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1.
Science ; 365(6458): 1079, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31515372

Assuntos
Pinças Ópticas
2.
J Chem Phys ; 151(1): 014901, 2019 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-31272182

RESUMO

The elasticity of dsDNA molecules is investigated by Monte Carlo simulations based on a coarse-grained model of DNA. The force-displacement (f-r) curves are computed under the constraints of the constant force (Gibbs) or the constant length (Helmholtz) ensemble. Particular attention was paid to the compressional (negative) and weak tensile forces. It was confirmed that simulations using the vector Gibbs ensemble fail to represent the compression behavior of polymers. Simulations using the scalar Gibbs protocol resulted in a qualitatively correct compressional response of DNA provided that the quadratic averages of displacements were employed. Furthermore, a well-known shortcoming of the popular Marko-Siggia relation for DNA elasticity at weak tensile forces is elucidated. Conversely, the function f-r from the simulation at the constant length constraint, as well as the new closed-form expressions, provides a realistic depiction of the DNA elasticity over the wide range of negative and positive forces. Merely a qualitative resemblance of the compression functions f-r predicted by the employed approaches supports the notion that the elastic response of DNA molecules may be greatly affected by the specifics of the experimental setups and the kind of averaging of the measured variable.


Assuntos
DNA/química , Microscopia de Força Atômica , Método de Monte Carlo , Pinças Ópticas
3.
Opt Express ; 27(11): 16184-16194, 2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31163802

RESUMO

Double-nanoholes fabricated by colloidal lithography were used for trapping single colloidal particles and single proteins. A gap separation of 60 nm between the cusps of the double-nanohole was achieved in a gold film of 70 nm thickness sputter coated onglass. The cusp separation was reduced steadily down to 10 nm by plasma etching the colloidal particles prior to sputter coating. Scanning electron microscopy was used to locate a particular double-nanohole and it was registered for later microscopy experiments. 30 nm polystyrene particles, the rubisco protein and bovine serum albumin were trapped using a laser focused through the aperture. Compared to other methods that require top-down nanofabrication, this approach is inexpensive and produces high-quality samples.


Assuntos
Técnicas Biossensoriais , Coloides/química , Nanopartículas/química , Nanotecnologia/métodos , Ribulose-Bifosfato Carboxilase/química , Soroalbumina Bovina/química , Ouro , Microscopia Eletrônica de Varredura , Pinças Ópticas , Poliestirenos/química
4.
Nat Commun ; 10(1): 2709, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221966

RESUMO

Protein folding can begin co-translationally. Due to the difference in timescale between folding and synthesis, co-translational folding is thought to occur at equilibrium for fast-folding domains. In this scenario, the folding kinetics of stalled ribosome-bound nascent chains should match the folding of nascent chains in real time. To test if this assumption is true, we compare the folding of a ribosome-bound, multi-domain calcium-binding protein stalled at different points in translation with the nascent chain as is it being synthesized in real-time, via optical tweezers. On stalled ribosomes, a misfolded state forms rapidly (1.5 s). However, during translation, this state is only attained after a long delay (63 s), indicating that, unexpectedly, the growing polypeptide is not equilibrated with its ensemble of accessible conformations. Slow equilibration on the ribosome can delay premature folding until adequate sequence is available and/or allow time for chaperone binding, thus promoting productive folding.


Assuntos
Modelos Moleculares , Biossíntese de Proteínas/fisiologia , Dobramento de Proteína , Proteínas de Bactérias/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Chaperonas Moleculares/metabolismo , Pinças Ópticas , Peptídeos/metabolismo , Domínios Proteicos/fisiologia , Ribossomos/metabolismo , Fatores de Tempo
5.
J Chem Phys ; 150(18): 185101, 2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31091907

