RESUMO
The formation of dynamic protein filaments contributes to various biological functions by clustering individual molecules together and enhancing their binding to ligands. We report such a propensity for the BTB domains of certain proteins from the ZBTB family, a large eukaryotic transcription factor family implicated in differentiation and cancer. Working with Xenopus laevis and human proteins, we solved the crystal structures of filaments formed by dimers of the BTB domains of ZBTB8A and ZBTB18 and demonstrated concentration-dependent higher-order assemblies of these dimers in solution. In cells, the BTB-domain filamentation supports clustering of full-length human ZBTB8A and ZBTB18 into dynamic nuclear foci and contributes to the ZBTB18-mediated repression of a reporter gene. The BTB domains of up to 21 human ZBTB family members and two related proteins, NACC1 and NACC2, are predicted to behave in a similar manner. Our results suggest that filamentation is a more common feature of transcription factors than is currently appreciated.
Assuntos
Domínio BTB-POZ , Fatores de Transcrição , Proteínas de Xenopus , Animais , Humanos , Núcleo Celular/metabolismo , Núcleo Celular/genética , Cristalografia por Raios X , Células HEK293 , Modelos Moleculares , Ligação Proteica , Multimerização Proteica , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/química , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Xenopus laevis , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/químicaRESUMO
Atomic force microscopy (AFM) can visualize the dynamics of single biomolecules under near-physiological conditions. However, the scanning tip probes only the molecular surface with limited resolution, missing details required to fully deduce functional mechanisms from imaging alone. To overcome such drawbacks, we developed a computational framework to reconstruct 3D atomistic structures from AFM surface scans, employing simulation AFM and automatized fitting to experimental images. We provide applications to AFM images ranging from single molecular machines, protein filaments, to large-scale assemblies of 2D protein lattices, and demonstrate how the obtained full atomistic information advances the molecular understanding beyond the original topographic AFM image. We show that simulation AFM further allows for quantitative molecular feature assignment within measured AFM topographies. Implementation of the developed methods into the versatile interactive interface of the BioAFMviewer software, freely available at www.bioafmviewer.com, presents the opportunity for the broad Bio-AFM community to employ the enormous amount of existing structural and modeling data to facilitate the interpretation of resolution-limited AFM images.
Assuntos
Nanotecnologia , Proteínas , Simulação por Computador , Microscopia de Força Atômica/métodos , Proteínas/química , SoftwareRESUMO
While many fields have contributed to biological physics, nanotechnology offers a new scale of observation. High-speed atomic force microscopy (HS-AFM) provides nanometre structural information and dynamics with subsecond resolution of biological systems. Moreover, HS-AFM allows us to measure piconewton forces within microseconds giving access to unexplored, fast biophysical processes. Thus, HS-AFM provides a tool to nourish biological physics through the observation of emergent physical phenomena in biological systems. In this review, we present an overview of the contribution of HS-AFM, both in imaging and force spectroscopy modes, to the field of biological physics. We focus on examples in which HS-AFM observations on membrane remodelling, molecular motors or the unfolding of proteins have stimulated the development of novel theories or the emergence of new concepts. We finally provide expected applications and developments of HS-AFM that we believe will continue contributing to our understanding of nature, by serving to the dialogue between biology and physics. This article is part of a discussion meeting issue 'Dynamic in situ microscopy relating structure and function'.
