利用内禀荧光探针研究蛋白相互作用中硫氧还蛋白的氧化还原态

细胞内蛋白质的氧化还原状态直接影响细胞的增殖、分化及凋亡,而氧化还原状态的改变对调控细胞的生存或死亡尤为重要。硫氧还蛋白(Thioredoxin, TRX)是一种广泛存在于生物体内的氧化还原调节蛋白,其在细胞内氧化还原状态的变化是发挥其氧化还原调控作用的重要过程。以TRX为对象并以其中的色氨酸残基(Trp)作为内禀荧光探针,利用蛋白质定点突变、SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)、荧光光谱和圆二色谱等技术和方法,研究TRX与谷胱甘肽过氧化物酶(glutathione peroxidase, GPX3)相互作用过程中氧化还原态的变化。通过观测TRX以及突变体中色氨酸荧光光谱的变化,研究蛋白相互作用的电子转移模式以及TRX氧化态-还原态之间的相互转化。结果表明氧化态的TRX与还原态的GPX3之间存在相互作用并发生电子交换,解释了二者之间电子传递模式为GPX3将电子传递给TRX,为揭示TRX在细胞信号传递过程中的物理化学机制提供了实验依据。     

Statistical physics of active processes in cells

Simple concepts from statistical physics are discussed to describe the transduction of chemical energy of a fuel to mechanical work on the molecular level. Such approaches can characterize general physical features of motor proteins that generate forces in the cell cytoskeleton. In integrated cellular systems such as cilia and hair bundles, cytoskeletal filaments and motors form complex structures and interact in large numbers. In such systems the interplay of filaments and motors can lead to emergent dynamic behaviors such as oscillating collective modes or to wave-like patterns. We discuss general aspects of such dynamic states and relate them to the dynamics of cytoskeletal structures in cells.     

Efficient Cellular Internalization and Transport of Bowl-Shaped Polydopamine Particles

In drug delivery applications, particle-based systems have been used widely due to their physicochemical properties such as size, shape, and surface charge to achieve desirable properties in intracellular environments. The way in which nanoparticles enter a biological cell is an important factor in determining their efficacy as drug carriers, their biodistribution, and toxicity. Most research thus far has focused on the comparison of spherical and rod-like particles on cellular internalization and transport. Here, the synthesis of bowl-shaped polydopamine (PDA) mesoporous nanoparticles with an average diameter of 200 nm and well-controlled radially oriented mesochannels are reported. By incubating bowl-shaped PDA nanoparticles and spherical nanoparticles with HeLa cells, their internalization behaviors are investigated using a suite of characterization techniques. Extensive experimental results demonstrate that bowl-shaped PDA nanoparticles adhere to the cell more efficiently and a faster rate of cellular uptake of bowl-shaped nanoparticles compared to their spherical counterparts. Overall, the cellular internalization behavior of particles is shape-dependent, and such information is crucial in designing nanoparticles for biomedical applications.     

Nonequilibrium thermodynamics and nonlinear kinetics in a cellular signaling switch

We develop a rigorous nonequilibrium thermodynamics for an open system of nonlinear biochemical reactions responsible for cell signal processing. We show that the quality of the biological switch consisting of a phosphorylation-dephosphorylation cycle, such as those in protein kinase cascade, is controlled by the available intracellular free energy from the adenosine triphosphate (ATP) hydrolysis in vivo: DeltaG=k(B)Tln(([ATP]/K(eq)[ADP]), where K(eq) is the equilibrium constant. The model reveals the correlation between the performance of the switch and the level of DeltaG. The result demonstrates the importance of nonequilibrium thermodynamics in analyzing biological information processing, provides its energetic cost, establishes an interplay between signal transduction and energy metabolism in cells, and suggests a biological function for phosphoenergetics in the ubiquitous phosphorylation signaling.     

Cell proliferation in normal and malignant transformed cells: thermodynamic model of signal transduction

We present a new thermodynamic model for the mechanism of activation and regulation of cell proliferation in the G1 stage. In this model, the interactions between growth factors and transmembrane proteins play a crucial role in cell growth control for a normal tissue-culture system. We calculate a phase diagram of normal and malignant transformed states of a cell signal transduction system. We propose thermodynamic reasons why cancer cells can continually grow without activation by the growth factors.     

