31 research outputs found

    Direct force measurements of subcellular mechanics in confinement using optical tweezers

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    During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue morphogenesis goes in hand with molecular and structural changes at the single cell level that result in variations of subcellular mechanical properties. As a consequence, even within the same cell, different organelles and compartments resist differently to mechanical stresses; and mechanotransduction pathways can actively regulate their mechanical properties. The ability of a cell to adapt to the microenvironment of the tissue niche thus is in part due to the ability to sense and respond to mechanical stresses. We recently proposed a new mechanosensation paradigm in which nuclear deformation and positioning enables a cell to gauge the physical 3D environment and endows the cell with a sense of proprioception to decode changes in cell shape. In this article, we describe a new method to measure the forces and material properties that shape the cell nucleus inside living cells, exemplified on adherent cells and mechanically confined cells. The measurements can be performed non-invasively with optical traps inside cells, and the forces are directly accessible through calibration-free detection of light momentum. This allows measuring the mechanics of the nucleus independently from cell surface deformations and allowing dissection of exteroceptive and interoceptive mechanotransduction pathways. Importantly, the trapping experiment can be combined with optical microscopy to investigate the cellular response and subcellular dynamics using fluorescence imaging of the cytoskeleton, calcium ions, or nuclear morphology. The presented method is straightforward to apply, compatible with commercial solutions for force measurements, and can easily be extended to investigate the mechanics of other subcellular compartments, e.g., mitochondria, stress-fibers, and endosomes.MK acknowledges financial support from the Spanish Ministry of Economy and Competitiveness through the Plan Nacional (PGC2018-097882-A-I00), FEDER (EQC2018-005048-P), Severo Ochoa program for Centres of Excellence in R&D (CEX2019-000910-S; RYC-2016-21062), from Fundació Privada Cellex, Fundació Mir-Puig, and from Generalitat de Catalunya through the CERCA and Research program (2017 SGR 1012), in addition to funding through ERC (MechanoSystems) and HFSP (CDA00023/2018). V.R. acknowledges support from the Spanish Ministry of Science and Innovation to the EMBL partnership, the Centro de Excelencia Severo Ochoa, MINECO's Plan Nacional (BFU2017-86296-P, PID2020-117011GB-I00) and Generalitat de Catalunya (CERCA). V.V. acknowledges support from the ICFOstepstone PhD Programme funded by the European Union's Horizon 2020 research and innovation program under Marie Skłodowska-Curie grant agreement 665884Peer ReviewedPostprint (published version

    Friction forces position the neural anlage

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    During embryonic development, mechanical forces are essential for cellular rearrangements driving tissue morphogenesis. Here, we show that in the early zebrafish embryo, friction forces are generated at the interface between anterior axial mesoderm (prechordal plate, ppl) progenitors migrating towards the animal pole and neurectoderm progenitors moving in the opposite direction towards the vegetal pole of the embryo. These friction forces lead to global rearrangement of cells within the neurectoderm and determine the position of the neural anlage. Using a combination of experiments and simulations, we show that this process depends on hydrodynamic coupling between neurectoderm and ppl as a result of E-cadherin-mediated adhesion between those tissues. Our data thus establish the emergence of friction forces at the interface between moving tissues as a critical force-generating process shaping the embryo

    Modulation-enhanced localization microscopy

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    Super-resolution fluorescence microscopy has become a powerful tool in cell biology to observe sub-cellular organization and molecular details below the diffraction limit of light. Super-resolution methods are generally classified into three main concepts: stimulated emission depletion (STED), single molecule localization microscopy (SMLM) and structured illumination microscopy (SIM). Here, we highlight the novel concept of modulation-enhanced localization microscopy (meLM) which we designate as the 4th super-resolution method. Recently, a series of modulation-enhanced localization microscopy methods have emerged, namely MINFLUX, SIMPLE, SIMFLUX, ModLoc and ROSE. Although meLM combines key ideas from STED, SIM and SMLM, the main concept of meLM relies on a different idea: isolated emitters are localized by measuring their modulated fluorescence intensities in a precisely shifted structured illumination pattern. To position meLM alongside state-of-the-art super-resolution methods we first highlight the basic principles of existing techniques and show which parts of these principles are utilized by the meLM method. We then present the overall novel super-resolution principle of meLM that can theoretically reach unlimited localization precision whenever illumination patterns are translated by an arbitrarily small distance.L R acknowledges support of a fellowship from 'la Caixa' Foundation (ID 100010434, LCF/BQ/IN18/11660032) and funding from the European Union´s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 713673. V R acknowledges support from the Spanish Ministry of Economy and Competitiveness through the Program 'Centro de Excelencia Severo Ochoa 2013-2017', the CERCA Programme/Generalitat de Catalunya, MINECO's Plan Nacional (BFU2017-86296-P) and support from the CRG Advanced Light Microscopy Facility. S W acknowledges support from the Spanish Ministry of Economy and Competitiveness through the 'Severo Ochoa' program for Centres of Excellence in R&D (SEV-2015-0522), from Fundació Privada Cellex, and from Generalitat de Catalunya through the CERCA program