Assuntos
Biofísica/métodos , Microscopia de Força Atômica/métodos , Fenômenos Biofísicos , Biofísica/instrumentação , Membrana Celular/química , Simulação por Computador , Proteínas Intrinsicamente Desordenadas/química , Proteínas de Membrana/química , Microscopia de Força Atômica/instrumentação , Modelos Moleculares , Proteínas Motores Moleculares/química , Nanotecnologia/instrumentação , Nanotecnologia/métodos , Dobramento de Proteína , Imagem Individual de Molécula , Biologia de Sistemas/métodosRESUMO
Publisher's notice concerning Nacho L.B. Munguira, Alfonso Barbas and Ignacio Casuso 2019 NanotechnologyThis article has been retracted by IOP Publishing following an institutional investigation by INSERM and AMU. The investigation concluded that the publication broke institutional rules and regulations. As a member of COPE, IOP Publishing agrees to honour the findings of the investigation and retract the paper, in line with COPE guidelines. (30 June 2020)The ß pore-forming toxins (ß-PFTs) are cytotoxic proteins produced as soluble monomers, which cluster and oligomerize at the membrane of the target host cells. Their initial oligomeric state, the prepore, is not cytotoxic. The ß-PFTs undergo a large structural transition to a second oligomeric state, the pore, which pierces the membrane of the host cell and is cytotoxic. Data from electrophysiology and vesicle permeabilization experiments have suggested the possibility of a correlation between the transition rate from prepore state to pore state and the levels of local crowding in the cluster of ß-PFT oligomers. Nevertheless, to this date, visualization and understanding at the molecular level are missing. We have addressed this issue using a panel of Atomic Force Microscopy (AFM) techniques and simulations. We describe the mechanism by which the rates of formation of the transmembrane pores correlate with the local levels of crowding for the ß-PFT lysenin and discuss possible biological and medical implications.
RESUMO
Pore-forming toxins form a family of proteins that act as virulence factors of pathogenic bacteria, but similar proteins are found in all kingdoms of life, including the vertebrate immune system. They are secreted as soluble monomers that oligomerize on target membranes in the so-called prepore state; after activation, they insert into the membrane and adopt the pore state. Lysenin is a pore-forming toxin from the earthworm Eisenida foetida, of which both the soluble and membrane-inserted structures are solved. However, the activation and membrane-insertion mechanisms have remained elusive. Here, we used high-speed atomic force microscopy to directly visualize the membrane-insertion mechanism. Changing the environmental pH from pH 7.5 to below pH 6.0 favored membrane insertion. We detected a short α-helix in the soluble structure that comprised three glutamic acids (Glu92, Glu94, and Glu97) that we hypothesized may represent a pH-sensor (as in similar toxins, e.g., Listeriolysin). Mutant lysenin still can form pores, but mutating these glutamic acids to glutamines rendered the toxin pH-insensitive. On the other hand, toxins in the pore state did not favor insertion of neighboring prepores; indeed, pore insertion breaks the hexagonal ordered domains of prepores and separates from neighboring molecules in the membrane. pH-dependent activation of toxins may represent a common feature of pore-forming toxins. High-speed atomic force microscopy with single-molecule resolution at high temporal resolution and the possibility of exchanging buffers during the experiments presents itself as a unique tool for the study of toxin-state conversion.
Assuntos
Membrana Celular/metabolismo , Toxinas Biológicas/metabolismo , Concentração de Íons de Hidrogênio , Modelos Moleculares , Porosidade , Conformação Proteica em alfa-Hélice , Toxinas Biológicas/químicaRESUMO
Atomic force microscopy (AFM) image acquisition is performed by raster-scanning a faint tip with respect to the sample by the use of a piezoelectric stage that is guided by a feedback system. This process implies that the resulting images feature particularities that distinguish them from images acquired by other techniques, such as the drift of the piezoelectric elements, the unequal image contrast along the fast- and the slow-scan axes, the physical contact between the tip of nondefinable geometry and the sample, and the feedback parameters. Recently, high-speed AFM (HS-AFM) has been introduced, which allows image acquisition about three orders of magnitude faster (500-100 ms frame rate) than conventional AFM (500 s to 100 s frame rate). HS-AFM produces image sequences, large data sets, which report biological sample dynamics. To analyze these movies, we have developed a software package that (i) adjusts individual scan lines and images to a common contrast and z-scale, (ii) filters specifically those scan lines where increased or insufficient force was applied, (iii) corrects for piezo-scanner drift, (iv) defines particle localization and angular orientation, and (v) performs particle tracking to analyze the lateral and rotation displacement of single molecules.
