Chan-Gi Pack

Local Nucleosome Dynamics Facilitate Chromatin Accessibility in Living Mammalian Cells

Genome information, which is three-dimensionally organized within cells as chromatin, is searched and read by various proteins for diverse cell functions. Although how the protein factors find their targets remains unclear, the dynamic and flexible nature of chromatin is likely crucial. Using a combined approach of fluorescence correlation spectroscopy, single-nucleosome imaging, and Monte Carlo computer simulations, we demonstrate local chromatin dynamics in living mammalian cells. We show that similar to interphase chromatin, dense mitotic chromosomes also have considerable chromatin accessibility. For both interphase and mitotic chromatin, we observed local fluctuation of individual nucleosomes (~50 nm movement/30 ms), which is caused by confined Brownian motion. Inhibition of these local dynamics by crosslinking impaired accessibility in the dense chromatin regions. Our findings show that local nucleosome dynamics drive chromatin accessibility. We propose that this local nucleosome fluctuation is the basis for scanning genome information.

Reciprocal interaction with G-actin and tropomyosin is essential for aquaporin-2 trafficking

Trafficking of water channel aquaporin-2 (AQP2) to the apical membrane and its vasopressin and protein kinase A (PKA)–dependent regulation in renal collecting ducts is critical for body water homeostasis. We previously identified an AQP2 binding protein complex including actin and tropomyosin-5b (TM5b). We show that dynamic interactions between AQP2 and the actin cytoskeleton are critical for initiating AQP2 apical targeting. Specific binding of AQP2 to G-actin in reconstituted liposomes is negatively regulated by PKA phosphorylation. Dual color fluorescence cross-correlation spectroscopy reveals local AQP2 interaction with G-actin in live epithelial cells at single-molecule resolution. Cyclic adenosine monophosphate signaling and AQP2 phosphorylation release AQP2 from G-actin. In turn, AQP2 phosphorylation increases its affinity to TM5b, resulting in reduction of TM5b bound to F-actin, subsequently inducing F-actin destabilization. RNA interference–mediated knockdown and overexpression of TM5b confirm its inhibitory role in apical trafficking of AQP2. These findings indicate a novel mechanism of channel protein trafficking, in which the channel protein itself critically regulates local actin reorganization to initiate its movement.

In vivo evidence for the fibrillar structures of Sup35 prions in yeast cells

Correlative light and electron microscopy provides support for the linear amalgamation of yeast prion proteins.

The regulator of the F1 motor: inhibition of rotation of cyanobacterial F1‐ATPase by the ε subunit

The chloroplast‐type F1 ATPase is the key enzyme of energy conversion in chloroplasts, and is regulated by the endogenous inhibitor ε, tightly bound ADP, the membrane potential and the redox state of the γ subunit. In order to understand the molecular mechanism of ε inhibition, we constructed an expression system for the α3β3γ subcomplex in thermophilic cyanobacteria allowing thorough investigation of ε inhibition. ε Inhibition was found to be ATP‐independent, and different to that observed for bacterial F1‐ATPase. The role of the additional region on the γ subunit of chloroplast‐type F1‐ATPase in ε inhibition was also determined. By single molecule rotation analysis, we succeeded in assigning the pausing angular position of γ in ε inhibition, which was found to be identical to that observed for ATP hydrolysis, product release and ADP inhibition, but distinctly different from the waiting position for ATP binding. These results suggest that the ε subunit of chloroplast‐type ATP synthase plays an important regulator for the rotary motor enzyme, thus preventing wasteful ATP hydrolysis.

Quantitative live-cell imaging reveals spatio-temporal dynamics and cytoplasmic assembly of the 26S proteasome

The 26S proteasome is a 2.5-MDa multisubunit protease complex that degrades polyubiquitylated proteins. Although its functions and structure have been extensively characterized, little is known about its dynamics in living cells. Here, we investigate the absolute concentration, spatio-temporal dynamics and complex formation of the proteasome in living cells using fluorescence correlation spectroscopy. We find that the 26S proteasome complex is highly mobile, and that almost all proteasome subunits throughout the cell are stably incorporated into 26S proteasomes. The interaction between 19S and 20S particles is stable even in an importin-α mutant, suggesting that the 26S proteasome is assembled in the cytoplasm. Furthermore, a genetically stabilized 26S proteasome mutant is able to enter the nucleus. These results suggest that the 26S proteasome completes its assembly process in the cytoplasm and translocates into the nucleus through the nuclear pore complex as a holoenzyme.

Single mother–daughter pair analysis to clarify the diffusion properties of yeast prion Sup35 in guanidine‐HCl‐treated [PSI+] cells

The yeast prion [PSI+] is a protein‐based heritable element, in which aggregates of Sup35 protein are transmitted to daughter cells in a non‐Mendelian manner. To elucidate the mechanism of the transmission, we have developed methods to directly analyse the dynamics of Sup35 fused with GFP in single mother–daughter pairs. As it is known that the treatment of yeast cells with guanidine hydrochloride (GuHCl) cures [PSI+] by perturbing Hsp104, a prion‐remodelling factor, we analysed the diffusion profiles of Sup35–GFP in GuHCl‐treated [PSI+] cells using fluorescence correlation spectroscopy (FCS). FCS analyses revealed that Sup35–GFP diffusion in the daughter cells was faster; that is, the Sup35–GFP particle was smaller, than that in the mother [PSI+] cells, and it eventually reached the diffusion profiles in [psi−] cells. We then analysed the flux of Sup35–GFP oligomers from mother to daughter [PSI+] cells in the presence of GuHCl, using a modified fluorescent recovery after photobleaching technique, and found that the flux of the diffuse oligomers was completely inhibited. The noninvasive methods described here can be applied to other protein‐based transmissible systems inside living cells.

