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Stressed cells shut down translation, release mRNA molecules from polysomes, and form stress granules (SGs) via a network of interactions that involve G3BP. Here we focus on the mechanistic underpinnings of SG assembly. We show that, under non-stress conditions, G3BP adopts a compact auto-inhibited state stabilized by electrostatic intramolecular interactions between the intrinsically disordered acidic tracts and the positively charged arginine-rich region. Upon release from polysomes, unfolded mRNAs outcompete G3BP auto-inhibitory interactions, engendering a conformational transition that facilitates clustering of G3BP through protein-RNA interactions. Subsequent physical crosslinking of G3BP clusters drives RNA molecules into networked RNA/protein condensates. We show that G3BP condensates impede RNA entanglement and recruit additional client proteins that promote SG maturation or induce a liquid-to-solid transition that may underlie disease. We propose that condensation coupled to conformational rearrangements and heterotypic multivalent interactions may be a general principle underlying RNP granule assembly.
Assuntos
Grânulos Citoplasmáticos/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Citoplasma/metabolismo , Células HeLa , Humanos , Conformação de Ácido Nucleico , Organelas/metabolismo , Fosforilação , RNA Mensageiro/metabolismo , Estresse Fisiológico/genéticaRESUMO
During osmotic changes of their environment, cells actively regulate their volume and plasma membrane tension that can passively change through osmosis. How tension and volume are coupled during osmotic adaptation remains unknown, as their quantitative characterization is lacking. Here, we performed dynamic membrane tension and cell volume measurements during osmotic shocks. During the first few seconds following the shock, cell volume varied to equilibrate osmotic pressures inside and outside the cell, and membrane tension dynamically followed these changes. A theoretical model based on the passive, reversible unfolding of the membrane as it detaches from the actin cortex during volume increase quantitatively describes our data. After the initial response, tension and volume recovered from hypoosmotic shocks but not from hyperosmotic shocks. Using a fluorescent membrane tension probe (fluorescent lipid tension reporter [Flipper-TR]), we investigated the coupling between tension and volume during these asymmetric recoveries. Caveolae depletion and pharmacological inhibition of ion transporters and channels, mTORCs, and the cytoskeleton all affected tension and volume responses. Treatments targeting mTORC2 and specific downstream effectors caused identical changes to both tension and volume responses, their coupling remaining the same. This supports that the coupling of tension and volume responses to osmotic shocks is primarily regulated by mTORC2.
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Tamanho Celular , Membranas/metabolismo , Osmose/fisiologia , Actinas/metabolismo , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Células HeLa , Humanos , Membranas/efeitos dos fármacos , Modelos Teóricos , Pressão Osmótica/fisiologiaRESUMO
Microgels are water-swollen, crosslinked polymers that are widely used as colloidal building blocks in scaffold materials for tissue engineering and regenerative medicine. Microgels can be controlled in their stiffness, degree of swelling, and mesh size depending on their polymer architecture, crosslink density, and fabrication method-all of which influence their function and interaction with the environment. Currently, there is a lack of understanding of how the polymer composition influences the internal structure of soft microgels and how this morphology affects specific biomedical applications. In this report, we systematically vary the architecture and molar mass of polyethylene glycol-acrylate (PEG-Ac) precursors, as well as their concentration and combination, to gain insight in the different parameters that affect the internal structure of rod-shaped microgels. We characterize the mechanical properties and diffusivity, as well as the conversion of acrylate groups during photopolymerization, in both bulk hydrogels and microgels produced from the PEG-Ac precursors. Furthermore, we investigate cell-microgel interaction, and we observe improved cell spreading on microgels with more accessible RGD peptide and with a stiffness in a range of 20â kPa to 50â kPa lead to better cell growth.
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Microgéis , Microgéis/química , Hidrogéis/química , Alicerces Teciduais/química , Polímeros , Polietilenoglicóis/química , AcrilatosRESUMO
Upon starvation or overcrowding, the nematode Caenorhabditis elegans enters diapause by forming a dauer larva, which can then further survive harsh desiccation in an anhydrobiotic state. We have previously identified the genetic and biochemical pathways essential for survival-but without detailed knowledge of their material properties, the mechanistic understanding of this intriguing phenomenon remains incomplete. Here we employed optical diffraction tomography (ODT) to quantitatively assess the internal mass density distribution of living larvae in the reproductive and diapause stages. ODT revealed that the properties of the dauer larvae undergo a dramatic transition upon harsh desiccation. Moreover, mutants that are sensitive to desiccation displayed structural abnormalities in the anhydrobiotic stage that could not be observed by conventional microscopy. Our advance opens a door to quantitatively assessing the transitions in material properties and structure necessary to fully understand an organism on the verge of life and death.
