Chemical Physics in Living Cells-using Light to Visualize and Control Intracellular Signal Transduction?

Cells are crowded microenvironments filled with macromolecules undergoing constant physical and chemical interactions. The physicochemical makeup of the cells affects various cellular responses, determines cell-cell interactions and influences cell decisions. Chemical and physical properties differ between cells and within cells. Moreover, these properties are subject to dynamic changes in response to environmental signals, which often demand adjustments in the chemical or physical states of intracellular molecules. Indeed, cellular responses such as gene expression rely on the faithful relay of information from the outside to the inside of the cell, a process termed signal transduction. The signal often traverses a complex path across subcellular spaces with variable physical chemistry, sometimes even influencing it. Understanding the molecular states of such signaling molecules and their intracellular environments is vital to our understanding of the cell. Exploring such intricate spaces is possible today largely because of experimental and theoretical tools. Here, we focus on one tool that is commonly used in chemical physics studies-light. We summarize recent work which uses light to both visualize the cellular environment and also control intracellular processes along the axis of signal transduction. We highlight recent accomplishments in optical microscopy and optogenetics, an emerging experimental strategy which utilizes light to control the molecular processes in live cells. We believe that optogenetics lends unprecedented spatiotemporal precision to the manipulation of physicochemical properties in biological contexts. We hope to use this work to demonstrate new opportunities for chemical physicists who are interested in pursuing biological and biomedical questions.     

The accumulation of phytoalexin in cucumber plant after stress

During the course of pathogens penetrating the plant cell, besides of chemical secretion, the pathogens may cause mechanical signal by the physical pressure on the plant cell. In the current study, we use the pressure as the stress signal to study the induction in plant resistance and the effect of accumulation of phytoalexin. We found that stress can induce the resistance in cucumber seeding significantly. Peptides contained RGD motif can specific block the adhesion between plant cell wall and plasma membrane. When breaking the plant cell wall and plasma membrane by using RGD peptides, the stress induction effect is almost absolutely eliminated. The results of assay with TLC and HPLC showed that stress stimulation could increase the accumulation of cucumber seeding phytoalexin. So, we can conclude that the accumulation of phytoalexin is one possible reason of improve the stress induced resistance. When block the adhesion between plant cell wall and plasma membrane by RGD, there are only part of accumulation of phytoalexin. The results suggest that stress induced resistance and accumulation of phytoalexin of plant is required for the adhesion of plant cell wall–plasma membrane.     

质谱及相关技术在信号传导方面的研究进展

细胞信号传导是近年来生命科学研究的热点之一。有关蛋白转录后修饰 (如蛋白质磷酸化、乙酰化、糖基化等 ) ,信号肽序列测定 ,信号传导途径和多通道调节方式 ,蛋白自折叠及构象变化 ,小分子脂类信号分子等研究由于质谱技术的快速发展而取得了突破性的进展     

Delivery of bioactive,gel-isolated proteins into live cells

The delivery of proteins into live cells is a promising strategy for the targeted modulation of protein-protein interactions and the manipulation of specific cellular functions. Cellular delivery can be facilitated by complexing the protein of interest with carrier molecules. Recently, an amphipatic peptide was identified, Pep-1 (KETWWETWWTE WSQPKKKRKV), which crosses the plasma membrane of many cell types to carry and deliver proteins as large as antibodies. Pep-1 effectively delivers proteins in solution; but Pep-1 is not suitable for delivering sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) isolated proteins because Pep-1 complexes with cargo proteins are destroyed by SDS. Here, we report cellular delivery of SDS-PAGE-isolated proteins, without causing cellular damage, by using a nonionic detergent, Triton X-100, as carrier. To determine the specificity of our method, we separated antibodies against different intracellular targets by nonreducing SDS-PAGE. Following electrophoresis, the antibody bands were detected by zinc-imidazole reverse staining, excised, in-gel refolded with Triton X-100, and eluted in detergent-free phosphate-buffered saline. When overlaid on cultured NIH 3T3 cells, the antibodies penetrated the cells localizing to their corresponding intracellular targets. These results are proof-of-principle for the delivery of gel-isolated bioactive proteins into cultured cells and suggest new ways for experimental protein therapy and for studying protein-protein interactions using gel-isolated protein.     

Development of planar patch clamp technology and its application in the analysis of cellular electrophysiology

A patch clamp chip, as a novel cell-based chip for electrophysiological recordings, has many prominent advantages such as high resolution, accuracy, high throughput and automation. It can be used to perform multivariate and real-time measurements of cell networks in situ. Therefore, this technology will dramatically promote the research on ionic channels, neuronal networks and the application of this technology in drug screening. This paper reviews the development of planar patch clamp technology and its applications in detail. The latest progress in the research of taste cells electrophysiology and taste transduction is also presented. Finally, this paper analyzes the methodology of neural chips. Based on the current research of our laboratory, the prospective applications of a patch clamp chip in the research of taste sensation and transduction mechanisms at molecular and cellular levels are discussed.     

N-myristoylated proteins,key components in intracellular signal transduction systems enabling rapid and flexible cell responses

N-myristoylation, one of the co- or post-translational modifications of proteins, has so far been regarded as necessary for anchoring of proteins to membranes. Recently, we have revealed that N^α-myristoylation of several brain proteins unambiguously regulates certain protein–protein interactions that may affect signaling pathways in brain. Comparison of the amino acid sequences of myristoylated proteins including those in other organs suggests that this regulation is involved in signaling pathways not only in brain but also in other organs. Thus, it has been shown that myristoylated proteins in cells regulate the signal transduction between membranes and cytoplasmic fractions. An algorithm we have developed to identify myristoylated proteins in cells predicts the presence of hundreds of myristoylated proteins. Interestingly, a large portion of the myristoylated proteins thought to take part in signal transduction between membranes and cytoplasmic fractions are included in the predicted myristoylated proteins. If the proteins functionally regulated by myristoylation, a posttranslational protein modification, were understood as cross-talk points within the intracellular signal transduction system, known signaling pathways could thus be linked to each other, and a novel map of this intracellular network could be constructed. On the basis of our recent results, this review will highlight the multifunctional aspects of protein N-myristoylation in brain.