People are judged by their actions, and these actions are coordinated by nerve cells and carried out by muscle cells. So an understanding of nerve and muscle is fundamental to our knowledge of how the human body functions.

This book provides an introductory account of how nerve and muscle cells work, suitable for students taking university courses in physiology, cell biology or preclinical medicine. It aims to give a straightforward exposition of the fundamentals of the subject, including particularly some of the experimental evidence upon which our conclusions are based. This edition includes new material reflecting the exciting discoveries that continue to be made in the field. So there is up-to-date detail on topics such as the ion channels involved in electrical activity and the molecular mechanisms of muscular contraction.

Structure of the cell membrane

All living cells are surrounded by a plasma membrane composed of lipids and proteins, whose main function is to control the passage of substances into and out of the cell. In general, the role of the lipids is to furnish a continuous matrix that is impermeable even to the smallest ions, in which proteins are embedded to provide selective pathways for the transport of ions and organic molecules both down and against the prevailing gradients of chemical activity. The ease with which a molecule can cross a cell membrane depends to some extent on its size, but more importantly on its charge and lipid solubility. Hence the lipid matrix can exclude completely all large water-soluble molecules and also small charged molecules and ions, but is permeable to water and small uncharged molecules like urea. The nature of the transport pathways is dependent on the specific function of the cell under consideration. In the case of nerve and muscle, the pathways that are functionally important in connection with the conduction mechanism are (1) the voltage-sensitive sodium and potassium channels peculiar to electrically excitable membranes, (2) the ligandgated channels at synapses that transfer excitation onwards from the nerve terminal, and (3) the ubiquitous sodium pump which is responsible in all types of cell for the extrusion of sodium ions from the interior.

Electrophysiological recording methods

Although the nervous impulse is accompanied by effects that can under specially favourable conditions be detected with radioactive tracers, or by optical and thermal techniques, electrical recording methods normally provide much the most sensitive and convenient approach. A brief account is therefore necessary of some of the technical problems that arise in making good measurements both of steady electrical potentials and rapidly changing ones.

In order to record the potential difference between two points, electrodes connected to a suitable amplifier and recording system must be placed at each of them. If the investigation is only concerned with action potentials, fine platinum or tungsten wires can serve as electrodes, but any bare metal surface has the disadvantage of becoming polarized by the passage of electric current into or out of the solution with which it is in contact. When, therefore, the magnitude of the steady potential at the electrode tip is to be measured, non-polarizable or reversible electrodes must be used, for which the unavoidable contact potential between the metal and the solution is both small and constant. The simplest type of reversible electrode is provided by coating a silver wire electrolytically with silver chloride, but for the most accurate measurements calomel (mercury/mercuric chloride) half-cells are best employed.

When the potential inside a cell is to be recorded, the electrode has to be very well insulated except at its tip, and so fine that it can penetrate the cell membrane with a minimum of damage and without giving rise to electrical leaks.

I was an undergraduate when I first saw a film of Dictyostelium aggregating, and decided that it was something I had to study. It would be great to say, 30-odd years later, that I had a clear vision of great issues in biology that could be addressed with such an organism, but actually I just thought it was neat. I suppose it had the correct combination of interest and mystery. Like hundreds, if not thousands of people before and since, I was fascinated by its regularity, its rhythms, and for someone schooled in bacteriophage, it seemed simple. If you are blessed with youth and think Dictyostelium development is simple, I hope this book will help you to realize your error without discouraging you.

The thousands who have been taken by this strange little organism include better minds than ours. John Tyler Bonner tells of being a young assistant professor at Princeton who one day received a call to show his slime mold films to Albert Einstein. John showed up with his 16 mm projector and he thinks Einstein and his colleagues were suitably impressed, but he does not actually know. John speaks wonderful French, but Einstein conversed in German.

When I first started to work on this organism, with Maurice and Raquel Sussman at Brandeis University, it was thought remarkable that a eukaryotic organism induced genes and enzymes, just like E. coli induced the gene for β-galactosidase.

