Biology subjects

Reina, F.

Publications and source records attributed to Reina, F..

6 recordsLinked to original sources

bioRxiv2026

Single-molecule reaction chambers reveal intramolecular protein kinase activation

Many protein kinases, ubiquitous signal transducers across all kingdoms of life, are activated by phosphorylation of one or more conserved residues in their activation loops. This reaction is widely considered to be intermolecular (trans) in nature, dependent on the transient dimerization of two kinase domains. However, since the substrate, product and reaction chemistry are identical, bulk solution kinase assays cannot distinguish between intra- and inter-molecular reactions. Discriminating between these two mechanisms is crucial because they impose different requirements on the cell for acute, high-fidelity signal transduction. Using single-molecule reaction chambers fabricated from DNA origami, we show that the paradigmatic insulin receptor kinase (IRK) undergoes intramolecular (cis) autophosphorylation with kinetics indistinguishable from those in free solution. Furthermore, we demonstrate that the bacterial serine/threonine kinase protein kinase B (PknB) undergoes intramolecular autophosphorylation, allosterically stimulated by intermolecular (trans) interactions. Our findings establish that intramolecular activation loop autophosphorylation is not only possible, but likely an ancient and conserved feature of protein kinase signaling. More broadly, our work provides new tools for single-molecule enzymology.

biochemistry↗

bioRxiv2025

Parameter Optimization for Iterative MINFLUX Microscopy enabled Single Particle Tracking

MINFLUX fluorescence microscopy is a recently introduced super-resolution approach for studying cellular structures and their dynamics with highest detail. Iterative MINFLUX (iMFX) performs Single Particle Tracking (SPT) at runtime. The ad hoc signal interpretation necessary to sustain the method relies on several parameters, which need to be optimized in relation to the sample under study, such as fluorescent lipid analogues in membranes, to ensure the fidelity of the measurement. We propose a parameter optimization strategy, an overview of the most important parameters, present a theoretical upper limit for trackable diffusion rates, and demonstrate iMFX-enabled SPT of fast ([<]DMSD[>] = 2.5m2/s) lateral Brownian motion of lipids in membranes.

biophysics↗

bioRxiv2024

Homogeneous large field-of-view and compact iSCAT-TIRF setup for dynamic single molecule measurements

Interferometric Scattering Microscopy (iSCAT) enables prolonged and high frame rate Single Particle Tracking (SPT) for single molecule dynamics studies. Typically, iSCAT setups employ scanning illumination schemes to achieve uniform sample illumination. However, this implementation limits the field of view (FoV) and maximum sampling rate, while increasing hardware requirements and setup size. We demonstrate the realization of a large (60{micro}m x 60{micro}m) uniformly illuminated FoV through a passive refractive optical element in the iSCAT illumination path. This scanning-free iSCAT microscopy setup is further combined with an objective based Total Internal Reflection Fluorescence Microscopy (TIRF) channel for a complementary fluorescence readout, a focus-lock system, and a tailored control platform via the open-source ImSwitch software, and has a compact footprint. As a proof-of-principle, we highlight the performance of the setup through the acquisition of iSCAT images with a uniform contrast and a [≤]10 nm localization precision throughout the whole FoV. The performance is further demonstrated through dynamic iSCAT SPT and imaging Fluorescence Correlation Spectroscopy of lipid diffusion in a model membrane system. Our iSCAT setup thus depicts an accurate and improved way of recording fast molecular dynamics in life sciences.

biophysics↗

bioRxiv2021

Lipid tracking at kilohertz sampling rates on live cell membranes through Interferometric Scattering microscopy