RESUMO

The dynamic behavior of bundles of actin filaments growing against a loaded obstacle is investigated through a generalized version of the standard multifilament Brownian Ratchet model in which the (de)polymerizing filaments are treated not as rigid rods but as semiflexible discrete wormlike chains with a realistic value of the persistence length. By stochastic dynamic simulations, we study the relaxation of a bundle of Nf filaments with a staggered seed arrangement against a harmonic trap load in supercritical conditions. Thanks to the time scale separation between the wall motion and the filament size relaxation, mimicking realistic conditions, this setup allows us to extract a full load-velocity curve from a single experiment over the trap force/size range explored. We observe a systematic evolution of steady nonequilibrium states over three regimes of bundle lengths L. A first threshold length Λ marks the transition between the rigid dynamic regime (L < Λ), characterized by the usual rigid filament load-velocity relationship V(F), and the flexible dynamic regime (L > Λ), where the velocity V(F, L) is an increasing function of the bundle length L at a fixed load F, the enhancement being the result of an improved level of work sharing among the filaments induced by flexibility. A second critical length corresponds to the beginning of an unstable regime characterized by a high probability to develop escaping filaments which start growing laterally and thus do not participate anymore in the generation of the polymerization force. This phenomenon prevents the bundle from reaching at this critical length the limit behavior corresponding to perfect load sharing.


Assuntos
Actinas/química , Maleabilidade , Modelos Químicos , Simulação de Dinâmica Molecular , Método de Monte Carlo , Pinças Ópticas , Polimerização , Processos Estocásticos
6.
Nat Commun ; 10(1): 2159, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31089141

RESUMO

Accurate DNA replication is tightly regulated in eukaryotes to ensure genome stability during cell division and is performed by the multi-protein replisome. At the core an AAA+ hetero-hexameric complex, Mcm2-7, together with GINS and Cdc45 form the active replicative helicase Cdc45/Mcm2-7/GINS (CMG). It is not clear how this replicative ring helicase translocates on, and unwinds, DNA. We measure real-time dynamics of purified recombinant Drosophila melanogaster CMG unwinding DNA with single-molecule magnetic tweezers. Our data demonstrates that CMG exhibits a biased random walk, not the expected unidirectional motion. Through building a kinetic model we find CMG may enter up to three paused states rather than unwinding, and should these be prevented, in vivo fork rates would be recovered in vitro. We propose a mechanism in which CMG couples ATP hydrolysis to unwinding by acting as a lazy Brownian ratchet, thus providing quantitative understanding of the central process in eukaryotic DNA replication.


Assuntos
DNA Helicases/metabolismo , Replicação do DNA , Proteínas de Drosophila/metabolismo , Modelos Moleculares , DNA Helicases/isolamento & purificação , Proteínas de Drosophila/isolamento & purificação , Fenômenos Magnéticos , Pinças Ópticas , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Imagem Individual de Molécula/métodos
7.
Soft Matter ; 15(16): 3397-3406, 2019 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-30933209

RESUMO

Swimming bacteria can be trapped for prolonged times at the surface of an impenetrable boundary. The subsequent surface confined motility is found to be very sensitive to the physico-chemical properties of the interfaces which determine the boundary conditions for the flow. The quantitative understanding of this complex dynamics requires detailed and systematic experimental data to validate theoretical models for both flagellar propulsion and interfacial dynamics. Using a combination of optical trapping and holographic imaging we study the 3D dynamics of wall entrapment of swimming bacteria that are sequentially released towards a surfactant-covered liquid-air interface. We find that an incompressible surfactant model for the interface quantitatively accounts for the observed normal and tangential speed of bacteria as they approach the boundary. Surprisingly we also find that, although bacteria circulate over the air phase in counterclockwise circular trajectories, typical of free-slip interfaces, the body axis is still tilted "nose down" as found for no-slip interfaces.


Assuntos
Ar , Bactérias , Água , Holografia , Pinças Ópticas , Propriedades de Superfície , Natação
8.
Analyst ; 144(9): 3136-3143, 2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-30941383

RESUMO

Nosema bombycis (Nb) is the pathogen that causes pebrine in silkworms. Aldehydes are effective disinfectants commonly used in sericulture. However, the precise mechanism of their action on Nb spores remains unclear. Here, we used laser tweezers Raman spectroscopy to investigate the effects of glutaraldehyde and formaldehyde on individual Nb spores, as well as phase contrast microscopy imaging to monitor the germination dynamics of individual treated spores, to acquire a deeper understanding of the mechanism of action of aldehydes and to provide a theoretical reference for establishing an effective strategy for disease control in sericulture. The positions of the Raman peaks remained constant during treatment. The Raman intensity was enhanced and the germination rate of the spores significantly decreased with treatment time. Tlag, the time when individual spores begin to germinate, and Tgerm, the time for complete germination, increased with enhanced treatment. The germination time (ΔTgerm) showed no significant difference from that for untreated spores. Heterogeneity was shown, which is relevant to the resistance of Nb spores to aldehydes. The results indicate that glutaraldehyde and formaldehyde do not destroy the spore wall and plasma membrane, do not cause the leakage of intracellular components, and might not damage the extrusion apparatus. The effects of aldehydes on Nb spores are mainly on the spore coat. They may block the external factors that stimulate spore germination. Single-cell analysis based on novel optical techniques reveals the action of chemical sporicides on microsporidia spores in real time and explains the heterogeneity of cell stress resistance. These applications of new techniques offer new insight into traditional disinfectants.