During the construction of a fruiting body, the social amoebae show impressive cooperativity. One of the problems that we face is to explain how such cooperativity and the altruism of the stalk cells evolved. There are numerous questions to be asked: what advantage does multicellularity provide? What is unique about the evolution of an organism that increases its size by aggregation rather than feeding and growth? How can parasitism be avoided in the developmental cycle? Can we learn anything that can be exploited as we study the development of these or other aggregative organisms? What is the relationship of the Dictyostelids to other multicellular developing organisms? Before asking such questions, certain fundamentals are required, beginning with the fact that the social amoebae are capable of three developmental cycles – microcyst, macrocyst, and fruiting body formation. Each developmental option stems from a trophic, or feeding, amoeboid cell population. Formation of the microcyst, the macrocyst, and the fruiting body are each highly programmed events. Only the last one receives great attention, but knowing about the first two is essential, because the microcyst and macrocyst pathways may be evolutionary precursors of the fruiting body.

A digression into ecology

The various species of social amoebae were first found in dung and later were recognized to be ubiquitous in forest soils. Soil as an environment has been thoroughly studied, and one of the results of this body of work shows that bacteria, the prey of Dictyostelium, are dispersed in patches in the soil.

Free-living Dictyostelium amoebae must be able to find their way toward prey or, in the face of starvation, toward each other. Dictyostelium amoebae share a chemotactic capacity with leukocytes and many other motile cells, and employ many of the same mechanisms during the detection of the chemotactic molecule, the activation of signal transduction pathways, and the mobilization of the cytoskeleton (Devreotes and Zigmond, 1988; Parent et al., 1998; Parent and Devreotes, 1999). Chemotactic molecules bind to cell-surface receptors and stimulate G protein-mediated signal transduction pathways in amoebae and in mammalian cells. Agonists are degraded to steepen gradients and to overcome the effects of adaptation. Despite evolutionary distance, the cytoskeletons of leukocytes and Dictyostelium employ similar cytoskeletal rearrangements to move in the right direction. The advantage of Dictyostelium in the study of chemotaxis, motility and aggregation is that the gene products involved in each event can be eliminated by mutation, and the contribution of each element can be studied. The biochemical advantages that stem from synchrony of development and quantities of material have also been invaluable. This is not to say that we understand completely how a cell perceives that a gradient exists or how it moves toward higher concentrations of cAMP, but we are beginning to understand how this complex process works.

Starvation is a crisis that the cells confront with immediate action. Polysomes are degraded; transcripts that are required for growth disappear; and the cell cycle halts. All of development is accomplished without the addition of new metabolic reserves, and so there is selective advantage in using the reserves that have been accumulated during the trophic phase as parsimoniously as possible, without wasting energy on constituents necessary only for growth. Slowly, after the onset of starvation, new protein synthesis begins – only after a few hours do many of the transcripts and proteins that will mediate aggregation appear. This earliest period of development leads to the induction of genes necessary for aggregation, and it is constructive to think of it as a separate and essential series of events. Chemotaxis and aggregation are not the earliest developmental events.

Cells can detect imminent starvation

In the laboratory, when development is induced by abruptly washing away nutrients, the onset of starvation is sudden. In the soil, depletion is more gradual and the cells have mechanisms to sense when hard times are approaching. There are two density-sensing mechanisms that function during the early stages of development. One mechanism is mediated by a molecule called prestarvation factor (PSF) and controls induction of certain very early genes (Rathi and Clarke, 1992). The other mechanism, mediated by a molecule called conditioned medium factor (CMF) (Gomer et al., 1991), helps the cells to assess density at a slightly later period – during aggregation (see below).

The first description of Dictyostelium, by the mycologist Oskar Brefeld, is 130 years old (Brefeld, 1869). Many of the features of these organisms that modern workers assume to be obvious – the phagocytic nature of the amoebae, the separation of growth and developmental cycles, the absence of cell fusion in the aggregate – were not apparent to early workers. Culture systems had not been developed and the ease of manipulation that now makes these organisms so attractive would not be used until the 1930s by Kenneth Raper (Raper, 1937).

