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Journal ArticleDOI

Multiparametric imaging of biological systems by force-distance curve-based AFM.

01 Sep 2013-Nature Methods (Nature Publ. Group)-Vol. 10, Iss: 9, pp 847-854
TL;DR: The principles and applications of advanced FD-based AFM tools for the quantitative multiparametric characterization of complex cellular and biomolecular systems under physiological conditions are discussed.
Abstract: A current challenge in the life sciences is to understand how biological systems change their structural, biophysical and chemical properties to adjust functionality. Addressing this issue has been severely hampered by the lack of methods capable of imaging biosystems at high resolution while simultaneously mapping their multiple properties. Recent developments in force-distance (FD) curve–based atomic force microscopy (AFM) now enable researchers to combine (sub)molecular imaging with quantitative mapping of physical, chemical and biological interactions. Here we discuss the principles and applications of advanced FD-based AFM tools for the quantitative multiparametric characterization of complex cellular and biomolecular systems under physiological conditions.
Citations
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Journal ArticleDOI
TL;DR: Overall, these data demonstrate that GSDMD is the direct and final executor of pyroptotic cell death.
Abstract: Pyroptosis is a lytic type of cell death that is initiated by inflammatory caspases. These caspases are activated within multi-protein inflammasome complexes that assemble in response to pathogens and endogenous danger signals. Pyroptotic cell death has been proposed to proceed via the formation of a plasma membrane pore, but the underlying molecular mechanism has remained unclear. Recently, gasdermin D (GSDMD), a member of the ill-characterized gasdermin protein family, was identified as a caspase substrate and an essential mediator of pyroptosis. GSDMD is thus a candidate for pyroptotic pore formation. Here, we characterize GSDMD function in live cells and in vitro We show that the N-terminal fragment of caspase-1-cleaved GSDMD rapidly targets the membrane fraction of macrophages and that it induces the formation of a plasma membrane pore. In vitro, the N-terminal fragment of caspase-1-cleaved recombinant GSDMD tightly binds liposomes and forms large permeability pores. Visualization of liposome-inserted GSDMD at nanometer resolution by cryo-electron and atomic force microscopy shows circular pores with variable ring diameters around 20 nm. Overall, these data demonstrate that GSDMD is the direct and final executor of pyroptotic cell death.

762 citations


Cites methods from "Multiparametric imaging of biologic..."

  • ...The full color range of the AFM topographs corresponds to a vertical scale (height) of 20 nm....

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  • ...E High-resolution AFM topographs and height profiles of GSDMDNterm oligomers (red curves)....

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  • ...In an initial control experiment, we adsorbed either GSDMD alone, or caspase-1 alone, or GSDMD and caspase-1 together to freshly cleaved mica, which we used as sample support in our AFM studies (Appendix Fig S7A–...

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  • ...The AFM was placed inside a home-built temperature controlled acoustic isolation box....

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  • ...To characterize the assembly of the transmembrane pores convincingly, we thus imaged GSDMD oligomers and pore formation on liposomes by atomic force microscopy (AFM)....

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Journal ArticleDOI
TL;DR: The basic principles, advantages and limitations of the most common AFM bioimaging modes are reviewed, including the popular contact and dynamic modes, as well as recently developed modes such as multiparametric, molecular recognition, multifrequency and high-speed imaging.
Abstract: Atomic force microscopy (AFM) is a powerful, multifunctional imaging platform that allows biological samples, from single molecules to living cells, to be visualized and manipulated. Soon after the instrument was invented, it was recognized that in order to maximize the opportunities of AFM imaging in biology, various technological developments would be required to address certain limitations of the method. This has led to the creation of a range of new imaging modes, which continue to push the capabilities of the technique today. Here, we review the basic principles, advantages and limitations of the most common AFM bioimaging modes, including the popular contact and dynamic modes, as well as recently developed modes such as multiparametric, molecular recognition, multifrequency and high-speed imaging. For each of these modes, we discuss recent experiments that highlight their unique capabilities.

649 citations

Journal ArticleDOI
TL;DR: It is demonstrated that phosphorylated or mutant aggregation prone recombinant tau undergoes LLPS, as does high molecular weight soluble phospho‐tau isolated from human Alzheimer brain, and it is suggested that LLPS represents a biophysical process with a role in multiple different neurodegenerative diseases.
Abstract: The transition between soluble intrinsically disordered tau protein and aggregated tau in neurofibrillary tangles in Alzheimer's disease is unknown. Here, we propose that soluble tau species can undergo liquid–liquid phase separation (LLPS) under cellular conditions and that phase‐separated tau droplets can serve as an intermediate toward tau aggregate formation. We demonstrate that phosphorylated or mutant aggregation prone recombinant tau undergoes LLPS, as does high molecular weight soluble phospho‐tau isolated from human Alzheimer brain. Droplet‐like tau can also be observed in neurons and other cells. We found that tau droplets become gel‐like in minutes, and over days start to spontaneously form thioflavin‐S‐positive tau aggregates that are competent of seeding cellular tau aggregation. Since analogous LLPS observations have been made for FUS, hnRNPA1, and TDP43, which aggregate in the context of amyotrophic lateral sclerosis, we suggest that LLPS represents a biophysical process with a role in multiple different neurodegenerative diseases.

