Introduction

Many different terms have been employed to describe different types of endocytosis, and these are listed by Chapman-Andresen (1962) and Jacques (1969), but in general endocytic phenomena fall into two broad categories, phagocytosis and pinocytosis. Phagocytosis describes the ingestion of particulate matter such as bacteria, latex beads and erythrocytes by specialised cells such as macrophages and certain unicellular organisms, whereas the more universal process of pinocytosis describes the engulfment of small droplets of extracellular fluid.

Sequence of events in pinocytosis

All types of pinocytosis show a common sequence of events (see Fig. 1).

1. Internalisation of plasma membrane. This may be triggered by attachment of some substance to the plasma membrane or by some other mechanism.

2. Translocation. Once the pinosome ‘pinches off’ from the plasma membrane, it migrates towards the perinuclear region. During this time many fusion events may occur. Initially these may be pinosome–pinosome fusions but subsequently pinosome–lysosome fusions take place, producing a secondary lysosome compartment which can also participate in the fusion sequence. The exposure of the pinosome contents to lysosomal enzymes results in the catabolism of any degradable material.

3. Lysosomal regression. Small molecules produced as a result of degradation escape through the lysosomal membrane, whereas any large non-biodegradable material remains trapped in the secondary lysosome compartment. Certain types of cell, especially unicellular organisms, have the ability to regurgitate material, but many mammalian cells accumulate material they cannot digest as residual bodies within the cell.

General characteristics

Alveolate coated vesicles of similar appearance have been observed in a broad spectrum of plant groups from high to low, and in many different cell types. However, in only three or four papers have these coated vesicles in plants been examined or considered in any detail; most of the reports of these organelles have consisted merely of brief remarks noting their presence and intracellular location in papers devoted primarily to other subjects. Many investigators have reported that coated vesicles occur predominantly in two regions of the plant cell, namely in the vicinity of dictyosomes and beneath the plasmalemma, and have suggested that they arise from cisternae of the former and fuse with the latter.

With the exception of the vesicles associated with the contractile vacuoles of certain algae, and the spiny coated vesicles of limited distribution in flowering plants, coated vesicles have proved to be remarkably uniform in morphology and size wherever encountered in the plant kingdom. The ordinary coated vesicles of plants are approximately 85–90 nm in diameter, including coat, and possess readily observable unit membrane structure surrounded on their cytoplasmic face-by an alveolate or reticulate layer. In median sections the coat exhibits radiating spokes or columellar projections about 25 nm long (Plate 1a and b). In tangential sections the coat can be seen to consist of polygonally packed ridges and in the most favorable views, of what appear to be pentagonally or hexagonally packed units (Plate 1c).

One of the most striking aspects of oogenesis is the remarkable growth that is undertaken by the oocyte. This is normally taken to be an adaptation for providing the embryo with the substances needed to sustain full development. In fact, with the exception of mammals, oocytes of most species achieve this by accumulating large quantities of reserve material generally referred to as yolk. The latter may either be derived from the maternal blood stream, in which case it is called heterosynthetic, or, alternatively, synthesised by the oocyte itself, and is then termed as autosynthetic (Schechtman, 1955).

Although coated vesicles may form in oocytes of numerous species, irrespective of their reproductive biology, it is quite apparent that micropinocytosis in oocytes plays a major role only when yolk is formed by heterosynthetic processes. In fact, as in other cell types, micropinocytosis in these instances provides a method of engulfing large molecules and sequestering them within an intracellular compartment which remains separated from the rest of the cytoplasm by a limiting membrane. However, substances ingested into the oocyte normally do not undergo intracellular digestion soon after uptake but are stored for a definite period of time prior to their final utilisation.

For the reasons outlined above, it is clear that certain species are better suited than others for studying the processes of micropinocytosis in oocytes (by biochemical as well as ultrastructural methods).

It is a usual assumption that the selective transport of proteins across cell plasma membranes is mediated by receptor molecules located in the membranes. The function of such receptors is to bind those molecules for which they have the correct stereospecificity. The binding process is usually saturable and occurs with a high affinity between receptor and protein. When the receptor–protein complex is subsequently internalised by endocytosis it may be either utilised in some metabolic function or translocated to a site suitable for exocytosis at the basolateral membrane. From there it may be absorbed by the vascular system.

