Abstract
The hallmark of eukaryotic cells is their segregation of key biological functions into discrete, membrane-bound organelles. Creating accurate models of their ultrastructural complexity has been difficult in part because of the limited resolution of light microscopy and the artifact-prone nature of conventional electron microscopy. Here we explored the potential of the emerging technology electron cryotomography to produce three-dimensional images of an entire eukaryotic cell in a near-native state. Ostreococcus tauri was chosen as the specimen because as a unicellular picoplankton with just one copy of each organelle, it is the smallest known eukaryote and was therefore likely to yield the highest resolution images. Whole cells were imaged at various stages of the cell cycle, yielding 3-D reconstructions of complete chloroplasts, mitochondria, endoplasmic reticula, Golgi bodies, peroxisomes, microtubules, and putative ribosome distributions in-situ. Surprisingly, the nucleus was seen to open long before mitosis, and while one microtubule (or two in some predivisional cells) was consistently present, no mitotic spindle was ever observed, prompting speculation that a single microtubule might be sufficient to segregate multiple chromosomes.
Highlights
The history of cell biology has been punctuated by major advances in imaging technologies
Cells were obtained from a strain of O. tauri isolated from the Thau lagoon (France) [18]
O. tauri’s growth was synchronized using a twelve-hour-light, twelve-hour-dark cycle that resulted in an average of one cell division per day
Summary
The history of cell biology has been punctuated by major advances in imaging technologies. Following the invention of the electron microscope in the early 1930s, what we would call the ‘‘conventional’’ specimen preparation methods of chemical fixation, dehydration, plastic-embedding, sectioning, and staining were developed to allow the visualization of biological material. Recent technological developments have created the opportunity to realize two major improvements: imaging cells in three dimensions (3-D) and in nearly-native states. Full 3-D reconstructions of the sample can be calculated Stacking such 3-D reconstructions of serial (thick) sections has made it possible to reconstruct even large regions of fixed cells [3]. It has become possible to image non-chemically-fixed cells in a more nearly native state through plunge-freezing. Plunge-freezing preserves cells in a life-like, ‘‘frozen-hydrated’’ state free of stains and with minimal artifacts [4]. Electron cryotomography (ECT) combines these two advances to produce 3-D reconstructions of nearly-native biological material to ‘‘molecular’’ resolution [5]
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