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Caenorhabditis elegans , Animais , Caenorhabditis elegans/metabolismo , LarvaRESUMO
Biophysical properties of cells such as intracellular mass density and cell mechanics are known to be involved in a wide range of homeostatic functions and pathological alterations. An optical readout that can be used to quantify such properties is the refractive index (RI) distribution. It has been recently reported that the nucleus, initially presumed to be the organelle with the highest dry mass density (ρ) within the cell, has in fact a lower RI and ρ than its surrounding cytoplasm. These studies have either been conducted in suspended cells, or cells adhered on 2D substrates, neither of which reflects the situation in vivo where cells are surrounded by the extracellular matrix (ECM). To better approximate the 3D situation, we encapsulated cells in 3D covalently-crosslinked alginate hydrogels with varying stiffness, and imaged the 3D RI distribution of cells, using a combined optical diffraction tomography (ODT)-epifluorescence microscope. Unexpectedly, the nuclei of cells in 3D displayed a higher ρ than the cytoplasm, in contrast to 2D cultures. Using a Brillouin-epifluorescence microscope we subsequently showed that in addition to higher ρ, the nuclei also had a higher longitudinal modulus (M) and viscosity (η) compared to the cytoplasm. Furthermore, increasing the stiffness of the hydrogel resulted in higher M for both the nuclei and cytoplasm of cells in stiff 3D alginate compared to cells in compliant 3D alginate. The ability to quantify intracellular biophysical properties with non-invasive techniques will improve our understanding of biological processes such as dormancy, apoptosis, cell growth or stem cell differentiation.
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Matriz Extracelular , Hidrogéis , Alginatos , Diferenciação Celular , Proliferação de CélulasRESUMO
The cell nucleus is a compartment in which essential processes such as gene transcription and DNA replication occur. Although the large amount of chromatin confined in the finite nuclear space could install the picture of a particularly dense organelle surrounded by less dense cytoplasm, recent studies have begun to report the opposite. However, the generality of this newly emerging, opposite picture has so far not been tested. Here, we used combined optical diffraction tomography and epi-fluorescence microscopy to systematically quantify the mass densities of cytoplasm, nucleoplasm, and nucleoli of human cell lines, challenged by various perturbations. We found that the nucleoplasm maintains a lower mass density than cytoplasm during cell cycle progression by scaling its volume to match the increase of dry mass during cell growth. At the same time, nucleoli exhibited a significantly higher mass density than the cytoplasm. Moreover, actin and microtubule depolymerization and changing chromatin condensation altered volume, shape, and dry mass of those compartments, whereas the relative distribution of mass densities was generally unchanged. Our findings suggest that the relative mass densities across membrane-bound and membraneless compartments are robustly conserved, likely by different as-of-yet unknown mechanisms, which hints at an underlying functional relevance. This surprising robustness of mass densities contributes to an increasing recognition of the importance of physico-chemical properties in determining cellular characteristics and compartments.
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Nucléolo Celular , Núcleo Celular , Cromatina , Citoplasma , Humanos , Gravidade EspecíficaRESUMO
Delivery of gold nanoparticles (GNPs) into live cells has high potentials, ranging from molecular-specific imaging, photodiagnostics, to photothermal therapy. However, studying the long-term dynamics of cells with GNPs using conventional fluorescence techniques suffers from phototoxicity and photobleaching. Here, we present a method for 3-D imaging of GNPs inside live cells exploiting refractive index (RI) as imaging contrast. Employing optical diffraction tomography, 3-D RI tomograms of live cells with GNPs are precisely measured for an extended period with sub-micrometer resolution. The locations and contents of GNPs in live cells are precisely addressed and quantified due to their distinctly high RI values, which was validated by confocal fluorescence imaging of fluorescent dye conjugated GNPs. In addition, we perform quantitative imaging analysis including the segmentations of GNPs in the cytosol, the volume distributions of aggregated GNPs, and the temporal evolution of GNPs contents in HeLa and 4T1 cells.
