1. I will arise and go now, for always night and day

2. I hear lake water lapping with low sounds by the shore;

3. While I stand on the roadway, or on the pavements gray,

4. I hear it in the deep heart’s core.

5. (William Butler Yeats, The Lake of Innisfree, 1888)

GENERAL IDEA

Here we venture beneath the surface of the cell membrane to explore some of the key biological processes that occur in the core of cells, which have been investigated using single-molecule biophysics techniques either in living samples or in physiologically relevant settings in the test tube.

Introduction

As we saw in the previous chapter, the cell membrane, with its various associated integrated protein complexes, is a key structure being the first point of contact for the cell with the outside world. However, the meat of the cellular machinery for metabolizing nutrients, manufacturing new molecular material, responding to signals detected at the cell membrane surface and for storing its genetic code are all located in the innards of the cell, either occurring in the cytoplasm often associated with a variety of cellular sub-structures or, in the case of eukaryotic cells, in specialized membrane-bound organelles. Previously, we discussed some details of two such organelles in the context of membrane-localized processes, the chloroplasts that perform photosynthesis in plant cells and mitochondria that generate the universal cellular fuel of ATP. Here, we will also extend the discussion to processes occurring in the cell nucleus, how the genetic code is packaged and ultimately replicated, and the means by which this code is converted into molecules of protein. But we will begin the chapter outside the nucleus and discuss the biophysical properties of the cytoplasm, and the mechanisms by which molecular cargo is controllably trafficked and sorted inside the cell.

GENERAL IDEA

Here we discuss the miscellaneous experimental techniques that allow us to monitor single biological molecules using physical approaches which do not rely primarily on visible light.

Introduction

There now exist several methods which permit measurement of the presence of single biological molecules using physical principles which do not rely primarily on the detection of visible light. These include a variety of scanning probe microscopy techniques, including atomic force microscopy, which are discussed in detail in the first section of this chapter. In addition, significant advances in our understanding of single-molecule biology have come from methods using electron microscopy, which is one of the pioneering techniques used for obtaining structural information on fixed single-molecule samples. Recent advances in the measurement of small ion currents through both solid-state and native physiological nanometre length scale pores have furthered our knowledge of many areas of single-molecule bioscience. Furthermore, Raman spectroscopy has now advanced to a level of sensitivity such that measurements of single biological molecules are feasible. And finally, there are several microscopy methods which allow us to deduce the position of single molecules using primarily infrared optical tweezers.

GENERAL IDEA

Here we take stock of the remarkable developments and innovations in biophysics that have allowed us to address very challenging and fundamental questions about the key cellular processes, and speculate where this might lead in the near future.

Introduction

The emergence of single-molecule cellular biophysics represents a coming-of-age of single-molecule bioscience. The first generation of single-molecule experiments resulted in some exceptionally pioneering developments in terms of the physics of techniques and novel analytical methods and in terms of significantly increasing our understanding of the functioning of isolated biological molecules. But now, as we have seen from myriad investigations discussed in this book, the next generation of single-molecule bioscience has opened up outstanding opportunities to study biological processes under physiologically highly appropriate conditions – in other words to gain enormous insight into how single molecules really function in the context of their native environment of the living cell. In this final chapter we survey the developments that have led us to this point, and ask the question ‘what next?’ As we will see, there is great potential to apply these novel technologies in areas that may have a large future impact on society, namely those of bionanotechnology and synthetic biology, fuel production for commerical use and single-molecule biomedicine.

GENERAL IDEA

Here we outline some of the key concepts and terminology in cell and molecular biology to orientate readers from a more physical science background.

Introduction

The classical biological view is that living organims are typically structured in a hierarchical manner in terms of physiological function relating to length scale. For example, a complex multi-cellular organism is composed of smaller units called organs which appear to be dedicated primarily to a subset of biological processes, and these may be further deconstructed into different tissues, and these tissues may be further sub-divided into smaller structural features consisting ultimately of individual cells, or some structural matrix secreted by cells. Single cells, whether part of a multi-cellular organism as in the human body or simply the organism itself as for unicellular life forms such as bacteria, can in turn be broken up conceptually into smaller subunits. In essence these are structural sub-cellular features which appear to work together to perform a narrow subset of biological functions, for example cell organelles such as the nucleus in certain cell types. Ultimately, smaller sub-cellular feautures can be perceived as collections of single biological molecules.

