Heat stress (HS) impacts the nuclear proteome and, subsequently, protein activities in different nuclear compartments. In Arabidopsis thaliana, a short exposure to 37 °C leads to loss of the standard tripartite architecture of the nucleolus, the most prominent nuclear substructure, and, consequently, affects the assembly of ribosomes. Here, we report a quantitative label-free LC‒MS/MS (Liquid Chromatography coupled to tandem Mass Spectrometry) analysis to determine the nuclear proteome of Arabidopsis at 22 °C, HS (37 °C for 4 and 24 h), and a recovery phase. This analysis identified ten distinct groups of proteins based on relative abundance changes in the nucleus before, during and after HS: Early, Late, Transient, Early Persistent, Late Persistent, Recovery, Early-Like, Late-Like, Transient-Like and Continuous Groups (EG, LG, TG, EPG, LPG, RG, ELG, LLG, TLG and CG, respectively). Interestingly, the RNA polymerase I subunit NRPA3 and other main nucleolar proteins, including NUCLEOLIN 1 and FIBRILLARIN 1 and 2, were detected in RG and CG, suggesting that plants require increased nucleolar activity and likely ribosome assembly to restore protein synthesis after HS.

The tumor suppressor p53 and its antagonists MDM2 and MDM4 integrate stress signaling. For instance, dysbalanced assembly of ribosomes in nucleoli induces p53. Here, we show that the ribosomal protein L22 (RPL22; eL22), under conditions of ribosomal and nucleolar stress, promotes the skipping of MDM4 exon 6. Upon L22 depletion, more full-length MDM4 is maintained, leading to diminished p53 activity and enhanced cellular proliferation. L22 binds to specific RNA elements within intron 6 of MDM4 that correspond to a stem-loop consensus, leading to exon 6 skipping. Targeted deletion of these intronic elements largely abolishes L22-mediated exon skipping and re-enables cell proliferation, despite nucleolar stress. L22 also governs alternative splicing of the L22L1 (RPL22L1) and UBAP2L mRNAs. Thus, L22 serves as a signaling intermediate that integrates different layers of gene expression. Defects in ribosome synthesis lead to specific alternative splicing, ultimately triggering p53-mediated transcription and arresting cell proliferation.

Cellular senescence, which is triggered by various stressors, manifests as irreversible cell cycle arrest, resulting in the disruption of multiple nuclear condensates. One of the affected structures is the nucleolus, whose tripartite layout, separated into distinct liquid phases, allows for the stepwise progression of ribosome biogenesis. The dynamic properties of dense fibrillar components, a sub-nucleolar phase, are crucial for mediating pre-rRNA processing. However, the mechanistic link between the material properties of dense fibrillar components and cellular senescence remains unclear. We established a significant association between cellular senescence and alterations in nucleolar materiality and characteristics, including the number, size, and sphericity of individual subphases of the nucleolus. Senescent cells exhibit reduced fibrillarin dynamics, aberrant accumulation of high-order protein assemblies, such as oligomers and fibrils, and increased dense fibrillar component density. Intriguingly, the addition of RNA-interacting entities mirrored the diminished diffusion of fibrillarin in the nucleolus during cellular senescence. Thus, our findings contribute to a broader understanding of the intricate changes in the materiality of the nucleolus associated with cellular senescence and shed light on nucleolar dynamics in the context of aging and cellular stress.

The Spalt transcriptional regulators participate in a variety of cell fate specification processes during development, regulating transcription through interactions with DNA AT-rich regions. Spalt proteins also bind to heterochromatic regions, and some of their effects require interactions with the NuRD chromatin remodeling and deacetylase complex. Most of the biological roles of Spalt proteins have been characterized in diploid cells engaged in cell proliferation. Here we address the function of Drosophila spalt genes in the development of a larval tissue formed by polyploid cells, the prothoracic gland, whose cells undergo several rounds of DNA replication without mitosis during larval development. We show that prothoracic glands depleted of spalt expression display severe changes in the size of the nucleolus, the morphology of the nuclear envelope and the disposition of the chromatin within the nucleus, leading to a failure in the synthesis of ecdysone. We propose that loss of ecdysone production in the prothoracic gland of spalt mutants is primarily caused by defects in nuclear pore complex function that occur as a consequence of faulty interactions between heterochromatic regions and the nuclear envelop.

Retinoblastoma is the most common pediatric intraocular malignant tumor affecting 1:15 000-1:20 000 live births. Even though the survival rate in developed countries is over 90 %, more efficient treatment options are needed for better vision salvage and reduction of the adverse effects. Therefore, we investigated fluorescein-labeled PL3 peptide targeting properties towards the Y79 retinoblastoma cell line in vitro. Through the application of cellular imaging and flow cytometry techniques, the PL3 peptide exhibited a rapid and specific internalization within Y79 cells, with subsequent translocation to the cell nuclei, showcasing notable accumulation in the nucleoli. This phenomenon was not present in other investigated cell lines and was not observable with similarly charged and length control peptide. However, the exact mechanism behind this Y79 cell line-specific nuclear and nucleolar targeting pattern remains elusive. In the future, this targeting process could facilitate specific treatment modalities of retinoblastoma with PL3 peptide-coupled drug delivery technologies.

