Microfluidics for biological measurements with single-molecule resolution

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A high-resolution magnetic tweezer for single-molecule measurements

Magnetic tweezers (MT) are single-molecule manipulation instruments that utilize a magnetic field to apply force to a biomolecule-tethered magnetic bead while using optical bead tracking to measure the biomolecule’s extension. While relatively simple to set up, prior MT implementations have lacked the resolution necessary to observe sub-nanometer biomolecular configuration changes. Here, we demonstrate a reflection-interference technique for bead tracking, and show that it has much better resolution than traditional diffraction-based systems. We enhance the resolution by fabricating optical coatings on all reflecting surfaces that optimize the intensity and contrast of the interference image, and we implement feedback control of the focal position to remove drift. To test the system, we measure the length change of a DNA hairpin as it undergoes a folding/unfolding transition.     

Two methods of temperature control for single-molecule measurements

Modern single-molecule biophysical experiments require high numerical aperture oil-immersion objectives in close contact with the sample. We introduce two methods of high numerical aperture temperature control which can be implemented on any microscope: objective temperature control using a ring-shaped Peltier device, and stage temperature control using a fluid flow cooling chip in close thermal contact with the sample. We demonstrate the efficacy of each system by showing the change in speed with temperature of two molecular motors, the bacterial flagellar motor and skeletal muscle myosin.     

Deconvolving single-molecule intensity distributions for quantitative microscopy measurements

In fluorescence microscopy, images often contain puncta in which the fluorescent molecules are spatially clustered. This article describes a method that uses single-molecule intensity distributions to deconvolve the number of fluorophores present in fluorescent puncta as a way to "count" protein number. This method requires a determination of the correct statistical relationship between the single-molecule and single-puncta intensity distributions. Once the correct relationship has been determined, basis histograms can be generated from the single-molecule intensity distribution to fit the puncta distribution. Simulated data were used to demonstrate procedures to determine this relationship, and to test the methodology. This method has the advantages of single-molecule measurements, providing both the mean and variation in molecules per puncta. This methodology has been tested with the avidin-biocytin binding system for which the best-fit distribution of biocytins in the sample puncta was in good agreement with a bulk determination of the avidin-biocytin binding ratio.     

Fluorescent gramicidin derivatives for single-molecule fluorescence and ion channel measurements.

Single-molecule spectroscopies in combination with single-channel patch-clamp measurements have the potential for providing new information on ion channel gating processes. Fluorescent gramicidin derivatives could provide a means for calibrating such experiments since the structure of the open channel is known and the mechanism of gating (peptide dimerization) is generally agreed. We describe here the synthesis and characterization of two pairs of gramicidin derivatives that should prove useful for such studies. They contain robust fluorophores, undergo resonance energy transfer (FRET) when they dimerize, and have single-channel properties close to those of the wild-type channel.     

Hydrodynamic effects in fast AFM single-molecule force measurements

Atomic force microscopy (AFM) allows the critical forces that unfold single proteins and rupture individual receptor–ligand bonds to be measured. To derive the shape of the energy landscape, the dynamic strength of the system is probed at different force loading rates. This is usually achieved by varying the pulling speed between a few nm/s and a few m/s, although for a more complete investigation of the kinetic properties higher speeds are desirable. Above 10 m/s, the hydrodynamic drag force acting on the AFM cantilever reaches the same order of magnitude as the molecular forces. This has limited the maximum pulling speed in AFM single-molecule force spectroscopy experiments. Here, we present an approach for considering these hydrodynamic effects, thereby allowing a correct evaluation of AFM force measurements recorded over an extended range of pulling speeds (and thus loading rates). To support and illustrate our theoretical considerations, we experimentally evaluated the mechanical unfolding of a multi-domain protein recorded at 30 m/s pulling speed.Abbrevations AFM atomic force micrcoscopy - pN piconewton - BR bacteriorhodopsin - DFS dynamic force spectroscopy - Ig27 immunoglobulin 27 - If27-8 immunoglobulin 27 octameric construct - BFP biomembrane force probe     