GENERAL IDEA

In this chapter we encounter the biophysical methods which can be used to measure forces exerted by single biological molecules, and also techniques which can allow us to manipulate single molecules controllably.

Introduction

There now exist several methods which permit highly controlled measurement and manipulation of the forces experienced by single biological molecules. These varied tools all come under the banner of force transduction devices, since they convert mechanical molecular forces into some form of amplified, measurable signal. They share other common features, for example, in general, the single molecules are not manipulated directly but are in effect physically conjugated, usually via one or more chemical links, to some form of adapter which is the the real force transduction element in the system. The principal forces which are used to manipulate the relevant adapter include optical, magnetic, electrical and mechanical forces, and in general all these forces are implemented in an environment of complex feedback electronics and stable, noise-minimizing microscope stages, for the purposes of both measurement and manipulation.

GENERAL IDEA

In this chapter we discuss the techniques which are available to the experimental scientist who wishes to visualize or detect single biological molecules primarily using visible light, both in the test tube and in the living cell.

Introduction

How can we ‘see’ something like a single biological molecule, which is of the order of a thousand million times smaller than a typical object in the macroscopic world that we visualize with our naked eyes? The region of the human eye responsible for detecting light, the retina, consists of two types of cells called rods and cones (rods differ by being 100 times more sensitive than cones but they respond more slowly, have less spatial resolution and do not discriminate colour), both of which can convert detected photons of light into electrical signals, conveyed via ion channels and nerve fibres into the brain. The resolving power of the human eye, the visual acuity, is a measure of the smallest angular separation that the eye can resolve, which for humans has a theoretical limit equivalent to ~0.01°, about 20 milliradians, determined by the limit of optical diffraction set by the wavelength of the incident light and the diameter of the aperture in front of the ‘imaging device’ (here set by the pupil of the eye), as shown in Figure 3.1A.

GENERAL IDEA

Here we discuss the conceptual foundations of single-molecule biophysics in the context of cellular biology. We provide an overview of existing ensemble average techniques used to study biological processes and consider the importance of single-molecule biology experiments.

Introduction

Some of the most talented physicists in modern science history have been led ultimately to address challenging questions of biology. This is exemplified in Erwin Schrödinger’s essay ‘What is Life?’ (see Schrödinger, 1944). It starts with the question ‘How can the events in space and time which take place within the spatial boundary of a living organism be accounted for by physics and chemistry?’ In other words, can we address the big questions of the life sciences from the standpoint of the physical sciences. The ~60 years following the publication of this seminal work has seen a vast increase in our understanding of biology at the molecular scale, and the physical sciences have played a key role in resolving many central problems. The biological problems have not been made easier by the absence of a compelling and consistent definition of ‘life’ – writing from the context for a meaning of ‘artificial life’, the American journalist Steven Levy noted 48 examples of definitions of life from eminent scientists, no two of which were the same (Levy, 1993). From a physical perspective, life is a means of trapping free energy (ultimately from the sun or, more rarely, geological thermal vents) into units of increased local order, which in effect locally decrease entropy, while the units maintain their status in situations which are generally far from thermal equilibrium.

GENERAL IDEA

Here we explore some of the pioneering single-molecule experiments that have increased our understanding of the processes that essentially involve foreign molecules external to cells either binding to the cell surface or being internalized by cells. Such molecules interact directly with the semipermeable membrane of the cell, making it an exceptionally lively and dynamic environment.