Assuntos
Processamento de Imagem Assistida por Computador , Microscopia de Força Atômica/instrumentação , SoftwareRESUMO
Clathrin-mediated endocytosis (CME) is a central trafficking pathway in eukaryotic cells regulated by phosphoinositides. The plasma membrane phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) plays an instrumental role in driving CME initiation. The F-BAR domain-only protein 1 and 2 complex (FCHo1/2) is among the early proteins that reach the plasma membrane, but the exact mechanisms triggering its recruitment remain elusive. Here, we show the molecular dynamics of FCHo2 self-assembly on membranes by combining minimal reconstituted in vitro and cellular systems. Our results indicate that PI(4,5)P2 domains assist FCHo2 docking at specific membrane regions, where it self-assembles into ring-like-shaped protein patches. We show that the binding of FCHo2 on cellular membranes promotes PI(4,5)P2 clustering at the boundary of cargo receptors and that this accumulation enhances clathrin assembly. Thus, our results provide a mechanistic framework that could explain the recruitment of early PI(4,5)P2-interacting proteins at endocytic sites.
Assuntos
Membrana Celular/metabolismo , Clatrina/metabolismo , Endocitose/genética , Proteínas de Ligação a Ácido Graxo/genética , Linhagem Celular Tumoral , Proteínas de Ligação a Ácido Graxo/metabolismo , HumanosRESUMO
Biological atomic force microscopy (AFM) is a fast growing and advancing field. This review's objective is to overview the state of the art and to retrace achievements of biological AFM as presented by past and present research, and wishes to give a (subjective) outlook where AFM may go in the upcoming years. The following areas of interest are discussed: High-resolution imaging, cell imaging, single molecule force spectroscopy, cell mechanical measurements, combined AFM instrumentation, and AFM instrumentation. Of all these topics, particular representative examples are shown, each of them standing for a variety of achievements by many research groups.
Assuntos
Microscopia de Força Atômica/métodos , Microscopia de Varredura por Sonda/métodosRESUMO
Membrane proteins diffuse within the membrane, form oligomers and supramolecular assemblies. Using high-speed atomic force microscopy, we present direct experimental measure of an in-membrane-plane interaction potential between membrane proteins. In purple membranes, ATP-synthase c-rings formed dimers that temporarily dissociated. C-ring dimers revealed subdiffusive motion, while dissociated monomers diffused freely. C-rings center-to-center distance probability distribution allowed the calculation and modeling of an in-membrane-plane energy landscape that presented repulsion at 80 Å, most stable dimer association at 103 Å (-3.5 k(B)T strength), and dissociation at 125 Å (-1 k(B)T strength). This first experimental data of nonlabeled membrane protein diffusion and the corresponding in-membrane-plane interaction energy landscape characterized membrane protein interaction with an attractive range of several k(B)T that reaches to a radius of â¼50 Å within the membrane plane.
Assuntos
Halobacterium salinarum/metabolismo , Proteínas de Membrana/metabolismo , Membrana Purpúrea/metabolismo , Bacteriorodopsinas/metabolismo , Microscopia de Força Atômica , Ligação Proteica , Membrana Purpúrea/ultraestrutura , TermodinâmicaRESUMO
Contact mode atomic force microscopy (AFM) is the most frequently used AFM imaging mode in biology. It is about 5-10 times faster than oscillating mode imaging (in conventional AFM setups), and provides topographs of biological samples with sub-molecular resolution and at a high signal-to-noise ratio. Unfortunately, contact mode imaging is sensitive to the applied force and intrinsic force drift: inappropriate force applied by the AFM tip damages the soft biological samples. We present a methodology that automatically searches for and maintains high resolution imaging forces. We found that the vertical and lateral vibrations of the probe during scanning are valuable signals for the characterization of the actual applied force by the tip. This allows automated adjustment and correction of the setpoint force during an experiment. A system that permanently performs this methodology steered the AFM towards high resolution imaging forces and imaged purple membrane at molecular resolution and live cells at high signal-to-noise ratio for hours without an operator.
