Biology subjects

Mangiarotti, A.

Publications and source records attributed to Mangiarotti, A..

9 recordsLinked to original sources

bioRxiv2025

Integrating experiments and simulations to unravel coacervate-membrane interactions: Insights into de-mixing and morphology modulation

Intrinsically disordered proteins and polypeptides can undergo liquid-liquid phase separation (LLPS) to form condensates/coacervates, which play numerous regulatory roles in the cell. Recently, the relevance of such LLPS occurring in the vicinity of membranes has been brought to light by several experimental studies. Membrane-adsorbed condensates are crucial for biomolecular localization, and in some cases, phase separation of proteins at the membrane surface induces significant changes in membrane morphology. A detailed microscopic understanding of the mechanisms behind these observations remains incomplete. Here we combine experiments and molecular simulations to unravel structural and dynamic features of the coacervate/membrane interface across scales. We study poly-Lysine/poly-Aspartate (K10/D10) coacervates as a prototype of phase-separated condensates with different unilamellar liposomes. Using a multiscale characterization approach that combines confocal microscopy, hyperspectral imaging, fluorescence recovery after photobleaching, and two complementary coarse-grained approaches, we show that the membrane-condensate affinity can be tuned by the anionic lipid content and quantified through the intrinsic contact angle - a material property derived from system geometry - both in vitro and in silico. We find that the membrane region in contact with the condensate displays a nearly two-fold reduced fluidity compared to the bare membrane. This is attributed to orientational ordering of lipid tails, resulting in decreased area per lipid. Moreover, we observed local lipid de-mixing induced by the coacervate adsorption. This study provides an effective framework for integrating experiment and computation to characterize the properties of coacervate/membrane interfaces that are critical to the functional impacts of these interactions.

biophysics↗

bioRxiv2025

Fluorescence-based mapping of dielectric permittivity of condensates and their environment

Biomolecular condensates, essential for cellular organization, possess mesoscale properties largely governed by hydrophobicity, influencing molecule partitioning and material characteristics like viscosity, surface tension, and hydration. While hydrophobicitys role is increasingly recognized, its impact on membrane-condensate interactions remains unexplored. Here, we introduce a novel approach, combining hyperspectral imaging of an environment-sensitive dye and phasor analysis, to quantitatively map the local dielectric permittivity of both condensates and their environment with pixel resolution as sensed by the dye. This robust method reveals a surprisingly broad range of condensate permittivities, from oil-like to water-like. Importantly, we uncover that membrane affinity is not dictated by condensate permittivity itself, but by the permittivity contrast with their surroundings. Indeed, membrane wetting affinity is found to scale linearly with this contrast, unveiling a unifying dielectric principle governing condensate-membrane interactions. Compatible with live-cell and in vitro imaging, this technique provides unprecedented insights into condensate biophysics and function and opens new avenues for studying biomolecular condensate biology.

biophysics↗

bioRxiv2025

Peptide-induced hydration of lipid bilayers modulates packing pattern and conformations of hydrocarbon chains - a potential pathway for peptide translocation?

Cell-penetrating peptides (CPPs) with a cationic-hydrophobic character are recognized as carriers for delivering various therapeutics and diagnostic agents across cell membranes and into the cells. Among the most studied CPPs, nona-arginine (R9) exhibits superior penetration compared to nona-lysine (K9), suggesting that the penetration ability depends not only on charge, distribution and concentration of peptides but also on the lipid membrane composition. However, for heptapeptides composed of arginine (R), lysine (K) and phenylalanine (F) residues, which show some CPPs properties, these interactions remain unexplored. This study sheds light on the adsorption of R5F2/K5F2 on model prokaryotic (PRO) and eukaryotic (EU) lipid membranes containing a zwitterionic lipid (phosphatidylcholine; PC) and an anionic lipid (either phosphatidylglycerol, PG, in the PRO model, or phosphatidylserine, PS in EU) at the 90:10 molar ratio. Using differential scanning calorimetry (DSC) and temperature-dependent UV-Vis spectroscopy, we observed peptide-induced changes in the interfacial water layer that affect the fluidity and rigidity of lipid bilayers. The distinct adsorption behavior of R5F2/K5F2 on PRO and EU lipid bilayers revealed the changes in lipid packing and hydrocarbon chain conformations as exclusively peptide-dependent features. The peptide-induced formation of vacancies in the non-polar bilayer part is consistent with partial leakage observed in giant unilamellar vesicles. The synchronized arrangement could represent a mechanism for the concerted translocation of CPPs, along with their potential cargo across the lipid membrane. This study provides new insights into the peptide-lipid interactions underlying CPPs functionality.