Assuntos
Desinfetantes/farmacologia , Formaldeído/farmacologia , Glutaral/farmacologia , Nosema/efeitos dos fármacos , Esporos Fúngicos/efeitos dos fármacos , Microscopia de Contraste de Fase/métodos , Pinças Ópticas , Análise de Célula Única/métodos , Análise Espectral Raman/métodos
9.
Biosens Bioelectron ; 133: 236-242, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30953882

RESUMO

Optical trapping of single particles or cells with the capability of in situ bio-sensing or genetic profiling opens the possibility of rapid screening of biological specimens. However, common optical tweezers suffer from the lack of long-range forces. Consequently, their application areas are predominantly limited to target manipulation instead of biological diagnostics. To solve this problem, we herein report an all-in-one approach by combining optical forces and convective drag forces generated through localized optothermal effect for long-range target manipulation. The device consists of a 2D array of gold coated polydimethylsiloxane (PDMS) micro-wells, which are immersed by colloidal particles or cell solution. Upon excitation of a 785-nm laser, the hydrodynamic convective force and optical forces will drag the targets of interest into their designated micro-wells. Moreover, the plasmonic thermal dissipation provides a constant temperature environment for following cell analysis procedures of cell isolation, lysis and isothermal nucleic acid amplification for the detection of genetic markers. With the merits of fabrication simplicity, short sample-to-answer cycle time and the compatibility with optical microscopes, the reported technique offers an attractive and highly versatile approach for on-site single cell analysis systems.


Assuntos
Técnicas Biossensoriais , Dimetilpolisiloxanos/química , Marcadores Genéticos , Análise de Célula Única , Coloides/química , Ouro/química , Lasers , Pinças Ópticas , Temperatura Ambiente
10.
Opt Lett ; 44(7): 1868-1871, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30933168

RESUMO

In advanced biomedicine and microfluidics, there is a strong desire to sort and manipulate various cells and bacteria based on miniaturized microfluidic chips. Here, by integrating fiber tweezers into a T-type microfluidic channel, we report an optofluidic chip to selectively trap Escherichia coli in human blood solution based on different sizes and shapes. Furthermore, we simulate the trapping and pushing regions of other cells and bacteria, including rod-shaped bacteria, sphere-shaped bacteria, and cancer cells based on finite-difference analysis. With the advantages of controllability, low optical power, and compact construction, the strategy may be possibly applied in the fields of optical separation, cell transportation, and water quality analysis.


Assuntos
Separação Celular/instrumentação , Miniaturização/instrumentação , Fibras Ópticas , Pinças Ópticas , Animais , Desenho de Equipamento , Eritrócitos/microbiologia , Escherichia coli/citologia , Humanos
11.
Sensors (Basel) ; 19(7)2019 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-30925721

RESUMO

We have developed a force sensing system to continuously evaluate the mechanical elasticity of micrometer-scale (a few hundred micrometers to a millimeter) live tissues. The sensing is achieved by measuring the deflection of force sensitive cantilevers through microscopic image analysis, which does not require electrical strain gauges. Cantilevers made of biocompatible polydimethylsiloxane (PDMS) were actuated by a piezoelectric actuator and functioned as a pair of chopsticks to measure the stiffness of the specimen. The dimensions of the cantilevers were easily adjusted to match the size, range, and stiffness of the zebrafish samples. In this paper, we demonstrated the versatility of this technique by measuring the mechanical elasticity of zebrafish embryos at different stages of development. The stiffness of zebrafish embryos was measured once per hour for 9 h. From the experimental results, we successfully quantified the stiffness change of zebrafish embryos during embryonic development.


