Fluorescence microscopy of single autofluorescent proteins for cellular biology

In this paper we review the applicability of autofluorescent proteins for single-molecule imaging in biology. The photophysical characteristics of several mutants of the Green Fluorescent Protein (GFP) and those of DsRed are compared and critically discussed for their use in cellular biology. The alternative use of two-photon excitation at the single-molecule level or Fluorescence Correlation Spectroscopy is envisaged for the study of individual autofluorescent proteins. Single-molecule experiments performed in live cells using eGFP and preferably eYFP fusion proteins are reviewed. Finally, the first use at the single-molecule level of citrine, a more photostable variant of the eYFP is reported when fused to a receptor for neurotransmitter in live cells. To cite this article: L. Cognet et al., C. R. Physique 3 (2002) 645–656.     

DNA based molecular motors

Most of the essential cellular processes such as polymerisation reactions, gene expression and regulation are governed by mechanical processes. Controlled mechanical investigations of these processes are therefore required in order to take our understanding of molecular biology to the next level. Single-molecule manipulation and force spectroscopy have over the last 15 years been developed into extremely powerful techniques. Applying these techniques to the investigation of proteins and DNA molecules has led to a mechanistic understanding of protein function on the level of single molecules. As examples for DNA based molecular machines we will describe single-molecule experiments on RNA polymerases as well as on the packaging of DNA into a viral capsid—a process that is driven by one of the most powerful molecular motors.     

Single-molecule imaging with x-ray free-electron lasers: dream or reality?

X-ray free-electron lasers (XFEL) are revolutionary photon sources, whose ultrashort, brilliant pulses are expected to allow single-molecule diffraction experiments providing structural information on the atomic length scale of nonperiodic objects. This ultimate goal, however, is currently hampered by several challenging questions basically concerning sample damage, Coulomb explosion, and the role of nonlinearity. By employing an original ab?initio approach, we address these issues showing that XFEL-based single-molecule imaging will be only possible with a few-hundred long attosecond pulses, due to significant radiation damage and the formation of preferred multisoliton clusters which reshape the overall electronic density of the molecular system at the femtosecond scale.     

Vortices in Low-Dimensional Magnetic Systems

Vortices are objects that are important to describe several physical phenomena. There are many examples of such objects in nature as in a large variety of physical situations like in fluid dynamics, superconductivity, magnetism, and biology. Historically, the interest in magnetic vortex-like excitations begun in the 1960s. That interest was mainly associated with an unusual phase-transition phenomenon in two-dimensional magnetic systems. More recently, direct experimental evidence for the existence of magnetic vortex states in nano-disks was found. The interest in such model was renewed due to the possibility of the use of magnetic nano-disks as bit elements in nano-scale memory devices. The goal of this study is to review some key points for the understanding of the vortex behavior and the progress that have been done in the study of vortices in low-dimensional magnetic systems.     

A quantum-mechanical treatment of Szilard's engine: Implications for the entropy of information

We present a quantum-mechanical analysis of Szilard's famous single-molecule engine, showing that it is analogous to the double-slit experiment. We further show that the energy derived from the engine's operation is provided by the act of observing the molecule's location. The engine can be operated with no increase in physical entropy, and the second law of thermodynamics does not compel us to relate physical entropy to informational entropy. We conclude that information per seis a subjective, idealized, concept separated from the physical realm. Physical entropy depends on physical objects and physical interactions, and any entropy change owing to observations is entirely a result of the entropy created in the physical apparatus by the process of observation.     

Physics of transport and traffic phenomena in biology: from molecular motors and cells to organisms

Traffic-like collective movements are observed at almost all levels of biological systems. Molecular motor proteins like, for example, kinesin and dynein, which are the vehicles of almost all intra-cellular transport in eukaryotic cells, sometimes encounter traffic jam that manifests as a disease of the organism. Similarly, traffic jam of collagenase MMP-1, which moves on the collagen fibrils of the extracellular matrix of vertebrates, has also been observed in recent experiments. Novel efforts have been made to utilize some uni-cellular organisms as “micro-transporters”. Traffic-like movements of social insects like ants and termites on trails are, perhaps, more familiar in our everyday life. Experimental, theoretical and computational investigations in the last few years have led to a deeper understanding of the generic or common physical principles involved in these phenomena. In this review we critically examine the current status of our understanding, expose the limitations of the existing methods, mention open challenging questions and speculate on the possible future directions of research in this interdisciplinary area where physics meets not only chemistry and biology but also (nano-)technology.     