We shall discuss in this chapter the various models which elaborate these basic concepts, with particular emphasis on the role and fate of the receptor molecule. Evidence for and against the involvement of receptors will be presented and an evaluation of the transport models will be attempted in the light of such evidence. Finally we shall propose some experimental approaches which might resolve some of the more ill-understood areas of the subject.

General properties of receptors

The concept of a ‘receptor’ was introduced about 100 years ago by Langley (1878). Langley's work on the response of certain muscle cells to nicotine led to the idea of a ‘receptive substance’ at the site of application of the drug. Later work by Ehrlich allowed a generalisation of the concept and the receptor became identified with the role of a recognition site for a specific region of a drug or ligand.

The underlying theme in numerous studies on coated vesicles is that these very distinctive and ubiquitous organelles are intimately involved in the selective uptake and transport of exogenous proteins by cells. Yet, even though many recent studies have helped elucidate the structure of these vesicles, it is still not clear what their precise transport functions are in most of the tissues where they have been identified.

Many of the best examples of coated vesicles being implicated in selective transport come from studies on tissues that transfer maternal immunoglobulins to the mammalian young. The tissues involved, depending on the particular species, include the yolk sac and chorioallantoic placenta of the foetus, and the small intestine of the newborn. In each tissue, absorptive cells are present which contain prominent populations of coated vesicles. Furthermore, considerable evidence has established that immunoglobulin transmission across each tissue is selective and, by implication, involves membrane carriers or receptors (Brambell, Halliday & Morris, 1958; Brambell, 1970). However, the exact site of the receptors and the degree of selection within cells are both ambiguous. Much of this uncertainty lies in the fact that all of the tissues serve a second, equally important function for the young, this being the relative non-selective uptake and intracellular digestion of proteins for nutrition. This review considers the physiology of these two functions and the possible roles of coated vesicles in each. It examines the evidence for the presence and locations of specific receptors for immunoglobulins and other proteins, particularly within coated vesicles.

The existence of coated vesicles (CVs) in neurons has been known since 1961 when Gray reported ‘complex vesicles’ in mossy fibre endings of the rat cerebellar cortex. He described them as spheres, 60–80 nm in diameter, surrounded by shells consisting of closely packed 15–20 nm vesicular bodies. This unusual structural organisation was probably only an apparent one. Today the structure assigned to CVs in neurons and other cells, which is based upon detailed analyses of isolated CVs, consists of a spherical lipid-bilayer vesicle enclosed by a protein coat composed of pentagonal and hexagonal subunits (Kanaseki & Kadota, 1969; Kadota & Kadota, 1973a, b; Pearse, 1975; Crowther, Finch & Pearse, 1976; Woods, Woodward & Roth, 1978). The comparison of CVs isolated from different tissues (Pearse, 1975, 1976; Woods et al., 1978) and in thin sections of isolated tissues (Nickel, Vogel & Waser, 1967) revealed no significant differences between the structure and chemical properties of CVs in neurons and in other cells.

For a time there was some doubt whether neuronal CVs were discrete organelles. Difficulty in resolving the three-dimensional organisation of the coat by goniometry led to the idea that the CVs seen in thin sections were fixation artifacts arising from the denaturation of microtubules or of the neuronal cytonet (Gray, 1972, 1975; Westrum & Gray, 1977). This idea, however, was not supported by the studies of CVs isolated from unfixed brain tissue cited above.

The first workers to use the term ‘coated vesicles’ in a definitive sense appear to have been Rosenbluth & Wissig (1963, 1964), in their case to describe characteristic vesicles in toad spinal ganglia which had the ability to endocytose exogenous ferritin. Other workers have used different names to describe the same type of vesicle in a wide variety of tissues and cell types. For example, in their now classic study of yolk protein uptake by oocytes of the mosquito, Roth & Porter (1964) described them as bristle-coated. Casley-Smith (1969), in studies on macrophages, referred to them as rhopheosomes or rhopheocytic vesicles, and in so doing was following a precedent set by Policard & Bessis (1958) in what was one of the earliest studies on these vesicles. Gray (1961) referred to them as complex vesicles, Palay (1963) as alveolate, Wolfe (1965) as acanthosomes, and Maunsbach (1963) as ‘possessing an amorphous coat’. In most cases these terms refer to a distinct organised layer on the cytoplasmic aspect of the vesicle membrane which is a characteristic feature of what are now commonly known as coated vesicles. Much interest has centred on them in relation to endocytosis and it is true to say that they are ubiquitous in tissues and cells actively engaged in protein uptake. In making such a statement I do not wish to imply that protein uptake is necessarily their sole function.

