Lysosomes

Professor Alfred Cuschieri

Department of Anatomy, University of Malta

Objectives

Identify lysosomes in electron micrographs

State the characteristics and functions of lysosomes

Explain how lysosomes are formed

Name the properties of lysosomal membranes

Explain the mechanisms and importance of phagocytosis

Give examples of phagocytic cells

Explain the importance of receptor-mediated endocytosis

Describe how lysosomal enzyme deficiency cause serious disease

Identify peroxisomes and state their characteristic features

Explain the importance of peroxidase generation and degradation in lysosomes

Name some diseases caused by deficient functions of lysosomes

Distinguish between melanosomes and melanin synthesis

Distinguish between melanosomes and melanocytes

Lysosomes

are membrane bound organelles

contain hydrolytic enzymes

have acidic contents (pH 4.5-5.5)

have electron-dense heterogeneous contents

digest ingested material and aged or damaged organelles

Lysosomal enzymes are hydrolases that catalyse the reaction: AB + H₂O à AH + BOH

Over 60 lysosomal enzymes are known.

There is a hydrolyse for each type of biological molecule. These include:

Peptidases – hydrolyse proteins

DNAases – hydrolyse DNA

RNAases – hydrolyse RNA

Lipases – hydrolyse lipids

Phosphatases – hydrolyse phosphates

Glucosidases – hydrolyse glycogen

Carboxylases - hydrolyse carboxyl groups

Sulphatases – hydrolyse sulphates

Esterases – hydrolyse esters

Acid phosphatase and b-glucuronidase have been used as histochemical markers for lysosomes.

The lysosome membrane has three special properties:

1. an ATP driven proton [H⁺] pump to maintain a low pH (4.5-5.5) in the lysosomal compartment.

The proton pump:

Has a ‘lollipop’ structure similar to the F¹ and F^o of mitochondria, and consisting of:

a head containing 6 polypeptide units

a stalk contains 5 polypeptide units

Is inhibited by n-ethylmaleimide

2. a glycoprotein coat, rich in carbohydrates, on its inner surface to protect it against hydrolysis by its own enzymes

3. transporter channels that transport break-down products such as amino acids, glucose, nucleotides and other small molecules out of the lysosome. These molecules may move out

a. by facilitated diffusion ;

b. by active transport;

c. by co-transport using the proton (H+) gradient to provide the energy for transport.

a.

The Formation of Lysosomes

a. The hydrolytic enzymes are formed in the RER

b. Enzymes have a terminal mannose–6-phosphate group as a marker to be packaged in lysosomes

c. They are packaged at the trans face of the Gogi complex (termed the Trans Golgi Network -TGN)

d. The newly formed vesicles that bud off are termed early endosomes or primary lysosomes. They are small and have homogeneous, electron-dense contents.

e. They early endosomes (primary lysosomes) may fuse with phagocytic or other vesicle to form late endosomes or secondary lysosomes.

Functions of Lysosomes

Lysosomes may be involved in the following pathways:

1. phagocytosis

2. autophagocytosis

3. Formation of endosomes

4. Receptor-mediated endocytosis

1 Phagocytosis

Phagocytosis is the process by which the cell engulfs particulate matter (>0.5 mm) from the extra cellular space, and digests it by lysosomal action. Three main cell types are capable of performing this function:

a. Macrophages

i. are widely distributed in the connective tissue around the body

ii. phagocytose and digest all particulate matter in the extracellular space, including micro-organisms, foreign particles and damaged cells.

iii. are derived from monocytes, circulating in the peripheral blood.

iv. may have different names in different tissues such as Kupffer cells in the liver, osteoclasts in bone, and microglia in the central nervous system.

b. Neutrophils in the peripheral blood, which phagocytose and digest microorganisms.

c. Eosinophils in the peripheral blood, which phagocytose and digest antigen-antibody complexes.

Macrophages have a gylcocalyx coat, rich in glycosaminoglycans, on their outer surface, which causes particulate matter to adhere to them. Neutrophils and eosinophils have specific receptors to recognise specific particles to be engulfed. (The specific molecules that bind to the receptors are called ligands).

