Valeria Levi

Glucocorticoids are among the most widely prescribed drugs globally due to their potent anti-inflammatory and immunosuppressive actions. These effects are primarily mediated by the glucocorticoid receptor (GR), a ligand-activated transcription factor that translocates from the cytoplasm to the nucleus to regulate hundreds of genes. Although nuclear entry is a prerequisite for its genomic response, the mechanisms governing this process remain unresolved; specifically, whether the receptor translocates as a monomer or a dimer remains a subject of significant controversy. Here, we employed the pair correlation function (pCF) approach to quantify the nuclear translocation of single fluorescent GR molecules in live cells. This minimally invasive method identifies correlations between intensity fluctuations generated by molecules moving from the cytoplasm into the nucleus. Our results demonstrate that GR’s quaternary structure and conformation modulate GR transport. While GR monomers rely exclusively on passive diffusion, GR dimers also utilize the microtubule-dynein machinery for active transport, proving that dimerization can precede nuclear import. Furthermore, the perinuclear vimentin network facilitates faster translocation by constraining actively transported dimers near nuclear pores. Collectively, our work reconciles contradicting reports regarding GR stoichiometry during import by demonstrating that both monomers and dimers translocate, albeit through distinct mechanisms. Importantly, these results reopen the door for a microtubule-dependent, heterocomplex-independent model of GR translocation, suggesting that the cytoskeleton is an integral, yet overlooked, component of the GR signaling pathway.

Maintaining energy homeostasis is a major challenge for plants in the current context of climate change. The Sucrose-non fermenting 1 (SNF1)-related kinase 1 (SnRK1) complex, a member of the SNF1-AMP-activated protein kinase (AMPK)-SnRK1 family of kinase complexes, is a central player in the regulation of cell energy homeostasis. The α-subunit of the complex, which possesses kinase activity and is known as SnRK1.1 or KIN10, plays a role in sensing energy status and coordinating metabolic reprogramming to counter any energy imbalance. The discovery of a dual and dynamic intracellular distribution of SnRK1.1 suggests that the activity and function of SnRK1 might be regulated by spatiotemporal changes. To investigate the spatiotemporal distribution of SnRK1.1, we developed a protocol to quantify its intracellular distribution using fluorescence confocal images acquired along the z-axis in plants expressing SnRK1.1–eGFP. Using the open-source software Fiji/ImageJ, we calculated the ratio between nuclear and non-nuclear SnRK1.1 fractions and defined this as the N/ER index. We validated our method by analyzing the response of SnRK1.1 to photosynthesis inhibition by DCMU, including changes in protein levels and phosphorylation status. In addition, comparison with results obtained using a commercial software-based approach confirmed the compatibility of the N/ER index with different segmentation and quantification tools. Originally designed for leaf tissue images, this protocol can be broadly applied to assess the role of intracellular spatiotemporal changes in a wide range of kinases or fluorescently tagged recombinant proteins. Finally, SnRK1.1 intracellular distribution may also serve as a proxy to assess changes in cellular energy status. One sentence summary New method to track SnRK1.1 distribution and changes in plant cell energy status

Intermediate filaments are intimately involved in the mechanical behavior of cells. Unfortunately, the resolution of optical microscopy limits our understanding of their organization. Here, we combined nanoscopy, single-filament tracking, and numerical simulations to inspect the dynamical organization of vimentin intermediate filaments in live cells. We show that a higher proportion of peripheral versus perinuclear vimentin pools are constrained in their lateral motion in the seconds time window, probably due to their cross-linking to other cytoskeletal networks. In a longer time scale, active forces become evident and affect similarly both pools of filaments. Our results provide a detailed description of the dynamical organization of the vimentin network in live cells and give some cues on its response to mechanical stimuli.

Several proteins necessary for mRNA production concentrate in intranuclear condensates, which are proposed to affect transcriptional output. The glucocorticoid receptor (GR) is a ligand-activated transcription factor that regulates the expression of hundreds of genes relevant to many physiological and pathological processes. As with all members of the steroid receptor family, GR forms condensates of unknown function. Here, we examine whether GR condensates are involved in transcription regulation using Airyscan super-resolution microscopy and nano-antibodies targeting initiation and elongating states of RNA polymerase II (Pol2). We observed subpopulations of GR condensates colocalizing with initiating and, surprisingly, elongating Pol2 foci. The analysis of GR mutants with different transcriptional outputs suggests a correlation between condensate formation capability and transcription initiation. Moreover, the number of GR molecules within initiation and elongation condensates appears to be linked to transcriptional activity. Taken together, our data suggests an involvement of GR condensates in transcription initiation and elongation.

Whether phase-separation is involved in the organization of the transcriptional machinery and if it aids or inhibits the transcriptional process is a matter of intense debate. In this Mini Review, we will cover the current knowledge regarding the role of transcriptional condensates on gene expression regulation. We will summarize the latest discoveries on the relationship between condensate formation, genome organization, and transcriptional activity, focusing on the strengths and weaknesses of the experimental approaches used to interrogate these aspects of transcription in living cells. Finally, we will discuss the challenges for future research.