Dissociation kinetics of an enzyme-inhibitor system using single-molecule force measurements

We report on an improved method to interpret single molecule dissociation measurements using atomic force microscopy. We describe an easy to use methodology to reject nonspecific binding events, as well as estimating the number of multiple binding events. The method takes nonlinearities in the force profiles into account that result from the deformation of the used polymeric linkers. This new method is applied to a relevant enzyme-inhibitor system, latent matrix metalloprotease 9 (ProMMP-9, a gelatinase), and its inhibitor, tissue inhibitor of metalloproteases 1 (TIMP 1), which are important players in cancer metastasis. Our method provides a measured kinetic off-rate of 0.010 ± 0.003 s(-1) for the dissociation of ProMMP9 and TIMP1, which is consistent with values measured by ensemble methods.     

Multiple-bond kinetics from single-molecule pulling experiments: evidence for multiple NCAM bonds

The kinetic parameters of single bonds between neural cell adhesion molecules were determined from atomic force microscope measurements of the forced dissociation of the homophilic protein-protein bonds. The analytical approach described provides a systematic procedure for obtaining rupture kinetics for single protein bonds from bond breakage frequency distributions obtained from single-molecule pulling experiments. For these studies, we used the neural cell adhesion molecule (NCAM), which was recently shown to form two independent protein bonds. The analysis of the bond rupture data at different loading rates, using the single-bond full microscopic model, indicates that the breakage frequency distribution is most sensitive to the distance to the transition state and least sensitive to the molecular spring constant. The analysis of bond failure data, however, motivates the use of a double-bond microscopic model that requires an additional kinetic parameter. This double-bond microscopic model assumes two independent NCAM-NCAM bonds, and more accurately describes the breakage frequency distribution, particularly at high loading rates. This finding agrees with recent surface-force measurements, which showed that NCAM forms two spatially distinct bonds between opposed proteins.     

Direct discrimination between models of protein activation by single-molecule force measurements

The limitations imposed on the analyses of complex chemical and biological systems by ensemble averaging can be overcome by single-molecule experiments. Here, we used a single-molecule technique to discriminate between two generally accepted mechanisms of a key biological process--the activation of proteins by molecular effectors. The two mechanisms, namely induced-fit and population-shift, are normally difficult to discriminate by ensemble approaches. As a model, we focused on the interaction between the nuclear transport effector, RanBP1, and two related complexes consisting of the nuclear import receptor, importin beta, and the GDP- or GppNHp-bound forms of the small GTPase, Ran. We found that recognition by the effector proceeds through either an induced-fit or a population-shift mechanism, depending on the substrate, and that the two mechanisms can be differentiated by the data.     

Free energy of membrane protein unfolding derived from single-molecule force measurements

Mechanical single-molecule techniques offer exciting possibilities to investigate protein folding and stability in native environments at submolecular resolution. By applying a free-energy reconstruction procedure developed by Hummer and Szabo, which is based on a statistical theorem introduced by Jarzynski, we determined the unfolding free energy of the membrane proteins bacteriorhodopsin (BR), halorhodopsin, and the sodium-proton antiporter NhaA. The calculated energies ranged from 290.5 kcal/mol for BR to 485.5 kcal/mol for NhaA. For the remarkably stable BR, the equilibrium unfolding free energy was independent of pulling rate and temperature ranging between 18 and 42 degrees C. Our experiments also revealed heterogeneous energetic properties in individual transmembrane helices. In halorhodopsin, the stabilization of a short helical segment yielded a characteristic signature in the energy profile. In NhaA, a pronounced peak was observed at a functionally important site in the protein. Since a large variety of single- and multispan membrane proteins can be tackled in mechanical unfolding experiments, our approach provides a basis for systematically elucidating energetic properties of membrane proteins with the resolution of individual secondary-structure elements.     

