Biology subjects

Belardi, B.

Publications and source records attributed to Belardi, B..

10 recordsLinked to original sources

bioRxiv2025

A balance between nucleating and elongating actin filaments controls deformation of protein condensates

Protein condensates use multivalent binding and surface tension to assemble actin filaments into diverse architectures, reminiscent of filopodia and stress fibers. During this process, nucleation of new filaments and elongation of existing filaments inherently compete for a shared pool of actin monomers. Here we show that a balance between these competing processes is required to deform condensates of VASP, an actin binding protein, into structures with high aspect ratios. Addition of magnesium, which promotes filament nucleation, helped actin to deform condensates into high aspect ratio structures. In contrast, addition of profilin, which inhibits filament nucleation, allowing existing filaments to elongate, caused actin to assemble into ring-like bundles that failed to substantially increase condensate aspect ratio. Computational modeling helped to explain these results by predicting that a group of short linear filaments, which can apply asymmetric pressure to the condensate boundary, is needed to increase condensate aspect ratio. In contrast, a small number of long filaments with the same total actin content should fail to overcome the droplet surface tension, forming a ring-like bundle. To test these predictions, we introduced gelsolin, which severed log filaments within rings, creating new barbed ends. The resulting set of shorter filaments regained the ability to deform condensates into high aspect ratio structures. Collectively, these results suggest that a balance of actin filament nucleation and elongation is required to deform protein condensates. More broadly, these findings illustrate how protein condensates can balance multiple kinetic processes to direct the assembly of diverse cytoskeletal architectures.

biophysics↗

bioRxiv2025

Light-Controlled Synthetic Communication Networks via Paired Connexon Nanopores

Living cells employ dynamic networks for intercellular communication and cooperation, leading to tissue-wide activity. One emerging challenge in the field of bottom-up synthetic biology is emulating such sophisticated behaviors in liposome-based synthetic cells (SCs). Fabricating communication networks in lipid bilayer-based SCs remains a challenge as signaling molecules must transit through two consecutive membranes to transfer information between different SCs. Here, we address this obstacle by engineering connexin channels that directly connect the lumens of adhering SC membranes. We focus on orthogonal channel-forming connexins, namely connexin 43 and connexin 32, and re-design their channel activity to be UV- and near IR-responsive, respectively. By combining engineered connexins into a single SC assembly, we demonstrate orthogonal transfer of reactive signaling molecules between SCs, giving rise to unique reaction products and network states in a wavelength-dependent manner - an important step toward synthetic communication networks.

synthetic biology↗

bioRxiv2023

Turn-On Protein Switches for Controlling Actin Binding in Cells

Within a shared cytoplasm, filamentous actin (F-actin) plays numerous and critical roles across the cell body. Cells rely on actin-binding proteins (ABPs) to organize F-actin and to integrate its polymeric characteristics into diverse cellular processes. Yet, the multitude of ABPs that engage with and shape F-actin make studying a single ABPs influence on cellular activities a significant challenge. Moreover, without a means of manipulating actin-binding subcellularly, harnessing the F-actin cytoskeleton for synthetic biology purposes remains elusive. Here, we describe a suite of designed proteins, Controllable Actin-binding Switch Tools (CASTs), whose actin-binding behavior can be controlled with external stimuli. CASTs were developed that respond to different external inputs, providing options for turn-on kinetics and enabling orthogonality. Being genetically encoded, we show that CASTs can be inserted into native protein sequences to control F-actin association locally and engineered into new structures to control cell and tissue shape and behavior.