Brefeld (1869) first observed Dictyostelium mucoroides while examining the fungal flora in horse dung, and then grew purer cultures in rabbit dung. Even with this difficult culture method, Brefeld realized that the amoebae were the trophic (feeding) form, and that they aggregated to give rise to fructifications. He named the species Dictyostelium (Dicty means net-like and stelium means tower) because the aggregation territories he observed looked like nets (Fig. 2.1) and the fruiting bodies like towers (Fig. 2.2). He added the qualifier mucoroides because the new organism resembled the fungus Mucor. This was a misnomer because closer examination by Brefeld (1884) established that his new species did not have the same sporangial walls as the fungus, but instead the spores were suspended in a drop of liquid. The germination of the spores led not to hyphae and a mycelium but to distinctly amoeboid cells, which the microscopes of the time were quite capable of resolving.

The tight aggregate elongates under control of the tip

The tight aggregate is at first a hemisphere, but soon elaborates a tip that becomes populated with prestalk cells. A large body of evidence indicates that prestalk cells move apically in the tight aggregate to establish the pattern, sorting out from the prespore cells as they go. The first sign of a tip is a signaling center in a mound, in which cells move radially around a center that does not yet form a morphologically distinct structure (Rietdorf et al., 1996; Siegert and Weijer, 1995; Sucgang et al., 1997). Often there will be more than one such center, but eventually, one predominates or, in large mounds, two tips form and the cell mass is subdivided. There is lingering confusion about what constitutes the tip because people use the word differently. In the text that follows, the tip refers to the most anterior part of the prestalk zone, which is usually suspended above the substratum. The word tip is also used to denote a signaling entity, and the morphological tip certainly fulfills such a role. We do not know whether the tip contains a specific group of cells, smaller than the physical structure, from which organizing signals emanate. The cells in the front of the slug are in constant motion, and it is hard to imagine how a few key cells at the tip could remain as a group.

The fusion of the prespore vesicles (PSVs) with the plasma membrane creates an immature spore. A number of further events must occur before the cell can be assured of protection from desiccation, osmotic shock, or the digestive tracts of soil creatures. The spore proteins, galuran, and cellulose must be organized and cross-linked in such a way that they protect the delicate amoebae within. The formation of the coat, about which we know quite a lot, still presents a number of problems (Lydan and Cotter, 1994; West and Erdos, 1990; West et al., 1996). We know that a number of the prespore proteins and the polysaccharide galuran are synthesized and modified in the early and intermediate stages of the secretory pathway, and then stored in a regulated secretory compartment, the PSV (Srinivasan et al., 2000). We do not know how the proteins of the spore coat interact with cellulose. There are alternative views regarding the extent of pre-assembly of proteins into specific complexes in the PSV. There is no information on how the spore coat is anchored to the plasma membrane. We are beginning to understand the mechanism that the spores use to maintain their dormancy, but new components in these pathways remain to be discovered. We do not know a great deal about how the spores detect the appearance of nutrients and launch the developmental program that leads to germination and the re-establishment of amoeboid life.

If Dictyostelium amoebae are not the Ferraris of moving cells, they are at least a respectable entrant in the cell motility Grand Prix. Amoebae can move as fast as 10–15 μm/min (Varnum and Soll, 1984). They do not do this in a random walk, but move directionally, up gradients of cAMP or folate. D. discoideum responds to gradients that vary by as little as 2% from the front to the back of a cell, as we will describe in Chapter 8. This astonishing chemical sensitivity raises a number of questions. How are signal transduction pathways connected to the cytoskeleton such that stimulation of chemotactic receptors leads to movement up a chemical gradient? How are the components of the cytoskeleton organized to promote sudden movement of cells? How does the mobilization of the cytoskeleton differ in the various protrusions, such as pseudopodia and fllopodia? How are responses terminated? In this chapter we will review how the actin-based cytoskeleton is mobilized during cell movement, while in Chapter 8 we will consider how it reorganizes to drive the cells up a gradient of cAMP, toward centers of aggregation.

