B Laser light is focused on the back aperture of the microscope objective, generating an intense Gaussian field at the sample just large enough to image a single E. The blue arrows point at replisomes with three polymerases and the red arrow indicates a replisome with six polymerases. E Raw blue and filtered red intensity for a putative single left panel and double middle panel replisome spot were compared with the intensity of a single surface-immobilized YPet in vitro right panel. Combined with the Fourier spectral analysis to find the brightness of a single YPet F , these data show that the in vivo steps were integer multiples of the intensity of a single YPet molecule and replisomes contain a mean of three polymerases.
B—F are adapted with permission from Reyes-Lamothe et al.
Structure and Mechanisms of SF1 DNA Helicases
The exchange is evident by the change in color of the fluorescence and length of the ssDNA. Arrows placed above the kymograph indicate the time points of the injections. G is adapted from Gibb et al. To reduce the background fluorescence even further and enable the visualization of individual labeled molecules at physiologically relevant concentrations, techniques have been introduced that rely on photoactivatable tags. After photoactivation of the protein near the surface, rapid diffusion of the unbound proteins away from the detection volume reduces background fluorescence, whereupon the bound molecules are imaged.
This method allowed the visualization of the micrometer-scale movement of replication forks, the spatiotemporal pattern of replication initiation along individual DNA molecules, and the dynamics of individual proteins at replication forks in undiluted cellular extracts Loveland et al. The drawback of this technique is the need for photoactivatable proteins. In an alternative method, LADye Geertsema et al. Only those proteins bound to their substrate are selectively activated, via a short-distance energy-transfer mechanism. Although the chemicals used to darken the fluorophores could potentially alter the behavior of the system, this approach has already allowed the observation of the sequence-independent interaction of interferon-inducible protein 16 with DNA and the sliding via diffusion of adenovirus protease on DNA in the presence of very high, micromolar concentrations of protein Geertsema et al.
PhADE and LADye have increased the useful concentration of proteins in in vitro single-molecule experiments to levels closer to in vivo conditions than ever before, thereby providing new insight into the behavior of DNA-interacting proteins at physiologically relevant concentrations.
The concentrations of most proteins inside living cells are well above the concentration limit that allows visualization using conventional single-molecule imaging methods Lewis et al. Therefore, similar techniques to reduce background fluorescence are used in vivo. In PAINT Sharonov and Hochstrasser, , the objects to be imaged are continuously targeted based on many cycles of transient association by fluorescent probes present in the solution, rather than having the fluorescent probe stably bound to the objects.
As a result, a fluorescent signal appears as a diffraction-limited spot on the object when a label briefly binds to it and is momentarily immobilized Fig. This method was used to track endogenous AMPA glutamate receptors on living neurons, revealing high receptor densities and reduced diffusion in synapses Giannone et al. FRET is the distance-dependent nonradiative energy transfer between two fluorescent molecules that occurs when the emission spectrum of one fluorophore overlaps with the absorption spectrum of the other.
By attaching two fluorophores with the appropriate spectral properties to two molecules of interest, association events and relative movements can be observed through smFRET Fig. By labeling a protein with two fluorophores at known positions within the protein, conformational changes and dynamics within a single molecule can be detected Fig.
Since the initial development of the method Ha et al. For example, by labeling the two heads of a kinesin with a FRET pair, it was shown that the kinesin waits for ATP in a one-head—bound state and makes brief transitions to a two-head—bound intermediate as it walks along the microtubule Mori et al. Further, smFRET has allowed the direct observation of the conformational dynamics of single amino-acid transporters during substrate transport Erkens et al.
Three successive FRET states were seen, corresponding to closure of the clamp, followed by clamp release from its loader, and diffusion on the DNA Cho et al. To enable in vivo fluorescence imaging, proteins are traditionally genetically fused to a fluorescent protein. The spectral properties and poor photostability of these fluorescent proteins, however, make their use in smFRET very challenging.
