Lysosomes: Structure, Forms, Functions and Other Details (With Diagram)

The existence of lysosomes was subtly suggested for the first time in the early 1950s by a series of experi­ments carried out by Nobel Prize Laureate Christian de Duve and his co-workers.

These experiments were designed to identify the cellular locus of the two en­zymes glucose-6-phosphatase and acid phosphatase.

Liver tissue homogenates were separated into nu­clear, mitochondrial, microsomal, and cytosol frac­tions by differential centrifugation and enzyme assays were performed on each of the col­lected fractions.

Although results with glucose-6- phosphatase clearly indicated that this enzyme was bound to particles sedimenting with the microsome fraction, observations on the distribution of add, phos­phatase were at first rather confusing.

The confusion centered around three seemingly peculiar but none­theless reproducible findings:

(1) The acid phosphatase activities of tissue homogenates prepared for centrif­ugation using a glass tube and close-fitting plunger (Dounce homogenizer) were about one-tenth the value observed when tissue was more vigorously dispersed using a Waring blender;

(2) The total (i.e., combined) enzyme activity of the isolated centrifugal fractions analyzed following differential centrifugation was about twice the activity of the original homogenate; and

(3) After storage for several days in a freezer, both the enzyme activity of the homogenate and the collected fractions, especially the mitochondrial fraction, increased dramatically (Table 19-1).

These observations were explained when de Duve and his colleagues showed that acid phosphatase ac­tivity was confined to sedimentable particles, the sur­rounding membranes of which limited the accessibility of the substrate (β-glycerophosphate) used in the en­zyme assay. Only when these membranes were dis­rupted and the acid phosphatase released from the particles was the enzyme activity demonstrable.

This occurred during vigorous dispersion in the Waring blender, which disrupted virtually all particles present. In contrast, only about 10% of the acid phosphatase-containing particles were disrupted by homogenization with the Dounce homogenizer, thus accounting for the low activity of the enzyme in the homogenate (Table 19-1). Some additional enzyme ac­tivity was released during and following centrifugal fractionation, but much larger quantities of enzyme were released by the membrane disruption that oc­curred during freezing and thawing.

At first, de Duve and his co-workers did not recog­nize that the acid phosphatase activity was associated with a distinct population of cellular particles. In­stead, on the basis of the observed “latent” activity of the mitochondrial fraction obtained by differential centrifugation (compare the values before and after freezing in Table 19-1), de Duve believed that acid phosphatase resided within the mitochondria.

How­ever, continued studies during the early 1950s in which the mitochondrial fraction was further divided centrifugally into a number of subtractions revealed that acid phosphatase was absent from fractions con­taining rapidly sedimenting mitochondria but was present in high concentrations in fractions containing slowly sedimenting mitochondria.

This observation, together with a newly developed appreciation of the potential contamination of sediments occurring dur­ing differential centrifugation, led de Duve to suspect that the acid phosphatase might, in fact, be associated with a special class of particles dis­tinct from the mitochondria.

Added credence was given to this idea by the finding that four other acid hydrolases, namely, β-glucuronidase, cathepsin, acid ribonuclease, and acid deoxyribonuclease, were dis­tributed through the centrifugal fractions in an identical manner. Thus, five hydrolytic enzymes, each hav­ing an acid pH optimum and acting on completely different substrates, appeared to be present in the same subcellular particle.

On the basis of the lytic ef­fects of all of these enzymes, de Duve named the parti­cles “lysosomes.” A number of additional enzymes have subsequently been identified in lysosomes (Table 19-2). Most of the chemical substances present in cells, including proteins, polysaccharides, nucleic ac­ids, and lipids, are degraded by these enzymes.

It is interesting to note that the initial postulation of the existence of lysosomes was made by de Duve purely on biochemical grounds. However, in 1955, A. Novikoff, working with de Duve, examined centrifugal fractions rich in acid phosphatase activity using trans­mission electron microscopy and provided the first morphological evidence supporting the existence of these particles. The lysosomes were identified as small, dense, membrane-enclosed particles distinct from the mitochondria.