Assuntos
Automação , Microscopia de Força Atômica/métodos , Membrana Purpúrea/ultraestrutura , Fenômenos Biomecânicos , Halobacterium salinarum/ultraestrutura , Epitélio Pigmentado da Retina/citologia , Fatores de Tempo , VibraçãoRESUMO
The increase in speed of the high-speed atomic force microscopy (HS-AFM) compared to that of the conventional AFM made possible the first-ever visualisation at the molecular-level of the activity of an antimicrobial peptide on a membrane. We investigated the medically prescribed but poorly understood lipopeptide Daptomycin under infection-like conditions (37 °C, bacterial lipid composition and antibiotic concentrations). We confirmed so far hypothetical models: Dap oligomerization and the existence of half pores. Moreover, we detected unknown molecular mechanisms: new mechanisms to form toroidal pores or to resist Dap action, and to unprecedently quantify the energy profile of interacting oligomers. Finally, the biological and medical relevance of the findings was ensured by a multi-scale multi-nativeness-from the molecule to the cell-correlation of molecular-level information from living bacteria (Bacillus subtilis strains) to liquid-suspended vesicles and supported-membranes using electron and optical microscopies and the lipid tension probe FliptR, where we found that the cells with a healthier state of their cell wall show smaller membrane deformations.
Assuntos
Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Daptomicina/farmacologia , Microscopia de Força Atômica , Antibacterianos/uso terapêutico , Bacillus subtilis/citologia , Bacillus subtilis/ultraestrutura , Membrana Externa Bacteriana/efeitos dos fármacos , Membrana Externa Bacteriana/ultraestrutura , Parede Celular/efeitos dos fármacos , Parede Celular/ultraestrutura , Daptomicina/uso terapêutico , Farmacorresistência Bacteriana , Humanos , Bicamadas Lipídicas , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Transmissão , Modelos BiológicosRESUMO
High-speed atomic force microscopy (HS-AFM) is becoming a reference tool for the study of dynamic biological processes. The spatial and time resolutions of HS-AFM are on the order of nanometers and milliseconds, respectively, and allow structural and functional characterization of biological processes at the single-molecule level. In this work we present contact-mode HS-AFM movies of purple membranes containing two-dimensional arrays of bacteriorhodopsin (bR). In high-resolution movies acquired at a 100 ms frame acquisition time, the substructure on individual bR trimers was visualized. In regions in between different bR arrays, dynamic topographies were observed and interpreted as motion of the bR trimers. Similarly, motion of bR monomers in the vicinity of lattice defects in the purple membrane was observed. Our findings indicate that the bR arrays are in a mobile association-dissociation equilibrium. HS-AFM on membranes provides novel perspectives for analyzing the membrane diffusion processes of nonlabeled molecules.
Assuntos
Bacteriorodopsinas/ultraestrutura , Microscopia de Força Atômica/métodos , Membrana Purpúrea/ultraestrutura , Gravação em Vídeo , Bacteriorodopsinas/química , Bacteriorodopsinas/metabolismo , Halobacterium salinarum , Movimento (Física) , Multimerização Proteica , Membrana Purpúrea/metabolismo , Fatores de TempoRESUMO
The advent of high-speed atomic force microscopy (HS-AFM) over the recent years has opened up new horizons for the study of structure, function and dynamics of biological molecules. HS-AFM is capable of 1000 times faster imaging than conventional AFM. This circumstance uniquely enables the observation of the dynamics of all the molecules present in the imaging area. Over the last 10 years, the HS-AFM has gone from a prototype-state technology that only a few labs in the world had access to (including ours) to an established commercialized technology that is present in tens of labs around the world. In this protocol chapter we share with the readers our practical know-how on high resolution HS-AFM imaging.
