Frequency modulation of the Min-protein oscillator by nucleoid-associated factors in Escherichia coli.

In E. coli, the Min-protein oscillator, together with the nucleoid occlusion (NO), destabilizes the Z-ring assembly away from the midcell to ensure faithful septation. These two inhibitory pathways are thought to be working independently for division site placement. Even though the Min-protein oscillator has been displayed by synthetic minimal systems, it is unclear the interplays of Min proteins and compartment geometry are sufficient to bolster oscillation stability in vivo. By probing if NO plays a role in the Min oscillation, we study the oscillation frequency in the anucleate and nucleoid-perturbed cells. Surprisingly, we found that the oscillation periods of the Min-protein oscillators were seriously deviated in the anucleate and nucleoid-perturbed cells, but the oscillation frequency either went up in the anucleate or down in the nucleoid-perturbed cells. Intriguingly, enhanced stability and reduced frequency were observed in the cells expressing the NO factor SlmA higher than the native level. Our results reveal an unanticipated role of the nucleoid in modulating the frequency and stability of Min-protein system. SlmA is indicated to facilitate such modulations, potentially via directly interacting with the Min-protein system. A fresh perspective is suggested that frequency modulation of Min-protein oscillator is mediated via the act of nucleoid-associated factors.

Gene expression in E. coli influences the position and motion of the lac operon and vicinal loci.

Transcription and translation of active genes play an important role in determining the global organization of the chromosome. To further elucidate this phenomenon, we examined how the expression of either the lacY or the cfp gene in the native lac operon influences adjacent chromosomal segments by fluorescently labeling loci upstream and downstream of the expressed gene. Based on the positions and motile behaviors of these loci, our results reveal that the local organization of the vicinal chromosomal segments and its position in the nucleoid are both influenced by gene expression. Furthermore, we found that the effects on local organization depend on whether the expressed gene encodes a membrane protein or a cytoplasmic protein. Our measurements showing the movement of loci toward the membrane and the correlation between the motions of the upstream and downstream loci support the conclusion that the expression of genes encoding membrane proteins greatly influences chromosome dynamics.

High-Copy-Number Plasmid Segregation-Single-Molecule Dynamics in Single Cells.

Bacterial high-copy-number (hcn) plasmids provide an excellent model to study the underlying physical mechanisms of DNA segment segregation in an intracellular context. Using two-color fluorescent repressor-operator systems and a synthetic repressible replication origin, we tracked the motion and segregation of single hcn plasmid molecules in individual cells. The plasmid diffusion dynamics revealed between-plasmid temporal associations (clustering) as well as entropic and elastic recoiling forces in the confined intracellular spaces outside of nucleoids. These two effects could be effectively used in models to predict the heterogeneity of segregation. Additionally, the motile behaviors of hcn plasmids provide quantitative estimates of entropic exclusion strength and dynamic associations between DNA segments. Overall, this study utilizes a, to our knowledge, novel approach to predict the polymer dynamics of DNA segments in spatially confined, crowded cellular compartments as well as during bacterial chromosome segregation.

Direct optical mapping of transcription factor binding sites on field-stretched λ-DNA in nanofluidic devices.

Mapping transcription factor (TF) binding sites along a DNA backbone is crucial in understanding the regulatory circuits that control cellular processes. Here, we deployed a method adopting bioconjugation, nanofluidic confinement and fluorescence single molecule imaging for direct mapping of TF (RNA polymerase) binding sites on field-stretched single DNA molecules. Using this method, we have mapped out five of the TF binding sites of E. coli RNA polymerase to bacteriophage λ-DNA, where two promoter sites and three pseudo-promoter sites are identified with the corresponding binding frequency of 45% and 30%, respectively. Our method is quick, robust and capable of resolving protein-binding locations with high accuracy (∼ 300 bp), making our system a complementary platform to the methods currently practiced. It is advantageous in parallel analysis and less prone to false positive results over other single molecule mapping techniques such as optical tweezers, atomic force microscopy and molecular combing, and could potentially be extended to general mapping of protein-DNA interaction sites.

Optimization of probe-laser focal offsets for single-particle tracking.

In optical tweezers applications, tracking a trapped particle is essential for force measurement. One of the most popular techniques for single-particle tracking is achieved by analyzing the forward and backward light pattern, scattered by the target particle trapped by a trap laser beam, of an additional probe-laser beam with different wavelength whose focus is slightly apart from the trapping center. However, the optimized focal offset has never been discussed. In this paper, we investigate the tracking range and sensitivity as a function of the focal offset between the trapping and the probe-laser beams. As a result, the optimized focal offsets are a 3.3-fold radius ahead and a 2.0-fold radius behind the trapping laser focus in the forward tracking and the backward tracking, respectively. The experimental result agrees well with a theoretical prediction using the Mie scattering theory.

Polarization modulation spectroscopy of single fluorescent nanodiamonds with multiple nitrogen vacancy centers.

A technique based on polarization modulation spectroscopy (PMS) has been developed to determine quantitatively the number of fluorophores in nanoparticles at the single-molecule level. The technique involves rotation of the polarization of the excitation laser on a millisecond time scale, leading to fluorescence intensity modulation. By taking account of the heterogeneous orientation among the dipoles of the fluorophores and simulating the modulation depth distribution with Monte Carlo calculations, we show that it is possible to deduce the ensemble average and number distribution of the fluorophores. We apply the technique to fluorescent nanodiamonds (FNDs) containing multiple nitrogen vacancy (NV) centers. Comparing the experimental and simulated modulation depth distributions of 11 nm FNDs, we deduce an average number of = 3, which is in good agreement with independent photon correlation measurements. The method is general, rapid, and applicable to other nanoparticles, polymers, and molecular complexes containing multiple and randomly orientated fluorophores as well.

Mass production and dynamic imaging of fluorescent nanodiamonds.

Fluorescent nanodiamond is a new nanomaterial that possesses several useful properties, including good biocompatibility, excellent photostability and facile surface functionalizability. Moreover, when excited by a laser, defect centres within the nanodiamond emit photons that are capable of penetrating tissue, making them well suited for biological imaging applications. Here, we show that bright fluorescent nanodiamonds can be produced in large quantities by irradiating synthetic diamond nanocrystallites with helium ions. The fluorescence is sufficiently bright and stable to allow three-dimensional tracking of a single particle within the cell by means of either one- or two-photon-excited fluorescence microscopy. The excellent photophysical characteristics are maintained for particles as small as 25 nm, suggesting that fluorescent nanodiamond is an ideal probe for long-term tracking and imaging in vivo, with good temporal and spatial resolution.

Optical trapping of a spherically symmetric sphere in the ray-optics regime: a model for optical tweezers upon cells.

Since their invention in 1986, optical tweezers have become a popular manipulation and force measurement tool in cellular and molecular biology. However, until recently there has not been a sophisticated model for optical tweezers on trapping cells in the ray-optics regime. We present a model for optical tweezers to calculate the optical force upon a spherically symmetric multilayer sphere representing a common biological cell. A numerical simulation of this model shows that not only is the magnitude of the optical force upon a Chinese hamster ovary cell significantly three times smaller than that upon a polystyrene bead of the same size, but the distribution of the optical force upon a cell is also much different from that upon a uniform particle, and there is a 30% difference in the optical trapping stiffness of these two cases. Furthermore, under a small variant condition for the refractive indices of any adjacent layers of the sphere, this model provides a simple approximation to calculate the optical force and the stiffness of an optical tweezers system.