Structure and Mechanisms of SF1 DNA Helicases
Therefore, observing smFRET in living cells requires new labeling, internalization, and imaging strategies. Significant progress in all these areas has been made in the last decade Sustarsic and Kapanidis, Fluorescently labeled DNA was internalized in living E. By electroporating a large fragment of DNA polymerase I Klenow fragment , doubly labeled on the fingers and thumb domains, FRET was measured between internalized, immobile Klenow fragment molecules. This study shows that the distance between the two domains is preserved in live cells Crawford et al. Perhaps the most rapidly developing single-molecule imaging technique is cryo-EM.
In cryo-EM, rapid freezing techniques vitrification provide immobilization of biological samples embedded in amorphous ice, preserving the structure of the samples in their native state. Using EM, these biological structures can be resolved down to the atomic level. The ability to obtain near-atomic resolution structures using cryo-EM was initially shown almost three decades ago Henderson et al. By now, cryo-EM is a firmly established tool to gain structural information on both purified and cellular systems.
Recent developments in both sample preparation and detection techniques have given access to resolutions as high as 2. With this information, structural rearrangements that occur upon binding of the kinesin motor domain to the microtubules could be identified Sindelar and Downing, In this mechanism, two domains of the helicase move in a pumpjack-like motion to translocate on DNA Yuan et al. This combination of techniques uses fluorescence microscopy to guide the search for specific features and locate areas worth recording and examining by cryo-EM.
Fluorescence imaging can furthermore provide valuable information about local variations in ice thickness, ice crystal contamination, or other defects that could affect cryo-EM data quality. In live CLEM, proteins in a living cell are first observed using fluorescence microscopy, followed by the observation of cellular structures, such as organelles or membranes, using cryo-EM in the same cell Kobayashi et al.
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With the combination of these two techniques, dynamic events can be observed in specific cellular structures. This potentially makes live CLEM a powerful method to provide functional and structural understanding of dynamic and complex events, such as nuclear envelope formation Haraguchi et al. Summarizing, single-molecule fluorescence imaging methods and fluorescence tagging strategies have matured to the point at which they can almost routinely be used to visualize biological processes, often in real time. Methods that allow the detection of individual molecules in high-concentration, crowded environments, combined with advances in specific and selective fluorescent labeling, pave the way to a precise interrogation of molecular processes inside living cells.
Combined with the advent of cryo-EM methods, in particular those that visualize cellular structures, we are now able to visualize the dynamics of individual proteins inside a living cell with access to the structural properties of their immediate environment. These methods will enable the field to study more and more complex systems in increasingly physiologically relevant environments. All molecular processes that support cellular activity arise from an intricate network of macromolecular interactions that take place in complex, crowded environments.
The great advances that have been made in single-molecule techniques are emphasizing a view of dynamic multiprotein systems that is not linear and deterministic, but highly stochastic van Oijen and Dixon, In this review, we compared in vitro and in vivo experiments on cytoskeletal motors. It is clear that increasing the complexity of the system, for example by having multiple kinesins and dyneins acting on the same cargo, changes their dynamics.
The composition of the replisome has previously been shown to be very stable and highly resistant to dilution Debyser et al. Single-molecule studies on the bacteriophage T7 and E.
This suggests a mechanism in which, in a low-concentration condition, a protein remains stably bound to a complex, while being exchanged rapidly in the presence of competing protein at high concentration. Such a perhaps counterintuitive concentration-dependent dissociative mechanism has recently also been reported for replication protein A RPA in S.
Further, in a study on the binding and unbinding kinetics of DNA transcription regulators in living E. The apparent paradox between stability under high dilution and plasticity at high concentrations can be rationalized through a network of many weak interactions Fig. Under dilute conditions, stochastic, transient disruptions of any one of the interactions within a protein complex will not result in dissociation of the protein, as it is held to the complex via the other bonds, and the interaction would be rapidly reform Fig.
Under more physiologically relevant protein concentrations, however, a protein can bind at a transiently vacated binding site and consequently compete out the original protein Fig. This phenomenon obeys fundamental chemical and thermodynamic principles and can be mathematically described Sing et al. This multisite exchange mechanism would allow components of multiprotein complexes to be easily replaced. In the case of the replisome, for example, this mechanism may represent a pathway through which a defective polymerase can easily be replaced, thereby insuring replication with a high fidelity.