Assuntos
Imageamento Tridimensional , Microscopia de Força Atômica/métodos , Bicamadas Lipídicas/química , Gravação em VídeoRESUMO
Many functions of the plasma membrane depend critically on its structure and dynamics. Observation of anomalous diffusion in vivo and in vitro using fluorescence microscopy and single particle tracking has advanced our concept of the membrane from a homogeneous fluid bilayer with freely diffusing proteins to a highly organized crowded and clustered mosaic of lipids and proteins. Unfortunately, anomalous diffusion could not be related to local molecular details given the lack of direct and unlabeled molecular observation capabilities. Here, we use high-speed atomic force microscopy and a novel analysis methodology to analyze the pore forming protein lysenin in a highly crowded environment and document coexistence of several diffusion regimes within one membrane. We show the formation of local glassy phases, where proteins are trapped in neighbor-formed cages for time scales up to 10 s, which had not been previously experimentally reported for biological membranes. Furthermore, around solid-like patches and immobile molecules a slower glass phase is detected leading to protein trapping and creating a perimeter of decreased membrane diffusion.
Assuntos
Bicamadas Lipídicas/química , Toxinas Biológicas/química , Vitrificação , DifusãoRESUMO
Atomic force microscopy (AFM) is the type of scanning probe microscopy that is probably best adapted for imaging biological samples in physiological conditions with submolecular lateral and vertical resolution. In addition, AFM is a method of choice to study the mechanical unfolding of proteins or for cellular force spectroscopy. In spite of 28 years of successful use in biological sciences, AFM is far from enjoying the same popularity as electron and fluorescence microscopy. The advent of high-speed atomic force microscopy (HS-AFM), about 10 years ago, has provided unprecedented insights into the dynamics of membrane proteins and molecular machines from the single-molecule to the cellular level. HS-AFM imaging at nanometer-resolution and sub-second frame rate may open novel research fields depicting dynamic events at the single bio-molecule level. As such, HS-AFM is complementary to other structural and cellular biology techniques, and hopefully will gain acceptance from researchers from various fields. In this review we describe some of the most recent reports of dynamic bio-molecular imaging by HS-AFM, as well as the advent of high-speed force spectroscopy (HS-FS) for single protein unfolding.
Assuntos
Fenômenos Mecânicos , Microscopia de Força Atômica/métodos , Imagem Molecular/métodos , Fenômenos Biomecânicos , Membrana Celular/metabolismoRESUMO
High-speed atomic force microscopy is a powerful tool for studying structure and dynamics of proteins. So far, however, high-speed atomic force microscopy was restricted to well-controlled molecular systems of purified proteins. Here we integrate an optical microscopy path into high-speed atomic force microscopy, allowing bright field and fluorescence microscopy, without loss of high-speed atomic force microscopy performance. This hybrid high-speed atomic force microscopy/optical microscopy setup allows positioning of the high-speed atomic force microscopy tip with high spatial precision on an optically identified zone of interest on cells. We present movies at 960 ms per frame displaying aquaporin-0 array and single molecule dynamics in the plasma membrane of intact eye lens cells. This hybrid setup allows high-speed atomic force microscopy imaging on cells about 1,000 times faster than conventional atomic force microscopy/optical microscopy setups, and allows first time visualization of unlabelled membrane proteins on a eukaryotic cell under physiological conditions. This development advances high-speed atomic force microscopy from molecular to cell biology to analyse cellular processes at the membrane such as signalling, infection, transport and diffusion.