Introduction

Highly efficient mechanisms exist that allow foreign molecules to bind to cell surfaces, ultimately evoking some form of signal response, and allow a variety of external molecules over a broad range of size, chemistry and charge to enter cells. At one level these mechanisms include processes involving receptor molecules embedded in the outer membrane of cells that bind to ligand molecules. Many of the stages of this detection and signal transduction process have canonical features, sometimes involving adapter molecules binding to the original ligand, as well as specific binding events and changes in molecular conformation which can often be transmitted over relatively long length scales of several nanometres spanning one or sometimes more lipid bilayer membranes and sometimes involving cooperative effects from other receptor molecules. Non-native particles which are internalized by cells range in size from single molecules to much larger heterogenous macromolecular complexes such as viruses. In this chapter we will encounter first-hand examples of how these processes have been investigated using single-molecule biophysics. These investigations reveal highly complex behaviours, indicating that the world outside the cell is just as important as the world on the inside.

GENERAL IDEA

Here we introduce the seminal biophysics investigations which have transformed our understanding of biology at the single-molecule level, and lay the foundations for describing single-biomolecule experimentation on functioning live cells

Introduction

An instructive exercise for those learning about single-molecule biophysics is to compile a list of one’s own top ten research papers of all time. The choice is obviously subject to a great deal of personal bias, and is a dynamic structure which may change with time, and some of the early, seminal papers on the list might later be superseded by subsequent incarnations with more of the original unresolved questions resolved (and maybe even with the ‘right’ answers, as opposed to what were perhaps novel but slightly incorrect ‘best guesses’ at the time!). Even so, as a process for understanding how, and why, single-molecule biophysics has evolved the way it has, and how it is likely to progress into the arena of far greater physiological relevance in the near future, the reader might find the exercise suprisingly fulfilling. In this chapter we discuss some of the strong candidates for this list of seminal papers, and lay the foundations for the remaining chapters in this book which describe real single-biomolecule experiments performed either on living cells or in an environment which has substantial physiological relevance.

GENERAL IDEA

In this chapter we encounter some key examples of single molecules and molecular complexes which are primarily integrated into the cell membrane performing a range of essential biological functions, and of the surrounding molecular architecture of the phospholipids, that have been studied extensively using exemplary single-molecule biophysics techniques

Introduction

Roughly 30% of all proteins are integrated into the membranes of cells, a significant proportion which is indicative of the importance of molecular processes which occur at the surfaces of cells. The cell membrane is an enormously important structure. It provides a physical support for seeding a vast array of complex surface chemistry reactions, as well as acting as the site for molecular detectors, pumps, channels and motors, not to mention its obvious function as a physical boundary to the cell. In the previous chapter, we discussed some of the important biological systems that deal with molecules and molecular complexes which spend significant periods outside the cell membrane boundary, and how single-molecule methods have dramatically improved our understanding of these processes. In this chapter, we will discuss several of the biological systems which are primarily associated directly with the cell membrane itself, and how single-molecule techniques have probed many of these in physiologically relevant settings, including both protein complexes integrated into the membrane and the makeup of the phosopholipid bilayer. These include molecular complexes which transport molecules across cell membranes, such as ion channels and protein transport nanopores, as well as some remarkable molecular machines which are involved in cell motility and cellular fuel manufacture.

Life, from the bottom up

Richard Feynman, celebrated physicist, science communicator and bongo-drum enthusiast, gave a lecture in Caltech, USA, a few days after Christmas 1959, that would come to be seen by future nanotechnologists as essentially prophetic. His talk was entitled ‘There’s plenty of room at the bottom’, and was concerned primarily with discussing the feasibility of a future ability to store information and to control and manipulate machines on a length scale which was tens of thousands of times smaller than that of the macroscopic world of things like typical books and electric motors of that day. It was essentially a clarion call to scientists and engineers to develop a new field, which would later be termed nanotechnology (see Taniguchi, 1974). But in one aside, Feynman alluded to the very small scale of biological systems, and how cells used these to do ‘all kinds of marvelous things’, which in its own small way has been wisely prescient for the subsequent seismic shifts in our understanding of how biological systems really work. We now know that the fundamental minimal functional unit which can adequately describe the properties of these systems is the single biological molecule. That is not to say that the constituent atoms at smaller length scales do not matter, nor the sub-atomic particles that make up the individual atoms, nor smaller still the quarks that make up the sub-atomic particles. Rather that, in general, we do not need to refer to a length scale smaller than the single molecule to understand biological processes.