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ATP-dependent, azide-sensitive rotation of the gamma subunit relative to the alpha3beta3 hexagonal ring of ATP synthase was observed with a single molecule imaging system. Thus, ATP synthase is a rotary motor enzyme, the first ever found.
Eukaryotic sequence-specific transcription factors (TFs) must find their cognate DNA targets hidden in genomic chromatin amid an excess of nonspecific sequences and degenerate motifs. Although static TF interactions with nucleosomal targets have been elucidated, how TFs efficiently search for cognate sites within native gene-sized chromatin domains has been unclear. Here we used purified Saccharomyces cerevisiae HIS3 minichromosomes and single-molecule imaging to compare association and dissociation kinetics of transcription activator GCN4 on chromatin and naked genomic DNA. GCN4 displays widespread and stable off-target binding on bare DNA because of entrapment by degenerate sites and interactions with nonspecific DNA of increasing length, indicative of one-dimensional (1D) diffusion. Nucleosome organization on the minichromosome reduces promiscuous GCN4 residence times by obstructing TF association and restricting 1D target search within nucleosome-free regions. Furthermore, the intrinsically disordered GCN4 activation domain independently enhances targeting efficiency and specificity by accelerating association-dissociation kinetics in vitro and in living cells. Altogether, both nucleosome organization and activation domains independently suppress promiscuous GCN4 binding, which, if unchecked, may cause aberrant cryptic transcription known to occur upon chromatin disruptions.
Global cellular responses induced by epidermal growth factor (EGF) receptor (EGFR) occur immediately with a less than 1% occupancy among tens of thousands of EGFR molecules on single cell surface. Activation of EGFR requires the formation of a signaling dimer of EGFR bound with a single ligand to each molecule. How sufficient numbers of signaling dimers are formed at such low occupancy rate is still not known. Here, we have analyzed the kinetics of EGF binding and the formation of the signaling dimer using single-molecule imaging and mathematical modeling. A small number of EGFR on the cell surface formed dimeric binding sites, which bound EGF two orders of magnitude faster than the monomeric binding sites. There was a positive cooperative binding of EGF to the dimeric binding sites through a newly discovered kinetic intermediate. These two mechanisms facilitate the formation of signaling dimers of EGF/EGFR complexes.
The binding change model for the F(1)-ATPase predicts that its rotation is intimately correlated with the changes in the affinities of the three catalytic sites for nucleotides. If so, subtle differences in the nucleotide structure may have pronounced effects on rotation. Here we show by single-molecule imaging that purine nucleotides ATP, GTP, and ITP support rotation but pyrimidine nucleotides UTP and CTP do not, suggesting that the extra ring in purine is indispensable for proper operation of this molecular motor. Although the three purine nucleotides were bound to the enzyme at different rates, all showed similar rotational characteristics: counterclockwise rotation, 120 degrees steps each driven by hydrolysis of one nucleotide molecule, occasional back steps, rotary torque of approximately 40 piconewtons (pN).nm, and mechanical work done in a step of approximately 80 pN.nm. These latter characteristics are likely to be determined by the rotational mechanism built in the protein structure, which purine nucleotides can energize. With ATP and GTP, rotation was observed even when the free energy of hydrolysis was -80 pN.nm/molecule, indicating approximately 100% efficiency. Reconstituted F(o)F(1)-ATPase actively translocated protons by hydrolyzing ATP, GTP, and ITP, but CTP and UTP were not even hydrolyzed. Isolated F(1) very slowly hydrolyzed UTP (but not CTP), suggesting possible uncoupling from rotation.
The 2003 International Prize for Biology was awarded to Shinya Inoue for his pioneering work in visualizing dynamic processes within living cells using the light microscope. He and his scientific descendants are now pushing light microscopy even further by developing new techniques such as imaging single molecules, visualizing processes in living animals, and correlating results from light and electron microscopy.
LukF and Hlg2 of staphylococcal gamma-hemolysin assemble into hetero-oligomeric pores on human red blood cells (HRBC). Here, we demonstrate, using a single-molecule imaging technique, that a W177T/R198T mutant of LukF, which exhibits no binding activity toward phosphatidylcholine, could form intermediate oligomers with Hlg2, including dimers, tetramers, and hexamer/heptamers, on HRBC. But, the mutant neither caused K(+) efflux nor lysed HRBC, indicating that functional pores were not formed. Hence, we conclude that the W177 and R198 residues are essential for proper pore-formation by staphylococcal gamma-hemolysin. We also suggest that the interaction between the W177 and R198 residues, and phosphatidylcholine on membranes is the key to the formation of functional pores.
