Biology subjects

Bui, T. T.

Publications and source records attributed to Bui, T. T..

3 recordsLinked to original sources

bioRxiv2025

Development of three-color FRET measurement of force-dependent sensing of RIAM-Vinculin interactions in focal adhesions

Forster resonance energy transfer (FRET) is a powerful technique for probing molecular interactions and conformational changes in biological systems. Cascade-FRET, a multistep energy transfer system involving three fluorophores, enables spatial and temporal mapping of molecular interactions. Here, we leveraged Cascade-FRET with time-correlated single photon counting fluorescence lifetime imaging microscopy (TCSPC-FLIM) to explore the putative interaction between Rap1-interacting Adapter Molecule (RIAM) and vinculin in focal adhesions. We developed a novel three-fluorophore Cascade-FRET system connected by flexible peptide linkers comprising mTurquoise2, mVenus, and mScarlet-I, validated using purified proteins, spectroscopic analysis, structural modeling, and negative staining transmission electron microscopy (TEM). Putative RIAM-vinculin interactions were explored in vinculin knockout mouse embryonic fibroblasts and revealed that RIAM binds to the N-terminus of vinculin in focal adhesions. This complex requires an intact microtubule cytoskeleton. Vinculin tension-sensing constructs quantified intracellular forces, with an average force of 2.95 {+/-} 0.97 pN per focal adhesion. These findings corroborate the mechanosensitive role of vinculin and its interaction with RIAM in a force-independent manner. This study demonstrates the utility of Cascade-FRET and TCSPC-FLIM in investigating multicomponent molecular complexes. Our findings provide novel insights into RIAM-vinculin interactions and their regulation by intracellular tension, paving the way for advanced applications of Cascade-FRET in dynamic cellular systems. Significance StatementFocal adhesions are used by all adherent cells to attach to their surroundings and transmit mechanical signals. This paper uses a biosensor to measure force changes within the focal adhesion-associated protein called vinculin. The fluorescence lifetime of the biosensor changes when force is applied across vinculin, allowing us to report on changes in force within developing focal adhesions. We show that another protein, RIAM, interacts with vinculin in a force-independent manner. This was achieved by developing a three-color cascade FRET model, showing how the three proteins--talin, vinculin, and RIAM--interact over time. This research has furthered our understanding of the order and mechanism in which these components assemble in cell adhesions.

biophysics↗

bioRxiv2018

Parallel evolution of frog antimicrobial peptides produces identical conformations but subtly distinct membrane and antibacterial activities

Frogs such as Rana temporaria and Litoria aurea secrete numerous closely related antimicrobial peptides (AMPs) as an effective chemical dermal defence. Despite the high similarity in physical properties and preference for adopting secondary amphipathic, -helix conformations in membrane mimicking milieu, their spectrum of activity and potency often varies considerably. Damage or penetration of the bacterial plasma membrane is considered essential for AMP activity and hence distinguishing apparently similar AMPs according to their behaviour in, and effects on, model membranes will inform understanding of species specific effective antimicrobial mechanisms. Here we use a combination of molecular dynamics simulations, circular dichroism and patch-clamp to investigate the basis for differing anti-bacterial activities in representative AMPs from each species; temporin L and aurein 2.5. Despite adopting near identical, -helix conformations in the steady-state in a variety of membrane models, these two AMPs can be distinguished both in vitro and in silico based on their dynamic interactions with model membranes; the greater conformational flexibility and the higher amplitude channel conductance induced offers a rationale for the greater potency and broader spectrum of activity of temporin L over aurein 2.5. Specific contributions from individual residues are identified that define the mechanisms of action of each AMP. Our findings suggest AMPs in frogs are examples of parallel evolution whose utility is based on apparently similar but subtly distinct mechanisms of action.

biophysics↗

bioRxiv2018

Minor sequence modifications in temporin B cause drastic changes in antibacterial potency and selectivity by fundamentally altering membrane activity

Antimicrobial peptides (AMPs) are a potential source of new molecules to counter the increase in antimicrobial resistant infections but a better understanding of their properties is required to understand their native function and for effective translation as therapeutics. Details of the mechanism of their interaction with the bacterial plasma membrane are desired since damage or penetration of this structure is considered essential for AMP activity. Relatively modest modifications to AMP primary sequence can induce substantial changes in potency and/or spectrum of activity but, hitherto, have not been predicted to substantially alter the mechanism of interaction with the bacterial plasma membrane. Here we use a combination of molecular dynamics simulations, circular dichroism, solid-state NMR and patch clamp to investigate the extent to which temporin B and its analogues can be distinguished both in vitro and in silico on the basis of their interactions with model membranes. Enhancing the hydrophobicity of the N-terminus and cationicity of the C-terminus in temporin B improves its membrane activity and potency against both Gram-negative and Gram-positive bacteria. In contrast, enhancing the cationicity of the N-terminus abrogates its ability to trigger channel conductance and renders it ineffective against Staphylococcus aureus while nevertheless enhancing its potency against Escherichia coli. Our findings suggest even closely related AMPs may target the same bacterium with fundamentally differing mechanisms of action.

biophysics↗