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Despite progress made in confocal microscopy, even fast systems still have insufficient temporal resolution for detailed live-cell volume imaging, such as tracking rapid movement of membrane vesicles in three-dimensional space. Depending on the shortfall, this may result in undersampling and/or motion artifacts that ultimately limit the quality of the imaging data. By sacrificing detailed information in the Z-direction, we propose a new imaging modality that involves capturing fast 'projections' from the field of depth and shortens imaging time by approximately an order of magnitude as compared to standard volumetric confocal imaging. With faster imaging, radiation exposure to the sample is reduced, resulting in less fluorophore photobleaching and potential photodamage. The implementation minimally requires two synchronized control signals that drive a piezo stage and trigger the camera exposure. The device generating the signals has been tested on spinning disk confocal and instant structured-illumination-microscopy (iSIM) microscopes. Our calibration images show that the approach provides highly repeatable and stable imaging conditions that enable photometric measurements of the acquired data, in both standard live imaging and super-resolution modes.This article has an associated First Person interview with the first author of the paper.
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Corantes Fluorescentes , Iluminação , Microscopia Confocal , FotodegradaçãoRESUMO
The dimeric focal adhesion protein talin contains up to 22 cryptic vinculin binding sites that are exposed by unfolding. Using a novel method to monitor the in situ dynamics of the talin dimer stretch, we find that in contrast to several prevalent talin dimer models the integrin-binding talin N-termini are separated by 162 ± 44 nm on average whereas as expected the C-terminal dimerization domains colocalize and are mobile. Using vinculin tagged by DHFR-TMP Atto655 label, we found that optimal vinculin and vinculin head binding occurred when talin was stretched to 180 nm, while the controls did not bind to talin. Surprisingly, multiple vinculins bound within a single second in narrowly localized regions of the talin rod during stretching. We suggest that talin stretches as an antiparallel dimer and that activates vinculin binding in a cooperative manner, consistent with the stabilization of folded talin by other binding proteins.
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Mechanotransduction is a critical function for cells, in terms of cell viability, shaping of tissues, and cellular behavior. In vitro, cellular level forces can stretch adhesion proteins that link extracellular matrix to the actin cytoskeleton exposing hidden binding sites. However, there is no evidence that in vivo forces produce significant in vivo stretching to cause domain unfolding. We now report that the adhesion protein, talin, is repeatedly stretched by 100-350 nm in vivo by myosin contraction of actin filaments. Using a functional EGFP-N-Talin1-C-mCherry to measure the length of single talin molecules, we observed that the C-terminal mCherry was normally displaced in the direction of actin flow by 90 to >250 nm from N-EGFP but only by 50-60 nm (talin's length in vitro) after myosin inhibition. Individual talin molecules transiently stretched and relaxed. Peripheral, multimolecular adhesions had green outside and red proximal edges. They also exhibited transient, myosin-dependent stretching of 50-350 nm for 6-16 s; however, expression of the talin-binding head of vinculin increased stretching to about 400 nm and suppressed dynamics. We suggest that rearward moving actin filaments bind, stretch, and release talin in multiple, stochastic stick-slip cycles and that multiple vinculin binding and release cycles integrate pulling on matrices into biochemical signals.
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Mecanotransdução Celular , Talina/biossíntese , Vinculina/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Adesão Celular , Linhagem Celular , Chlorocebus aethiops , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Talina/metabolismoRESUMO
This study investigates differences in focal adhesion (FA) morphology and Talin cleavage levels between transformed and non-transformed cell lines. Utilizing fluorescently tagged wild-type Talin and Talin mutants with calpain cleavage site mutations, FA structures were visualized. Mutations in different Talin cleavage sites showed varying impacts on FA morphology and distribution across melanoma cell lines (Meljuso, A375P, A2058) and a non-transformed cell line (HFF). Western blot analysis, ratiometric fluorescence intensity-based measurements, and FRAP experiments revealed higher Talin cleavage levels within FAs of transformed cell lines compared to non-transformed cells. Additionally, growth assays indicated that reducing calpain cleavage levels attenuated transformed cell growth. These findings suggest that Talin cleavage level is crucial for FA morphology and assembly, with higher levels observed in transformed cells, influencing their growth dynamics.
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The lens mitochondrion of the tree shrew, located along the optical pathway between the lens and photoreceptors, has been investigated. The results suggest that the lens mitochondrion acts as a quasi-bandgap or imperfect photonic crystal. Interference effects cause a shift in the focus and introduce wavelength-dependent behavior similar to dispersion. Optical channels within the mitochondrion form a mild waveguide, preferentially propagating light within certain compartments. The lens mitochondrion also functions as an imperfect UV-shielding interference filter. Overall, this study provides insights into the dual role of the lens mitochondrion and the complex behavior of light within biological systems.
