Introduction

‘This Small Particle acts as the digestive tract of the living cell. Its enzymes dissolve the substances ingested by the cell and under certain circumstances can dissolve the cell itself.’

Such was the definition given by de Duve in 1963 of the role of the lysosome. All future work stemmed from this definition and it is now agreed that the lysosomes are tiny bags filled with a powerful digestive juice capable of breaking down most of the constituents of living matter.

Most of the work done on lysosomes has been on rat liver cells, although the evidence that is accumulating supports the hypothesis that they are universal in animal cells.

Only a few plant tissues have been investigated for lysosomes.

Lysosome Morphology

Cytological and biochemical studies with the aid of the electron microscope have shown the lysosome to be a cytoplasmic organelle, spherical in shape with a mean diameter of about 0.4 microns and an average density of about 1.15, and a single, limiting membrane, but no definite internal structure. Associated with the lysosome are a number of easily soluble acid hydrolases with acid pH optima but there are no oxidative enzymes as one finds in mitochondria. These lysosomal enzymes are released by treatment of the lysosome with agents known to affect the binding of lipin to protein.

The latency of lysosomal enzymes is considered to be due to the presence of this limiting membrane barrier of lipid-protein which restricts the accessibility of the internal hydrolases to any external substrate. The presence of such a membrane was deduced from experiments concerning the action of lecithinase and proteolytic enzymes on the particles. After treatment with these enzymes all hydrolase activities were released simultaneously and in a fully active form, from the particles. One of the biochemical characters of the lysosome is this structure - linked enzyme latency. In studying acid phosphatase activity, de Duve found that the substrate beta-glycerophosphate did not readily penetrate the membrane of the lysosome unless the particle had been subjected to an agent such as acid pH or high temperature (37°C.). Presumably these treatments increased the permeability of the membrane by altering its structure.

Whereas enzyme action is not confined to lysosomal particles, the activation of an acid hydrolase at a particular cytoplasmic site may be considered indicative of the presence of a lysosome, especially if the page 37 particular enzyme has been already demonstrated biochemically to be associated only with a lysosomal fraction. The presence of the acid hydrolases in association with a particle limited by a single membrane, allows a positive identification of lysosomes. To quote Gahan (1967)

‘It has been discussed that the histochemical identification of a lysosome in a particular tissue ideally should rest upon the evidence demonstrating the presence of two or more acid hydrolases contained within a particle limited by a single membrane’.

While the presence of lysosomes in animal tissues has been well established, the same is not true for lysosomes in plant tissues. Using the presence of acid phosphatase located in particles as a criterion, lysosomes have been recorded in the embryo of Triticum vulgare (single membrane observed), root meristem of Vicia faba, root meristem and epidermis of Allium cepa, and pollen grain of Tradescantia bracteata. Cytochemical demonstrations of the activity of acid phosphatase have also been made in the fungus Botrytis cinerea.

Role of the Lysosome: Digestion

All the major classes of biologically active compounds, including proteins, nucleic acids and polysaccharides, were shown to be susceptible to action by the enzymes contained in lysosomes. The number of acid hydrolases discovered stands at ten: acid phosphatase, cathepsin, acid deoxy ribonuclease, acid ribonuclease, beta-glucuronidase, arylsulfatases A and B, phosphoprotein phosphatase, beta - galactosidase, beta - N - acetylglucosaminidase, and alpha - mannosidase. But all are in different proportions.

Considered as a group, the enzymes present in the lysosome have one major function: a lytic, or digestive one. Hence the name ‘lysosome’ (meaning lytic body) that de Duve gave to the body.

De Duve first concluded that the membrane must act as a shield between the enzymes and the rest of the cell. The digestive processes, he deduced, must be confined within the limits of the membrane, and the substances to be digested must somehow be taken up in the particles. He then endeavoured to find out how this took place and then to look for those conditions that might lead to the release of the enzymes inside the cell and the dissolution of the cell.

De Duve noticed that lysosomes are not readily distinguishable in any type of cell and cannot be identified solely on the basis of their appearance. They come in a bewildering assortment of shapes and sizes, even in a single type of cell. This polymorphism is now understandable: their digestive activities cause them to be filled with a variety of substances and objects in an advanced state of disintegration and it is their contents that determine their shape, size and density.

To discuss de Duve's work, one must explain the phenomenon of pinocytosis. Not all of the substances that nurture a cell require digestion by lysosomes. In higher animals tissue cells receive most of page 38 their nutrients from the bloodstream in the form of small molecules absorbed through the cell membrane and requiring no digestion in the cell. Some materials, however, are too bulky for direct absorption and too complex chemically for immediate utilisation. Cells are able to engulf such large molecules and even bodies as big as bacteria or other cells by a process referred to as ‘endocytosis’ or ‘pinocytosis’. A portion of the cell membrane first attaches itself to the ‘prey’ and then appears to be sucked inward to form a small internal pocket containing the prey. The pocket pinches free from the cell membrane and drifts off into the cell interior, now forming a phagosome.