Coated vesicles varying considerably in morphology have been described in various types of cells and a survey of the literature leads one to consider it probable that more than one functional entity has been given this name. Friend & Farquhar (1967) in a widely quoted paper present evidence for two types of coated vesicles in the rat vas deferens: a large type (> 100 nm in diameter) which they equate with heterophagosomes and to which they ascribe the functions of protein uptake and membrane resorption, and a smaller variety (< 75 nm in diameter) which they consider to be primary lysosomes. Kallio, Garant & Minkin (1971) described the ruffled border of active osteoclasts in fish and rats. They drew attention to deep imaginations and villous extensions of the plasma membrane which possess a coat of repeating units along their cytoplasmic surface. The presence of numerous smooth-walled and coated vesicles in the cytoplasm deep to the ruffled border was also noted. The authors suggest that the participate coat on the surface cytoplasmic membrane may be of a different nature to that present on the coated vesicles. Tangential sections of the coating on the cytoplasmic aspect of the surface membrane failed to reveal the polygonal pattern characteristic of the coated vesicles in the apical cytoplasm and it was thought possible that while the coated vesicles played a part in protein resorption, the particles on the ruffled border may represent sites of enzymatic activity connected with bone demineralisation.

Coated vesicles are structurally distinct cellular organelles with a variable geometry limited by, amongst other factors, Eulers theorem. They occur in organisms as diverse as pigs and Protozoa. Just as other organelles, e.g. lysosomes and microtubules, became the object of increased research effort subsequent to their first successful isolation, we believe that coated vesicles, which have recently been isolated successfully and consistently in different laboratories, are poised ready for examination by a range of refined molecular and immunological techniques.

Our aims for this volume include the gathering together of relevant information which will act as a reference for researchers in and entering this field. We will definitely produce the best source book on the subject, for the simple reason that there is no other! The fact that we can make this claim reflects what we think is an extremely serious imbalance. Whereas we would not wish to promote the use of weight of published material as a yardstick for the quality of the science it contains, we cannot help but notice that the published volumes on lysosomes and mitochondria already fill complete shelves in biomedical reference libraries; we wish to go a small way towards redressing this balance by producing one volume on coated vesicles.

A critic might be tempted to suggest that there is as yet no volume on coated vesicles because not enough is known about them to make one worthwhile.

Ample evidence indicates that coated vesicles are commonly occurring structures in different cell types in a diversity of animal species. Coated vesicles have been demonstrated in cells under physiological and experimental conditions, in vivo and in vitro, as well as in pathological situations. It is necessary, therefore, to examine the possibility that coated vesicles are ubiquitous structures and to record their occurrence in different animals, different cell types, and under varying cell-environmental conditions.

In a recent article an attempt has been made to review the information pertinent to coated vesicles and their function (Nevorotin, 1977). From an analysis of this and of further original observations it may be concluded that there are several distinct groups of coated vesicles. These all share a similar if not identical structural organisation of an external lattice structure and membranous vesicle wall, but differ in their vesicular contents, the route the coated vesicle apparently follows within the cytoplasm, and their contribution to cellular metabolism.

This chapter presents a comparative analysis of coated vesicles and their function but deals only briefly with the literature in the specific areas dealt with in later chapters.

Morphological aspects: general comments

There is more than one ultrastructurally distinct entity which has been termed a coated vesicle. The structure generally observed and tentatively related here to a principal class is characterised, when viewed in median ultrathin section, as a sphere bounded by unit membrane, with several projections, 15–20 nm in length, radiating from the external surface.

The literature on coated vesicles has been dogged with nomenclature problems and Bowers (1964) lists a plethora of synonyms for the organelle. Since coated vesicles were first described, terms such as complex vesicles, fuzzy vesicles, spiny vesicles, bristle-coated vesicles, dense-rimmed vesicles, alveolate vesicles and acanthosomes have all been employed. Because the majority of the data contained in this volume refers to the one organelle we have used the single term ‘coated vesicle’ throughout. We propose that for this organelle the use of all the other terms be dropped.