Assuntos
Aquaporinas/química , Membrana Celular/ultraestrutura , Escherichia coli/ultraestrutura , Proteínas do Olho/química , Cristalino/ultraestrutura , Microscopia de Força Atômica/métodos , Microscopia de Fluorescência/métodos , Animais , Aquaporinas/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Células Imobilizadas , Escherichia coli/química , Proteínas do Olho/metabolismo , Cristalino/metabolismo , Microscopia de Força Atômica/instrumentação , Microscopia de Fluorescência/instrumentação , Simulação de Dinâmica Molecular , Cultura Primária de Células , Ovinos , Carneiro Doméstico , Imagem com Lapso de TempoRESUMO
The mechanical unfolding of the muscle protein titin by atomic force microscopy was a landmark in our understanding of single-biomolecule mechanics. Molecular dynamics simulations offered atomic-level descriptions of the forced unfolding. However, experiment and simulation could not be directly compared because they differed in pulling velocity by orders of magnitude. We have developed high-speed force spectroscopy to unfold titin at velocities reached by simulation (~4 millimeters per second). We found that a small ß-strand pair of an immunoglobulin domain dynamically unfolds and refolds, buffering pulling forces up to ~100 piconewtons. The distance to the unfolding transition barrier is larger than previously estimated but is in better agreement with atomistic predictions. The ability to directly compare experiment and simulation is likely to be important in studies of biomechanical processes.
Assuntos
Conectina/química , Desdobramento de Proteína , Análise Espectral/métodos , Simulação de Dinâmica MolecularRESUMO
Junctional microdomains, paradigm for membrane protein segregation in functional assemblies, in eye lens fiber cell membranes are constituted of lens-specific aquaporin-0 tetramers (AQP0(4)) and connexin (Cx) hexamers, termed connexons. Both proteins have double function to assure nutrition and mediate adhesion of lens cells. Here we use high-speed atomic force microscopy to examine microdomain protein dynamics at the single-molecule level. We found that the adhesion function of head-to-head associated AQP0(4) and Cx is cooperative. This finding provides first experimental evidence for the mechanistic importance for junctional microdomain formation. From the observation of lateral association-dissociation events of AQP0(4), we determine that the enthalpic energy gain of a single AQP0(4)-AQP0(4) interaction in the membrane plane is -2.7 k(B)T, sufficient to drive formation of microdomains. Connexon association is stronger as dynamics are rarely observed, explaining their rim localization in junctional microdomains.
Assuntos
Aquaporinas/química , Conexinas/química , Proteínas do Olho/química , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/química , Animais , Membrana Celular/química , Membrana Celular/metabolismo , Conexinas/metabolismo , Cristalino/química , Cristalino/metabolismo , Proteínas de Membrana/metabolismo , Microscopia de Força Atômica , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , OvinosRESUMO
For cells to function properly, membrane proteins must be able to diffuse within biological membranes. The functions of these membrane proteins depend on their position and also on protein-protein and protein-lipid interactions. However, so far, it has not been possible to study simultaneously the structure and dynamics of biological membranes. Here, we show that the motion of unlabelled membrane proteins can be characterized using high-speed atomic force microscopy. We find that the molecules of outer membrane protein F (OmpF) are widely distributed in the membrane as a result of diffusion-limited aggregation, and while the overall protein motion scales roughly with the local density of proteins in the membrane, individual protein molecules can also diffuse freely or become trapped by protein-protein interactions. Using these measurements, and the results of molecular dynamics simulations, we determine an interaction potential map and an interaction pathway for a membrane protein, which should provide new insights into the connection between the structures of individual proteins and the structures and dynamics of supramolecular membranes.
Assuntos
Proteínas de Membrana/química , Movimento , Porinas/química , Difusão , Humanos , Bicamadas Lipídicas/química , Microscopia de Força Atômica , Simulação de Dinâmica Molecular , Mapas de Interação de ProteínasRESUMO
For surface analysis of biological molecules, atomic force microscopy (AFM) is an appealing technique combining data acquisition under physiological conditions, for example buffer solution, room temperature and ambient pressure, and high resolution. However, a key feature of life, dynamics, could not be assessed until recently because of the slowness of conventional AFM setups. Thus, for observing bio-molecular processes, the gain of image acquisition speed signifies a key progress. Here, we review the development and recent achievements using high-speed atomic force microscopy (HS-AFM). The HS-AFM is now the only technique to assess structure and dynamics of single molecules, revealing molecular motor action and diffusion dynamics. From this imaging data, watching molecules at work, novel and direct insights could be gained concerning the structure, dynamics and function relationship at the single bio-molecule level.