De Duve distinguished four conditions of lysosome states: ‘storage granules’, digestive vacuoles, residual bodies and ‘autophagic vacuoles’. The first three are directly involved in the main digestive process (refer Fig. 1). The storage granule is the original form of the lysosome. De Duve postulated that the enzymes in the granule presumably were produced by the ribosomes associated with the endoplasmic reticulum, but he did not comment on the origin of the lysosome membrane. A storage granule fuses with a phagosome to form a digestive vacuole. Digestion products diffuse through the membrane into the cell. The digestive vacuole can continue its digestive activity, gradually accumulating indigestable material until it becomes a residual body, which may then be eliminated by fusion with the cell membrane. The distinguishing feature of the autophagic vacuole is the material digested: parts of the cell itself, such as mitochondria and portions of the endoplasmic reticulum.

Another mode of action of the lysosome involves the actual rupture of the lysosome membrane inside the cell and the digestion of the cell as a whole by the released enzymes. Such ruptures take place fairly quickly in dead cells, e.g. cells suddenly deprived of oxygen or exposed to cell poisons of certain kinds.

Formation of Lysosomes

There is much speculation concerning the formation of lysosomes. It is known that protein synthesis in the cell cytoplasm occurs at ribosomal sites, and the assumption that this is the site of hydrolytic enzymes is also certain.

It is thought that the hydrolases may pass from the ribosomal sites of synthesis to the lysosomes either by the formation of lysosomes directly from the endoplasmic reticulum or via the Golgi apparatus.

Wolman and Weiner (1963) suggested that lysosomes are derived from the endoplasmic reticulum by a change in its structure in which the membrane is folded into a globular body surrounded by polar lipids, with the hydrophilic groups pointed inwards. By this means, the enzymes normally situated on the outer surface of the membrane are enclosed in vesicles formed by the inversion of the membranes.

Dalton (1962) found that small vesicles appeared to arise from page 39 the endoplasmic reticulum but considered these vesicles to join the Golgi vacuoles in pancreas cells.

FIG. 1: Intracellular Digestion. a.v. autophagic vacuole; d.v. digestive vacuole; e.r. endoplasmic reticulum; I. lysosome; ph. phagosome; r.b. residual body; s.g. storage granule. (after de Duve).

Benedetti and Leplus (1958) working with erythroblasts of chickens infected with erythroblastosis virus found granules like lysosomes in appearance, in, or adjacent to the highly developed Golgi zone. There is, according to them, some indication of continuity between Golgi membrane and membrane of the granule, i.e. the granule appears to be like an enlarged Golgi vacuole. They suggest the Golgi apparatus is a site for segregation of lysosomal hydrolases.

It is unlikely that all of the lysosomal hydrolases are contained within each lysosome since, e.g. rat liver lysosomes do not appear to behave as enzymically homogeneous particles (de Duve, 1963).

Constituent enzymes seem to show a slightly different distribution pattern when the particles are analysed for enzyme content.

Novikoff (1964) considered that it was difficult to distinguish between secretion-transporting vesicles and those vacuoles separating from the Golgi, which he feels are likely to be lysosomes. He demonstrated an experimental procedure which changes the distribution of the Golgi apparatus which correspondingly changes the lysosome distribution. Novikoff even suggested that lysosomal hydrolases may be transported to larger lysosomes by Golgi vesicles. This was based on studies of the uptake of exogenous proteins (horse-radish peroxidase) by rat kidney cells. He reported that proteins gained access by pinocytosis into the cell, thereby forming pinocytotic vacuoles and migrating into the centre of the cell and acquiring acid hydrolases on passing close to the Golgi region. Novikoff did stress, however, that this hypothesis was not adequately supported by experimental data.

Brandes (1965), working on Euglena gracilis, showed hydrolase activities demonstrable at the Golgi sites. This supported the idea that enzyme-rich vesicles, presumed to be lysosomes, originated as terminal dilations of the Golgi cisternae, which in many instances also possessed intense enzyme activity.

On the other hand Ogawa and Shinonaga (1962) found acid phosphatase activity to be associated intimately with the smooth membrane system of the cell. They considered the lysosomes to be specially differentiated organelles originating from the smooth endoplasmic reticulum.

