Biology subjects

Fatti, E.

Publications and source records attributed to Fatti, E..

4 recordsLinked to original sources

bioRxiv2026

Non-contact direct sensing of material properties of biomolecular condensate using Scanning Ionic Conductance Microscopy

Biomolecular condensates are important regulators of cellular compartmentalization and biochemical processes. Understanding their material properties is critical to elucidate how they control molecular organization and dynamics within cells. However, quantitatively probing these properties remains challenging due to the wide range of length scales, concentrations, and timescales over which condensates operate, as well as the limited force ranges accessible to current nanoscale mechanical mapping methods. We explored the use of a non-contact 3D imaging tool Scanning Ion Conductance Microscopy (SICM) for stiffness measurements of liquid-liquid phase-separated biomolecular condensates. We focus on the Dhh1 protein, which is a regulator of cytoplasmic processing bodies (PBs) membrane-less cytoplasmic condensates that control the storage and degradation of untranslated mRNA. In our study, we investigate the properties of mCherry2- or His-mCherry2-tagged full-length Dhh1 and N- or C-terminus tail-deletion constructs, as well as the catalytically inactive mutant DQAD, under different pH and incubation times. We mapped both spatial and temporal changes in the material properties of the condensates, highlighting the capabilities of the instrument. We found that the removal of either of the two tails led to an increase in condensate stiffness upon shifting the pH from a stress-associated cellular environment (pH 6.5) to physiological conditions (pH 7.5). Additionally, the choice of protein tags led to vastly different results depending on the pH where mCherry2-Dhh1 exhibited a stiffening going from pH 6.0 to 6.5 while the double-tagged His-mCherry2 did not. Our measurements are verified and corroborated by established techniques such as optical tweezer-based fusion assays and fluorescence recovery after photobleaching (FRAP). Furthermore, we were able to track the same biomolecular condensate sample for up to 20 days getting insights on the ageing and evolution of the condensates. Overall, our study demonstrates the applicability of SICM for direct measurement of the material properties of biomolecular condensate.

biophysics↗

bioRxiv2025

The Heterogeneous Solution Ensemble of the DEAD-box Protein Dhh1 Reveals a Modular Architecture

DEAD-box proteins such as Dhh1 play essential roles in RNA metabolism and the formation of biomolecular condensates, with a modular architecture comprising folded domains and disordered regions. To elucidate how this architecture shapes conformational dynamics in solution, we combined solution scattering experiments and multi-scale simulations on core and full-length constructs. Enhanced-sampling simulations captured a dynamic ensemble of core conformations stabilized by transient interdomain contacts that underpin functional regulation. Coarse-grained modelling revealed that disordered tails behave as independent modules exerting minimal influence on core dynamics. This integrated approach reveals a modular organization balancing structural heterogeneity and functional specificity, providing a framework for studying DEAD-box proteins in phase separation.

biophysics↗

bioRxiv2024

The dark side of fluorescent protein tagging: the impact ofprotein tags on biomolecular condensation

Biomolecular condensation has emerged as an important mechanism to control various cellular processes through the formation of membraneless organelles. Fluorescent protein tags have been extensively used to study the formation and the properties of condensates in vitro and in vivo, but there is evidence that tags may perturb the condensation properties of proteins. In this study, we carefully assess the effects of protein tags on the yeast DEAD-box ATPase Dhh1, a central regulator of processing bodies (P-bodies), which are biomolecular condensates involved in mRNA metabolism. We show that fluorescent tags as well as a poly-histidine tag greatly affect Dhh1 condensation in vitro and lead to condensates with different dynamic properties. Tagging of Dhh1 with various fluorescent proteins in vivo alters the number of P-bodies upon glucose starvation and some tags even show constitutive P-bodies in non-stressed cells. These data raise concerns about the accuracy of tagged protein condensation experiments, highlighting the need for caution when interpreting the results. Significance StatementO_LIFluorescent tags are extensively used in protein condensation studies although their effect in condensate dynamics has not been carefully investigated. C_LIO_LITags affect the condensation propensity and dynamics of Dhh1 in vitro and P-body numbers in vivo. C_LIO_LITags may generally alter the behavior of proteins in biomolecular condensates and their use needs to be carefully evaluated and controlled. C_LI

cell biology↗

bioRxiv2021

DEAD-box RNA Helicases Act as Nucleotide Exchange Factors for Casein Kinase 2

DDX RNA helicases promote RNA processing but DDX3X is also known to activate casein kinase 1 {varepsilon} (CK1{varepsilon}). Here we show that not only is protein kinase stimulation a latent property of other DDX proteins towards CK1{varepsilon}, but that this extends to casein kinase 2 (CK22) as well. CK22 enzymatic activity is stimulated by a variety of DDX proteins and we identify DDX1/24/41/54 as physiological activators required for full kinase activity in vitro and in Xenopus embryos. Mutational analysis of DDX3X reveals that CK1 and CK2 kinase stimulation engages its RNA binding-but not catalytic motifs. Mathematical modelling of enzyme kinetics and stopped-flow spectroscopy converge that DDX proteins function as nucleotide exchange factor towards CK22 that reduce unproductive reaction intermediates and substrate inhibition. Our study reveals protein kinase stimulation by nucleotide exchange as a new principle in kinase regulation and an evolved function of DDX proteins.

biochemistry↗