616 citations


Cites methods from "Multiparametric imaging of biologic..."

  • ...…of 1-day-old p-tau441 droplets (in buffer in absence of PEG) attached to a glass surface by force-distance curve-based atomic force microcopy (Dufrêne et al, 2013), we found a rather high rigidity of the droplets, which by AFM resisted scanning forces of > 250 pN, and that small tau…...

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  • ...Imaging parameters included a scan rate of 0.4–0.6 Hz, sampling rate of 2 kHz, maximum imaging force of 200–300 pN and oscillating the AFM stylus at a vertical amplitude of 400 nm....

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  • ...To further explore the rigidity of the mature tau droplets, we employed atomic force microscopy (AFM)....

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  • ...When imaging the topography of 1-day-old p-tau441 droplets (in buffer in absence of PEG) attached to a glass surface by force-distance curve-based atomic force microcopy (Dufrêne et al, 2013), we found a rather high rigidity of the droplets, which by AFM resisted scanning forces of > 250 pN, and that small tau aggregates protruded up to 0.5 lm as stiff structures from the droplet surface (Fig 4F, Appendix Fig S3)....

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  • ...AFM cantilevers (PFQNM-LC from Bruker) had nominal spring constants of 0.08 N/m, had resonance frequencies of 60 kHz in buffer, and carried a SiO2 stylus with a nominal apex radius of 65 nm....

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Journal ArticleDOI
01 Jan 2019
TL;DR: The potential of combining AFM with complementary techniques, including optical microscopy and spectroscopy of mechanosensitive fluorescent constructs, super-resolution microscopy, the patch clamp technique and the use of microstructured and fluidic devices to characterize the 3D distribution of mechanical responses within biological systems and to track their morphology and functional state as discussed by the authors.
Abstract: Mechanobiology emerges at the crossroads of medicine, biology, biophysics and engineering and describes how the responses of proteins, cells, tissues and organs to mechanical cues contribute to development, differentiation, physiology and disease. The grand challenge in mechanobiology is to quantify how biological systems sense, transduce, respond and apply mechanical signals. Over the past three decades, atomic force microscopy (AFM) has emerged as a key platform enabling the simultaneous morphological and mechanical characterization of living biological systems. In this Review, we survey the basic principles, advantages and limitations of the most common AFM modalities used to map the dynamic mechanical properties of complex biological samples to their morphology. We discuss how mechanical properties can be directly linked to function, which has remained a poorly addressed issue. We outline the potential of combining AFM with complementary techniques, including optical microscopy and spectroscopy of mechanosensitive fluorescent constructs, super-resolution microscopy, the patch clamp technique and the use of microstructured and fluidic devices to characterize the 3D distribution of mechanical responses within biological systems and to track their morphology and functional state. Mechanobiology describes how biological systems respond to mechanical stimuli. This Review surveys basic principles, advantages and limitations of applying and combining atomic force microscopy-based modalities with complementary techniques to characterize the morphology, mechanical properties and functional response of complex biological systems to mechanical cues.

387 citations

Journal ArticleDOI
TL;DR: Understanding of the mechanisms governing bacterial adhesion at the single-cell level is summarized, including the physical forces experienced by a cell before reaching the surface, the first contact with a surface and the transition from reversible to permanent adhesion.
Abstract: The formation of multicellular microbial communities, called biofilms, starts from the adhesion of a few planktonic cells to the surface. The transition from a free-living planktonic lifestyle to a sessile, attached state is a multifactorial process that is determined by biological, chemical and physical properties of the environment, the surface and the bacterial cell. The initial weak, reversible interactions between a bacterium and a surface strengthen to yield irreversible adhesion. In this Review, we summarize our understanding of the mechanisms governing bacterial adhesion at the single-cell level, including the physical forces experienced by a cell before reaching the surface, the first contact with a surface and the transition from reversible to permanent adhesion.