In recent years, sophisticated centrifugal methods have been devised for obtaining preparations that are rich in lysosomes. Nearly all preparations obtained by differential centrifugation are contaminated with quantities of mitochondria. Although the average sedi­mentation coefficient of mitochondria is greater than that of lysosomes, mitochondria are polydisperse with respect to size, so that the smaller mitochondria in­variably sediment with the lysosomes.

Moreover, in tissues containing peroxisomes, the range of sedimen­tation coefficients for these organelles is almost iden­tical to that of the lysosomes. Consequently, it is virtu­ally impossible to obtain lysosome preparations that do not also contain peroxisomes. Somewhat greater success is obtained when isopycnic density gradient centrifugation is used in the last stages of the isola­tion procedure, for the equilibrium densities of lysoso­mes (1.22 g/cm³), mitochondria (1.19 g/cm³), and peroxisomes (1.23-1.25 g/cm³) in sucrose density gra­dients are slightly different.

Most density gradient procedures used to prepare lysosomes are modifica­tions of the technique developed by W. C. Schneider (Fig. 19-1). Using this technique, most of the mito­chondria are banded isopycnically at a density of about 1.19 g/cm³, whereas most of the lysosomes form a separate zone at about 1.22 g/cm³ and can be recov­ered independently from the density gradient.

By far the greatest purity of lysosomes is obtained from tissues of animals previously treated with Triton WR-1339 (a polyethylene glycol derivative of polymer­ized p-tert-octyl phenol), Dextran (a polymer of glu­cose), and Thorotrast (colloidal thorium hydroxide). These compounds are rapidly incorporated in large quantities by the cell’s lysosomes, significantly alter­ing their density. For example, the incorporation of Triton WR-1339 reduces the average density of the lysosome from 1.22 to about 1.10 g/cm³.

It is interesting to note that although the density of lysosomes incor­porating Triton WR-1339 is significantly reduced, their size is increased; the result is that Triton WR- 1339-loaded lysosomes have the same sedimentation coefficient as normal lysosomes but have a lower den­sity.

The latent enzymatic effect originally noted by de Duve and his co-workers is still employed as a major criterion in evaluating the effectiveness of any lyso­some isolation. Accordingly, the lysosome preparation is incubated under the appropriate conditions with the hydrolase substrate before and after treatments known to disrupt the lysosome membranes.

If the original preparation contains intact lysosomes, then no substrate is hydrolyzed before treatment (most substrates of the lysosomal hydrolases are unable to permeate the lysosome’s membrane); however, disrup­tion of the membrane (by sonification, repeated freez­ing and thawing, addition of lytic agents such as bile salts, digitonin, Triton X-100, etc.) and release of the lysosomal enzymes are quickly followed by hydrolysis of the added substrates.

Structure and Forms of Lysosomes:

Lysosomes are a structurally heterogeneous group of organelles and vary dramatically in size and morphol­ogy. As a result, it is difficult to identify lysosomes strictly on the basis of morphological criteria. When lysosome-rich fractions were initially isolated centrifugally by de Duve and Novikoff and examined with the electron microscope, it was found that the suspected lysosomes were generally about the same size as small mitochondria.

Typically, they varied in diameter from about 0.1 to 0.8 μm, were bounded by a single mem­brane, and were usually somewhat electron-dense. Identification of lysosomes in sections of whole cells is considerably more difficult because other small, dense organelles are also bounded by a single membrane.

The application of cytochemical procedures at the level of the electron microscope in which the lyso­somes are identified on the basis of their enzyme con­tent is much more reliable. Notable among such proce­dures is that introduced in 1952 by G. Gomori and that is routinely employed in variously modified forms for the identification of lysosomes on the basis of their high acid phosphatase content.

