The nucleolus, a membraneless organelle crucial for ribosome production, has a unique nanoscale structure whose organization is responsive to cell signals and disease progression. Here, we highlight t Show more
The nucleolus, a membraneless organelle crucial for ribosome production, has a unique nanoscale structure whose organization is responsive to cell signals and disease progression. Here, we highlight the potential of Expansion Microscopy (ExM) for capturing intricate spatial and functional information about membraneless organelles such as the nucleolus and nuclear foci. We apply dual protein Expansion Microscopy (dual-proExM) in combination with click Expansion Microscopy (click-ExM) to capture images at the highest resolution reported for the nucleolus of ∼45 ± 2 nm. Inhibition of nucleolar processes triggers a nucleolar stress response, causing distinct structural rearrangements whose molecular basis is an area of active investigation. We investigate time-dependent changes in nucleolar structure and function under nucleolar stress induced by oxaliplatin, actinomycin D, and other platinum-based compounds. Our findings reveal new stages that occur prior to the complete sequestration of RNA Pol I into nucleolar caps, shedding light on the early mechanisms of the nucleolar stress response. RNA transcription is linked to nanoscale protein rearrangements using a combination of click-ExM and pro-ExM, revealing locations of active transcripts during the early stages of nucleolar stress reorganization. With prolonged stress, fibrillarin and NPM1 segregate from the nucleolus into nucleoplasmic foci that are for the first time imaged at nanometer resolution. In addition to revealing new morphological information about the nucleolus, this study demonstrates the potential of ExM for imaging membraneless organelles with nanometer-scale precision. Show less
The ribosome is a macromolecular complex composed of RNA and proteins that interact
through an integrated and interconnected network to preserve its ancient core activities. In this review,
we emphasi Show more
The ribosome is a macromolecular complex composed of RNA and proteins that interact
through an integrated and interconnected network to preserve its ancient core activities. In this review,
we emphasize the pivotal role played by RNA-binding proteins as a driving force in the evolution
of the current form of the ribosome, underscoring their importance in ensuring accurate protein
synthesis. This category of proteins includes both ribosomal proteins and ribosome biogenesis
factors. Impairment of their RNA-binding activity can also lead to ribosomopathies, which is a
group of disorders characterized by defects in ribosome biogenesis that are detrimental to protein
synthesis and cellular homeostasis. A comprehensive understanding of these intricate processes is
essential for elucidating the mechanisms underlying the resulting diseases and advancing potential
therapeutic interventions. Show less
Recent evidence showed that a variety of DNA damaging agents including 5-FU and L-OHP impairs ribosomal biogenesis activating a ribosomal stress pathway. Here, we demonstrate that in lung and colon ca Show more
Recent evidence showed that a variety of DNA damaging agents including 5-FU and L-OHP impairs ribosomal biogenesis activating a ribosomal stress pathway. Here, we demonstrate that in lung and colon cancer cell lines devoid of p53, the efficacy of 5-FU and L-OHP chemotherapy depends on rpL3 status. Specifically, we demonstrate that ribosomal stress induced by 5-FU and L-OHP is associated to up-regulation of rpL3 and its accumulation as ribosome-free form. We show that rpL3 participates in the cell response to chemotherapy acting as a critical regulator of cell cycle, apoptosis Show less