Biophysical properties of single rotavirus particles account for the functions of protein shells in a multilayered virus

The functions performed by the concentric shells of multilayered dsRNA viruses require specific protein interactions that can be directly explored through their mechanical properties. We studied the stiffness, breaking force, critical strain and mechanical fatigue of individual Triple, Double and Single layered rotavirus (RV) particles. Our results, in combination with Finite Element simulations, demonstrate that the mechanics of the external layer provides the resistance needed to counteract the stringent conditions of extracellular media. Our experiments, in combination with electrostatic analyses, reveal a strong interaction between the two outer layers and how it is suppressed by the removal of calcium ions, a key step for transcription initiation. The intermediate layer presents weak hydrophobic interactions with the inner layer that allow the assembly and favor the conformational dynamics needed for transcription. Our work shows how the biophysical properties of the three shells are finely tuned to produce an infective RV virio

Optomechanical devides for mechanobiological fingerprinting

Resumen del trabajo presentado en el Frontiers of Nanomechanical Systems (FSN2021), celebraod de forma virtual del 19 al 21 de enero de 2021Twenty years have passed since the first detection of biomolecular recognition using micromechanical systems[1]. In the last two decades, micro- nanomechanical systems have been refined to achieve amazing detection limits in force and mass that have enabled different schemes for ultrasensitive measurements of biological interactions as well as new ways of biological spectrometry. More recently, these figures of merit have been improved by coupling optical cavities to mechanical systems. In this talk, I will review the use of micro- nanomechanical systems for mechanobiological fingerprinting of biological entities, particularizing in the contributions of our group [2]. An essential core of this topic is the discussion about the mechanical coupling between a biological particle and a mechanical resonator, an issue that it is has been often oversimplified. We show that the biomechanical coupling that emerges between a bioparticle and a mechanical resonator is more complex than previously expect and it can give rise to different interaction regimes, in which the resonator response is dominated by different physical parameters of the analyte [3-4]. In particular, we will show experiments done with a variety of micro- nano- optomechanical systems using different measurement schemes where the mass, the stiffness and even the vibration modes of single biological entities can be measured with high sensitivity. It is now widely appreciated the essential role of mechanics in relevant biological processes and how disease can be revealed as changes in the mechanical properties of biological matter. I am pretty sure that future developments in optomechanical devices will contribute for major understanding of diseases as well as for new avenues in diagnosis and therapy

Effects of energy metabolism on the mechanical properties of breast cancer cells

Resumen del trabajo presentado en el Simposio Nuevas fronteras y retos en Magnetismo de la XXXVIII Reunión Bienal de la Real Sociedad Española de Física, celebrada en Murcia (España), del 11 al 15 de julio de 202

Effects of energy metabolism on the mechanical properties of breast cancer cells

Tumorigenesis induces actin cortex remodeling, which makes cancerous cells softer. Cell deformability is largely determined by myosin-driven cortical tension and actin fiber architecture at the cell cortex. However, it is still unclear what the weight of each contribution is, and how these contributions change during cancer development. Moreover, little attention has been paid to the effect of energy metabolism on this phenomenon and its reprogramming in cancer. Here, we perform precise two-dimensional mechanical phenotyping based on power-law rheology to unveil the contributions of myosin II, actin fiber architecture and energy metabolism to the deformability of healthy (MCF-10A), noninvasive cancerous (MCF-7), and metastatic (MDA-MB-231) human breast epithelial cells. Contrary to the perception that the actin cortex is a passive structure that provides mechanical resistance to the cell, we find that this is only true when the actin cortex is activated by metabolic processes. The results show marked differences in the nature of the active processes that build up cell stiffness, namely that healthy cells use ATP-driven actin polymerization whereas metastatic cells use myosin II activity. Noninvasive cancerous cells exhibit an anomalous behavior, as their stiffness is not as affected by the lack of nutrients and ATP, suggesting that energy metabolism reprogramming is used to sustain active processes at the actin cortex.This work was supported by the European Union’s Horizon 2020 research and innovation program under European Research Council Grant 681275-LIQUIDMASS-ERCCoG-2015, under Grant Agreement No. 731868 – VIRUSCAN and by the Spanish Science, Innovation and Universities Ministry through project CELLTANGLE reference RTI2018-099369-B-I00; by the Comunidad de Madrid (iLUNG B2017/BMD-3884) with support from EU (FEDER, FSE) and Ramón y Cajal grant RYC-2017-21640 to P.M.K. The service from the X-SEM Laboratory is funded by MCIU (CSIC13-4E-1794) and EU (FEDER, FSE)