Dictyostelium is one of the few organisms with impressive motility and tractable genetics. The cells move toward folate during growth and toward cAMP during development. The amoebae are useful for optical observation, so that with a few tricks, the movements of macromolecules within the cells can be observed by a variety of microscopic methods.

Deciding when migration has gone on long enough

One of the decisions that a slug must make is when to form a fruiting body. It is possible to trick slugs into migrating toward light until they expire, having used all of their reserves. The slug offers the organism escape from noxious environments, dispersal, and perhaps protection from nematodes, but ultimately the major protection is the creation of a resistant spore, placed so that it can be dispersed. This transition is accomplished late in development by a series of elaborate cell maneuvers. The slug begins with a set of partially differentiated prespore cells, all contiguous in the rear of the structure, and at the end has put these cells, fully encapsulated, into a loosely held sphere at the top of a stalk.

Ammonia, as we learned in Chapter 10, causes the slugs to refrain from culmination. They migrate away from it, so it may have a negative chemotactic effect. Sussman, White, and Schindler determined that 108 cells contain about 5 mg of protein, and that during the course of development about 2 mg of this protein is degraded, eventually releasing a substantial amount of ammonia (Schindler and Sussman, 1977; White and Sussman, 1961). Similar observations were made by Gregg et al. (1954). Wilson and Rutherford (1978) measured the amounts of ammonia in tissue slices and found that it accumulated at the end of development.

Books

A number of books have been produced previously on Dictyostelium and its relatives. Some of these remain important sources of information on techniques and earlier work, and should be available in university libraries.

John Bonner has written a classic volume on Dictyostelium (1967), and this is an important source for early experiments and details that might get overlooked.

Kenneth Raper has also written well on Dictyostelium, and his 1984 book contains much material that is not covered elsewhere.

L. S. Olive has written a book on the mycetozoans, for those interested in the relatives of Dictyostelium, or at least in species with a similar life style.

William Loomis has been a longtime contributor and has written and edited several books that are listed below.

As a technical help, a Methods in Cell Biology book, edited by J. Spudich has much value. This provides recipes for media and detailed methods for growing and developing Dictyostelium.

An excellent book by D. Wessels and D. R. Soil describes microscopy and imaging methods for the study cell movement. Several chapters concern Dictyostelium.

The most recent book dedicated to Dictyostelium is the result of a meeting held in Sendai in Japan, and is an excellent source of recent reviews. It can be ordered through the Kyoto Dictyostelium Group Website, which is listed below. A relatively unknown monograph by Hagiwara deserves more attention.

As they collect in the mound, cells begin the transcription of cell type-specific genes, develop adhesion mechanisms, and secrete an acellular covering called the sheath. The loose mound goes on to form a compact hemispherical aggregate and prepares the anterior-posterior pattern of the future slug. The mound has been called the crucible of cellular differentiation and morphogenesis. In earlier days of Dictyostelium research, it was difficult to detect the complex gene induction events and cellular movements that occur within the mound, but as markers of cell-type differentiation have improved, the nature of the differentiation events has begun to unfold. Great efforts in microscopy, notably by the laboratories of Weijer, Siegert, McNally, and Soll, have detected elaborate coordinated movements of cells, not just during aggregation, but within the confines of the aggregate. In this chapter we will examine some of the early events that occur after the amoebae aggregate – how they secrete a covering and how they induce new genes in the loose aggregate. The aggregate is where most prestalk- and prespore-specific genes are first transcribed. This subject encompasses a large and complex literature, and an attempt will be made to put this in context. Following an examination of how the pattern of prestalk and prespore cells forms, the cellular adhesion mechanisms that Dictyostelium employs to hold cells together will be analyzed.