Non-Markovian properties and multiscale hidden Markovian network buried in single molecule time series.

We present a novel scheme to extract a multiscale state space network (SSN) from single-molecule time series. The multiscale SSN is a type of hidden Markov model that takes into account both multiple states buried in the measurement and memory effects in the process of the observable whenever they exist. Most biological systems function in a nonstationary manner across multiple timescales.

Palmitoylated Ras proteins traffic through recycling endosomes to the plasma membrane during exocytosis.

Ras proteins regulate cell growth, death, and differentiation, and it is well established that this functional versatility is accomplished through their different subcellular localizations. Palmitoylated H- and N-Ras are believed to localize at the perinuclear Golgi and plasma membrane (PM). Notably, however, recycling endosomes (REs) also localize to a perinuclear region, which is often indistinguishable from the Golgi.

Multiple-state reactions between the epidermal growth factor receptor and Grb2 as observed by using single-molecule analysis.

Phosphorylation of the cytoplasmic tyrosine residues of the epidermal growth factor receptor (EGFR) upon binding of EGF induces recognition of various intracellular signaling molecules, including Grb2. Here, the reaction kinetics between EGFR and Grb2 was analyzed by visualizing single molecules of Grb2 conjugated to the fluorophore Cy3 (Cy3-Grb2). The plasma membrane fraction was purified from human epithelial carcinoma A431 cells after stimulation with EGF and attached to coverslips.

Covalent immobilization of epidermal growth factor molecules for single-molecule imaging analysis of intracellular signaling.

We have developed cell stimulative system by covalently immobilized signalling molecules on the surface of coverslips on which cells are later cultured. N-(6-maleimidocaproyloxy)sulfo-succinimide (sulfo-EMCS) cross-links the amino-terminal of epidermal growth factors (EGF) with the thiol-modified glass surface without degrading EGF's physiological activity. The glass surface was covered up to about 1.

Optical bioimaging: from living tissue to a single molecule: single-molecule visualization of cell signaling processes of epidermal growth factor receptor.

Single-molecule imaging is an ideal technology to study molecular mechanisms of biological reactions in vitro. Recently, this technology has been extended to real-time observation of fluorescent dye-labeled molecules in living cells. Total internal reflection fluorescence microscopy is the major technique for this purpose.

Protein phosphorylation regulates actomyosin-driven vesicle movement in cell extracts isolated from the green algae, Chara corallina.

In Characean cells endoplasmic streaming stops upon membrane depolarization accompanied by Ca(2+) entry. We investigated the mechanism of this cessation of endoplasmic streaming by reconstituting the vesicle movement in vitro. In a living cell of Chara corallina, there are a number of vesicles moving along actin cables.