Hydrodynamic assisted multiparametric particle spectrometry

The real-time analysis of single analytes in flow is becoming increasingly relevant in cell biology. In this work, we theoretically predict and experimentally demonstrate hydrodynamic focusing with hollow nanomechanical resonators by using an interferometric system which allows the optical probing of flowing particles and tracking of the fundamental mechanical mode of the resonator. We have characterized the hydrodynamic forces acting on the particles, which will determine their velocity depending on their diameter. By using the parameters simultaneously acquired: frequency shift, velocity and reflectivity, we can unambiguously classify flowing particles in real-time, allowing the measurement of the mass density: 1.35 ± 0.07 g·mL-1 for PMMA and 1.7 ± 0.2 g·mL-1 for silica particles, which perfectly agrees with the nominal values. Once we have tested our technique, MCF-7 human breast adenocarcinoma cells are characterized (1.11 ± 0.08 g·mL-1) with high throughput (300 cells/minute) observing a dependency with their size, opening the door for individual cell cycle studies.This work was supported by the European Union’s Horizon 2020 research and innovation program under European Research Council Grant 681275-LIQUIDMASS-ERC-CoG-2015, project CELLTANGLE reference RTI2018-099369-B-I00, project MOMPs reference TEC2017-89765-R, Asociación Elena Torres (contract OTR04893) and FERO Foundation (Grant OTR06145). The service from the Micro and Nanofabrication Laboratory (MiNa) and X-SEM Laboratory, is funded by MCIU (CSIC13-4E-1794) and EU (FEDER, FSE).Peer reviewe

Mechano-Optical Analysis of Single Cells with Transparent Microcapillary Resonators

The study of biophysical properties of single cells is becoming increasingly relevant in cell biology and pathology. The measurement and tracking of magnitudes such as cell stiffness, morphology, and mass or refractive index have brought otherwise inaccessible knowledge about cell physiology, as well as innovative methods for high-throughput labelfree cell classification. In this work, we present hollow resonator devices based on suspended glass microcapillaries for the simultaneous measurement of single-cell buoyant mass and reflectivity with a throughput of 300 cells/minute. In the experimental methodology presented here, both magnitudes are extracted from the devices’ response to a single probe, a focused laser beam that enables simultaneous readout of changes in resonance frequency and reflected optical power of the devices as cells flow within them. Through its application to MCF-7 human breast adenocarcinoma cells and MCF-10A nontumorigenic cells, we demonstrate that this mechano-optical technique can successfully discriminate pathological from healthy cells of the same tissue type.This work was supported by the European Union’s Horizon 2020 research and innovation program under European Research Council Grant 681275-LIQUIDMASS-ERC-CoG2015 and by the Spanish Science, Innovation and Universities Ministry through project CELLTANGLE reference RTI2018- 099369-B-I00, project MOMPs reference TEC2017-89765-R and Ramon y Cajal grant RYC-2017-21640 to P.M.K.; by the ́ Comunidad de Madrid (iLUNG B2017/BMD-3884) with support from EU (FEDER, FSE). E.G.-S. received funding from Fundacion General CSIC (Programa ComFuturo), as ́ well as Marie-Sklodowska Curie Actions (H2020-MSCA-IF2015) under NOMBIS project (703354).The service from the Micro and Nanofabrication Laboratory (MiNa) and X-SEM Laboratory, is funded by MCIU (CSIC13-4E-1794) and EU (FEDER, FSE). We have received support for part of the publication fee by CSIC open access publication support initiative through URICI.Peer reviewe

Biophysical properties of single rotavirus particles account for the functions of protein shells in a multilayered virus

The functions performed by the concentric shells of multilayered dsRNA viruses require specific protein interactions that can be directly explored through their mechanical properties. We studied the stiffness, breaking force, critical strain and mechanical fatigue of individual Triple, Double and Single layered rotavirus (RV) particles. Our results, in combination with Finite Element simulations, demonstrate that the mechanics of the external layer provides the resistance needed to counteract the stringent conditions of extracellular media. Our experiments, in combination with electrostatic analyses, reveal a strong interaction between the two outer layers and how it is suppressed by the removal of calcium ions, a key step for transcription initiation. The intermediate layer presents weak hydrophobic interactions with the inner layer that allow the assembly and favor the conformational dynamics needed for transcription. Our work shows how the biophysical properties of the three shells are finely tuned to produce an infective RV virio