Assuntos
Materiais Biocompatíveis/química , Módulo de Elasticidade , Embrião não Mamífero/fisiologia , Peixe-Zebra/crescimento & desenvolvimento , Animais , Dimetilpolisiloxanos/química , Desenvolvimento Embrionário , Análise de Elementos Finitos , Pinças Ópticas
12.
Int J Biol Macromol ; 130: 1018-1024, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30844457

RESUMO

Here, we use single molecule force spectroscopy performed with optical tweezers in order to investigate the interaction between Caffeine and the DNA molecule for various different concentrations of the alkaloid and under two distinct ionic strengths of the surrounding buffer. We were able to determine the mechanical changes induced on the double-helix structure due to Caffeine binding, the binding mode and the binding parameters of the interaction. The results obtained show that Caffeine binds to DNA by outside the double-helix with a higher affinity at lower ionic strengths. On the other hand, a considerable cooperativity was found only for sufficient high ionic strengths, suggesting that Caffeine may binding forming dimers and/or trimers along the double-helix under this condition. Finally, it was also shown that Caffeine stabilizes the DNA double-helix upon binding, preventing force-induced DNA melting.


Assuntos
Cafeína/farmacologia , DNA/química , Conformação de Ácido Nucleico/efeitos dos fármacos , Algoritmos , Cafeína/química , Modelos Teóricos , Estrutura Molecular , Desnaturação de Ácido Nucleico/efeitos dos fármacos , Pinças Ópticas , Análise Espectral
13.
RNA ; 25(4): 472-480, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30705137

RESUMO

In vitro reconstitution studies have shown that ribosome assembly is highly cooperative and starts with the binding of a few ribosomal (r-) proteins to rRNA. It is unknown how these early binders act. Focusing on the initial stage of the assembly of the large subunit of the Escherichia coli ribosome, we prepared a 79-nucleotide-long region of 23S rRNA encompassing the binding sites of the early binders uL4 and uL24. Force signals were measured in a DNA/RNA dumbbell configuration with a double optical tweezers setup. The rRNA fragment was stretched until unfolded, in the absence or in the presence of the r-proteins (either uL4, uL24, or both). We show that the r-proteins uL4 and uL24 individually stabilize the rRNA fragment, both acting as molecular clamps. Interestingly, this mechanical stabilization is enhanced when both proteins are bound simultaneously. Independently, we observe a cooperative binding of uL4 and uL24 to the rRNA fragment. These two aspects of r-proteins binding both contribute to the efficient stabilization of the 3D structure of the rRNA fragment under investigation. We finally consider implications of our results for large ribosomal subunit assembly.


Assuntos
RNA Bacteriano/química , RNA Ribossômico 23S/química , Proteínas Ribossômicas/genética , Ribossomos/química , Pareamento de Bases , Sequência de Bases , Fenômenos Biomecânicos , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Pinças Ópticas , Biogênese de Organelas , Biossíntese de Proteínas , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico 23S/genética , RNA Ribossômico 23S/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo
14.
Nanoscale ; 11(9): 3945-3951, 2019 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-30762052

RESUMO

Understanding the folding mechanism of knotted and slipknotted proteins has attracted considerable interest. Due to their topological complexity, knotted and slipknotted proteins are predicted to fold slowly and involve large topological barriers. Molecular dynamics simulation studies suggest that a slipknotted conformation can serve as an important intermediate to help greatly reduce the topological difficulty during the folding of some knotted proteins. Here we use a single molecule optical tweezers technique to directly probe the folding of a small slipknotted protein AFV3-109. We found that stretching AFV3-109 can lead to the untying of the slipknot and complete unfolding of AFV3-109. Upon relaxation, AFV3-109 can readily refold back to its native slipknot conformation with high fidelity when the stretching force is lower than 6 pN. The refolding of AFV3-109 occurs in a sharp two-state like transition. Our results indicate that, different from knotted proteins, the folding of a slipknotted protein like AFV3-109 can be fast, and may not necessarily involve a high topological barrier.