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Focal adhesions (FAs) control cell shape and motility, which are important processes that underlie a wide range of physiological functions. FA dynamics is regulated by cytoskeleton, motor proteins and small GTPases. Kinectin is an integral endoplasmic reticulum (ER) membrane protein that extends the ER along microtubules. Here, we investigated the influence of the ER on FA dynamics within the cellular lamella by disrupting the kinectin-kinesin interaction by overexpressing the minimal kinectin-kinesin interaction domain on kinectin in cells. This perturbation resulted in a morphological change to a rounded cell shape and reduced cell spreading and migration. Immunofluorescence and live-cell imaging demonstrated a kinectin-dependent ER extension into the cellular lamella and ER colocalisation with FAs within the cellular lamella. FRAP experiments showed that ER contact with FAs was accompanied with an increase in FA protein recruitment to FAs. Disruption of the kinectin-kinesin interaction caused a reduction in FA protein recruitment to FAs. This suggests that the ER supports FA growth within the cellular lamella. Microtubule targeting to FAs is known to promote adhesion disassembly; however, ER contact increased FA size even in the presence of microtubules. Our results suggest a scenario whereby kinectin-kinesin interaction facilitates ER transport along microtubules to support FA growth.
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Retículo Endoplasmático/metabolismo , Adesões Focais/metabolismo , Cinesinas/metabolismo , Proteínas de Membrana/metabolismo , Movimento Celular/fisiologia , Células HeLa , Humanos , Integrina beta3/metabolismo , Microtúbulos/metabolismoRESUMO
Recent studies show that tumor cells are vulnerable to mechanical stresses and undergo calcium-dependent apoptosis (mechanoptosis) with mechanical perturbation by low-frequency ultrasound alone. To determine if tumor cells are particularly sensitive to mechanical stress in certain phases of the cell cycle, inhibitors of the cell-cycle phases are tested for effects on mechanoptosis. Most inhibitors show no significant effect, but inhibitors of mitosis that cause microtubule depolymerization increase the mechanoptosis. Surprisingly, ultrasound treatment also disrupts microtubules independent of inhibitors in tumor cells but not in normal cells. Ultrasound causes calcium entry through mechanosensitive Piezo1 channels that disrupts microtubules via calpain protease activation. Myosin IIA contractility is required for ultrasound-mediated mechanoptosis and microtubule disruption enhances myosin IIA contractility through activation of GEF-H1 and RhoA pathway. Further, ultrasound promotes contractility-dependent Piezo1 expression and localization to the peripheral adhesions where activated Piezo1 allows calcium entry to continue feedback loop. Thus, the synergistic action of ultrasound and nanomolar concentrations of microtubule depolymerizing agents can enhance tumor therapies.
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The members of the Rab family of small GTPases are molecular switches that regulate distinct steps in different membrane traffic pathways. In addition to this canonical function, Rabs can play a role in other processes, such as cell adhesion and motility. Here, we reveal the role of the small GTPase Rab18 as a positive regulator of directional migration in chemotaxis, and the underlying mechanism. We show that knockdown of Rab18 reduces the size of focal adhesions (FAs) and influences their dynamics. Furthermore, we found that Rab18, by directly interacting with the endoplasmic reticulum (ER)-resident protein kinectin-1, controls the anterograde kinesin-1-dependent transport of the ER required for the maturation of nascent FAs and protrusion orientation toward a chemoattractant. Altogether, our data support a model in which Rab18 regulates kinectin-1 transport toward the cell surface to form ER-FA contacts, thus promoting FA growth and cell migration during chemotaxis.