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Imageamento Tridimensional/métodos , Nanopartículas Metálicas/química , Tomografia/métodos , Fluorescência , Ouro/química , Células HeLa , Humanos , Nanopartículas Metálicas/ultraestruturaRESUMO
The mechanical properties of biological tissues are increasingly recognized as important factors in developmental and pathological processes. Most existing mechanical measurement techniques either necessitate destruction of the tissue for access or provide insufficient spatial resolution. Here, we show for the first time to our knowledge a systematic application of confocal Brillouin microscopy to quantitatively map the mechanical properties of spinal cord tissues during biologically relevant processes in a contact-free and nondestructive manner. Living zebrafish larvae were mechanically imaged in all anatomical planes during development and after spinal cord injury. These experiments revealed that Brillouin microscopy is capable of detecting the mechanical properties of distinct anatomical structures without interfering with the animal's natural development. The Brillouin shift within the spinal cord remained comparable during development and transiently decreased during the repair processes after spinal cord transection. By taking into account the refractive index distribution, we explicitly determined the apparent longitudinal modulus and viscosity of different larval zebrafish tissues. Importantly, mechanical properties differed between tissues in situ and in excised slices. The presented work constitutes the first step toward an in vivo assessment of spinal cord tissue mechanics during regeneration, provides a methodical basis to identify key determinants of mechanical tissue properties, and allows us to test their relative importance in combination with biochemical and genetic factors during developmental and regenerative processes.
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Larva/fisiologia , Fenômenos Mecânicos , Microscopia , Medula Espinal/diagnóstico por imagem , Medula Espinal/crescimento & desenvolvimento , Peixe-Zebra , Animais , Fenômenos Biomecânicos , Elasticidade , Processamento de Imagem Assistida por Computador , Larva/crescimento & desenvolvimento , Medula Espinal/fisiologia , ViscosidadeRESUMO
Parkinson's disease (PD) is a common neurodegenerative disease. However, therapeutic methods of PD are still limited due to complex pathophysiology in PD. Here, optical measurements of individual neurons from in vitro PD model using optical diffraction tomography (ODT) are presented. By measuring 3D refractive index distribution of neurons, morphological and biophysical alterations in in-vitro PD model are quantitatively investigated. It was found that neurons show apoptotic features in early PD progression. The present approach will open up new opportunities for quantitative investigation of the pathophysiology of various neurodegenerative diseases. © 2017 International Society for Advancement of Cytometry.
Assuntos
Biofísica/métodos , Neurônios/ultraestrutura , Doença de Parkinson/diagnóstico por imagem , Contagem de Células/métodos , Linhagem Celular , Humanos , Neurônios/patologia , Doença de Parkinson/patologiaRESUMO
Illumination coherence plays a major role in various imaging systems, from microscopy, metrology, digital holography, optical coherence tomography, to ultrasound imaging. Here, we present a systematic study on the effects of degrees of spatiotemporal coherence of an illumination (DSTCI) on imaging quality of interferometric microscopy. An optical field with arbitrary DSTCI was decomposed into wavelets with constituent spatiotemporal frequencies, and the effects on image quality were quantitatively investigated. The results show the synergistic effects on reduction of speckle noise when DSTCI is decreased. This study presents a method to systematically control DSTCI, and the result provides an essential reference on the effects of DSTCI on the imaging quality. We believe that the presented methods and results can be implemented in various imaging systems for characterizing and improving the imaging quality.
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We found an error in Fig. 1 of our article "White-light Quantitative Phase Imaging Unit." Here we publish the revised figure.
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We present a time-multiplexing structured illumination control technique for optical diffraction tomography (ODT). Instead of tilting the angle of illumination, time-multiplexed sinusoidal illumination is exploited using a digital micromirror device (DMD). The present method effectively eliminates unwanted diffracted beams from binary DMD patterns, which deteriorates the image quality of the ODT in the previous binary Lee hologram method. We experimentally show the feasibility and advantage of the present method by reconstructing three-dimensional refractive index distributions of various samples and comparing with a conventional Lee hologram method.
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Microfluidic mixing plays a key role in various fields, including biomedicine and chemical engineering. To date, although various approaches for imaging microfluidic mixing have been proposed, they provide only quantitative imaging capability and require exogenous labeling agents. Quantitative phase imaging techniques, however, circumvent these problems and offer label-free quantitative information about concentration maps of microfluidic mixing. We present the quantitative phase imaging of microfluidic mixing in various types of polydimethylsiloxane microfluidic channels with different geometries; the feasibility of the present method was validated by comparing it with the results obtained by theoretical calculation based on Fick's law.