In the Gomori method, the tissue to be examined is incubated at pH 5.0 in a medium containing β-glycerophosphate (a substrate for acid phosphatase) and a lead salt (such as lead ni­trate). Phosphate enzymatically cleaved from the sub­strate during incubation combines with the lead ions to form insoluble lead phosphate, which precipitates at the locus of enzyme activity.

Because the lead phos­phate is electron-dense, electron microscopy reveals the lysosomes as dark, granular organelles (Fig. 19- 2). For identification with the light microscope, am­monium sulfide may be used to convert the lead phos­phate to lead sulfide, which appears black. The Gomori reaction may be carried out with fixed and sectioned material, as well as with fresh tissue, albeit with reduced efficiency as a result of some enzyme in- activation during and following fixation.

Several different lysosomal forms have been identi­fied within individual cells, including:

(1) Primary lyso­somes,

(2) Secondary lysosomes, and

(3) Residual bodies.

Primary Lysosomes:

Primary lysosomes, or proto- lysosomes, are newly produced organelles bounded by a single membrane and believed to be derived from the trans face of the Golgi apparatus. Al­though varying somewhat in size, primary lysosomes are typically about 100 nm in diameter. Primary lysosomes are virgin particles in that their digestive en­zymes have not yet taken part in hydrolysis.

Secondary Lysosomes:

Two different kinds of sec­ondary lysosomes can be identified: heterophagic vacuoles (also called heterolysosomes or phagolyso­somes) and autophagic vacuoles (also called auto- lysosomes). Heterophagic vacuoles are formed by the fusion (see below) of primary lysosomes with cytoplas­mic vacuoles containing extracellular substances brought into the cell by any of a variety of endocytic processes. Following fusion, the hy­drolases of the primary lysosomes are released into the vacuole (variously called a phagosome or endosome). Autophagic vacuoles contain particles isolated from the cell’s own cytoplasm, including mitochon­dria, microbodies, and smooth and rough fragments of the endoplasmic reticulum.

The autodigestion of cellu­lar organelles is a normal event during cell growth and repair and is especially prevalent in differentiating and dedifferentiating tissues and tissues under stress. Autophagic vacuoles containing partially de­graded mitochondria are shown in Figure 19-3.

The formation of heterophagic and autophagic vacuoles is soon followed by enzymatic digestion of the vacuolar contents. As digestion proceeds, it becomes increas­ingly difficult to identify the nature of the original secondary lysosome and the more general term diges­tive vacuole is used to describe the organelle at this stage.

Residual Bodies:

Endocytosed substances and parts of autophagocytosed organelles that are not digested within the secondary lysosomes and transferred to the cytoplasm are retained (usually temporarily) within the vacuoles as residues.

Lysosomes containing such residues are called residual bodies (sometimes also called telolysosomes or dense bodies). Residual bodies are large, irregular in shape, and are usually quite electron-dense. The undigested residues often take the form of whorls of membranes, grains, amor­phous masses, ferritinlike or hemosiderinlike particles, or myelin figures (Fig. 19-4). Residual bodies of­ten fail to display the degree of hydrolytic activity associated with the primary and secondary lysosomes.

Formation and Function of Lysosomes (the “Vacuolar System”):

Lysosomal enzymes are concerned with the degrada­tion of metabolites and not with cellular synthetic or transfer reactions. On the basis of numerous cytochemical observations, it is generally believed that pri­mary lysosomes are formed by budding from the trans face of the Golgi apparatus. The primary lysosomes are dispatched as either smooth or coated vesicles hav­ing a diameter of about 50-100 nm. After budding, the coat is lost. The acid hydrolases that are destined for lysoso­mes are synthesized by ribosomes of the rough ER in the vicinity of the Golgi bodies.

Some of these hydrolases are discharged into the lumenal phase of the ER and others remain anchored in the ER membranes. Through either the dispatchment of tiny vesicles from the ER or via direct communication through cisternae, the hydrolases make their way to the cis face of the Golgi body. After purification and processing in successive Golgi cisternae, the hydro­lases are released from the trans face of the Golgi ap­paratus in the form of primary lysosomes.