synthetic biology↗

bioRxiv2022

Light-Activated Assembly of Connexon Nanopores in Synthetic Cells

During developmental processes and wound healing, activation of living cells occurs with spatiotemporal precision and leads to rapid release of soluble molecular signals, allowing communication and coordination between neighbors. Non-living systems capable of similar responsive release hold great promise for information transfer in materials and site-specific drug delivery. One non-living system that offers a tunable platform for programming release is synthetic cells. Encased in a lipid bilayer structure, synthetic cells can be outfitted with molecular conduits that span the bilayer and lead to material exchange. While previous work expressing membrane pore proteins in synthetic cells demonstrated content exchange, user-defined control over release has remained elusive. In mammalian cells, connexon nanopore structures drive content release and have garnered significant interest since they can direct material exchange through intercellular contacts. Here, we focus on connexon nanopores and present activated release of material from synthetic cells in a light-sensitive fashion. To do this, we re-engineer connexon nanopores to assemble after post-translational processing by a protease. By encapsulating proteases in light-sensitive liposomes, we show that assembly of nanopores can be triggered by illumination, resulting in rapid release of molecules encapsulated within synthetic cells. Controlling connexin nanopore activity provides an opportunity for initiating communication with extracellular signals and for transferring molecular agents to the cytoplasm of living cells in a rapid, light-guided manner.

synthetic biology↗

bioRxiv2021

Extracellular Electron Transfer Enables Cellular Control of Cu(I)-catalyzed Alkyne-Azide Cycloaddition

Extracellular electron transfer (EET) is an anaerobic respiration process that couples carbon oxidation to the reduction of metal species. In the presence of a suitable metal catalyst, EET allows for cellular metabolism to control a variety of synthetic transformations. Here, we report the use of EET from the model electroactive bacterium Shewanella oneidensis for metabolic and genetic control over Cu(I)-catalyzed Alkyne-Azide Cycloaddition (CuAAC). CuAAC conversion under anaerobic and aerobic conditions was dependent on live, actively respiring S. oneidensis cells. In addition, reaction progress and kinetics could be further manipulated by tailoring the central carbon metabolism of S. oneidensis. Similarly, CuAAC activity was dependent on specific EET pathways and could be manipulated using inducible genetic circuits controlling the expression of EET-relevant proteins including MtrC, MtrA, and CymA. EET-driven CuAAC also exhibited modularity and robustness in ligand tolerance and substrate scope. Furthermore, the living nature of this system could be exploited to perform multiple reaction cycles without requiring regeneration, something inaccessible to traditional chemical reductants. Finally, S. oneidensis enabled bioorthogonal CuAAC membrane labelling on live mammalian cells without affecting cell viability, suggesting that S. oneidensis can act as a dynamically tunable biocatalyst in complex environments. In summary, our results demonstrate how EET can expand the reaction scope available to living systems by enabling cellular control of CuAAC.

bioengineering↗

bioRxiv2020

Biased localization of actin binding proteins by actin filament conformation

The assembly of actin filaments into distinct cytoskeletal structures plays a critical role in cell physiology, but how proteins localize differentially to these structures within a shared cytoplasm remains unclear. Here, we show that the actin-binding domains of accessory proteins can be sensitive to filament conformational changes. Using a combination of live cell imaging and in vitro single molecule binding measurements, we show that tandem calponin homology domains (CH1-CH2) can be mutated to preferentially bind actin networks at the front or rear of motile cells, and we demonstrate that the affinity of CH1-CH2 domain mutants varies as actin filament conformation is altered by perturbations that include stabilizing drugs, physical constraints, and other binding proteins. These findings suggest that conformational heterogeneity of actin filaments in cells could help to direct accessory binding proteins to different actin cytoskeletal structures through a biophysical feedback loop.

biophysics↗

bioRxiv2020

Molecular height measurement by cell surface optical profilometry (CSOP)