How eukaryotes specify their replication origins is an important unanswered question. Here, we analyze the replicative organization of yeast rDNA, which consists of approximately 150 identical repeats, each containing a potential origin. Using DNA combing and single-molecule imaging, we show that functional rDNA origins are clustered and interspersed with large domains where initiation is silenced. This repression is largely mediated by the Sir2p histone-deacetylase. Increased origin firing in sir2 Delta mutants leads to the accumulation of circular rDNA species, a major determinant of yeast aging. We conclude that rDNA replication is regulated epigenetically and that Sir2p may promote genome stability and longevity by suppressing replication-dependent rDNA recombination.
We used single-molecule imaging techniques and measured the one-dimensional diffusion of LacI repressor proteins along elongated DNA to address the long-standing puzzle of why some proteins find their targets faster than allowed by 3D diffusion. Our analysis of the LacI transcription factor's diffusion yielded four main results: (1) LacI diffuses along nonspecific sequences of DNA in the form of 1D Brownian motion; (2) the observed 1D diffusion coefficients D1vary over an unexpectedly large range, from 2.3x10(-12) cm2/s to 1.3x10(-9) cm2/s; (3) the lengths of DNA covered by these 1D diffusions vary from 120 nm to 2920 nm; and (4) the mean values of D1 and the diffusional lengths indeed predict a LacI target binding rate 90 times faster than the 3D diffusion limit.
A single-molecule-imaging technique, atomic force microscopy (AFM) was applied to the analyses of the genome architecture of Staphylococcus aureus. The staphylococcal cells on a cover glass were subjected to a mild lysis procedure that had maintained the fundamental structural units in Escherichia coli. The nucleoids were found to consist of fibrous structures with diameters of 80 and 40 nm. This feature was shared with the E. coli nucleoid. However, whereas the E. coli nucleoid dynamically changed its structure to a highly compacted one towards the stationary phase, the S. aureus nucleoid never underwent such a tight compaction under a normal growth condition. Bioinformatic analysis suggested that this was attributable to the lack of IHF that regulate the expression of a nucleoid protein, Dps, required for nucleoid compaction in E. coli. On the other hand, under oxidative conditions, MrgA (a staphylococcal Dps homolog) was over-expressed and a drastic compaction of the nucleoid was detected. A knock-out mutant of the gene encoding the transcription factor (perR) constitutively expressed mrgA, and its nucleoid was compacted without the oxidative stresses. The regulatory mechanisms of Dps/MrgA expression and their biological significance were postulated in relation to the nucleoid compaction.
Single-molecule imaging techniques were used to reveal the binding of individual cyclic adenosine 3',5'-monophosphate molecules to heterotrimeric guanine nucleotide-binding protein coupled receptors on the surface of living Dictyostelium discoideum cells. The binding sites were uniformly distributed and diffused rapidly in the plane of the membrane. The probabilities of individual association and dissociation events were greater for receptors at the anterior end of the cell. Agonist-induced receptor phosphorylation had little effect on any of the monitored properties, whereas G protein coupling influenced the binding kinetics. These observations illustrate the dynamic properties of receptors involved in gradient sensing and suggest that these may be polarized in chemotactic cells.
mDia1, a Rho effector, belongs to the Formin family of proteins, which shares the conserved tandem FH1-FH2 unit structure. Formins including mDia1 accelerate actin nucleation while interacting with actin filament fast-growing ends. Here our single-molecule imaging revealed fast directional movement of mDia1 FH1-FH2 for tens of microns in living cells. The movement of mDia1 FH1-FH2 was blocked by actin-perturbing drugs, and the speed of mDia1 FH1-FH2 movement appeared to correlate with actin elongation rates. In vitro, mDia1 FH1-FH2 associated persistently with the growing actin barbed end. mDia1 probably moves processively along the growing end of actin filaments in cells, and Formins may be a molecular motility machinery that is independent from motor proteins.
Efficient collection and detection of fluorescence coupled with careful minimization of background from impurities and Raman scattering now enable routine optical microscopy and study of single molecules in complex condensed matter environments. This ultimate method for unraveling ensemble averages leads to the observation of new effects and to direct measurements of stochastic fluctuations. Experiments at cryogenic temperatures open new directions in molecular spectroscopy, quantum optics, and solid-state dynamics. Room-temperature investigations apply several techniques (polarization microscopy, single-molecule imaging, emission time dependence, energy transfer, lifetime studies, and the like) to a growing array of biophysical problems where new insight may be gained from direct observations of hidden static and dynamic inhomogeneity.