Furthermore this concentration-dependent exchange could provide easy access to other potential binding partners, like repair polymerases Sutton, The upregulation of these repair polymerases will increase their copy number and stimulate the dissociation of Pol III through the multisite exchange mechanism, thereby guaranteeing fast DNA repair. Stability versus plasticity. A Under dilute conditions, transient disruption of any one of the weak interactions holding a complex together would be followed by its rapid reformation, preventing complete dissociation of the protein from the complex.
This rapid microscopic reassociation would allow a protein to remain stably bound to the complex. B If, however, there are competing proteins in close proximity to the complex, one of these can bind at a transiently vacated binding site and consequently be at a sufficiently high local concentration to compete out the original protein. Single-molecule tools have enabled experimental access to the dynamic behavior of complex biomolecular systems under physiologically relevant conditions.cpanel.builttospill.reclaimhosting.com/d-o-n-d-e.php
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An important next direction is to further develop the single-molecule methods to study larger, more complex systems. The in vitro use of force- and fluorescence-based tools described in this review has matured to a point at which the complexity of the systems under study seems limitless. Single-molecule studies of complex biochemical systems have already significantly changed our view of the dynamic behavior of molecular systems. The role of stochastic processes in how biological macromolecules move and interact with one another has significant impact on how biochemical processes are controlled.
Instead of deterministic pathways, multiprotein complexes seem to perform their tasks by choosing from a multitude of pathways, each made possible by the constellation of weak and strong interactions that hold such a complex together. Applications of these tools within cells are still comparatively limited, however, in their ability to monitor structural and functional properties in real time at the single-molecule level. Further development of these tools and new labeling approaches are needed to further elucidate the molecular gymnastics of these complexes in vivo and bridge the gap between in vitro studies and observations inside living cells.
The authors would like to thank Dr. Slobodan Jergic, Dr. Andrew Robinson, and Mr. Jacob Lewis for their contributions to the figures. Monachino and L. November 4, Volume , No. Skip to main content. Spenkelink , Antoine M. Enrico Monachino. DOI: Introduction Single-molecule approaches are transforming our understanding of cell biology. Push, pull, poke, and prod: Mechanical single-molecule techniques The folding of proteins into functional structures, the manner with which they undergo conformational transitions, and their interactions between binding partners are all complex processes that are strictly ruled by the shape of the free-energy landscapes describing the thermodynamics of the system.
AFM AFM is a scanning probe microscopy technique that allows visualization of the surface topography of a sample at subnanometer resolution.
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Figure 1. Figure 2. OT In OT also called optical traps , a tightly focused laser beam is diffracted by a dielectric particle, resulting in a force that traps the particle nearby the focus of the laser. MT MT are conceptually similar to OT: a magnetic field is used to trap a superparamagnetic bead that is bound to one end of the molecule of interest Fig. What you see is what you get: Imaging techniques Fluorescence imaging Mechanical single-molecule techniques allow the precise measurement of force and energy changes and have, therefore, been invaluable to studies on protein folding, DNA stability, and protein—DNA interactions.
Total internal reflection fluorescence TIRF One of the first methods introduced to increase the useful concentration range of single-molecule fluorescence imaging was TIRF microscopy.
Figure 3. Local activation of dye LADye , photoactivation, diffusion, and excitation PhADE , and point accumulation for imaging in nanoscale topography PAINT To reduce the background fluorescence even further and enable the visualization of individual labeled molecules at physiologically relevant concentrations, techniques have been introduced that rely on photoactivatable tags.
Single-molecule fluorescence resonance energy transfer smFRET FRET is the distance-dependent nonradiative energy transfer between two fluorescent molecules that occurs when the emission spectrum of one fluorophore overlaps with the absorption spectrum of the other. Figure 4. Outlook Single-molecule tools have enabled experimental access to the dynamic behavior of complex biomolecular systems under physiologically relevant conditions.