Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2022.07.15.500159

The difference in FRET efficiency fluctuation at each end of the actin filament

Mitani, T.·Mashiko, R.·Nezasa, M.·Nishikata, I.·Kamimura, K.·Honda, H.·Fujiwara, I.
View original source ↗Export metadata ↓

Abstract

Actin filaments are involved in various cell motility processes. Actin polymerisation is primarily governed by monomer association and dissociation occurring at the rapid-growing end called the barbed end, which generates the force to push the plasma membrane forward. Individual actin filaments bind to one nucleotide and its hydrolysis energy is used to maintain the filamentous form by changing the characteristics of the subunits. The asymmetry of the individual actin filaments is also important for detecting the asymmetry of the cell. However, asymmetry at the subunit level including conformational and temporal changes in actin filaments has not been visualized yet. Here, we used "Forster (or fluorescence) resonance energy transfer (FRET)-actin filament" by copolymerising an equal amount of donor and acceptor labelled actin. FRET efficiency change was measured along each actin filament under a light microscope. The FRET efficiency was lower near the end region than in the interior regions. Fluctuations in the FRET efficiency (fFRET) were used to monitor local flexibilities along each actin filament. The fFRET was larger near the end region than the interior region. Our quantified data showed that spatial change of fFRET along actin filaments was rapidly decayed from the barbed end from near the pointed end toward internal region, suggesting that the behaviour of actin subunits near ends is affected from each end. Our result revealed that actin filaments have different orientations locally. These orientations appear when actin forms filaments, which may contribute the cell motility.

Explore related subjects

biophysicsPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Mitani, T., Mashiko, R., Nezasa, M., Nishikata, I., Kamimura, K., Honda, H., Fujiwara, I.. 2022-07-15. The difference in FRET efficiency fluctuation at each end of the actin filament. https://doi.org/10.1101/2022.07.15.500159

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Mechanism of molecular recognition revealed through dynamic drug binding pathways to SARS-CoV-2 main protease

Characterization of drug-binding pathways remains experimentally limited by transient intermediates and computationally challenging due to long timescales intractable for conventional molecular dynamics. To address these challenges, we combined solution NMR titrations with weighted ensemble (WE) enhanced sampling simulations to resolve atomistic pathways of nirmatrelvir binding to the SARS-CoV-2 main protease. NMR titration revealed residue-dependent heterogeneity spanning fast, intermediate, and slow exchange regimes. WE simulations complement the NMR by providing insights into unassigned residues and adding time-resolved and three-dimensional structural context. We map key interactions along two distinct binding pathways, provide dynamic explanations for residues involved in resistance, and capture unique backbone conformations compared to those sampled in unbound or bound states. Our comprehensive binding model is consistent with a combined conformational selection and induced fit mechanism in which early transient contacts are made with residues E47 and L50 and allosteric motions are centered around residue V204 of the distal domain. This synergistic application of WE and titration NMR enables a more comprehensive characterization of drug binding than either method alone, providing an integrated framework that may have broader applicability to defining structure-kinetic relationships and guiding design of next-generation inhibitors.

biophysics↗
bioRxiv2026

Fibers and Glasses are Competing Material States in FUS Protein Condensation

Dense, well-ordered material states of proteins form the amyloid fibers that are a hallmark of neurodegenerative disease in the brain. Beyond forming amyloid fibers, some of these proteins can also adopt other material states termed condensates which are initially liquid-like but evolve to a soft, glassy phase. Fiber growth requires a large supply of monomers and, thus, it is often speculated that fibers emerge from within a dense condensate as it ages and its microscopic dynamics slow into a glassy state. Here, we use the well-established model system Fused in Sarcoma (FUS) to directly observe, quantify and theoretically describe fiber growth and its interplay with condensates. We report the discovery that fibers grow overwhelmingly in the dilute phase surrounding the condensates while the condensates concurrently evolve to a glassy arrested solid. The resulting protein fibers and glassy condensates are both distinct solid-like phases that coexist but do not directly interconvert. Taken together, these findings reveal that there are two competitive aging pathways in FUS condensation that are linked through phase separation kinetics.

biophysics↗
bioRxiv2026

A Minimally Perturbative DARPin Probe for Quantitative Fluorescence Imaging of the Human TCR-CD3 Complex

Fluorescence microscopy is a powerful tool for dissecting the molecular mechanisms of T-cell antigen recognition in living cells, but its quantitative insight critically depends on non-perturbative, high-quality probes. Here, we repurpose a small (~15 kDa) CD3epsilon-binding DARPin (designed ankyrin repeat proteins) to a fluorescent label for T-cell receptor (TCR)/CD3 complexes on primary human CD8+ T-cells, with the aim of generating a powerful tool for quantitative analysis, single-molecule tracking, and advanced imaging of TCR dynamics. We show that the DARPin binds CD3{varepsilon} with high affinity and selectivity and using single molecule tracking and brightness analysis, we characterize the TCR-CD3 diffusion behavior and show that the DARPin binds to both CD3epsilon; subunits. Importantly, labeling preserves antigen sensitivity: on supported lipid bilayers presenting cognate pMHC, T-cells remain responsive, assemble synapses, form TCR microclusters, and initiate signaling similar to unlabeled controls. We further demonstrate compatibility with lattice light-sheet microscopy for volumetric imaging of T-cell - APC interactions in living cells. Together, these results establish DARPins as versatile, minimally perturbative probes for high resolution, quantitative studies of T cell synapse organization and signaling.

biophysics↗