Analysis of cell surface molecular distributions and cellular signaling by flow cytometry

Flow cytometry is a fast analysis and separation method for large cell populations, based on collection and processing of optical signals gained on a cell-by-cell basis. These optical signals are scattered light and fluorescence. Owing to its unique potential ofStatistical data analysis and sensitive monitoring of (micro)heterogeneities in large cell populations, flow cytometry—in combination with microscopic imaging techniques—is a powerful tool to study molecular details of cellular signal transduction processes as well. The method also has a widespread clinical application, mostly in analysis of lymphocyte subpopulations for diagnostic (or research) purposes in diseases related to the immune system. A special application of flow cytometry is the mapping of molecular interactions (proximity relationships between membrane proteins) at the cell surface, on a cell-by-cell basis. We developed two approaches to study such questions; both are based ondistance-dependent quenching of excited state fluorophores (donors) by fluorescent or dark (nitroxide radical) acceptors via Förstertype dipole-dipole resonance energy transfer (FRET) and long-range electron transfer (LRET) mechanisms, respectively. A critical evaluation of these methods using donor- or acceptor-conjugated monoclonal antibodies (or their Fab fragments) to select the appropriate cell surface receptor or antigen will be presented in comparison with other approaches for similar purposes. The applicability of FRET and LRET for two-dimensional antigen mapping as well as for detection of conformational changes in extracellular domains of membrane-bound proteins is discussed and illustrated by examples of several lymphoma cell lines. Another special application area of flow cytometry is the analysis of different aspects of cellular signal transduction, e.g., changes of intracellular ion (Ca²⁺, H⁺, Na⁺) concentrations, regulation of ion channel activities, or more complex physiological responses of cell to external stimuli via correlated fluorescence and scatter signal analysis, on a cell-by-cell basis. This way different signaling events such as changes in membrane permeability, membrane potential, cell size and shape, ion distribution, cell density, chromatin structure, etc., can be easily and quickly monitored over large cell populations with the advantage of revealing microheterogeneities in the cellular responses. Flow cytometry also offers the possibility to follow the kinetics of slow (minute- and hour-scale) biological processes in cell populations. These applications are illustrated by the example of complex flow cytometric analysis of signaling in extracellular ATP-triggered apoptosis (programmed cell death) of murine thymic lymphocytes.     

DNA dynamics and organization in sub-micron scale: Bacterial chromosomes and plasmids in vivo and in vitro

Decades of research have witnessed that the mechanical properties of DNA and the associated protein interactions are crucial to the structural organizations and dynamic behaviors of bacterial DNA molecules. Packaged in a restricted cellular space and respective compositions, the entropic force due to confinement is a major factor driving the basal structural formation of the bacterial chromosome and the distribution of plasmids. The dynamic responses of the DNA-protein interactions also modulate the DNA organization when the cellular functions proceed. In this review, we survey the in-vivo and in-vitro evidence and the current understandings of the cellular phenomena of DNA molecules in the sub-micron scale of the bacterial cellular environment. The physical nature and constraints of DNA in cells and the related biological factors, especially the effects induced by gene expressions, are also discussed. We anticipate that by the methodological advances in vivo and in vitro as well as their combinations, the fundamental insights of the coupling from the physical environment and DNA-protein interactions on bacterial DNA dynamics and organization may be revealed.     

A cell-centered approach to developmental biology

Explaining embryonic development of multicellular organisms requires insight into complex interactions between genetic regulation and physical, generic mechanisms at multiple scales. As more physicists move into developmental biology, we need to be aware of the “cultural” differences between the two fields, whose concepts of “explanations” and “models” traditionally differ: biologists aiming to identify genetic pathways and expression patterns, physicists tending to look for generic underlying principles.Here we discuss how we can combine such biological and physical approaches into a cell-centered approach to developmental biology. Genetic information can only indirectly influence the morphology and physiology of multicellular organisms. DNA translates into proteins and regulatory RNA sequences, which steer the biophysical properties of cells, their response to signals from neighboring cells, and the production and properties of extracellular matrix (ECM). We argue that in many aspects of biological development, cells’ inner workings are irrelevant: what matter are the cell's biophysical properties, the signals it emits and its responses to extracellular signals. Thus we can separate questions about genetic regulation from questions about development. First, we ask what effects a gene network has on cell phenomenology, and how it operates. We then ask through which mechanisms such single-cell phenomenology directs multicellular morphogenesis and physiology. This approach treats the cell as the fundamental module of development.We discuss how this cell-centered approach—which requires significant input from computational biophysics—can assist and supplement experimental research in developmental biology. We review cell-centered approaches, focusing in particular on the Cellular Potts Model (CPM), and present the Tissue Simulation Toolkit which implements the CPM.     