This con­cept, depicted diagrammatically in Figure 19-5, is supported by a large number of observations made with a variety of tissues. The process is reminiscent of the formation of zymogen granules for secretion by Golgi bodies. Because of the intimate association be­tween Golgi bodies, ER, and primary lysosomes, re­gions of cells containing these organelles are some­times called “GERL” (i.e., “Golgi-Endoplasmic Reticulum-Lysosome”) complexes.

The synthesis of the lysosomal enzymes involves the preliminary assembly of a signal sequence that di­rects the large ribosomal subunit to its appropriate docking site on the endoplasmic reticulum. Following this, the elongating polypeptide is discharged through the membrane into the intracisternal space. Virtually all of the lysosomal enzymes are glycoproteins. Core glycosylation occurs during translation but final processing occurs in the cis Golgi cisterna.

Using histochemical procedures it is possible to lo­calize lysosomal enzyme activity in the Golgi appa­ratus; this, of course, supports the view that lysoso­mal enzymes progressively transit through at least some of the Golgi cisternae. The carbohydrate por­tions of the lysosomal enzymes include an uncommon mannose-6-phosphate-containing oligosaccharide. Re­cent studies indicate that the Golgi membranes contain a specific receptor for mannose-6-phosphate and it is believed that these receptors play an important role in directing the lysosomal hydrolases into newly forming primary lysosomes.

Heterophagy:

Extracellular materials brought into the cell by endocytosis are enclosed within vacuoles called endosomes. These materials may later be re­jected unaltered by exocytosis, or the endosome may fuse with one or more primary lysosomes that empty their digestive hydrolases into the newly formed par­ticle (now called a secondary lysosome, see Fig. 19-5). Lysosomal digestion of endocytosed material is termed heterophagy.

The fusion of primary lysosomes with endosomes has been demonstrated in vivo in a number of tissues using various exogenous markers introduced into the organism. These markers, which include horseradish peroxidase, ferritin, and hemoglobin, are engulfed by the tissue cells and are later detected within second­ary lysosomes along with lysosomal hydrolases.

Z. A. Cohn and B. Benson employed ³H-labeled leucine to trace the fate of newly synthesized hydro­lases in peritoneal phagocytes of mice. They found that pinocytic activity was greatly increased when these cells were incubated in blood serum. Autoradio­graphic analysis revealed that the labeled hydrolases appeared first in the Golgi region of the cells and later within pinocytic vesicles or endosomes.

Their observa­tions support the proposal that secondary lysosomes are formed by the fusion of endosomes and primary lysosomes. Moreover, Cohn and Benson also found that the rate at which hydrolases were produced by the cells was related to the level of pinocytic activity, suggesting that the production of primary lysosomes may somehow be regulated by endocytosis (see be­low).

In some cells, several small primary lysosomes may fuse with a single large endosome; in other cells, large primary lysosomes sequentially fuse with a number of small endosomes.

The contents of the secondary lyso­some change dramatically with time as:

(1) The con­tents of the lysosome are enzymatically degraded,

(2) New materials are introduced through fusion of addi­tional endosomes, and

(3) Additional hydrolases are added by the fusion of new primary lysosomes.

The hydrolases in the secondary lysosome break down the endocytosed materials, producing a variety of useful substances (e.g., amino acids, sugars, etc.) as well as some useless waste products. It is generally agreed that usable materials make their way across the mem­brane of the secondary lysosome and enter the cytosol, where they participate in cellular metabolism.

This transfer probably takes the form of passive diffu­sion or facilitated or active transport. Eventually, di­gestion and absorption are terminated) leaving only residues and denatured enzymes within the vacuole, which is now referred to as a residual body. In many cells, residual bodies fuse with the plasma membrane, and this is followed by exocytosis (Fig. 19-5).