The fluorescence lifetime of ethidium bromide (EB), a widely used fluorescent dye, has been monitored in water solution versus trehalose concentration in order to learn how the presence of the disaccharide modifies the hydration of EB, which is used in this study as a model probe. The interest in trehalose, a naturally occurring bioprotector (osmolyte), stems from its ability to stabilize biomolecules under stress situations. The observed effects of trehalose on EB fluorescence properties have been compared with those induced by sucrose and glucose. Lifetime measurements have been performed by frequency domain fluorometry (2-40 MHz), and from the analysis of their changes versus sugar concentration, the EB-sugar interaction parameters have been obtained. The effect of trehalose on the EB decay in glasses has also been studied both by exploring the heterogeneity of lifetime decay and by single-molecule imaging. Trehalose appears to be more efficient in changing the EB fluorescence parameters, such as the emission lifetime, and it leads to a degree of heterogeneity higher than that induced by the other sugars. When EB is embedded in trehalose glasses, the heterogeneity of the emission and of the bleaching time is further enhanced.
The regulation of DNA replication and transcription is achieved by dynamic structural changes of chromatin in which a series of proteins will acquire accessibility to specific regions of the DNA strand. A combination of biochemistry and nano-technology is essential to address questions regarding the structural basis for such macromolecular mechanisms. In the present study, we established an efficient salt-dialysis method of chromatin reconstitution and employed atomic force microscopy (AFM) as a single-molecule-imaging technique, to monitor the efficiency of the reconstitution. At first, the reconstitution efficiency with short DNA molecules of several kilo-base pairs was low, although the salt dialysis yielded a "beads-on-a-string" structure of oligonucleosomes with each nucleosome trapping 158+/-27 bp DNA. However, the efficiency for nucleosome formation became higher when longer DNA molecules with a super-helical constraint were used. A statistical analysis of the obtained AFM images identified a first-order relationship between the efficiency of the reconstitution and the length of the super-coiled DNA used. A high efficiency of approximately 290 bp/nucleosome that is close to the in vivo situation was obtained with a approximately 100 kbp template DNA. This enabled the structure-function studies of long chromatin molecules under well-defined conditions.
Atomic force microscopy was used to study adsorption and adhesion peculiarities of interferon alpha-2a on glass and mica surfaces. The specific protein adsorption behavior as a function of the pH value was illustrated on mica by single molecule imaging, while adhesion forces between interferon molecules and inner surfaces of borosilicate glass vials were measured directly under aqueous buffer conditions by force microscopy. We found that the adhesion force on Schott FIOLAX Type I plus was reduced by 40% of the total adhesion force measured on Schott FIOLAX, a standard type I borosilicate glass quality. These results reflect the anticipated superiority of the special "Type I plus" coating over undesired protein adsorption to glass. In addition, this study gives insight into a new method to predict unintended protein adsorption to glass container walls and to characterize the adsorption process by force measurement.
The combination of high-resolution tantalum/tungsten (Ta/W) shadowing at very low specimen temperature (-250 degrees C) under ultrahigh vacuum (less than 2 x 10(-9) mbar) with circular harmonic image averaging revealed details on the surface structure of mitochondrial creatine kinase (Mi-CK) molecules with a resolution less than 2.5 nm. Mi-CK octamers exhibit a cross-like surface depression dividing the square shaped projection of 10 x 10 nm into four equally sized subdomains, which correspond to the four dimers forming the octameric Mi-CK molecule. By a combination of positive staining (with uranyl acetate) and heavy metal shadowing, internal structures as well as the surface relief of Mi-CK were visualized at the same time at high resolution. Computational image analysis revealed only a single projection class of molecules, but the ability of Mi-CK to form linear filaments, as well as geometrical considerations concerning the formation of octamers by four equal, asymmetric dimers, suggest the existence of at least two distinct faces on the molecule. By image processing of Mi-CK filaments a side view of the octamer differing from the top-bottom projections of single molecules became evident showing a funnel-like access each form the top and bottom of the octamer connected by a central channel. The general structure of the Mi-CK octamer described here is relevant to the localization of the molecule at the inner-outer mitochondrial contact sites and to the function of Mi-CK as an "energy channeling" molecule.
Single-molecule AFM imaging of single-stranded and double-stranded DNA molecules self-assembled from solution onto a HOPG electrode surface is reported. The interaction of DNA with the hydrophobic surface induced DNA aggregation, overlapping, intra- and intermolecular interactions. Controlling the electrode potential and using the phase images as a control method, to confirm the correct topographical characterization, offers the possibility to enlarge the capability of AFM imaging of DNA immobilized onto conducting substrates, such as HOPG. The application of a potential of +300 mV (versus AgQRE) to the HOPG enhanced the robustness and stability of the adsorbed DNA molecules, increasing the electrostatic interaction between the positively charged electrode surface and the negatively charged DNA sugar-phosphate backbone.
The emerging nanomaterial, quantum dots or QDs, offers numerous potential applications in the biological area. As cell labeling probes, QDs become now an alternative of existing organic fluorescent dyes and fluorescent proteins. In this short review, we cover typical and successful applications of QDs as fluorescent probes in cell labeling and genomic diagnosis. As a future important application, biomolecular detection at a single molecule level utilizing QDs is also discussed.