Biology subjects

Alberti, S.

Publications and source records attributed to Alberti, S..

31 records · Page 2Linked to original sources

bioRxiv2020

Quantitative phase microscopy enables precise and efficient determination of biomolecular condensate composition

Biomolecular condensates are membrane-less organelles made of multiple components, often including several distinct proteins and nucleic acids. However, current tools to measure condensate composition are limited and cannot capture this complexity quantitatively, as they either require fluorescent labels, which we show can perturb composition, or can distinguish only 1-2 components. Here, we describe a label-free method based on quantitative phase microscopy to measure the composition of condensates with an arbitrarily large number of components. We first validate the method empirically in binary mixtures, revealing sequence-encoded density variation and complex aging dynamics for condensates composed of full-length proteins. In simplified multi-component protein/RNA condensates, we uncover a regime of constant condensate density and a large range of protein:RNA stoichiometry when varying average composition. The unexpected decoupling of density and composition highlights the need to determine molecular stoichiometry in multi-component condensates. We foresee this approach enabling the study of compositional regulation of condensate properties and function.

biophysics↗

bioRxiv2020

Rapid characterisation of protein phase behaviour using droplet microfluidics

The assembly of intracellular proteins into biomolecular condensates is a fundamental process underlying the organisation of intracellular space and the regulation of many cellular processes. Mapping and characterising phase behaviour of biomolecules is essential to understand the mechanisms of condensate assembly, and to develop therapeutic strategies targeting biomolecular condensate systems. A central concept for characterising phase-separating systems is the phase diagram. Phase diagrams are typically built from numerous individual measurements sampling different parts of the parameter space. However, even when performed in microwell plate format, this process is slow, low throughput and requires significant sample consumption. To address this challenge, we present here a combinatorial droplet microfluidic platform, termed PhaseScan, for rapid and high-resolution acquisition of multidimensional biomolecular phase diagrams. Using this platform, we characterise the phase behaviour of a wide range of systems under a variety of conditions and demonstrate that this approach allows the quantitative characterisation of the effect of small molecules on biomolecular phase transitions.

biophysics↗

bioRxiv2020

Reentrant liquid condensate phase of proteins is stabilized by hydrophobic and non-ionic interactions

Many cellular proteins demix spontaneously from solution to form liquid condensates. These phase-separated systems have wide-ranging roles in health and disease. Elucidating the molecular driving forces underlying liquid-liquid phase separation (LLPS) is therefore a key objective for understanding biological function and malfunction. Here we show that proteins implicated in cellular LLPS, including FUS, TDP-43, Brd4, Sox2, and Annexin A11, which form condensates at low salt concentrations, can reenter a phase-separated regime at high salt concentrations. By bringing together experiments and simulations, we demonstrate that phase separation in the high-salt regime is driven by hydrophobic and non-ionic interactions, and is mechanistically distinct from the low-salt regime, where condensates are additionally stabilized by electrostatic forces. Our work thus provides a new view on the cooperation of hydrophobicity and non-ionic interactions as non-specific driving forces for the condensation process, with important implications for aberrant function, druggability, and material properties of biomolecular condensates.

biophysics↗

bioRxiv2020

Single particle zeta-potential measurements reveal the role of electrostatics in protein condensate stability

Liquid-liquid phase separation underlies the formation of biological condensates. Physically, such systems are microemulsions which have a general propensity to fuse and coalesce; however, many condensates persist as independent droplets inside cells. This stability is crucial for their functioning, but the physicochemical mechanisms that control the emulsion stability of condensates remain poorly understood. Here, by combining single-condensate zeta potential measurements, optical microscopy, tweezer experiments, and multiscale molecular modelling, we investigate how the forces that sustain condensates impact their stability against fusion. By comparing PR25:PolyU and FUS condensates, we show that a higher condensate surface charge correlates with a lower fusion propensity, and that this behavior can be inferred from their zeta potentials. We reveal that overall stabilization against fusion stems from a combination of repulsive forces between condensates and the effects that surface electrostatics have on lowering surface tension, thus shedding light on the molecular determinants of condensate coalescence.

biophysics↗

bioRxiv2020

Biomolecular condensates undergo a generic shear-mediated liquid-to-solid transition

A wide range of systems containing proteins have been shown to undergo liquid-liquid phase separation (LLPS) forming membraneless compartments, such as processing bodies1, germ granules2, stress granules3 and Cajal bodies4. The condensates resulting from this phase transition control essential cell functions, including mRNA regulation, cytoplasm structuring, cell signalling and embryogenesis1-4. RNA-binding Fused in Sarcoma (FUS) protein is one of the most studied systems in this context, due to its important role in neurodegenerative diseases5-7. It has recently been discovered that FUS condensates can undergo an irreversible phase transition which results in fibrous aggregate formation6. Gelation of protein condensates is generally associated with pathology. One case where liquid-to-solid transition (LST) of liquid-liquid phase separated proteins is functional, however, is that of silk spinning8,9, which is largely driven by shear, but it is not known what factors control the pathological gelation of functional condensates. Here we show that four proteins and one peptide system not related to silk, and with no function associated with fibre formation, have a strong propensity to undergo LST when exposed to even low levels of mechanical shear comparable to those found inside a living cell, once present in their liquid-liquid phase separated forms. Using microfluidics to control the application of mechanical shear, we generated fibres from single protein condensates and characterized their structures and material properties as a function of shear stress. Our results inform on the molecular grammar underlying protein LST and highlight generic backbone-backbone hydrogen bonding constraints as a determining factor in governing this transition. Taken together, these observations suggest that the shear plays an important role in the irreversible phase transition of liquid-liquid phase separated droplets, shed light on the role of physical factors in driving this transition in protein aggregation related diseases, and open a new route towards artificial shear responsive biomaterials.