The specific details of the lateral diffusion dynamics in cellular plasma membrane are an open topic in modern biophysics. Many studies have documented several different behaviours, including free (Brownian) motion, confined diffusion, transiently confined (hop) diffusion, anomalous diffusion, and combinations thereof. Here we have employed Interferometric Scattering Microscopy (ISCAT) to explore the lateral diffusion dynamics in the plasma membrane of living cells of a biotinylated lipid analogue that had been labelled with streptavidin-coated gold nanoparticles (20 and 40nm in diameter) at a sampling rate of 2kHz. The data was analysed with an unbiased statistics-driven mean squared displacement analysis pipeline that was designed to identify both the most likely diffusion mode for a specific data set, and the best fit parameters of the most likely model. We found that the prevalent diffusion mode of the tracked lipids, independent of the particle size, is compartmentalized diffusion, although the use of the larger tags resulted in tighter confinement and reduced diffusion rates. Through our analysis and comparison with simulated data, we quantify significant physical parameters, such as average compartment size, dynamic localization uncertainty, and the diffusion rates. We hereby further demonstrate the use of a confinement strength metric that makes it possible to compare diffusivity measurements across techniques and experimental conditions. Statement of SignificanceThis work offers new details on the data analysis of lipid diffusion on cellular membranes in vitro, through Interferometric Scattering microscopy. With this technique, we performed single particle tracking (SPT) experiments at 2kHz sampling rate. We analyzed the data through an unbiased statistics-driven protocol. The data shows that the diffusion motion of the tracked lipids follows mainly the "hopping" diffusion behaviour, whereby transient confinement zones hinder the particle dynamics. Matching the experimental data with diffusion simulations, we have been able to verify the physical parameters inferred by the experimental data analysis. Finally, we showcase a framework to compare SPT data with other techniques, to offer a complete overview of plasma membrane dynamics.

biophysics↗

bioRxiv2021

TRAIT2D: a Software for Quantitative Analysis of Single Particle Diffusion Data

Single Particle Tracking (SPT) is one of the most widespread techniques to evaluate particle mobility in a variety of situations, such as in cellular and model membrane dynamics. The proposed TRAIT2D Python library is developed to provide object tracking, trajectory analysis and produce simulated datasets with graphical user interface. The tool allows advanced users to customise the analysis to their requirements. Availability and implementation: the software has been coded in Python, and can be accessed from: https://github.com/Eggeling-Lab-Microscope-Software/TRAIT2D, or the pypi and condaforge repositories. A comprehensive user guide is provided at https://eggeling-lab-microscope-software.github.io/TRAIT2D/.

bioinformatics↗

bioRxiv2017

Complementary studies of lipid membrane dynamics using iSCAT and STED microscopy

Observation techniques with high spatial and temporal resolution, such as single-particle tracking (SPT) based on interferometric Scattering (iSCAT) microscopy, and fluorescence correlation spectroscopy applied on a super-resolution STED microscope (STED-FCS), have revealed new insights of the molecular organization of membranes. While delivering complementary information, there are still distinct differences between these techniques, most prominently the use of fluorescent dye-tagged probes for STED-FCS and a need for larger scattering gold nanoparticle tags for iSCAT. In this work we have used lipid analogues tagged with a hybrid fluorescent tag - gold nanoparticle construct, to directly compare the results from STED-FCS and iSCAT measurements of phospholipid diffusion on a homogeneous Supported Lipid Bilayer (SLB). These comparative measurements showed that while the mode of diffusion remained free, at least at the spatial (>40 nm) and temporal (50 [≤] t [≤] 100 ms) scales probed, the diffussion coefficient was reduced by 20- to 60-fold when tagging with 20 and 40 nm large gold particles as compared to when using dye-tagged lipid analogues. These FCS measurements of hybrid fluorescent tag - gold nanoparticle labeled lipids also revealed that commercially supplied streptavidin-coated gold nanoparticles contain large quantities of free streptavidin. Finally, the values of apparent diffusion coefficients obtained by STED-FCS and iSCAT differed by a factor of 2-3 across the techniques, while relative differences in mobility between different species of lipid analogues considered were identical in both approaches. In conclusion, our experiments reveal that large and potentially crosslinking scattering tags introduce a significant slow-down in diffusion on SLBs but no additional bias, and our labeling approach creates a new way of exploiting complementary information from STED-FCS and iSCAT measurements.

biophysics↗