Candida albicans is the most prevalent fungal pathogen associated with candidemia. Similar to other fungi, the complex life cycle of C. albicans has been challenging to study with high-resolution microscopy due to its small size. We employed ultrastructure expansion microscopy (U-ExM) to directly visualise sub-cellular structures at high resolution in the yeast and during its transition to hyphal growth. NHS-ester pan-labelling in combination with immunofluorescence (IF) via snapshots of various mitotic stages provided a comprehensive map of nucleolar and mitochondrial segregation dynamics and enabled the resolution of inner and outer plaque of spindle pole bodies (SPBs). Analyses of microtubules (MTs) and SPBs suggest that C. albicans displays side-by-side SPB arrangement with a short mitotic spindle and longer astral MTs (aMTs) at the pre-anaphase stage. Modifications to the established U-ExM protocol enabled the expansion of six other human fungal pathogens, revealing that the side-by-side SPB configuration is a plausible conserved feature shared by many fungal species. We highlight the power of U-ExM to investigate sub-cellular organisation at high resolution and low cost in poorly studied and medically relevant microbial pathogens.

Stem cell loss in aging and disease is associated with nuclear deformation. Yet, how nuclear shape influences stem cell homeostasis is poorly understood. We investigated this connection using Drosophila germline stem cells, as survival of these stem cells is compromised by dysfunction of the nuclear lamina, the extensive protein network that lines the inner nuclear membrane and gives shape to the nucleus. To induce nuclear distortion in germline stem cells, we used the GAL4-UAS system to increase expression of the permanently farnesylated nuclear lamina protein, Kugelkern, a rate limiting factor for nuclear growth. We show that elevated Kugelkern levels cause severe nuclear distortion in germline stem cells, including extensive thickening and lobulation of the nuclear envelope and nuclear lamina, as well as alteration of internal nuclear compartments. Despite these changes, germline stem cell number, proliferation, and female fertility are preserved, even as females age. Collectively, these data demonstrate that disruption of nuclear architecture does not cause a failure of germline stem cell survival or homeostasis, revealing that nuclear deformation does not invariably promote stem cell loss.

The nucleolar enzyme sirtuin 7 (SIRT7) promotes cancer progression in certain malignancies, likely in part by controlling ribosome biosynthesis. Recently, we discovered that SIRT7 destabilizes the cyclin dependent kinase inhibitor 2A (CDKN2A, known as ARF) within the nucleolus, aiding cancer progression. We propose that targeting nucleolar SIRT7 offers promise for new anti-cancer therapies.

In budding yeast, the nucleolus serves as the site to sequester Cdc14, a phosphatase essential for mitotic exit. Nucleolar proteins Tof2, Net1, and Fob1 are required for this sequestration. Although it is known that these nucleolar proteins are SUMOylated, how SUMOylation regulates their activity remains unknown. Here, we show that Tof2 exhibits cell-cycle-regulated nucleolar delocalization and turnover. Depletion of the nuclear small ubiquitin-like modifier (SUMO) protease Ulp2 not only causes Tof2 polySUMOylation, nucleolar delocalization, and degradation but also leads to Cdc14 nucleolar release and activation. This outcome depends on polySUMOylation and the activity of downstream enzymes, including SUMO-targeted ubiquitin ligase and Cdc48/p97 segregase. We further developed a system to tether SUMO machinery to Tof2 and generated a SUMO-deficient tof2 mutant, and the results indicate that Tof2 polySUMOylation is necessary and sufficient for its nucleolar delocalization and degradation. Together, our work reveals a polySUMO-dependent mechanism that delocalizes Tof2 from the nucleolus to facilitate mitotic exit.

The effects of genotoxic agents on DNA and the processes involved in their removal have been thoroughly studied; however, very little is known about the mechanisms governing the reinstatement of cellular activities after DNA repair, despite restoration of the damage-induced block of transcription being essential for cell survival. In addition to impeding transcription, DNA lesions have the potential to disrupt the precise positioning of chromatin domains within the nucleus and alter the meticulously organized architecture of the nucleolus. Alongside the necessity of resuming transcription mediated by RNA polymerase 1 and 2 transcription, it is crucial to restore the structure of the nucleolus to facilitate optimal ribosome biogenesis and ensure efficient and error-free translation. Here, we examine the current understanding of how transcriptional activity from RNA polymerase 2 is reinstated following DNA repair completion and explore the mechanisms involved in reassembling the nucleolus to safeguard the correct progression of cellular functions. Given the lack of information on this vital function, this Review seeks to inspire researchers to explore deeper into this specific subject and offers essential suggestions on how to investigate this complex and nearly unexplored process further.