Fluorescence from Diffusing Single Molecules Illuminates Biomolecular Structure and Dynamics

This review provides an account of single-molecule fluorescence methodologies for freely diffusing molecules applied to a diverse array of biological problems pertaining to biomolecular folding and assembly. We describe the principles of confocal fluorescence microscopy to detect and analyze fluorescence bursts from diffusing single molecules. These methods including single-molecule fluorescence resonance energy transfer, coincidence, correlations and polarization offer a powerful means to uncover hidden information about conformational sub-populations and interconversion dynamics of biomolecular systems in a wide range of timescales. We offer several key examples to illustrate how these methodologies have been extremely useful in teasing out structural and dynamical aspects of many important biomolecular systems.     

Single-molecule detection using continuous wave excitation of two-photon fluorescence

Two-photon fluorescence (TPF) is one of the most important discoveries for biological imaging. Although a cw laser is known to excite TPF, its application in TPF imaging has been very limited due to the perceived low efficiency of excitation. Here we directly excited fluorophores with an IR cw laser used for optical trapping and achieved single-molecule fluorescence sensitivity: discrete stepwise photobleaching of enhanced green fluorescent proteins was observed. The single-molecule fluorescence intensity analysis and on-time distribution strongly indicate that a cw laser can generate TPF detectable at the single-molecule level, and thus opens the door to single-molecule TPF imaging using cw lasers.     

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.     

The Effect of Physical Form of DNA on ExonucleaseIII Activity Revealed by Single-molecule Observations

Single-molecule studies have revealed molecular behaviors usually hidden in the ensemble and time averaging of bulk experiments. Single-molecule measurement that can control physical form of individual DNA molecules is a powerful method to obtain new knowledge about correlation between DNA-tension and enzyme activity. Here we study the effect of physical form of DNA on exonucleaseIII (ExoIII) reaction. ExoIII has a double-stranded DNA specific 3′→5′ exonuclease activity and the digestion is distributive. We observed the ExoIII digestion of individual stretched DNA molecules from the free ends. The sequentially captured photographs demonstrated that the digested DNA molecule linearly shortened with the reaction time. We also carried out the single-molecule observation under random coiled form by pausing the buffer flow. The digestion rates obtained from both single-molecule experiments showed that the digestion rate under the stretched condition was two times higher than the random coiled condition. The correlation between physical form of DNA and digestion rate of ExoIII was clearly demonstrated by single-molecule observations.     

Binary quantum logic and generating semigroups

A refined definition of basic concepts for logic describing physical systems is proposed. Within the suggested formalism of generating semigroups the active logic of questions and passive logic of answers are introduced. The objects for which both logics are isomorphic are called self-adequate. It is shown that the assumption of self-adequacy implies that the object is either quantum or classical. The possibility of application of the theory to non-self-adequate objects is discussed.     

单分子物理与化学的新进展

本文对新兴边缘学科-单分子物理与化学的一些研究进展进行简要综述。在对单分子科学中几类基本实验技术如扫描隧道显微术和光镊技术等作了简要介绍之后,重点评述了单分子实验技术和研究方法在物理、化学、生物和分子电子学等学科领域的应用和影响。基于扫描隧道显微术和电子结构计算,列举了最近几个关于单分子高分辨表征、单分子器件和单分子量子调控等方面的研究实例。最后对单分子物理与化学的发展前景进行了展望。     

Energy transformation in biological molecular motors

Biological molecular motors transform the metabolic free energy into the directed movement. The physical principles governing this transformation are very different from the principles underlying the manmade macroscopic motors. Theoretical analysis shows that the internal thermal diffusion in motor proteins is a key element of the process, and the chemical energy performs no mechanical work directly but instead it is used for rectifying the diffusion. A few specific motor systems are considered to illustrate the general principle. The principle of rectified thermal diffusion has recently received a great support from the single-molecule studies.     

High pressure studies on the misfolding and aggregation of p53 in cancer and of α-synuclein in Parkinson’s disease

ABSTRACT

High pressure research can be used to aid in answering why some polypeptide chains reversibly unfold and others adopt a misfolding conformation culminating in aggregation. This topic is one of the fundamental questions in biology that we seek to understand, and for that, we have been experimentally applying pressure to proteins for the last 20 years. Here, we exemplify how pressure research should be used to identify preamyloidogenic protein intermediates as well as to dissociate supramolecular complexes. We believe that the applications of high pressure to understand the behavior of proteins are diverse and can help biologists answer fundamental questions of biomedical relevance.     