346 citations

References
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Journal ArticleDOI
12 May 1978-Science
TL;DR: The force required to separate two cells is shown to be greater than the expected electrical forces between cells, and of the same order of magnitude as the forces required to pull gangliosides and perhaps some integral membrane proteins out of the cell membrane.
Abstract: A theoretical framework is proposed for the analysis of adhesion between cells or of cells to surfaces when the adhesion is mediated by reversible bonds between specific molecules such as antigen and antibody, lectin and carbohydrate, or enzyme and substrate. From a knowledge of the reaction rates for reactants in solution and of their diffusion constants both in solution and on membranes, it is possible to estimate reaction rates for membrane-bound reactants. Two models are developed for predicting the rate of bond formation between cells and are compared with experiments. The force required to separate two cells is shown to be greater than the expected electrical forces between cells, and of the same order of magnitude as the forces required to pull gangliosides and perhaps some integral membrane proteins out of the cell membrane.

4,058 citations

Journal ArticleDOI
TL;DR: The atomic force microscope (AFM) is not only used to image the topography of solid surfaces at high resolution but also to measure force-versus-distance curves as discussed by the authors, which provide valuable information on local material properties such as elasticity, hardness, Hamaker constant, adhesion and surface charge densities.

3,281 citations

Journal ArticleDOI
TL;DR: A single-molecule study of the substrate and oxidation-dependent adhesive properties of dopa is reported, in which dopa exploits a remarkable combination of high strength and chemical multifunctionality to accomplish adhesion to substrates of widely varying composition.
Abstract: The glue proteins secreted by marine mussels bind strongly to virtually all inorganic and organic surfaces in aqueous environments in which most adhesives function poorly. Studies of these functionally unique proteins have revealed the presence of the unusual amino acid 3,4-dihydroxy-L-phenylalanine (dopa), which is formed by posttranslational modification of tyrosine. However, the detailed binding mechanisms of dopa remain unknown, and the chemical basis for mussels' ability to adhere to both inorganic and organic surfaces has never been fully explained. Herein, we report a single-molecule study of the substrate and oxidation-dependent adhesive properties of dopa. Atomic force microscopy (AFM) measurements of a single dopa residue contacting a wet metal oxide surface reveal a surprisingly high strength yet fully reversible, noncovalent interaction. The magnitude of the bond dissociation energy as well as the inability to observe this interaction with tyrosine suggests that dopa is critical to adhesion and that the binding mechanism is not hydrogen bond formation. Oxidation of dopa, as occurs during curing of the secreted mussel glue, dramatically reduces the strength of the interaction to metal oxide but results in high strength irreversible covalent bond formation to an organic surface. A new picture of the interfacial adhesive role of dopa emerges from these studies, in which dopa exploits a remarkable combination of high strength and chemical multifunctionality to accomplish adhesion to substrates of widely varying composition from organic to metallic.

1,859 citations

Journal ArticleDOI
TL;DR: This work reports the stiffness of live metastatic cancer cells taken from the body fluids of patients with suspected lung, breast and pancreas cancer, and shows that nanomechanical analysis correlates well with immunohistochemical testing currently used for detecting cancer.
Abstract: Change in cell stiffness is a new characteristic of cancer cells that affects the way they spread1,2. Despite several studies on architectural changes in cultured cell lines1,3, no ex vivo mechanical analyses of cancer cells obtained from patients have been reported. Using atomic force microscopy, we report the stiffness of live metastatic cancer cells taken from the body (pleural) fluids of patients with suspected lung, breast and pancreas cancer. Within the same sample, we find that the cell stiffness of metastatic cancer cells is more than 70% softer, with a standard deviation over five times narrower, than the benign cells that line the body cavity. Different cancer types were found to display a common stiffness. Our work shows that mechanical analysis can distinguish cancerous cells from normal ones even when they show similar shapes. These results show that nanomechanical analysis correlates well with immunohistochemical testing currently used for detecting cancer.

1,744 citations

Journal ArticleDOI
TL;DR: In this paper, the authors calculated the thermal noise of a cantilever with a free end by considering all possible vibration modes of the cantilevers and showed that if the end is supported by a hard surface, no thermal fluctuations of the deflection are possible.
Abstract: Thermal fluctuations of the cantilever are a fundamental source of noise in atomic force microscopy. We calculated thermal noise using the equipartition theorem and considering all possible vibration modes of the cantilever. The measurable amplitude of thermal noise depends on the temperature, the spring constant K of the cantilever and on the method by which the cantilever deflection is detected. If the deflection is measured directly, e.g. with an interferometer or a scanning tunneling microscope, the thermal noise of a cantilever with a free end can be calculated from square root kT/K. If the end of the cantilever is supported by a hard surface no thermal fluctuations of the deflection are possible. If the optical lever technique is applied to measure the deflection, the thermal noise of a cantilever with a free end is square root 4kT/3K. When the cantilever is supported thermal noise decreases to square root kT/3K, but it does not vanish.

1,535 citations