In some ceils (especially those of higher organisms), residual bodies accumulate within the cytoplasm or continue to increase in size, eventually interfering with the nor­mal activities of the cell and resulting in cell death. Progressive lysosome engorgement is believed to be involved in the aging process.

Autophagy:

The isolation and digestion of portions of a cell’s own cytoplasmic constituents by its lysoso­mes occurs in normal cells and is termed autophagy (Fig. 19-5). The phenomenon is most dramatic in the tissues of organs undergoing regression (e.g., changes in the uterus following delivery, during meta­morphosis in insects, etc.).

Autophagic vacuoles con­taining partially degraded mitochondria, smooth and rough endoplasmic reticulum, micro-bodies, glycogen particles, or other cytoplasmic structures are fre­quently observed in tissue sections examined with the electron microscope. Cellular autophagy results in a continuous turnover of mitochondria in liver tissue. The half-life of the liver mitochondrion is about 10 days and corresponds to the destruction of one mito­chondrion per liver cell every 15 minutes.

Distribution of Lysosomes:

Since their initial discovery in mammalian liver, lyso­somes have been identified in many different cells and tissues; some of these are listed in Table 19-3. The greatest variety of tissues found to contain lysosomes occurs in animals. Although most studies have been carried out using mammalian tissues, lysosomes have been identified in insects, marine invertebrates, fish, amphibians, reptiles, and birds.

Lysosomes are partic­ularly numerous in epithelial cells of absorptive, secre­tory, and excretory organs (liver, kidneys, etc.). They are also present in large numbers in the epithelial cells of the intestines, lungs, and uterus. Phagocytic cells and cells of the reticuloendothelial system (e.g., bone marrow, spleen, and liver) have also been found to contain large numbers of lysosomes. Few lyso­somes occur in muscle cells or in acinar cells of the pancreas. Lysosomes are produced by certain cells in tissue culture (HeLa cells, monocytes, lymphoctyes, etc.). Although it has a number of functions not shared by lysosomes of animal cells, the large vacuole of many plant cells is a modified lysosome. Some of the various roles played by the lysosomes are summa­rized in Table 19-4.

Leukocytes, especially granulocytes, are a particu­larly rich source of lysosomes, and this is related to their physiological role as scavengers of microorgan­isms or other foreign particles in the blood. Following phagocytosis of a bacterium by a leukocyte, numerous lysosomes fuse with the endocytic vacuole containing the microorganism and initiate its digestion. The lyso­somes of granular leukocytes are especially large and readily visible by light microscopy. Once the lysosome content of the leukocyte is exhausted, the white blood cell dies.

Plant Vacuoles:

Many plant cells contain one or more vacuoles, which possess some of the properties of lysosomes. In immature and actively dividing plant cells the vacuoles are quite small. As the cells mature, the vacuoles coalesce to form larger compartments. Ma­ture cells of higher plants usually have a larger, cen­tral vacuole that may occupy as much as (occasionally more than) 80% of the total cell volume.

The membrane enclosing the plant cell vacuole is called the tonoplast. Like lysosomes, plant cell vacu­oles contain hydrolytic enzymes. In addition, they usu­ally contain sugars, salts, acids, and nitrogenous com­pounds such as alkaloids and anthrocyanin pigments. The pH of the plant vacuole may be as high as 9 or 10 due to large quantities of alkaline substances or as low as 3 due to the accumulation of quantities of acids (e.g., citric, oxalic, and tartaric acids).

The plant vacuole is the major contributor to the turgor that provides support for the indi­vidual plant cell and contributes to the rigidity of the leaves and younger parts of the plant. Water accumu­lation in the vacuole as a result of the osmotic effects of the dissolved substances causes the vacuole to ex­pand, pushing outward against the cytoplasm and cell wall. When there is a lack of water the turgor dimin­ishes and these results in wilting.