Comparison between slide and flow cytophotometric DNA measurements in breast tumors

Nuclear DNA analysis was performed in 37 human mammary adenocarcinomas in order to elucidate the difficulties and pitfalls connected with the interpretation of DNA histograms obtained using different methodologic approaches. For each tumor, DNA profiles were obtained by means of slide microspectrophotometry on a fine needle aspirate, slide cytophotometry on a 4-micron histologic section and flow cytometry on a suspension prepared from a cube of fresh tissue. When the DNA histograms were interpreted according to criteria usually applied to discriminate low-grade malignant tumors from high-grade malignant tumors, some tumors classified as euploid by one method were classified as aneuploid by another method. The main reasons for this weak correlation seem to be in specimen preparation and in tumor cell representation within the specimen between the methods. Another reason is that slide and flow techniques exhibit different sensitivities for malignancy-associated nuclear DNA changes: minor alterations of the DNA content of the tumor stemlines seem to be more exactly reported by means of the flow technique whereas structural alterations of the nuclear chromatin seem to be more sensitively recorded by means of the slide technique. It is suggested that thorough control of each step of the various DNA analysis procedures and the use of information obtainable by slide and flow techniques taken together may significantly improve the prognostic value of DNA measurements.     

In-cell single-molecule FRET measurements reveal three conformational state changes in RAF protein

BackgroundThe structures of proteins are intimately related to their functions. Significant efforts have been dedicated to the structural investigation of proteins, mainly those of purified proteins in in vitro environments. Proteins function in living cells and thus protein structures must be regulated by interactions with various molecules, some of which participate in reaction networks, depending on the states, conditions, or actions of the cell. Therefore, it is very important to understand the structural behavior of proteins in living cells.MethodsSingle-molecule Förster resonance energy transfer (smFRET) measurements were conducted using the alternative laser excitation (ALEX) technique. smFRET distributions of cytosolic Rapidly Accelerated Fibrosarcoma (RAF) proteins in living HeLa cells were obtained with exclusion of the negative effects of photobleached fluorophores and incompletely labeled proteins on smFRET.ResultssmFRET histograms of wildtype (wt) RAF in live cells exhibited two major peaks, whereas that of the S621A mutant, which has been thought to have an expanded structure, was almost single-peaked. A population shift involving the peaks for wt RAF was detected upon epidermal growth factor stimulation. Spontaneous transitions between the conformational states corresponding to the two peaks were also detected using the FRET-two-channel kernel-based density distribution estimator method in comparison to static double-stranded DNA samples.ConclusionsCytosolic CRAF has at least three conformational states; in addition to the closed and open forms, the fully-open form was distinctly specified. Based on the results, we propose a speculative structural model for CRAF.General significanceStructural distribution and changes to proteins in live cells as a result of intracellular interactions were successfully identified. smFRET using ALEX is applicable to any other cytosolic proteins.     

Regeneration and Agrobacterium-mediated transformation of multiple lily cultivars

To pursue genetic improvement of lily, efficiency of both regeneration and transformation from callus cultures induced from different explants were evaluated in multiple cultivars. Thirty-five callus lines induced from filaments or styles and one control callus line derived from bulb scales of in total twenty lily cultivars representing Lilium longiflorum, Oriental × Trumpet and Longiflorum × Asiatic hybrids were maintained on a medium with 8.3 μM picloram (PIC). In this study, they were tested for their regeneration potential by transferring them onto a regeneration medium supplemented with 0.4 μM PIC and 0.044 μM 6-benzyladenine. Regeneration was obtained in all cultivars examined and the percentage varied from zero to 89 % in the 36 callus lines. Regeneration frequency was significantly influenced by the genotype (cultivar). Subculturing the calli every 4 weeks by refreshing the regeneration medium contributed positively to bulblet formation, when compared to an eight week subculture frequency. It was found that the regeneration ability generally decreased with an increasing age of the callus cultures for all cultivars. The origin of the callus (style or filament) did not lead to significant differences in regeneration frequency, but there was an interaction between callus origin and genotype. Calli of eight randomly chosen cultivars were co-cultivated with Agrobacterium tumefaciens strain AGL0 carrying binary vectors with the gus gene as reporter and putative transgenic plants were produced. GUS histochemical assays demonstrated transient and stable expression of the gus gene in both calli and regenerated lily plants. Transient expression frequencies ranged from 0.3 to 20.6 % while stable transformation was much lower, only 1.4 % as the maximum.     