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Membrana Celular/metabolismo , Quimiotaxia/genética , Retículo Endoplasmático/metabolismo , Adesões Focais/metabolismo , Proteínas de Membrana/genética , Proteínas rab de Ligação ao GTP/genética , Transporte Biológico , Adesão Celular , Linhagem Celular Tumoral , Membrana Celular/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Adesões Focais/ultraestrutura , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Fosforilação , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Proteínas rab de Ligação ao GTP/antagonistas & inibidores , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
For individual cells in tissues to create the diverse forms of biological organisms, it is necessary that they must reliably sense and generate the correct forces over the correct distances and directions. There is considerable evidence that the mechanical aspects of the cellular microenvironment provide critical physical parameters to be sensed. How proteins sense forces and cellular geometry to create the correct morphology is not understood in detail but protein unfolding appears to be a major component in force and displacement sensing. Thus, the crystallographic structure of a protein domain provides only a starting point to then analyze what will be the effects of physiological forces through domain unfolding or catch-bond formation. In this review, we will discuss the recent studies of cytoskeletal and adhesion proteins that describe protein domain dynamics. Forces applied to proteins can activate or inhibit enzymes, increase or decrease protein-protein interactions, activate or inhibit protein substrates, induce catch bonds and regulate interactions with membranes or nucleic acids. Further, the dynamics of stretch-relaxation can average forces or movements to reliably regulate morphogenic movements. In the few cases where single molecule mechanics are studied under physiological conditions such as titin and talin, there are rapid cycles of stretch-relaxation that produce mechanosensing signals. Fortunately, the development of new single molecule and super-resolution imaging methods enable the analysis of single molecule mechanics in physiologically relevant conditions. Thus, we feel that stereotypical changes in cell and tissue shape involve mechanosensing that can be analyzed at the nanometer level to determine the molecular mechanisms involved.
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Membrana Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/fisiologia , Mecanotransdução Celular/fisiologia , Animais , HumanosRESUMO
α-Actinins, a family of critical cytoskeletal actin-binding proteins that usually exist as anti-parallel dimers, play crucial roles in organizing the framework of the cytoskeleton through crosslinking the actin filaments, as well as in focal adhesion maturation. However, the molecular mechanisms underlying its functions are unclear. Here, by mechanical manipulation of single human α-actinin 1 using magnetic tweezers, we determined the mechanical stability and kinetics of the functional domains in α-actinin 1. Moreover, we identified the force-dependence of vinculin binding to α-actinin 1, with the demonstration that force is required to expose the high-affinity binding site for vinculin binding. Further, a role of the α-actinin 1 as molecular shock absorber for the cytoskeleton network is revealed. Our results provide a comprehensive analysis of the force-dependent stability and interactions of α-actinin 1, which sheds important light on the molecular mechanisms underlying its mechanotransmission and mechanosensing functions.
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Actinina/metabolismo , Actinina/química , Citoesqueleto/metabolismo , Humanos , Cinética , Nanopartículas de Magnetita/química , Mecanotransdução Celular/genética , Mecanotransdução Celular/fisiologia , Vinculina/químicaRESUMO
We present a vector implementation for nonlinear filters that allows an efficient execution on graphics processing units. These filters are popular to suppress shot noise arising in low light conditions. Having them available at the end of the visualization stage makes this setup particularly suitable for handling the postprocessing stages for light microscopy. Without vector acceleration, these filters constitute a bottleneck since real time or frequent update volume rendering has become available on desktop workstations. Even simple averaging operations can push the overall system's performance noticeably below the original frame rate.
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Integrin adhesions assemble and mature in response to ligand binding and mechanical factors, but the molecular-level organization is not known. We report that â¼100-nm clusters of â¼50 ß3-activated integrins form very early adhesions under a wide variety of conditions on RGD surfaces. These adhesions form similarly on fluid and rigid substrates, but most adhesions are transient on rigid substrates. Without talin or actin polymerization, few early adhesions form, but expression of either the talin head or rod domain in talin-depleted cells restores early adhesion formation. Mutation of the integrin binding site in the talin rod decreases cluster size. We suggest that the integrin clusters constitute universal early adhesions and that they are the modular units of cell matrix adhesions. They require the association of activated integrins with cytoplasmic proteins, in particular talin and actin, and cytoskeletal contraction on them causes adhesion maturation for cell motility and growth.
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Integrina beta3/metabolismo , Oligopeptídeos/metabolismo , Talina/metabolismo , Actinas/metabolismo , Algoritmos , Animais , Sítios de Ligação , Adesão Celular , Movimento Celular , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Ligantes , Camundongos , Mutação , Ligação Proteica , Estresse MecânicoRESUMO
Increasing evidence has shown that mechanical cues from the environment play an important role in cell biology. Mechanotransduction or the study of how cells can sense these mechanical cues, and respond to them, is an active field of research. However, it is still not clear how cells sense and respond to mechanical cues. Thus, new tools are being rapidly developed to quantitatively study cell mechanobiology. Particularly, force measurement tools such as micropillar substrates have provided new insights into the underlying mechanisms of mechanosensing. In this chapter, we provide detailed protocol for fabrication, characterization, functionalization, and use of the micropillar substrates.