Further evidence interpreted to indicate the formation of lysosomes from the Golgi complex is given in the study of sebaceous glands from adult male rats (Brandes, 1965). In an electron microscope study of the localization of glycerophophatase and esterases, Brandes found that in undifferentiated cells the Golgi apparatus was poorly developed There was no evidence of secretory activity nor any reaction for hydrolases. In differentiating cells secretory activity was indicated by the formation of vacuoles and a highly developed Golgi apparatus which was positive for acid hydrolase activity.

The two hydrolases appeared limited to small granules similar to Golgi vesicles, and to larger bodies which could have been lysosomes. On the basis of this evidence, it was suggested that the Golgi apparatus plays an important part in the formation of lysosomes in sebaceous cells.

In review of this section, the available evidence would suggest that lysosomes may be formed directly from the endoplasmic reticulum, perhaps by invagination of the membrane, or by the production of vesicles from the Golgi cisternae. Whether either or both of these possibilities in fact operates is argumentative at this stage.

Cell Death

The difficulty in assessing evidence concerning the role of lysosomes in cell death is that although the cells may give the appearance of dying and there is free hydrolase activity present in the cells, it is necessary to determine whether

De Duve coined the term ‘suicide-bags’ for lysosomes with respect to cell death.

Brandes (1965) using electron microscope studies of esterase and glycerophosphatase on the sebaceous cells of the adult rat, showed that in fully mature cells about to disintegrate, the lysosomes enlarge and appear to become ruptured. When the stage of actual cellular lysis occurs, the lysosomes are no longer visible and scattered glycerophosphatase-rich particles are seen in all parts of the cell. page 41 These are presumed to have been derived from the ruptured lysosomes.

Of the several cytological changes described as correlated with ageing the most widely accepted is the accumulation of lipofuscin granules. Novikoff found the appearance of lipofuscin granules in the Golgi zone and suggested that these granules may be altered lysosomes. He noticed that in aged cells the Golgi apparatus fragmented and disappeared whilst lipofuscin accumulated.

Lipofuscin granules have been seen to accumulate with age in human myocardium. These lipofuscin-rich granules show high acid phosphatase and cathepsin activity and so may be conceived as being altered lysosomes in which metabolic waste materials have accumulated.

Sulkin and Kuntz (1952) noted that as lipofuscin accumulated in the autonomic ganglia of ageing men and dogs the Golgi apparatus fragmented and largely disappeared.

Dalton and Felix (1957) reported granules adjacent to Golgi membranes and vacuoles in human neurons. They postulate they are altered Golgi vesicles, lysosomes, in transition to lipofuscin granules.

Jarrett and Spearman (1962), basing their consideration on the role of lysosomes in the autolysis of the contents of the epidermal cells prior to normal keratinisation, suggested that abnormal keratinisation found in the condition of psoriasis may result from the failure of the lysosomes to release their enzymes. That the lysosomes may not rupture in the usual way has been shown by the electron microscope.

The problem of the possible role of lysosomes in cell death has also been considered in respect of plant tissues. It is known that on differentiation of meristematic cells into primary xylem cells, the differentiating cells gradually lose their cell contents, a change accompanied by a markedly increased impermeability of the cell wall. Using glycerophosphatase as a marker for lysosome-like particles in the roots of Vicia faba, Gahan (1967) found that in undifferentiated meristematic cells the glycerophosphatase activity was confined to particular sites, but with the onset of differentiation, as demonstrated by the elongation of the cells accompanied by changes in the structure of the cell wall, the acid phosphatase activity was no longer solely particulate, but was also diffuse. On further differentiation, the loss of cytoplasmic contents was accompanied by the presence of acid phosphatase activity right throughout the cell.

The author observed a similar situation in the cells of the root cap of Allium cepa where the innermost cell adjacent to the meristematic region showed a particulate reaction for acid phosphatase, whereas the outermost cells of the root cap showed a diffuse reaction.

The above observations may support the concept of the self-digestion of the cell contents by the release of the hydrolytic enzymes from the lysosomes.

From the accumulated data it is not possible to make a definitive statement concerning the role of lysosomes in cell death. It seems page 42 established that the hydrolase activity may be realeased into the cell, resulting in the breakdown of the cell contents, but in no set of results is there a definite state where release of the hydrolases occurs in an intact cell. Although, this may be the case with the plant cells considered above.

Lysosomes and Cell Division

Robbins and Gonatas (1964), when studying mitosis in Hela cells, found the acid phosphatase - containing granules aggregated circumferentially in packets during prophase and metaphase. This was contrary to the polarised distribution of individual granules during interphase.

In a study of the effects of spindle inhibitors, colchicine and vinblastine sulfate on Hela cells, Robbins and Gonatas found marked changes in the nature and behaviour of the lysosomal particles.

