RESUMO
Single-molecule localization microscopy enables far-field imaging with lateral resolution in the range of 10 to 20 nanometres, exploiting the fact that the centre position of a single-molecule's image can be determined with much higher accuracy than the size of that image itself. However, attaining the same level of resolution in the axial (third) dimension remains challenging. Here, we present Supercritical Illumination Microscopy Photometric z-Localization with Enhanced Resolution (SIMPLER), a photometric method to decode the axial position of single molecules in a total internal reflection fluorescence microscope. SIMPLER requires no hardware modification whatsoever to a conventional total internal reflection fluorescence microscope and complements any 2D single-molecule localization microscopy method to deliver 3D images with nearly isotropic nanometric resolution. Performance examples include SIMPLER-direct stochastic optical reconstruction microscopy images of the nuclear pore complex with sub-20 nm axial localization precision and visualization of microtubule cross-sections through SIMPLER-DNA points accumulation for imaging in nanoscale topography with sub-10 nm axial localization precision.
Assuntos
Fluorescência , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Nanotecnologia/métodos , Imagem Individual de Molécula/métodos , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , DNA/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Células HeLa , Humanos , Processamento de Imagem Assistida por Computador/métodos , Camundongos , Microtúbulos/metabolismo , Fotometria/métodosRESUMO
Gram-positive bacteria use their adhesive pili to attach to host cells during early stages of a bacterial infection. These extracellular hair-like appendages experience mechanical stresses of hundreds of picoNewtons; however, the presence of an internal isopeptide bond prevents the pilus protein from unfolding. Here, we describe a method to interfere with nascent pili proteins through a peptide that mimics one of the ß-strands of the molecule. By using AFM-based force spectroscopy, we study the isopeptide bond formation and the effect of the peptide in the elasticity of the pilus protein. This method could be used to afford a new strategy for mechanically targeted antibiotics by simply blocking the folding of the bacterial pilus protein.
Assuntos
Proteínas de Fímbrias/metabolismo , Desdobramento de Proteína/efeitos dos fármacos , Imagem Individual de Molécula/métodos , Proteínas de Bactérias/metabolismo , Fímbrias Bacterianas/metabolismo , Bactérias Gram-Positivas/metabolismo , Microscopia de Força Atômica/métodos , Peptídeos/farmacologia , Dobramento de Proteína , Streptococcus pyogenes/química , Streptococcus pyogenes/metabolismo , Estresse MecânicoRESUMO
In this work we use single molecule force spectroscopy performed with optical tweezers in order to characterize the complexes formed between the anticancer drug Pixantrone (PIX) and the DNA molecule, at two very different ionic strengths. Firstly, the changes of the mechanical properties of the DNA-PIX complexes were studied as a function of the drug concentration in the sample. Then, a quenched-disorder statistical model of ligand binding was used in order to determine the physicochemical (binding) parameters of the DNA-PIX interaction. In particular, we have found that the PIX molecular mechanism of action involves intercalation into the double helix, followed by a significant compaction of the DNA molecule due to partial neutralization of the phosphate backbone. Finally, this scenario of interaction was quantitatively compared to that found for the related drug Mitoxantrone (MTX), which binds to DNA with a considerably higher equilibrium binding constant and promotes a much stronger DNA compaction. The comparison performed between the two drugs can bring clues to the development of new (and more efficient) related compounds.
Assuntos
Antineoplásicos/química , DNA/química , Substâncias Intercalantes/química , Isoquinolinas/química , Ligantes , Pinças Ópticas , Imagem Individual de Molécula/métodosRESUMO
The combination of single-molecule fluorescence imaging and electrophysiology provides a powerful tool to explore the stoichiometry and functional properties of ionic channels, simultaneously. Here, we describe a typical SC-SMD experiment from the preparation of plasmids containing the genes encoding for the channels of interest fused to fluorescent proteins to the use of the SC-SMD system for simultaneous patch clamping and single-molecule determinations.
Assuntos
Fenômenos Eletrofisiológicos , Ativação do Canal Iônico , Canais Iônicos/metabolismo , Técnicas de Patch-Clamp , Imagem Individual de Molécula , Expressão Gênica , Células HEK293 , Humanos , Canais Iônicos/genética , Técnicas de Patch-Clamp/instrumentação , Técnicas de Patch-Clamp/métodos , Imagem Individual de Molécula/métodos , TransfecçãoRESUMO
Thy-1 and αvß3 integrin mediate bidirectional cell-to-cell communication between neurons and astrocytes. Thy-1/αvß3 interactions stimulate astrocyte migration and the retraction of neuronal prolongations, both processes in which internal forces are generated affecting the bimolecular interactions that maintain cell-cell adhesion. Nonetheless, how the Thy-1/αvß3 interactions respond to mechanical cues is an unresolved issue. In this study, optical tweezers were used as a single-molecule force transducer, and the Dudko-Hummer-Szabo model was applied to calculate the kinetic parameters of Thy-1/αvß3 dissociation. A novel experimental strategy was implemented to analyze the interaction of Thy-1-Fc with nonpurified αvß3-Fc integrin, whereby nonspecific rupture events were corrected by using a new mathematical approach. This methodology permitted accurately estimating specific rupture forces for Thy-1-Fc/αvß3-Fc dissociation and calculating the kinetic and transition state parameters. Force exponentially accelerated Thy-1/αvß3 dissociation, indicating slip bond behavior. Importantly, nonspecific interactions were detected even for purified proteins, highlighting the importance of correcting for such interactions. In conclusion, we describe a new strategy to characterize the response of bimolecular interactions to forces even in the presence of nonspecific binding events. By defining how force regulates Thy-1/αvß3 integrin binding, we provide an initial step towards understanding how the neuron-astrocyte pair senses and responds to mechanical cues.