biophysics↗

bioRxiv2019

Protein products of non-stop mRNA disrupt nucleolar homeostasis

Mutations that cause ribosome stalling or impair the cells protective response to stalling have been demonstrated to cause neurodegeneration, yet the mechanisms underlying these pathologies remain poorly understood. Here we investigated the fate of defective proteins translated from stall-inducing, nonstop mRNA that escape ubiquitylation by the Ribosome-associated Quality Control (RQC) E3 ligase LTN1. We found that nonstop protein products accumulated in nucleoli and this localization was driven by polylysine tracts produced by translation of the poly(A) tail of nonstop mRNA. Nucleolar sequestration increased the solubility of invading proteins but disrupted nucleoli, altering their dynamics, morphology, and resistance to stress. Changes in nucleolar morphology are consistent with a simple physical model in which LTN1 impairment enhances the inter-molecular interactions of nucleolar components. Our work elucidates how failure to degrade the protein products of stalled translation may affect distal cellular processes and will inform studies on the pathology of neurodegenerative disease.

cell biology↗

bioRxiv2019

Loss of ELK1 has differential effects on age-dependent organ fibrosis and integrin expression

ETS domain-containing protein-1 (ELK1) is a transcriptional repressor important in regulating v{beta}6 integrin expression. v{beta}6 integrins activate the profibrotic cytokine Transforming Growth Factor {beta}1 (TGF{beta}1) and are increased in the alveolar epithelium in Idiopathic Pulmonary Fibrosis (IPF). IPF is a disease associated with ageing and therefore we hypothesised that aged animals lacking Elk1 globally would develop spontaneous fibrosis in organs where v{beta}6-mediated TGF{beta} activation has been implicated.\n\nHere we identify that Elk1-knockout (Elk1-/0) mice aged to one year developed spontaneous fibrosis in the absence of injury in both the lung and the liver but not in the heart or kidneys. The lungs of Elk1-/0 aged mice demonstrated increased collagen deposition, in particular collagen 31, located in small fibrotic foci and thickened alveolar walls. Despite the liver having relatively low global levels of ELK1 expression, Elk1-/0 animals developed hepatosteatosis and fibrosis. The loss of Elk1 also had differential effects on Itgb1, Itgb5 and Itgb6 genes expression in the four organs potentially explaining the phenotypic differences in these organs. To understand the potential causes of reduced ELK1 in human disease we exposed human cells and murine lung slices to cigarette smoke extract which lead to reduced ELK1 expression which may explain the loss of ELK1 in human disease.\n\nThese data support a fundamental role for ELK1 in protecting against the development of progressive fibrosis via transcriptional regulation of beta integrin subunit genes, and demonstrate that loss of ELK1 can be caused by cigarette smoke.

pathology↗

bioRxiv2019

Small molecules for modulating protein driven liquid-liquid phase separation in treating neurodegenerative disease

Neurodegenerave diseases such as amyotrophic lateral sclerosis (ALS) are oten associated with mutaons in proteins that are associated with stress granules. Stress granules are condensates formed by liquid-liquid phase separaon which, when aberrant, can lead to altered condensaon behaviours and disease phenotypes. Here, we identified lipoamide, a small molecule which specifically prevents cytoplasmic condensaon of stress granule proteins. Thermal proteome profiling showed that lipoamide preferentially stabilises intrinsically disordered domain-containing proteins. These include SRSF1 and SFPQ, stress granule proteins necessary for lipoamide activity. The redox state of SFPQ correlates with its condensate-dissolving behaviour, in concordance with the importance of the dithiolane ring for lipoamide activity. In animals, lipoamide ameliorates aging-associated aggregaon of a stress granule reporter, improves neuronal morphology, and recovers motor defects caused by expression of ALS-associated FUS and TDP-43 mutants. In conclusion, lipoamide is a well-tolerated small molecule modulator of stress granule condensaon and dissection of its molecular mechanism identified a cellular pathway for redox regulaon of stress granule formaon.

cell biology↗

bioRxiv2019

Proteome-wide signatures of function in highly diverged intrinsically disordered regions

