35 research outputs found

    Mechanical traits of isolated nuclei inspected via force spectroscopy

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    The larger stiffness of the nuclei when compared to the rest of the cell imposes a key restriction to cell deformability and their capability to traverse interstices. In contrast, cancerous cells have been reported to exhibit larger and poorly-defined nuclear shapes. Upon probing the mechanical properties of these abnormal nuclei, membrane rigidities were found to be below that of normal nuclei. A plausible explanation is an altered distribution of the nuclear chromatin. This argument is in line with the increased migration capabilities of invasive nuclei and their enhanced adaptability to the abnormal forces these cells experiment. As a response to mechanical stresses, the normal function of the nuclei is altered and can induce changes such as gene expression alteration in the cell. Despite the obvious relevance of the nuclear mechanical traits, few works report data directly acquired on nuclear membranes without any participation from the plasma membrane, which is bound to induce alterations that may disrupt results yielded by high sensitivity tests such as those performed using optical tweezers. In the present work, optical tweezers are used alongside force spectroscopy to test the mechanical traits of isolated nuclear membranes. Membranes’ Young moduli and, therefore, stiffnesses are calculated by performing indentation/retraction cycles inducing gentle deformation on the membranes using an optically trapped microbead. Nuclear membrane responses are studied as a function of the frequency with which cycles were performed to highlight possible dependency on the time lapse over which the perturbation is applied. Additionally, drastic pushing of the trapped bead against the membranes and pulling motions were performed to trigger more dramatic mechanical responses from the nuclei. During those perturbations, maximum indentation depth and maximum tension could be measured from simultaneously acquired confocal microscopy images.Ayudas para la recualificación del Sistema Universitario español modalidad Margarita Salas. NextGenerationEU (UE) & Ministerio de Universidades, Gobierno de España. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

    Annexin A4 trimers are recruited by high membrane curvatures in giant plasma membrane vesicles

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    The plasma membrane (PM) of eukaryotic cells consists of a crowded environment comprised of a high diversity of proteins in a complex lipid matrix. The lateral organization of membrane proteins in the PM is closely correlated with biological functions such as endocytosis, membrane budding and other processes which involve protein mediated shaping of the membrane into highly curved structures. Annexin A4 (ANXA4) is a prominent player in a number of biological functions including PM repair. Its binding to membranes is activated by Ca2+ influx and it is therefore rapidly recruited to the cell surface near rupture sites where Ca2+ influx takes place. However, the free edges near rupture sites can easily bend into complex curvatures and hence may accelerate recruitment of curvature sensing proteins to facilitate rapid membrane repair. To analyze the curvature sensing behavior of curvature inducing proteins in crowded membranes, we quantifify the affinity of ANXA4 monomers and trimers for high membrane curvatures by extracting membrane nanotubes from giant PM vesicles (GPMVs). ANXA4 is found to be a sensor of negative membrane curvatures. Multiscale simulations, in which we extract molecular information from atomistic scale simulations as input to our macroscopic scale simulations, furthermore predicted that ANXA4 trimers generate membrane curvature upon binding and have an affinity for highly curved membrane regions only within a well defined membrane curvature window. Our results indicate that curvature sensing and mobility of ANXA4 depend on the trimer structure of ANXA4 which could provide new biophysical insight into the role of ANXA4 in membrane repair and other biological processes. This journal i

    Label-free optical interferometric microscopy to characterize morphodynamics in living plants

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    During the last century, fluorescence microscopy has played a pivotal role in a range of scientific discoveries. The success of fluorescence microscopy has prevailed despite several shortcomings like measurement time, photobleaching, temporal resolution, and specific sample preparation. To bypass these obstacles, label-free interferometric methods have been developed. Interferometry exploits the full wavefront information of laser light after interaction with biological material to yield interference patterns that contain information about structure and activity. Here, we review recent studies in interferometric imaging of plant cells and tissues, using techniques such as biospeckle imaging, optical coherence tomography, and digital holography. These methods enable quantification of cell morphology and dynamic intracellular measurements over extended periods of time. Recent investigations have showcased the potential of interferometric techniques for precise identification of seed viability and germination, plant diseases, plant growth and cell texture, intracellular activity and cytoplasmic transport. We envision that further developments of these label-free approaches, will allow for high-resolution, dynamic imaging of plants and their organelles, ranging in scales from sub-cellular to tissue and from milliseconds to hours

    Interdisciplinary Synergy to Reveal Mechanisms of Annexin-Mediated Plasma Membrane Shaping and Repair

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    The plasma membrane surrounds every single cell and essentially shapes cell life by separating the interior from the external environment. Thus, maintenance of cell membrane integrity is essential to prevent death caused by disruption of the plasma membrane. To counteract plasma membrane injuries, eukaryotic cells have developed efficient repair tools that depend on Ca2+- and phospholipid-binding annexin proteins. Upon membrane damage, annexin family members are activated by a Ca2+ influx, enabling them to quickly bind at the damaged membrane and facilitate wound healing. Our recent studies, based on interdisciplinary research synergy across molecular cell biology, experimental membrane physics, and computational simulations show that annexins have additional biophysical functions in the repair response besides enabling membrane fusion. Annexins possess different membrane-shaping properties, allowing for a tailored response that involves rapid bending, constriction, and fusion of membrane edges for resealing. Moreover, some annexins have high affinity for highly curved membranes that appear at free edges near rupture sites, a property that might accelerate their recruitment for rapid repair. Here, we discuss the mechanisms of annexin-mediated membrane shaping and curvature sensing in the light of our interdisciplinary approach to study plasma membrane repair
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