The physical dimensions of proteins and glycans on cell surfaces can critically affect cell function, for example by preventing close contact between cells and limiting receptor accessibility. However, high-resolution measurements of molecular heights on native cell membranes have been difficult to obtain. Here we present a simple and rapid method that achieves nanometer height resolution by localizing fluorophores at the tip and base of cell surface molecules and determining their separation by radially averaging across many molecules. We use this method, which we call cell surface optical profilometry (CSOP), to quantify height of key multi-domain proteins on a model macrophage and cancer cell, as well as to capture average protein and glycan heights on native cell membranes. We show that average height of a protein is significantly smaller than its contour length due to thermally driven bending and rotation on the membrane and that height strongly depends on local surface and solution conditions. We find that average height increases with cell surface molecular crowding, while it decreases with solution crowding by solutes, both of which we confirm with molecular dynamics simulations. We also use experiments and simulations to determine the height of an epitope based on the location of an antibody, which allows CSOP to profile various proteins and glycans on a native cell surface using antibodies and lectins. This versatile method for profiling cell surfaces has the potential to advance understanding of the molecular landscape of cells and its role in cell function.

biophysics↗

bioRxiv2019

A weak link with actin organizes tight junctions to control epithelial permeability

In vertebrates, epithelial permeability is regulated by the tight junction (TJ) formed by specialized adhesive membrane proteins, adaptor proteins, and the actin cytoskeleton. Despite the TJs critical physiological role, a molecular-level understanding of how TJ assembly sets the permeability of epithelial tissue is lacking. Here, we identify a 28-amino acid sequence in the TJ adaptor protein ZO-1 that is responsible for actin binding and show that this interaction is essential for TJ permeability. In contrast to the strong interactions at the adherens junction, we find that the affinity between ZO-1 and actin is surprisingly weak, and we propose a model based on kinetic trapping to explain how affinity could affect TJ assembly. Finally, by tuning the affinity of ZO-1 to actin, we demonstrate that epithelial monolayers can be engineered with a spectrum of permeabilities, which points to a new target for treating transport disorders and improving drug delivery.

cell biology↗

bioRxiv2019

Tuning the affinity of tandem calponin homology domains

Tandem calponin homology (CH1-CH2) domains are common actin-binding domains in proteins that interact with and organize the actin cytoskeleton. Despite regions of high sequence similarity, CH1-CH2 domains can have remarkably different actin-binding properties, with disease-associated point mutants known to increase as well as decrease affinity for f-actin. To investigate features that affect CH1-CH2 affinity for f-actin in cells and in vitro, we perturbed the utrophin actin-binding domain by making point mutations at the CH1-CH2 interface, replacing the linker domain, and adding a PEG polymer to CH2. Consistent with a previous model describing CH2 as a steric negative regulator of actin binding, we find that utrophin CH1-CH2 affinity is both increased and decreased by modifications that change the effective openness of CH1 and CH2 in solution. We also identified interface mutations that caused a large increase in affinity without changing solution openness, suggesting additional influences on affinity. Interestingly, we also observe non-uniform sub-cellular localization of utrophin CH1-CH2 that depends on the N-terminal flanking region but not on bulk affinity. These observations provide new insights into how small sequence changes, such as those found in diseases, can affect CH1-CH2 binding properties.

biochemistry↗

bioRxiv2018

Claudin-4 Reconstituted in Unilamellar Vesicles is Sufficient to Form Tight Interfaces that Partition Membrane Proteins

Tight junctions have been hypothesized to act as molecular fences in the plasma membrane of epithelial cells, helping to form differentiated apical and basolateral domains. While this fence function is believed to arise from the interaction of four-pass transmembrane claudins, the complexity of tight junctions has made direct evidence of their role as a putative diffusion barrier difficult to obtain. Here we address this challenge by reconstituting claudin-4 into giant unilamellar vesicles using microfluidic jetting. We find that reconstituted claudin-4 is sufficient to form adhesive interfaces between unilamellar vesicles without accessory proteins present in vivo. By controlling the molecular composition of the inner and outer leaflets of jetted membranes, we show that claudin-4-mediated interfaces can drive partitioning of extracellular membrane proteins but not of inner or outer leaflet lipids. Our findings indicate that homotypic interactions of claudins and their small size can contribute to the polarization of epithelial cells.

cell biology↗