Assuntos
Pinças Ópticas , Proteínas/química , Cinética , Microscopia de Força Atômica , Simulação de Dinâmica Molecular , Método de Monte Carlo , Desnaturação Proteica , Dobramento de Proteína , Proteínas/metabolismo , Termodinâmica
15.
Biosens Bioelectron ; 129: 107-117, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30685705

RESUMO

As an efficient tool in the multiplexed detection of biomolecules, bead-array could achieve separation-free detection to multiple targets, making it suitable to analyze valuable and scarce samples like antigen and antibody from living organism. Herein, we propose a spectral-optical-tweezer-assisted fluorescence multiplexing system to analyze biomolecule-conjugated bead-array. Using optical tweezer, we trapped and locked beads at the focus to accept stimulation, offering a stable and optimized analysis condition. Moving the system focus and scanning the sample slide, we achieved emissions collection to QDs-encoded bead-array after the multiplexed detection. The emission spectra of fluorescence were collected and recorded by the spectrometer. By recognizing locations of decoding peaks and counting the intensities of label signals of emission spectra, we achieved qualitative and quantitative detection to targets. As proof-of-concept studies, we use this system to carry out multiplexed detection to various types of anti-IgG in the single sample and the detection limit reaches 1.52 pM with a linear range from 0.31 to 10 nM. Through further optimization of experimental conditions, we achieved specific detection to target IgG with sandwich method in human serum and the detection limit reaches as low as 0.23 pM with a linear range from 0.88 to 28 pM, validating the practical application of this method in real samples.


Assuntos
Técnicas Biossensoriais/instrumentação , Corantes Fluorescentes/química , Imunoglobulina G/sangue , Pinças Ópticas , Pontos Quânticos/química , Desenho de Equipamento , Fluorescência , Humanos , Limite de Detecção
16.
Biomolecules ; 9(1)2019 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-30641944

RESUMO

In the past three decades, the ability to optically manipulate biomolecules has spurred a new era of medical and biophysical research. Optical tweezers (OT) have enabled experimenters to trap, sort, and probe cells, as well as discern the structural dynamics of proteins and nucleic acids at single molecule level. The steady improvement in OT's resolving power has progressively pushed the envelope of their applications; there are, however, some inherent limitations that are prompting researchers to look for alternatives to the conventional techniques. To begin with, OT are restricted by their one-dimensional approach, which makes it difficult to conjure an exhaustive three-dimensional picture of biological systems. The high-intensity trapping laser can damage biological samples, a fact that restricts the feasibility of in vivo applications. Finally, direct manipulation of biological matter at nanometer scale remains a significant challenge for conventional OT. A significant amount of literature has been dedicated in the last 10 years to address the aforementioned shortcomings. Innovations in laser technology and advances in various other spheres of applied physics have been capitalized upon to evolve the next generation OT systems. In this review, we elucidate a few of these developments, with particular focus on their biological applications. The manipulation of nanoscopic objects has been achieved by means of plasmonic optical tweezers (POT), which utilize localized surface plasmons to generate optical traps with enhanced trapping potential, and photonic crystal optical tweezers (PhC OT), which attain the same goal by employing different photonic crystal geometries. Femtosecond optical tweezers (fs OT), constructed by replacing the continuous wave (cw) laser source with a femtosecond laser, promise to greatly reduce the damage to living samples. Finally, one way to transcend the one-dimensional nature of the data gained by OT is to couple them to the other large family of single molecule tools, i.e., fluorescence-based imaging techniques. We discuss the distinct advantages of the aforementioned techniques as well as the alternative experimental perspective they provide in comparison to conventional OT.


Assuntos
Pinças Ópticas , Proteínas/química , DNA/química , Dispositivos Lab-On-A-Chip , Microscopia de Fluorescência
17.
Opt Lett ; 44(2): 319-322, 2019 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-30644890

RESUMO

We propose novel plasmonic tweezers based on silver V-type nanoantennas placed on a conducting ground layer, which can effectively mitigate the plasmonic heating effect and thus enable subwavelength plasmonic trapping in the near-infrared region. Using the centroid algorithm to analyze the motion of trapped spheres, we can experimentally extract the value of optical trapping potential. The result confirms that the plasmonic tweezers have a dual-mode subwavelength trapping capability when the incident laser beam is linearly polarized along two orthogonal directions. We have also performed full-wave simulations, which agree with the experimental data very well in terms of spectral response and trapping potential. It is expected that the dual-mode subwavelength trapping can be used in non-contact manipulations of a single nanoscale object, such as a biomolecule or quantum dot, and find important applications in biology, life science, and applied physics.


