Biology subjects

Capitanio, M.

Publications and source records attributed to Capitanio, M..

6 recordsLinked to original sources

bioRxiv2026

Extracellular Matrix Stiffness Controls Actin Cytoskeletal Tension

Mechanical properties of the extracellular matrix (ECM) regulate cell behavior and strongly impact the cytoskeleton, yet direct measurements of actin tension across different ECM stiffnesses are lacking. Using a genetically encoded FRET-based actin tension sensor, we show that cells cultured on stiffer substrates exhibit increased spreading, reduced multicellular aggregation, and higher molecular tension within filamentous actin. Complementary measurements using an -actinin tension sensor support increased cytoskeletal mechanical loading. These results provide direct evidence that ECM stiffness modulates intracellular actin tension during mechanotransduction.

biophysics↗

bioRxiv2023

Optimization of highly inclined Illumination for diffraction-limited and super-resolution microscopy

In HILO microscopy, a highly inclined and laminated light sheet is used to illuminate the sample, thus drastically reducing background fluorescence in wide-field microscopy, but maintaining the simplicity of the use of a single objective for both illumination and detection. Although the technique has become widely popular, particularly in single-molecule and super-resolution microscopy, a limited understanding of how to finely shape the illumination beam and how this impact on the image quality complicates setting HILO to fit the experimental needs. In this work, we build up a simple and comprehensive guide to optimize the beam shape and alignment in HILO and to predict its performance in conventional fluorescence and super-resolution microscopy. We model the beam propagation through Gaussian optics and validate the model through far- and near-field experiments, thus characterizing the main geometrical features of the beam. Further, we fully quantify the impact of a progressive reduction of the inclined beam thickness on the image quality of both diffraction-limited and super-resolution images and we show that the most relevant impact is obtained by reducing the beam thickness to sub-cellular dimensions (< 3 m). Based on this, we present a simple optical solution to reduce the inclined beam thickness down to 2.6 m while keeping a field-of-view dimension suited for cell imaging and allowing an increase in the number of localizations in super-resolution imaging of up to 2.6 folds.

biophysics↗

bioRxiv2021

Particle localization using local gradients and its application to nanometer stabilization of a microscope

Accurate localization of single particles plays an increasingly important role in a range of biological techniques, including single molecule tracking and localization-based superresolution microscopy. Such techniques require fast and accurate particle localization algorithms as well as nanometer-scale stability of the microscope. Here, we present a universal method for three-dimensional localization of single labeled and unlabeled particles based on local gradient calculation of microscopy images. The method outperforms current techniques in high noise conditions, and it is capable of nanometer accuracy localization of nano- and micro-particles with sub-ms calculation time. By localizing a fixed particle as fiducial mark and running a feedback loop, we demonstrate its applicability for active drift correction in sensitive nanomechanical measurements such as optical trapping and superresolution imaging. A multiplatform open software package comprising a set of tools for local gradient calculation in brightfield and fluorescence microscopy is shared to the scientific community.

biophysics↗

bioRxiv2021

Probing mechanotransduction in living cells by optical tweezers and FRET-based molecular force microscopy

Cells sense mechanical signals and forces to probe the external environment and adapt to tissue morphogenesis, external mechanical stresses, and a wide range of diverse mechanical cues. Here, we propose a combination of optical tools to manipulate single cells and measure the propagation of mechanical and biochemical signals inside them. Optical tweezers are used to trap microbeads that are used as handles to manipulate the cell plasma membrane; genetically encoded FRET-based force sensors inserted in F-actin and alpha-actinin are used to measure the propagation of mechanical signals to the cell cytoskeleton; while fluorescence microscopy with single molecule sensitivity can be used with a huge array of biochemical and genetic sensors. We describe the details of the setup implementation, the calibration of the basic components and preliminary characterization of actin and alpha-actinin FRET-based force sensors.

biophysics↗

bioRxiv2020

α-catenin regulates cell junction fluidity by cooperative mechanosensing

-catenin is a crucial protein at cell junctions that provides connection between the actin cytoskeleton and the cell membrane. At adherens junctions (AJs), -catenin forms heterodimers with {beta}-catenin that are believed to resist force on F-actin. Outside AJs, -catenin forms homodimers that directly connect the cell membrane to the actin cytoskeleton, but their mechanosensitive properties are inherently unknown. Surprisingly, by using ultra-fast laser tweezers we found that a single -{beta}-catenin heterodimer does not resist force but instead slips along F-actin in the direction of force. Conversely, the action of 5 to 10 -{beta}-catenin heterodimers together with force applied toward F-actin pointed end engaged a molecular switch in -catenin, which unfolded and strongly bound F-actin as a cooperative catch bond. Similarly, an -catenin homodimer formed an asymmetric catch bond with F-actin triggered by protein unfolding under force. Our data suggest that -catenin clustering together with intracellular tension engage a fluid-to-solid phase transition at the membrane-cytoskeleton interface.

biophysics↗

bioRxiv2019

Single molecule mechanics resolves the earliest events in force generation by cardiac myosin

Key steps of cardiac mechanochemistry, including the force-generating working stroke and the release of phosphate (Pi), occur rapidly after myosin-actin attachment. An ultra-high-speed optical trap enabled direct observation of the timing and amplitude of the working stroke, which can occur within <200 s of actin binding by {beta}-cardiac myosin. The initial actomyosin state can sustain loads of at least 4.5 pN and proceeds directly to the stroke or detaches before releasing ATP hydrolysis products. The rates of these processes depend on the force. The time between binding and stroke is unaffected by 10 mM Pi which, along with other findings, indicates the stroke precedes phosphate release. After Pi release, Pi can rebind enabling reversal of the working stroke. Detecting these rapid events under physiological loads provides definitive indication of the dynamics by which actomyosin converts biochemical energy into mechanical work.

biochemistry↗