Intrinsically disordered regions make up a large part of the proteome, but the sequence-to-function relationship in these regions is poorly understood, in part because the primary amino acid sequences of these regions are poorly conserved in alignments. Here we use an evolutionary approach to detect molecular features that are preserved in the amino acid sequences of orthologous intrinsically disordered regions. We find that most disordered regions contain multiple molecular features that are preserved, and we define these as \"evolutionary signatures\" of disordered regions. We demonstrate that intrinsically disordered regions with similar evolutionary signatures can rescue function in vivo, and that groups of intrinsically disordered regions with similar evolutionary signatures are strongly enriched for functional annotations and phenotypes. We propose that evolutionary signatures can be used to predict function for many disordered regions from their amino acid sequences.

genomics↗

bioRxiv2018

Filament formation by the translation factor eIF2B regulates protein synthesis in starved cells

Cells exposed to starvation have to adjust their metabolism to conserve energy and protect themselves. Protein synthesis is one of the major energy-consuming processes and as such has to be tightly controlled. The mechanism by which starved cells regulate the process of protein synthesis is largely unknown. Here, we report that the essential translation initiation factor eIF2B forms filaments in starved budding yeast cells. We demonstrate that filamentation is triggered by starvation-induced acidification of the cytosol, which is caused by an influx of protons from the extracellular environment. We show that filament assembly by eIF2B is necessary for rapid and efficient downregulation of translation. Importantly, this mechanism does not require the kinase Gcn2. Furthermore, analysis of site-specific variants of eIF2B suggests that eIF2B assembly results in enzymatically inactive filaments that promote stress survival and fast recovery of cells from starvation. We propose that translation regulation through protein assembly is a widespread mechanism that allows cells to adapt to fluctuating environments.

cell biology↗

bioRxiv2018

Adaptive reorganization of the cytoplasm upon stress in budding yeast

Yeast cells, when exposed to stress, can enter a protective state in which cell division, growth and metabolism are downregulated. They remain viable in this state until nutrients become available again. How cells enter this protective survival state and what happens at a cellular and subcellular level is largely unknown. In this study, we used electron tomography to investigate the stress-induced ultrastructural changes in the cytoplasm of yeast cells. After ATP depletion, we observed a significant cytosolic compaction and an extensive cytoplasmic reorganization, as well as the emergence of distinct membrane-bound and membrane-less organelles. By using correlative light and electron microscopy (CLEM), we further demonstrate that one of these membrane-less organelles is generated by the reversible polymerization into large bundles of filaments of the eukaryotic translation initiation factor 2B (eIF2B), an essential enzyme in the initiation of protein synthesis. The changes we observe are part of a stress-induced survival strategy, allowing yeast cells to save energy, protect proteins from degradation, and inhibit protein functionality by forming assemblies of said proteins.

cell biology↗

bioRxiv2018

Intracellular mass density increase is accompanying but not sufficient for stiffening and growth arrest of yeast cells

Many organisms, including yeast cells, bacteria, nematodes and tardigrades, endure harsh environmental conditions, such as nutrient scarcity, or lack of water and energy for a remarkably long time. The rescue programs that these organisms launch upon encountering these adverse conditions include reprogramming their metabolism in order to enter a quiescent or dormant state in a controlled fashion. Reprogramming coincides with changes in the macromolecular architecture and changes in the physical and mechanical properties of the cells. However, the cellular mechanisms underlying the physical-mechanical changes remain enigmatic. Here, we induce metabolic arrest of yeast cells by lowering their intracellular pH. We then determine the differences in the intracellular mass density and stiffness of active and metabolically arrested cells using optical diffraction tomography and atomic force microscopy. We show that an increased intracellular mass density is associated with an increase in stiffness when the growth of yeast is arrested. However, increasing the intracellular mass density alone is not sufficient for maintenance of the growth-arrested state in yeast cells. Our data suggest that the cytoplasm of metabolically arrested yeast displays characteristics of solid. Our findings constitute a bridge between the mechanical behavior of the cytoplasm and the physical and chemical mechanisms of metabolically arrested cells with the ultimate aim of understanding dormant organisms.

biophysics↗

bioRxiv2017

Local Nucleation Of Microtubule Bundles Through Tubulin Concentration Into A Condensed Tau Phase

Non-centrosomal microtubule bundles play important roles in cellular organization and function. Although many diverse proteins are known that can bundle microtubules, biochemical mechanisms by which cells could locally control the nucleation and formation of microtubule bundles are understudied. Here, we demonstrate that concentration of tubulin into a condensed, liquid-like compartment composed of the unstructured neuronal protein tau is sufficient to nucleate microtubule bundles. We show that under conditions of macro-molecular crowding, tau forms liquid drops. Tubulin partitions into these drops, efficiently increasing tubulin concentration and driving the nucleation of microtubules. These growing microtubules form bundles enclosed in a liquid sheath of tau. Our data suggest that condensed compartments of microtubule bundling proteins could promote the local formation of microtubule bundles in neurons by acting as non-centrosomal microtubule nucleation centers, and that liquid-like tau encapsulation could provide both stability and plasticity to long axonal microtubule bundles.

cell biology↗