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
Spinal muscular atrophy is an autosomal recessive neuromuscular disease caused by mutations in the multifunctional protein Survival of Motor Neuron, or SMN. Within the nucleus, SMN localizes to Cajal Show more
Spinal muscular atrophy is an autosomal recessive neuromuscular disease caused by mutations in the multifunctional protein Survival of Motor Neuron, or SMN. Within the nucleus, SMN localizes to Cajal bodies, which are associated with nucleoli, nuclear organelles dedicated to the first steps of ribosome biogenesis. The highly organized structure of the nucleolus can be dynamically altered by genotoxic agents. RNAP1, Fibrillarin, and nucleolar DNA are exported to the periphery of the nucleolus after genotoxic stress and, once DNA repair is fully completed, the organization of the nucleolus is restored. We find that SMN is required for the restoration of the nucleolar structure after genotoxic stress. During DNA repair, SMN shuttles from the Cajal bodies to the nucleolus. This shuttling is important for nucleolar homeostasis and relies on the presence of Coilin and the activity of PRMT1. Show less