biophysics↗

bioRxiv2024

Lipid packing and cholesterol content regulate membrane wetting by biomolecular condensates.

Biomolecular condensates play a pivotal role in cellular processes by interacting with membranes and leading to wetting transitions and to mutual remodeling. Using a combination of hyperspectral imaging, phasor analysis, and fluid-elastic parameter measurements, we investigated how membrane lipid packing affects condensate wetting. Our results show that it is not only the membrane phase state, but rather the degree of lipid packing that determines the condensate affinity for membranes. Increasing lipid chain length or cholesterol content enhances lipid packing, thereby decreasing condensate affinity. This regulatory mechanism is consistent across various condensate-membrane systems, underscoring the critical role of the membrane interface. Additionally, protein adsorption promotes extensive membrane remodeling, including tube and double-membrane sheet formation. This work provides a novel mechanism by which membrane composition fine-tunes condensate wetting, highlighting its potential impact on cellular functions and organelle interactions.

biophysics↗

bioRxiv2024

Extracellular vesicle mobility in collagen I hydrogels is modulated by RGD-binding integrins

Extracellular vesicles (EVs) are a diverse population of membrane structures produced and released by cells into the extracellular space for the intercellular trafficking of cargo molecules. They are implicated in various biological processes, including angiogenesis, immunomodulation, and cancer cell signaling. While much research has focused on their biogenesis or their effects on recipient cells, less is understood about how EVs are capable of traversing diverse tissue environments and crossing biological barriers. Their interactions with extracellular matrix components are of particular interest, as such interactions govern diffusivity and mobility, providing a potential basis for organotropism. To start to untangle how EV-matrix interactions affect diffusivity, we use highspeed epifluorescence microscopy, single particle tracking, and confocal reflectance microscopy to analyze particle mobility and localization in extracellular matrix-mimicking hydrogels composed of collagen I. EVs are compared with synthetic liposomes and extruded plasma membrane vesicles to better understand the importance of membrane composition on these interactions. By treating EVs with trypsin to digest surface proteins, we determine that proteins are primarily responsible for EV immobilization in collagen I hydrogels. We next use a synthetic peptide competitive inhibitor to narrow down the identity of the proteins involved to argynylglycylaspartic acid (RGD) motif-binding integrins, which interact with unincorporated or denatured non-fibrillar collagen. Moreover, the effect of integrin inhibition with RGD peptides has strong implications for the use of RGD-peptide-based drugs to treat certain cancers, as integrin inhibition appears to increase EV mobility, improving their ability to infiltrate tissue-like environments.

biophysics↗

bioRxiv2024

Photoswitchable endocytosis of biomolecular condensates in giant vesicles

Interactions between membranes and biomolecular condensates can give rise to complex phenomena such as wetting transitions, mutual remodeling, and endocytosis. In this study, we demonstrate a light-triggered manipulation of condensate engulfment using giant vesicles containing photoswitchable lipids. UV irradiation increases the membrane area, facilitating a rapid condensate endocytosis, which can be reverted by blue light. The affinity of the protein-rich condensates to the membrane and the reversibility of the engulfment processes is quantified from confocal microscopy images. The degree of engulfment, whether partial or complete, depends on the initial membrane excess area and the relative sizes of vesicles and condensates. Theoretical estimates suggest that utilizing the light-induced excess area to increase the vesicles-condensate adhesion interface is energetically more favorable than the energy gain from folding the membrane into invaginations and tubes. Our overall findings demonstrate that membrane-condensate interactions can be easily and quickly modulated via light, providing a versatile system for building platforms to control cellular events and design intelligent drug delivery systems for cell repair.