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Biofísica/métodos , Mecanotransdução Celular , Animais , Técnicas Biossensoriais , Adesão Celular , Sobrevivência Celular , Fibroblastos/citologia , Processamento de Imagem Assistida por ComputadorRESUMO
Matrix rigidity sensing regulates a large variety of cellular processes and has important implications for tissue development and disease. However, how cells probe matrix rigidity, and hence respond to it, remains unclear. Here, we show that rigidity sensing and adaptation emerge naturally from actin cytoskeleton remodelling. Our in vitro experiments and theoretical modelling demonstrate a biphasic rheology of the actin cytoskeleton, which transitions from fluid on soft substrates to solid on stiffer ones. Furthermore, we find that increasing substrate stiffness correlates with the emergence of an orientational order in actin stress fibres, which exhibit an isotropic to nematic transition that we characterize quantitatively in the framework of active matter theory. These findings imply mechanisms mediated by a large-scale reinforcement of actin structures under stress, which could be the mechanical drivers of substrate stiffness-dependent cell shape changes and cell polarity.
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Actinas/fisiologia , Citoesqueleto/fisiologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fenômenos Biomecânicos , Células Alimentadoras , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Força Atômica , Modelos Biológicos , Ratos , Reologia/métodosRESUMO
Cell membranes may fold up into three-dimensional nanoperiodic cubic structures in biological systems. Similar geometries are well studied in other disciplines such as mathematics, physics and polymer chemistry. The fundamental function of cubic membranes in biological systems has not been uncovered yet; however, their appearance in specialized cell types indicates a role as structural templates or perhaps direct physical entities with specialized biophysical properties. The mitochondria located at the inner segment of the retinal cones of tree shrew (Tupaia glis and Tupaia belangeri) contain unique patterns of concentric cristae with a highly ordered membrane arrangement in three dimensions similar to the photonic nanostructures observed in butterfly wing scales. Using a direct template matching method, we show that the inner mitochondrial membrane folds into multi-layered (8 to 12 layers) gyroid cubic membrane arrangements in the photoreceptor cells. Three-dimensional simulation data demonstrate that such multi-layer gyroid membrane arrangements in the retinal cones of a tree shrew's eye can potentially function as: (i) multi-focal lens; (ii) angle-independent interference filters to block UV light; and (iii) a waveguide photonic crystal. These theoretical results highlight for the first time the significance of multi-layer cubic membrane arrangements to achieve near-quasi-photonic crystal properties through the simple and reversible biological process of continuous membrane folding.
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Biomembranes are traditionally viewed as flat phospholipid-bilayer sheets delineating the cell boundaries and dividing the cell into multiple subcellular organelles with specialized functions. However, biological membranes may also fold up into three-dimensional nanoperiodic arrangements, termed cubic membranes. This type of geometry is mathematically well described and extensively studied in lipidic cubic phase systems. This chapter will (1) summarize similarities and dissimilarities between cubic membranes and cubic phases; (2) provide an update on the experimental data describing the role of lipids, proteins and electrostatic charges on the biogenesis of cubic membranes; and (3) discuss their potential function in intracellular macromolecular transport and as optical filters, as well as potential practical applications such as gene delivery vehicles.
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OBJECTIVES: The purpose of this study was to develop a transesophageal probe that: 1) enables on-line representation of the spatial structures of the heart, and 2) enables navigation of medical instruments. BACKGROUND: Whereas transthoracic real-time 3-dimensional (3D) echocardiography could recently be implemented, there is still no corresponding transesophageal system. Transesophageal real-time 3D echocardiography would have great potential for numerous clinical applications, such as navigation of catheters. METHODS: The newly developed real-time 3D system is based on a transesophageal probe in which multiple transducers are arranged in an interlaced pattern on a rotating cylinder. This enables continuous recording of a large echo volume of 70 mm in length and a sector angle of 120 degrees . The presentation of the volume-reconstructed data is made with a time lag of <100 ms. The frame rate is up to 20 Hz. In addition to conventional imaging, the observer can obtain a stereoscopic image of the structures examined with red/blue goggles. RESULTS: It was shown in vitro on ventricle- and aorta-form agar models and in vivo that the system enables excellent visualization of the 3D structures. Shape, spatial orientation, and the navigation of various catheters (e.g., EPS-catheter, Swan-Ganz-catheter), stents, or atrial septal defect occluders could be recorded on-line and stereoscopically depicted. The size of the echo sector enables a wide field of view without changing the position of the probe. CONCLUSIONS: Transesophageal real-time 3D echocardiography can be technically realized with the system presented here. The in vitro and in vivo studies show particularly the potential for navigation in the heart and large vessels on the basis of stereoscopic images.