This article has been reviewed according to Science X's editorial process and policies. Editors have highlighted the following attributes while ensuring the content's credibility: For decades, ribosomes have been under the lens of many scientists. Ribosomes are highly sophisticated and complex molecular machines that allow cells to synthesize proteins.
However, putting ribosomes together requires intricate assembly processes. While many underlying mechanisms for building ribosomes have been discovered ex situ (outside their native cellular context), our understanding of how ribosomes are assembled in situ has so far remained limited. Through a collaboration between the research groups of Christoph Müller, Sara Cuylen-Häring and Julia Mahamid at EMBL Heidelberg, researchers have now pushed these limits.
By combining cryo-electron tomography (cryo-ET) with correlative light and electron microscopy (CLEM), they were able to image the nucleolus—the site of ribosome assembly inside cells—at sufficiently high resolution to visualize the ribosome assembly pathway inside intact human cells for the first time. The results—a collection of molecular snapshots—yielded subnanometer-resolution in-cell structures of tens of intermediates along the ribosome assembly pathways, complementing and expanding previous insights into ribosome assembly obtained from ex situ structures. The new results have now been published in the journal Science.
EMBL researchers could not rely on traditional structural biology approaches to get a closer look at this complex cellular assembly line. Past studies on pre-ribosomes—structural ribosome precursors—employed approaches that required the researchers to take the ribosome components out of the cell. This, in some cases, caused components that only bound each other temporarily to separate.
The difficulty with visualizing this process inside the cell, however, lies in where ribosomes are made: the nucleolus, an extremely dense, low-contrast compartment inside the cell nucleus, which is technically challenging to visualize with cryo-ET. The scientists therefore combined cryo-ET with cryo-CLEM to shed light on these gloomy cellular factories. "Using this combined approach helped us immensely in identifying where this process actually happens," said Xiaohan Zhao, first author and predoctoral fellow in Müller's group.
"Cryo-electron tomography allowed us to observe the pre-ribosomes not by isolating them from the cells, but really within the nucleoli." Both techniques are well established. Mahamid's research group has been at the forefront of cryo-ET development, and cryo-CLEM is now widely used by researchers at EMBL. Whole human cells are too thick to image directly by electron microscopy, so they are first flash-frozen and cut into thin slices to make them accessible for observation under the microscope, generating images called tomograms.
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