Flexible and dynamic nucleosome fiber in living mammalian cells.

Genomic DNA is organized three dimensionally within cells as chromatin and is searched and read by various proteins by an unknown mechanism; this mediates diverse cell functions. Recently, several pieces of evidence, including our cryomicroscopy and synchrotron X-ray scattering analyses, have demonstrated that chromatin consists of irregularly folded nucleosome fibers without a 30-nm chromatin fiber (i.e., a polymer melt-like structure). This melt-like structure implies a less physically constrained and locally more dynamic state, which may be crucial for protein factors to scan genomic DNA. Using a combined approach of fluorescence correlation spectroscopy, Monte Carlo computer simulations, and single nucleosome imaging, we demonstrated the flexible and dynamic nature of the nucleosome fiber in living mammalian cells. We observed local nucleosome fluctuation (~50 nm movement per 30 ms) caused by Brownian motion. Our in vivo-in silico results suggest that local nucleosome dynamics facilitate chromatin accessibility and play a critical role in the scanning of genome information.

Live cell single-molecule detection in systems biology.

In this review, we describe technology and use of single‐fluorophore imaging and detection in living cells with regard to application in systems biology and medicine. Because all biological reactions occur under aqueous conditions, the realization of single‐fluorophore imaging using an optical microscope has led to the direct observation of biological molecules at work. Today, we can observe single molecules in individual living cells and even higher multicellular organisms. Using single‐molecule imaging, we can determine the absolute values of kinetic and dynamic parameters of molecular reactions as a whole and during fluctuations and distribution. In addition, identification of the coordinate of single molecules has enabled super‐localization techniques to virtually improve spatial resolution of optical microscopy. Single‐molecule detection that depends on point detection instead of imaging is also useful in detecting concentrations, diffusive movements, and molecular interactions in living cells, especially in the cytoplasm. The precise and absolute values of positional, kinetic, and dynamic parameters that are determined by single‐molecule imaging and detection in living cells constitute valuable data on unitary biological reactions, because they are obtained without destroying the integrity of complex cellular systems. Moreover, most parameters that are determined by single‐molecule measurements can be substituted directly into equations that describe kinetic and dynamic models in systems biology and medicine. WIREs Syst Biol Med 2012, 4:183–192. doi: 10.1002/wsbm.161

[PSI+] aggregate enlargement in rnq1 nonprion domain mutants, leading to a loss of prion in yeast

[PIN+] is the prion form of the Rnq1 protein of unknown function in Saccharomyces cerevisiae. A glutamine/asparagine (Q/N)‐rich C‐terminal domain is necessary for the propagation of [PIN+], whereas the N‐terminal region is non‐Q/N‐rich and considered the nonprion domain. Here, we isolated numerous single‐amino‐acid mutations in Rnq1, phenotypically similar to Rnq1Δ100, which inhibit [PSI+] propagation in the [PIN+] state, but not in the [pin−] state, when overproduced. The dynamics of the prion aggregates was analyzed by semi‐denaturing detergent‐agarose gel electrophoresis and fluorescence correlation spectroscopy. The results indicated that [PSI+] aggregates were enlarged in mother cells and, instead, not apparently transmitted into daughter cells. Under these conditions, the activity of Hsp104, a known prion disaggregase, was not affected when monitored for the thermotolerance of the rnq1 mutants. These [PSI+]‐inhibitory rnq1 mutations did not affect [PIN+] propagation itself when over‐expressed from a strong promoter, but instead destabilized [PIN+] when expressed from the weak authentic RNQ1 promoter. The majority of these mutated residues are mapped to the surface, and on one side, of contiguous α‐helices of the nonprion domain of Rnq1, suggesting its involvement in interactions with a prion or a factor necessary for prion development.

Interaction of a Small Heat Shock Protein of the Fission Yeast, Schizosaccharomyces pombe, with a Denatured Protein at Elevated Temperature

We have expressed, purified, and characterized one small heat shock protein of the fission yeast Schizosaccharomyces pombe, SpHsp16.0. SpHsp16.0 was able to protect citrate synthase from thermal aggregation at 45 °C with high efficiency. It existed as a hexadecameric globular oligomer near the physiological growth temperature. At elevated temperatures, the oligomer dissociated into small species, probably dimers. The dissociation was completely reversible, and the original oligomer reformed immediately after the temperature dropped. Large complexes of SpHsp16.0 and denatured citrate synthase were observed by size exclusion chromatography and electron microscopy following incubation at 45 °C and then cooling. However, such large complexes did not elute from the size exclusion column incubated at 45 °C. The denatured citrate synthase protected from aggregation was trapped by a GroEL trap mutant at 45 °C. These results suggest that the complex of SpHsp16.0 and denatured citrate synthase at elevated temperatures is in the transient state and has a hydrophobic nature. Analyses of the interaction between SpHsp16.0 and denatured citrate synthase by fluorescence cross-correlation spectrometry have also shown that the characteristics of SpHsp16.0-denatured citrate synthase complex at the elevated temperature are different from those of the large complex obtained after the shift to lowered temperatures.