Lysosome Precursors in Bacteria:

Although bacterial cells do not possess lysosomes, they do contain a variety of hydrolases that are be­lieved to be localized in the space between the plasma membrane and the cell wall. These hydrolases may be synthesized by ribosomes attached to the plasma membrane and then dispatched through it. The bacte­rial hydrolases play a digestive role, breaking down complex substrates in the cell’s environment and pro­viding smaller molecules required for cell growth. Bacterial hydrolases also participate in sporulation and autolysis.

Although the latter process destroys the individual cells involved, it is highly beneficial to the bacterial population as a whole, for it provides for the survival of a small number of cells under unfavor­able environmental conditions. In-folding of the bacte­rial membrane to form internalized extracellular pockets containing both hydrolases and their sub­strates would provide the “evolutionary link” with ly­sosomes of animal and plant cells.

Regulation of Lysosome Production:

The mechanism proposed for primary lysosome formation is strikingly similar to that pro­posed for zymogen granule formation in pancreatic cells and other instances of secretory protein synthe­sis. This similarity does not seem so unusual when one considers the following. The enzymatic contents of primary lysosomes are discharged into vacuoles (i.e., endosomes) that are derived from the plasma mem­brane and that contain extracellular materials. Conse­quently, the mechanism is similar to secretion except that the extracellular space into which the secretory products pass is internalized.

In secretory cells, the production of new secretory products is regulated by a feedback mechanism in which secretion itself acts as a stimulus for the production of additional secretory materials. The experiments of Cohn and Benson de­scribed above demonstrate the relationship that exists between endocytic activity and lysosomal enzyme syn­thesis. It has therefore been suggested that the pas­sage of endocytic vesicles into the Golgi regions of the cell is followed by the discharge of some primary lysosomes and that this triggers the synthesis of new acid hydrolases.

Disposition and Action of the Lysosomal Hydrolases:

Many of the lysosome’s enzymes are released into the surrounding environment when these organelles are physically or chemically disrupted. Those enzymes that are so readily solubilized are believed to be lo­cated in the interior of the organelle. Other lysosomal hydrolases cannot be solubilized or are extracted with great difficulty and are thought to be an integral part of the lysosome membrane together with other pro­teins and lipids. Some of the enzymes known to be present in lysosomes are listed in Table 19-2; it is to be noted that while this list is extensive, it is by no means complete.

All the substrates of lysosomal enzymes are either polymers or complex compounds and include proteins, DNA, RNA, polysaccharides, carbohydrate side chains of glycoproteins and glycolipids, lipids, and phosphates. The lysosomal breakdown of proteins into amino acids illustrates how these enzymes act in con­cert. The initial hydrolysis of protein is effected by cathepsins D and E and also by collagenase. These en­zymes cleave peptide bonds and produce peptide fragments of varying length.

The peptides, together with previously undigested proteins, are further hydrolyzed to individual amino acids by cathepsins A and B. Cathepsin C, arylamidase, and the lysosomal dipeptidases act on specific peptides, producing addi­tional amino acids.

The breakdown of DNA and RNA is initiated by the enzymes acid deoxyribonuclease and acid ribonuclease. The resulting oligonucleotides are then degraded first by phosphodiesterase and then by acid phospha­tase, producing nucleosides and inorganic phosphate. Lysosomes also possess all the enzymes necessary for hydrolysis of lipids and polysaccharides.

Some lysosomal enzymes are part of the membrane encasing the organelle. Among the enzymes found to be integral parts of the lysosome membrane are acetylglucosaminidase, glucosidase, and sialidase. Arylsulfatase, acid phosphatase, ribo- nuclease, and glucuronidase may also be bound to the membrane under certain conditions.

Enzymes freed from disrupted lysosomes exhibit a wide variation in stability. Some are particularly re­sistant to autolysis and retain their activity for months when appropriately refrigerated; others lose their activity only a few hours following tissue disrup­tion. Of the lysosomal enzymes isolated and charac­terized to date, most are glycoproteins, including cathepsin C, acid deoxyribonuclease, glucuronidase, and acetylglucosaminidase.