide better three-dimensional resolution and improved preservation of the subcellular components. In particular, the combination of cryofixation/freeze substitution and electron tomography have allowed plant cell biologists to image organelles and macromolecular complexes in their native cellular context with unprecedented three-dimensional resolution （4-7 nm）. Until now, electron tomography has been applied in plant cell biology for the study of cytokinesis, Golgi structure and trafficking, formation of plant endosome/prevacuolar compartments, and organization of photosynthetic membranes. We discuss in this review the new insights that these tomographic studies have brought to the plant biology field.展开更多
文摘Unicellular cyanobacteria Synechocystis 6803 were fixed using high-pressure freezing （HPF） and freeze substitution without any chemical cross-linkers. Immunoelectron microscopy of these cells showed that five sequential enzymes of the Calvin cycle （phosphoriboisomerase, phosphoribulokinase, ribulose-1,5-bisphosphate carboxylase/oxygenase （RuBisCO）, 3-phosphoglyceratekinase and glyceraldehyde-3-phosphate dehydrogenase） and the catalytic portion of the chloroplast H^＋-ATP synthase （CF1） are located adjacent to the thylakoid membranes. Cell-free extracts of Synechocystis were processed by ultracentrifugation to isolate thylakoid fractions sedimenting at 40 000, 90 000, and 150 000 g. Among these, the 150 000-g fraction showed the highest linked activity of the above five sequential Calvin cycle enzymes and also the highest coordinated activity of light and dark reactions as assessed by ribose-5-phosphate （R-5-P） ＋ADP dependent CO2 fixation. Immunogold labeling of this membrane fraction confirmed the presence of the above five enzymes as well as the catalytic portion of the CF1 ATP synthase. Notably, the protein A-gold labeling of the thylakoids was observed without use of chemical cross-linkers and in spite of the normal washing steps used during standard immunolabeling. The results showed that soluble Calvin cycle enzymes might be organized along the thylakoid membranes.
基金Supported by US National Foundation grant MCB-0619736 to MSO
文摘This review focuses on the contribution of electron tomography-based techniques to our understanding of cellular processes in plant cells. Electron microscopy techniques have evolved to provide better three-dimensional resolution and improved preservation of the subcellular components. In particular, the combination of cryofixation/freeze substitution and electron tomography have allowed plant cell biologists to image organelles and macromolecular complexes in their native cellular context with unprecedented three-dimensional resolution （4-7 nm）. Until now, electron tomography has been applied in plant cell biology for the study of cytokinesis, Golgi structure and trafficking, formation of plant endosome/prevacuolar compartments, and organization of photosynthetic membranes. We discuss in this review the new insights that these tomographic studies have brought to the plant biology field.