    What can we learn from single molecule trajectories?

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    Diffusing membrane constituents are constantly exposed to a variety of forces that influence their stochastic path. Single molecule experiments allow for resolving trajectories at extremely high spatial and temporal accuracy, thereby offering insights into en route interactions of the tracer. In this review we discuss approaches to derive information about the underlying processes, based on single molecule tracking experiments. In particular, we focus on a new versatile way to analyze single molecule diffusion in the absence of a full analytical treatment. The method is based on comprehensive comparison of an experimental data set against the hypothetical outcome of multiple experiments performed on the computer. Since Monte Carlo simulations can be easily and rapidly performed even on state-of-the-art PCs, our method provides a simple way for testing various - even complicated - diffusion models. We describe the new method in detail, and show the applicability on two specific examples: firstly, kinetic rate constants can be derived for the transient interaction of mobile membrane proteins; secondly, residence time and corral size can be extracted for confined diffusion

    A reconstituted mammalian APC-kinesin complex selectively transports defined packages of axonal mRNAs

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    Through the asymmetric distribution of messenger RNAs (mRNAs), cells spatially regulate gene expression to create cytoplasmic domains with specialized functions. In neurons, mRNA localization is required for essential processes such as cell polarization, migration, and synaptic plasticity underlying long-term memory formation. The essential components driving cytoplasmic mRNA transport in neurons and mammalian cells are not known. We report the first reconstitution of a mammalian mRNA transport system revealing that the tumor suppressor adenomatous polyposis coli (APC) forms stable complexes with the axonally localized β-actin and β2B-tubulin mRNAs, which are linked to a kinesin-2 via the cargo adaptor KAP3. APC activates kinesin-2, and both proteins are sufficient to drive specific transport of defined mRNA packages. Guanine-rich sequences located in 3'UTRs of axonal mRNAs increase transport efficiency and balance the access of different mRNAs to the transport system. Our findings reveal a minimal set of proteins sufficient to transport mammalian mRNAs.This work was funded by the Spanish Ministry of Economy and Competitiveness (MINECO) (BFU2017-85361-P, BFU2014-54278-P, and BFU2015-62550-ERC), Juan de la Cierva-Incorporación Program (IJCI-2015-25994), the Human Frontiers in Science Program (HFSP) (RGY0083/2016), and the European Research Council (ERC) (H2020-MSCA-IF-2014-659271). V.R. acknowledges support from the Spanish Ministry of Economy and Competitiveness through the Program “Centro de Excelencia Severo Ochoa 2013–2017,” the CERCA Programme/Generalitat de Catalunya, MINECO’s Plan Nacional (BFU2017-86296-P). S.W. acknowledges support from the Spanish Ministry of Economy and Competitiveness through the “Severo Ochoa” program for Centres of Excellence in R&D (SEV-2015-0522), from Fundació Privada Cellex, and from Generalitat de Catalunya through the CERCA program. We further acknowledge support of the Spanish Ministry of Economy and Competitiveness (MEIC) to the EMBL partnership, “Centro de Excelencia Severo Ochoa” (SEV-2012-0208) and the CERCA Programme/Generalitat de Cataluny

    Cortical flow-driven shapes of nonadherent cells

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    Nonadherent polarized cells have been observed to have a pearlike, elongated shape. Using a minimal model that describes the cell cortex as a thin layer of contractile active gel, we show that the anisotropy of active stresses, controlled by cortical viscosity and filament ordering, can account for this morphology. The predicted shapes can be determined from the flow pattern only; they prove to be independent of the mechanism at the origin of the cortical flow, and are only weakly sensitive to the cytoplasmic rheology. In the case of actin flows resulting from a contractile instability, we propose a phase diagram of three-dimensional cell shapes that encompasses nonpolarized spherical, elongated, as well as oblate shapes, all of which have been observed in experiment