Detecting protein-induced folding of the U4 snRNA kink-turn by single-molecule multiparameter FRET measurements

The kink-turn (k-turn), a new RNA structural motif found in the spliceosome and the ribosome, serves as a specific protein recognition element and as a structural building block. While the structure of the spliceosomal U4 snRNA k-turn/15.5K complex is known from a crystal structure, it is unclear whether the k-turn also exists in this folded conformation in the free U4 snRNA. Thus, we investigated the U4 snRNA k-turn by single-molecule FRET measurements in the absence and presence of the 15.5K protein and its dependence on the Na(+) and Mg(2+) ion concentration. We show that the unfolded U4 snRNA k-turn introduces a kink of 85 degrees +/- 15 degrees in an RNA double helix. While Na(+) and Mg(2+) ions induce this more open conformation of the k-turn, binding of the 15.5K protein was found to induce the tightly kinked conformation in the RNA that increases the kink to 52 degrees +/- 15 degrees . By comparison of the measured FRET distances with a computer-modeled structure, we show that this strong kink is due to the k-turn motif adopting its folded conformation. Thus, in the free U4 snRNA, the k-turn exists only in an unfolded conformation, and its folding is induced by binding of the 15.5K protein.     

Biophysical nanotools for single-molecule dynamics

The focus of the cell biology field is now shifting from characterizing cellular activities to organelle and molecular behaviors. This process accompanies the development of new biophysical visualization techniques that offer high spatial and temporal resolutions with ultra-sensitivity and low cell toxicity. They allow the biology research community to observe dynamic behaviors from scales of single molecules, organelles, cells to organoids, and even live animal tissues. In this review, we summarize these biophysical techniques into two major classes: the mechanical nanotools like dynamic force spectroscopy (DFS) and the optical nanotools like single-molecule and super-resolution microscopy. We also discuss their applications in elucidating molecular dynamics and functionally mapping of interactions between inter-cellular networks and intra-cellular components, which is key to understanding cellular processes such as adhesion, trafficking, inheritance, and division.     

Fluorophores for single-molecule localization microscopy

Super-resolution fluorescence microscopy allows for obtaining images with a resolution of 10–20 nm, far exceeding the diffraction limit of conventional optical microscopy (200–350 nm), and provides an opportunity to study in detail the subcellular structures and individual proteins in both living and fixed cells. Among these methods, single-molecule localization microscopy (SMLM) has become widespread. SMLM techniques are based on special fluorophores capable of photoswitching. The paper presents a classification of such fluorophores and describes their photoswitching mechanisms and successful practical applications. We discuss recent progress and prospects for the development of new effective labels suitable for SMLM.     

Monitoring neural progenitor fate through multiple rounds of division in an intact vertebrate brain

The behaviour of neural progenitors in the intact vertebrate brain and spinal cord is poorly understood, chiefly because of the inaccessibility and poor optical qualities inherent in many model systems. To overcome these problems we have studied the optically superior brain of the zebrafish embryo and have monitored the in vivo behaviour of fluorescently labelled neural progenitors and their daughter cells throughout a substantial period of hindbrain development. We find the majority (84%) of hindbrain neurons are born from progenitor divisions that generate two neurons and 68% of reconstructed lineage trees contained no asymmetric stem cell-like divisions. No progenitors divided in the manner expected of a classic stem cell; i.e. one that repeatedly self-renews and generates a differentiated cell type by asymmetric division. We also analysed the orientation of progenitor divisions relative to the plane of the ventricular zone (VZ) and find that this does not correlate with the fate of the daughter cells. Our results suggest that in this vertebrate system the molecular determinants that control whether a cell will become a neuron are usually not linked to a mechanism that generates asymmetric divisions.