Assuntos
Nanotecnologia/instrumentação , Pinças Ópticas
18.
Methods Mol Biol ; 1860: 95-114, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30317500

RESUMO

Intracellular membrane fusion mediates material and information exchange among different cells or cellular compartments with high accuracy and spatiotemporal resolution. Fusion is driven by ordered folding and assembly of soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptors (SNAREs) and regulated by many other proteins. Understanding regulated SNARE assembly is key to dissecting mechanisms and physiologies of various fusion processes and their associated diseases. Yet, it remains challenging to study regulated SNARE assembly using traditional ensemble-based experimental approaches. Here, we describe our new method to measure the energy and kinetics of neuronal SNARE assembly in the presence of α-SNAP, using a single-molecule manipulation approach based on high-resolution optical tweezers. Detailed experimental protocols and methods of data analysis are shown. This approach can be widely applied to elucidate the effects of regulatory proteins on SNARE assembly and membrane fusion.


Assuntos
Pinças Ópticas , Proteínas SNARE/metabolismo , Imagem Individual de Molécula/métodos , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , Biotinilação , Reagentes para Ligações Cruzadas/química , Cinética , Fusão de Membrana , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Ligação Proteica , Dobramento de Proteína , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas SNARE/química , Proteínas SNARE/isolamento & purificação , Imagem Individual de Molécula/instrumentação , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/química , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/isolamento & purificação
19.
Methods Mol Biol ; 1860: 145-159, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30317502

RESUMO

Force spectroscopy allows the manipulation of single molecules and the characterization of their properties and interactions thereby rendering it a powerful tool for biological sciences. Force spectroscopy at the level of individual molecules requires force resolution in the piconewton regime as achieved by optical tweezers (OT), magnetic tweezers (MT), and atomic force microscopy (AFM) with AFM providing the largest force range from tenth of piconewton to several micronewton. In membrane probe spectroscopy the commonly used sharp cantilever tip is replaced by a lipid-coated glass sphere. This technique expands the scope of force spectroscopy to processes at and between lipid bilayers, like the formation of coiled coils between SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) proteins as well as subsequent membrane fusion. To this end, two solid-supported membranes equipped with SNARE proteins or fusion peptides are separately deposited on a flat glassy surface and on a micrometer glass sphere attached to the end of a tipless AFM cantilever. These two membranes are rapidly brought into contact until a defined force is reached. The AFM deflection readout is used to monitor the distance between the two bilayers, which allows to observe and identify fusion processes of the two lipid membranes, while the forces needed to separate the two surfaces give insights into the formation of SNARE complexes. By changing the contact pressure one can access fusion kinetics and to some extent reconstruct the energy landscape of membrane fusion. In this chapter we describe the preparation of membrane-coated colloidal probes attached to AFM cantilevers, experimental procedures, and necessary data analysis to perform membrane probe spectroscopy in the presence of fusogenic peptides or proteins.


Assuntos
Bicamadas Lipídicas/metabolismo , Fusão de Membrana , Proteínas SNARE/metabolismo , Análise Espectral/métodos , Bicamadas Lipídicas/química , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Microscopia de Força Atômica/instrumentação , Microscopia de Força Atômica/métodos , Pinças Ópticas , Domínios Proteicos , Proteolipídeos/química , Proteolipídeos/metabolismo , Proteínas SNARE/química , Análise Espectral/instrumentação
20.
FEBS Lett ; 593(3): 296-307, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30575960

RESUMO

Cytoplasmic dynein, a microtubule-based motor protein, is responsible for many cellular functions ranging from cargo transport to cell division. The various functions are carried out by a single isoform of cytoplasmic dynein, thus requiring different forms of motor regulation. A possible pathway to regulate motor function was revealed in optical trap experiments. Switching motor function from single steps to processive runs could be achieved by changing Mg2+ and ATP concentrations. Here, we confirm by single molecule total internal reflection fluorescence microscopy that a native cytoplasmic dynein dimer is able to switch to processive runs of more than 680 consecutive steps or 5.5 µm. We also identified the ratio of Mg2+ -free ATP to Mg.ATP as the regulating factor and propose a model for dynein processive stepping.


Assuntos
Trifosfato de Adenosina/química , Citoplasma/química , Dineínas/química , Pinças Ópticas , Trifosfato de Adenosina/metabolismo , Animais , Citoplasma/metabolismo , Dineínas/metabolismo , Suínos
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