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Here, we present a novel microscopic technique for measuring wavelength-dependent three-dimensional (3-D) distributions of the refractive indices (RIs) of microscopic samples in the visible wavelengths. Employing 3-D quantitative phase microscopy techniques with a wavelength-swept source, 3-D RI tomograms were obtained in the range of 450 - 700 nm with a spectral resolution of a few nanometers. The capability of the technique was demonstrated by measuring the hyperspectral 3-D RI tomograms of polystyrene beads, human red blood cells, and hepatocytes. The results demonstrate the potential for label-free molecular specific 3-D tomography of biological samples.
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Tomografia Óptica/métodos , Linhagem Celular Tumoral , Eritrócitos/citologia , Humanos , Imageamento Tridimensional , IluminaçãoRESUMO
We introduce the white-light quantitative phase imaging unit (WQPIU) as a practical realization of quantitative phase imaging (QPI) on standard microscope platforms. The WQPIU is a compact stand-alone unit which measures sample induced phase delay under white-light illumination. It does not require any modification of the microscope or additional accessories for its use. The principle of the WQPIU based on lateral shearing interferometry and phase shifting interferometry provides a cost-effective and user-friendly use of QPI. The validity and capacity of the presented method are demonstrated by measuring quantitative phase images of polystyrene beads, human red blood cells, HeLa cells and mouse white blood cells. With speckle-free imaging capability due to the use of white-light illumination, the WQPIU is expected to expand the scope of QPI in biological sciences as a powerful but simple imaging tool.
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Herein is presented an optical diffraction tomography (ODT) technique for measuring 3-D refractive index (RI) maps of optical plastic lenses. A Mach-Zehnder interferometer was used to measure multiple complex optical fields of a plastic lens immersed in RI-matching oil at various rotational orientations. From this, ODT was used to reconstruct a 3-D RI distribution of the plastic lens with unprecedented RI sensitivity (Δn=4.21×10(-5) and high resolution (12.8 µm). As a demonstration, 3-D RI distributions of a 2 mm-diameter borosilicate sphere and a 5 mm-diameter plastic lens were reconstructed. Defects in the lens, generated by pulsed laser ablation, were also detected using the present method.
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Lentes , Plásticos , Tomografia Óptica/métodos , Interferometria , Refratometria , Tomografia Óptica/instrumentaçãoRESUMO
An erratum is presented to correct a typographical error on Fig. 1 in [Opt. Express 22(9), 10398 (2014)].
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We present a powerful and cost-effective method for active illumination using a digital micromirror device (DMD) for quantitative phase-imaging techniques. Displaying binary illumination patterns on a DMD with appropriate spatial filtering, plane waves with various illumination angles are generated and impinged onto a sample. Complex optical fields of the sample obtained with various incident angles are then measured via Mach-Zehnder interferometry, from which a high-resolution 2D synthetic aperture phase image and a 3D refractive index tomogram of the sample are reconstructed. We demonstrate the fast and stable illumination-control capability of the proposed method by imaging colloidal spheres and biological cells. The capability of high-speed optical diffraction tomography is also demonstrated by measuring 3D Brownian motion of colloidal particles with the tomogram acquisition rate of 100 Hz.
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We present an optical holographic micro-tomographic technique for imaging both the three-dimensional structures and dynamics of biological cells. Optical light field images of a sample, illuminated by a plane wave with various illumination angles, are measured in a common-path interferometry, and thus both the three-dimensional refractive index tomogram and two-dimensional dynamics of live biological cells are measured with extremely high sensitivity. The applicability of the technique is demonstrated through quantitative and measurements of morphological, chemical, and mechanical parameters at the individual cell level.
Assuntos
Células/citologia , Holografia , Interferometria/métodos , Luz , Refratometria/métodos , Tomografia Óptica/métodos , Tomografia Computadorizada por Raios XRESUMO
A simple and practical method to measure three-dimensional (3-D) refractive index (RI) distributions of biological cells is presented. A common-path self-reference interferometry consisting of a compact set of polarizers is attached to a conventional inverted microscope equipped with a beam scanning unit, which can precisely measure multiple 2-D holograms of a sample with high phase stability for various illumination angles, from which accurate 3-D optical diffraction tomograms of the sample can be reconstructed. 3-D RI tomograms of nonbiological samples such as polystyrene microspheres, as well as biological samples including human red blood cells and breast cancer cells, are presented.