Multiple Ligand and Cell-Dependent States of Lateral Mobility of Plasmalemmal G Protein-Coupled Cholecystokinin Receptors

Lateral movement of receptor molecules in the plane of the plasmalemma has important implications for signal transduction and receptor regulation, yet mechanisms affecting such movement are not well understood. We have studied the lateral mobility of the G protein-coupled cholecystokinin (CCK) receptor expressed in the natural milieu of the rat pancreatic acinar cell and in a model cell system, the CHO-CCKR cell, after occupation with fluorescent agonist and antagonist. Lateral diffusion characteristics were distinct in each type of cell and for receptors occupied by each type of ligand, fluorescent agonist, rhodamine-Gly-[(Nle^28,31)CCK-26-33], and fluorescent antagonist, rhodamine-Gly-[(D-Trp³⁰,Nle^28,31)CCK-26-32]-phenethyl ester. Multiple states of mobility were detected for CCK receptors. The slowest population of mobile receptors on the CHO-CCKR cells moved at similar rates when occupied by both antagonist and agonist, while the faster-moving populations moved at a faster rate when occupied with antagonist than with agonist. The fastest component of mobile receptors may reflect unconstrained interactions of the antagonist-occupied receptors with signaling or anchoring structures, while the slowest component may represent the fraction of ligand-occupied receptors that ultimately undergo internalization. The intermediate mobility states may reflect receptor interactions with signal transduction and regulatory machinery. While only a single population of mobile receptors was demonstrable on the acinar cells, increased ligand concentrations (agonist and antagonist) resulted in increased percentages of mobile receptors, suggesting a stoichiometric limitation of immobilizing molecular constraints. Inhibition of protein kinase C had no significant effect on the lateral mobility of agonist-occupied CCK receptors.     

Radiation response of cell organelles

The cellular responses to various form of radiation, including ionizing- and UV-irradiation or exposure to electromagnetic fields is manifested as irreversible and reversible structural and functional changes to cells and cell organelles. Moreover, beside the morphological signs related to cell death, there are several reversible alterations in the structure of different cell organelles. The radiation-induced changes in the supramolecular organization of the membranes, including plasma membrane, and different cell organelle membranes, play a significant role in the development of acute radiation injury. These signs of radiation-induced reversible perturbation biological membranes reflect changes in the organization and/or composition of the glycocalix, modified activity and/or distribution of different membrane domains, including enzymes and binding sites. The observed changes of the cell surface micromorphology and the alteration of intercellular connections are closely related to the reorganization of the cytoskeletal elements in the irradiated cells. The mitochondria, endoplasmic reticulum, Golgi-complex, the lysosomal system have long been considered to be direct intracellular targets of irradiation. The listed morphological alterations of nuclear chromatin (e.g. changes of fine structure, altered number of nucleolar organizing regions and micronuclei, development of chromosome aberrations) may originate from the radiation-induced damage to the supramolecular organization of DNA and/or nucleus specific proteins. These endpoints of radiation effects resulted as direct consequence(s) of absorbed radiation energy, and indirectly altered intra-, intercellular communication or modified signal transduction. Some complementary data suggest that all these effects are not strictly specific to radiation and may be best considered as general stress responses, similar to those observed after application of various injurious agents and treatments to cells. Moreover, they may be equally responsible for direct degradation of supramolecular component of cells, altered signal transduction, or changes in the amount or ratio of any extracellular mediators upon irradiation. Nevertheless, qualitative and/or quantitative evaluation of any changes of chromosomes by different techniques (morphological analysis of metaphase chromosomes, fluorescent in situ hybridization, development of micronuclei etc.) are useful biological indicators as well as "biological dosimeters" of radiation injury. It is suggested, that some modern methods such as immunohistochemical detection of different proteins, specific markers of cell organelles and cytoskeleton, inspection of distribution of cell surface charged sites and different membrane domains and application of tracer substances may all be included into protocols for evaluation of cell alterations induced by different types and intensities of radiation.