Against such a background it was not surprising to observe recently the beginnings of a rift developing over choice of a name for the major protein composing the polygonal structure on these vesicles' outer (cytoplasmic) surface (Matus, 1976; Pearse & Bretscher, 1976). The name proferred by Matus for this protein is dependent on Gray's (1972) interpretation of the vesicle coat as a fixation artifact. Gray had observed that the coated vesicles in his preparations of sectioned material for transmission electron microscopy were surrounded by an electron-lucent area. He concluded that material which had occupied this area in vivo was contracted during preparation for electron microscopy onto the surface of smooth vesicles giving rise to the polygonally patterned surface structure. Biochemical evidence and fine-structural information based on improved fixation methods make the artifact hypothesis hardly tenable.

Pearse's (1975) work which showed that the polygonal structure was composed for the most part of a single protein (mol. wt 180,000), has since been repeated and confirmed by several independent groups working on coated vesicles isolated from several different tissues.

This book is concerned with the dynamic mechanisms involved in the defence of plant cells against attack by parasitic bacteria and fungi. Thus I scarcely discuss those plant features such as bark and cuticle which play an obvious role in defence, but which are essentially static contributors. Circumvent these barriers and the ability of apparently undifferentiated parenchyma to defend itself is revealed. Furthermore, this ability is dependent upon particular genes in plant and parasite which interact after infection. My interest is with the processes by which plant cells perceive the approach of an intruder and occasionally permit, but commonly discourage, its further progress. How do the genes of host and parasite communicate to determine the outcome of attempted parasitism? Is there a universal defence mechanism in all plants, and, if so, what is it? What contribution does the much studied process of phytoalexin formation make to the defence of plants?

Research on the physiology of host–parasite relationships has been prolific in recent years and a number of multi-author treatises are being published on different aspects of this work. Hopefully, this monograph will make a useful contribution by presenting a shorter and personal view of those parts of this research which bear directly upon the processes of resistance in plants. My envisaged readership comprises research workers in the subject, and University teachers and their advanced students in plant pathology, botany and plant biochemistry.

Parasitic fungi and bacteria require general attributes to fit themselves for the parasitic, rather than the saprophytic, habit, and possess special features which enable them to be virulent on particular taxonomic groupings of host plants. The general properties of parasites include the ability to enter a plant by a specialized route, the capacity to synthesize enzymes for breaching barriers presented by cuticles and cell walls, the ability to obtain nutrients from the host, possibly via haustoria, and perhaps, the capacity to tolerate or to metabolize anti-microbial compounds in the host plant. Fine degrees of specialization might sometimes depend upon success or failure of these processes, but it should be clear from Chapters 1, 2 and 3 that the fate of parasitism is usually dependent upon the matching of very specific information, determined by complementary genes in host and parasite and expressed after penetration into host cells. The major question for this chapter concerns the intermolecular means by which the single genes in host and parasite express themselves. In what form is the information based on the DNA in both partners compared?

It seems logical that expression of specificity is mediated by nucleic acids, peptides and proteins, although lipoproteins, polysaccharides, glycoproteins and glycolipids may also be able to act as mediation factors. The essential feature of these molecules is that they are made up of many subunits arranged in particular sequences and thus suited to recognize matching molecules in the other partner in the host–parasite relationship.

Many micro-organisms are dispersed in air currents or in splash droplets caused by rain, and thereby arrive on leaves and stems. Other fungi and bacteria move in soil water before encountering roots or persist as resting stages until roots grow into their vicinity. Plants can then influence micro-organisms around their surfaces by physical and chemical means, thus starting an interaction which must be followed by entry of the parasite into the plant by a specialized route, if parasitism is to have a chance of success. This chapter is concerned with the few attempts that have been made to assess quantitatively the contributions to the success or failure of parasitism of these primary interactions between potential parasites and hosts.

EFFECT OF ROOTS ON PARASITES IN THE SOIL

The principal effect of roots on organisms in the soil is a general stimulation of germination and growth, and this is particularly important for parasites most of which remain dormant until contacted by their living substrates. Fungal parasites lie dormant in soil as a number of different types of resting body such as sclerotia, oospores, chlamydospores, basidiospores and hyphal fragments. Their dormancy is considered to be of two types, constitutive or exogenous (Sussman, 1966). Constitutive dormancy is thought to be maintained by internal factors in the fungus, and is particularly important in basidiospores. Experimentally, constitutive dormancy can be broken, in at least a small proportion of spores, by temperature shocks, treatment with certain chemicals, and proximity to other micro-organisms and some plant roots in culture.