Single-molecule Detection of Reactive Oxygen Species: Application to Photocatalytic Reactions

In this review, we have focused on the oxidation reactions of single dye molecules by reactive oxygen species (ROS). The methodologies for the single-molecule detection of ROS, such as hydroxyl radical (HO^•), singlet oxygen (O₂(a¹Δ_g)), and hydrogen peroxide (H₂O₂), have been introduced together with examples. In particular, a successful application using the single-molecule fluorescence technique for the investigation of the TiO₂ photocatalytic oxidation reactions is demonstrated in detail.     

Physical methods for genetic transformation of fungi and yeast

The production of transgenic fungi is a routine process. Currently, it is possible to insert genes from other fungi, viruses, bacteria and even animals, albeit with low efficiency, into the genomes of a number of fungal species. Genetic transformation requires the penetration of the transgene through the fungal cell wall, a process that can be facilitated by biological or physical methods. Novel methodologies for the efficient introduction of specific genes and stronger promoters are needed to increase production levels. A possible solution to this problem is the recently discovered shock-wave-mediated transformation. The objective of this article is to review the state of the art of the physical methods used for genetic fungi transformation and to describe some of the basic physics and molecular biology behind them.     

Reply to da Rocha and Rodrigues' comments on the orientation congruent algebra and twisted forms in electrodynamics

The recent claim by da Rocha and Rodrigues that the nonassociative orientation congruent algebra (???? algebra) and native Clifford algebra are incompatible with the Clifford bundle approach is false. The new native Clifford bundle approach, in fact, subsumes the ordinary Clifford bundle one. Associativity is an unnecessarily too strong a requirement for physical applications. Consequently, we obtain a new principle of nonassociative irrelevance for physically meaningful formulas. In addition, the adoption of formalisms that respect the native representation of twisted (or odd) objects and physical quantities is required for the advancement of mathematics, physics, and engineering because they allow equations to be written in sign‐invariant form. This perspective simplifies the analysis of, resolves questions about, and ends needless controversies over the signs, orientations, and parities of physical quantities.     

Bio-macromolecular dynamic structures and functions,illustrated with DNA,antibody, and lipoprotein

Bio-macromolecules, such as proteins and nucleic acids, are the basic materials that perform fundamental activities required for life. Their structural heterogeneities and dynamic personalities are vital to understand the underlying functional mechanisms of bio-macromolecules. With the rapid development of advanced technologies such as single-molecule technologies and cryo-electron microscopy(cryo-EM), an increasing number of their structural details and mechanics properties at molecular level have significantly raised awareness of basic life processes. In this review, firstly the basic principles of single-molecule method and cryo-EM are summarized, to shine a light on the development in these fields. Secondly, recent progress driven by the above two methods are underway to explore the dynamic structures and functions of DNA, antibody,and lipoprotein. Finally, an outlook is provided for the further research on both the dynamic structures and functions of bio-macromolecules, through single-molecule method and cryo-EM combining with molecular dynamics simulations.     

The role of gravitational fields in stellar physics and the evolution of the universe

The assertion that the gravitational field is a material matter with all the attributes of any other matter (energy density, pressure, four-velocity of the elements, and interaction of the elements with one another and with other material objects) is shown to modify the physical notions of the dynamics of bodies, Riemannian space, the internal structure of a star, the evolution of the universe, etc. Instead of the black holes of the geometrized approach to the theory of gravitation, we have objects with a researchable internal structure, which explains, e.g., the observed attenuation of the boundaries of the emission spectra of matter that falls into supermassive objects. The problem of dark matter is explained. The generally accepted understanding of matter (without adding any free parameters to the theory) is shown to allow a scenario of the universe that is permanently pulsating between the states of the maximum and minimum density of matter. The scenario is shown to be in good agreement with the recent observational data.     

Crystallinity and degradation of silk: correlations between analytical signatures and physical condition on ageing

Adequately characterising the physical condition of historic textiles, and understanding the microstructural changes that occur in these materials, is essential when considering appropriate conservation, display and storage strategies. Our work has concentrated on developing non-destructive or micro-destructive methodologies that will permit this for one of the most important historic fibres, silk. We have been able to demonstrate that correlations can be drawn between the physical deterioration of silk samples and certain measurable spectroscopic, chromatographic and chemical signatures. Understanding the way in which these signatures arise then allows the microstructural changes within the crystalline and amorphous content of the fibres to be investigated and more fully interpreted. The techniques developed by our group and by other researchers in the field include polarised FTIR-ATR (Pol-ATR) and near infrared (NIR) spectroscopy, HPLC microsampling analyses and pH measurements. The results of these analyses correlate to measurable mechanical properties and thus suggest that the physical state of historic silk fabrics might be adequately characterised for conservation purposes by such indirect micromethodology. PACS 81.05.Lg; 82.80.Gk; 87.14.Ee