biophysics↗

bioRxiv2023

Wetting by biomolecular condensates increases membrane lipid packing and dehydration

Membrane wetting by biomolecular condensates recently emerged as a key phenomenon in cell biology, playing an important role in a diverse range of processes across different organisms. However, an understanding of the molecular mechanisms behind condensate formation and interaction with lipid membranes is still missing. To study this, we exploited the properties of the dyes ACDAN and LAURDAN as nano-environmental sensors in combination with phasor analysis of hyperspectral and lifetime imaging microscopy. Using glycinin as a model condensate-forming protein and giant vesicles as model membranes, we obtained vital information on the process of condensate formation and membrane wetting. Our results reveal that glycinin condensates display differences in water dynamics when changing the salinity of the medium as a consequence of rearrangements in the secondary structure of the protein. Remarkably, analysis of membrane-condensates interaction with protein as well as polymer condensates indicated a correlation between increased wetting affinity and enhanced lipid packing. This is demonstrated by a decrease in the dipolar relaxation of water across all membrane-condensate systems, suggesting a general mechanism to tune membrane packing by condensate wetting.

biophysics↗

bioRxiv2022

Curli fibers in Escherichia coli biofilms: the influence of water availability on amyloid structure and properties

E. coli biofilms consist of bacteria embedded in a self-produced matrix mainly made of protein fibers and polysaccharides. The curli amyloid fibers found in the biofilm matrix are promising versatile building blocks to design sustainable bio-sourced materials. To exploit this potential, it is crucial to understand i) how environmental cues during biofilm growth influence the molecular structure of these amyloid fibers, and ii) how this translates at higher length scales. To explore these questions, we studied the effect of water availability during biofilm growth on the conformation and functions of curli. We used microscopy and spectroscopy to characterize the amyloid fibers purified from biofilms grown on nutritive substrates with different water contents, and micro-indentation to measure the rigidity of the respective biofilms. The purified curli amyloid fibers present differences in the yield, structure and functional properties upon biofilm growth conditions. Fiber packing and {beta}-sheets content correlate with their hydrophobicity and chemical stability, and with the rigidity of the biofilms. Our study highlights how E. coli biofilm growth conditions impact curli structure and functions contributing to macroscopic materials properties. These fundamental findings infer an alternative strategy to tune curli structure, which will ultimately benefit to engineer hierarchical and functional curli-based materials. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=169 SRC="FIGDIR/small/517345v3_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@124ddd2org.highwire.dtl.DTLVardef@15f234dorg.highwire.dtl.DTLVardef@106a65forg.highwire.dtl.DTLVardef@194c53f_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2022

Membrane wetting, molding and reticulation by protein condensates

Cells compartmentalize their components in liquid-like condensates, which can be reconstituted in vitro. Although these condensates interact with membrane-bound organelles, the potential of membrane remodeling and the underlying mechanisms are not well understood. Here, we demonstrate that interactions between protein condensates (including hollow ones) and membranes can lead to remarkable morphological transformations and describe these with theory. Modulation of solution salinity or membrane composition drives the condensate-membrane system through two wetting transitions, from dewetting, through a broad regime of partial wetting, to complete wetting. A new phenomenon, namely fingering or ruffling of the condensate-membrane interface is observed when sufficient membrane area is available, producing intricately curved structures. The observed morphologies are governed by the interplay of adhesion, membrane elasticity, and interfacial tension. Our results highlight the relevance of wetting in cell biology, and pave the way for the design of synthetic membrane-droplet based biomaterials and compartments with tunable properties.

biophysics↗