After treatment for fifteen hours with the inhibitors, the lysosomes were circumferentially dispersed instead of in their normally polarised, juxtanuclear position. Furthermore, they appeared to be larger than their normal counterparts in untreated cells due, apparently, to their being clustered and not individually resolvable in the optical microscope. The general behaviour of these lysosomes after treatment with the spindle inhibitors is identical to that normally observed in the untreated mitotic cell.

Holt (1968) also found changes in the distribution of lysosomes in rat liver cells during mitosis. At interphase the lysosomes occupied their typical position, but moved to a juxtanuclear position at the beginning of prophase. During metaphase they became uniformly distributed throughout the spindle. At anaphase, as the chromosomes moved to opposite poles, so a cluster of lysosomes preceded each chromosome set to the poles where they formed a ‘cap’. The lysosomes remained in the ‘cap’ until the end of telophase, after which they returned to their normal location in the cytoplasm.

The behaviour of lysosomes during mitosis has been considered also by Maggi (1966) in her studies upon Hela cells. Lysosomes were identified by the reaction for acid phosphatase when it was found that after incubating the cells for five minutes the number of granules varied from ten to thirteen per cell. The number of particles increased to nineteen for prophase, twenty-one for metaphase, seventeen for anaphase, and twenty-nine for telophase. It was concluded that during division the permeability of the lysosomal membranes to the substrate employed was increased, so enabling a more rapid penetration by the substrate. Thus, there would seem to be some involvement of the lysosomal particles during mitosis, possibly relating to the dissolution of the nuclear membrane and of the spindle.

A further point arising from the observations of Maggi (1966) was that at telophase an approximately equal number of particles passed to each of the two daughter cells, the sum of the two page 43 populations of lysosomes equalling the maximal number found during interphase. This suggests that a synthesis of particles probably occurred during interphase.

Allison and Mallucci (1964) considered that the lysosomes were involved in the initiation of mitosis due to a release of an unspecified activator from the lysosomes or to one of the released hydrolases inactivating a repressor. However it has also been suggested that lysosomes may be responsible for chromosome breakage. The results of Allison and Paton (1965) showed that treatment of human embryonic lung cells in tissue culture with photosensitising agents allowed high frequency of chromosome breaks on irradiating the cells with light from a high intensity tungsten source. It was claimed that the effects of the photosensitising agents (neutral red and acridine), were observed only at lysosomal sites, from which it was concluded that a cytoplasmic event was giving rise to a structural alteration in the chromosomes. It was suggested that deoxyribonuclease from the lysosomes might be the agent responsible for the chromosome breaks. This is difficult to explain in the light of results of Chevremont et al., (1960), who grew mammalian cells in tissue culture in the presence of acid deoxyribonuclease. This treatment inhibited cell division, but allowed DNA synthesis to proceed and did not appear to alter the nuclear DNA. Thus, while it is tempting to implicate lysosomal activity in mitosis and chromosome behaviour, via the release of lysosomal enzymes, some caution must be observed in evaluating this field, which at present is rather limited.

Action of Hormones on Lysosomes

Little work has been done from this aspect, although Cohen et al., (1964) studying the ovary of the rat, observed the localisation of acid phosphatase and noted a variability of lysosomal activity depending on the particular stage of the oestrus cycle. During oestrus the number of small, enzymically active particles increased, with the epithelial cells containing numerous large lysosomes. As the cycle continued the number of particles decreased, until they disappeared with the beginning of the new cycle. Although this study was performed at the level of the optical microscope only, Cohen suggested that the large lysosomes may be equivalent to cytolysomes.

The work of Scheib (1963) would indicate a possible involvement of the sex hormones during regression of the Mullerian ducts of male chick embryos where he claims both androgen and estrogen caused the release of lysosomal hydrolases.

Summary

When a new cell particle is discovered, attempts are made to link its activities with a range of functions, and the lysosome has proved no exception.

Cytological and chemical studies have contributed to extending the lysosome concept from one of ‘a bag of hydrolytic enzymes’ to one in which the lysosome has assumed a physiological importance. Despite the wide range of studies, it is still too early to be definite about the role of the lysosome. The nature of the enzymes associated with it led de Duve (1963) to postulate a role in degradation rather than in biosynthesis and histochemical findings would point strongly to a role in intracellular digestion. This, in fact, may prove to be the essence of all lysosomal functions, for while there are changes in the behaviour pattern of the particles under a wide range of cellular activities, such as mitosis, virus infection and physiological cycles, the underlying function of the lysosomes is still likely to be one of digestion. However, more data is necessary concerning the behaviour of lysosomes in conditions such as cell death and cell division, in cellular metabolism and ageing, before a final answer can be obtained to the question, ‘What is the role of the lysosome?’