Phagocytosis involves:

a. Adhesion of the particle to the glycocalyx or specific receptor on the plasma membrane

b. Extrusion of pseudopodia to surround the particle. This is mediated by actin filaments.

c. Formation of a phagocytic vacuole containing the engulfed particle

d. Fusion with a primary lysosome

Autophagocytosis

This is the process in which old or damaged organelles are broken down. It occurs in practically all cells as a recycling system. It is most marked in cells that are not replaced, such as neurons.

Autophagocytosis involves:

a. The organelle is surrounded by vesicles, which coalesce

b. The coalesced vesicles form a membrane surrounding the organelle. This is called an autophagic vacuole.

c. The autophagic vacuole fuses with one or more primary lysosomes to form secondary lysosomes.

d. Residual bodies are lysosomes with partially undigested material

Endocytosis and exocytosis

Endocytosis is the uptake of extra cellular fluid by infolding of the plasma membrane, and formation of a vesicle containing the extra cellular material. This process was formerly called pinocytosis.

Exocytosis is the reverse of pinocytosis, i.e. the extrusion of fluid contained in vesicles into the extra cellular space.

Endocytosis and exocytosis are important mostly for membrane flow. For example, exocytosis replaces the plasma membrane removed by phagocytosis. Similarly, exocytosis of secretion vesicles must be balanced by endocytosis.

Receptor-mediated endocytosis

Receptor-mediated endocytosis is the process whereby cells that have a specific receptor take up specific macromolecules.

This process is used for the uptake of hormones, growth factors, antibodies, lipoproteins etc

The receptors are integral membrane proteins

The molecule that binds to the receptor is termed the ligand.

The process of receptor-mediated endocytosis involves the following steps:

1 Binding of the ligand to the receptor

2 Lateral diffusion of the ligand-receptor complex

3 Accumulation of clathrin, adaptor protein and dynamin on the cytoplasmic surface of the plasma membrane at a particular site

4 Formation of a pit, and accumulation of the ligand receptor complex at the site of clathrin acculmulation

5 Deepening of the pit and formation of clathrin-coated vesicles containing the ligand-receptor complex

6 The vesicles lose their clathrin coat

7 The vesicles fuse with a primary lysosome (early endosome) and the ligand is cleaved from the receptor

Familial hypercholesterolaemia is a condition in which there is defective binding of low density lipoprotein (LDL) to its receptor. Receptor-mediated endocytosis of the LDL does not occur, and it accumulates as high levels in the blood.

The process of receptor-mediated endocytosis was first described through the study of LDL uptake by cultured fibroblasts.

Three molecules are involved in forming the coat on the cytoplasmic face of coated vesicles:

1. Clathrin

§ A molecule that has a triskelion (three-pronged) structure.

§ Under appropriate conditions it forms a hexagonal (geodesic) latticework on the cytoplasmic surface of the plasma membrane,

§ It causes the membrane to invaginate and form a coated pit and vesiclevesicle

2. Adaptor protein

§ Binds to the cytoplasmic end of the transmembrane receptor

§ Mediates the attachment of clathrin to the plasma membrane

§ Regulates clathrin assembly and disassembly

§ Is itself regulated by phosphorylation and dephosphorylation

3. Dynamin

§ Incorporates a GTPase that provides the energy for formation of the coated pit and vesicle

§ Undergoes a configuration change that brings about closure of the vesicle

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Gangliosidosis is an example of a Lysosomal Storage Disease

Gangliosidosis:

Is caused by deficiency of the enzymes b-galactosidase.

Is associated with inability to break down the terminal galactose from GM1 ganglioside

Results in accumulation of GM₁ ganglioside in lysosomes, which are consequently greatly enlarged.

Enlarged lysosomes accumulate in the cells of several tissues, the most important of which are:

Involvement of neurons of the central nervous system

-fits, psychomotor deterioration, mental retardation

Enlargement of liver & spleen

Widening of bones: deposition in marrow

Vacuolated lymphocytes deposition in lymphocytes

This disease is inherited as an autosomal recessive disorder.

Other Lysosomal Storage Diseases

There are several lysosomal storage disorders. All are associated with a deficiency of a particular lysosomal enzyme, resulting in accumulation of an undigested substrate within the lysosomes. The following are a few examples of lysosomal storage disease with the associated substrate:

Pompe à glycogen

Hunter disease à heparan & dermatan sulphates

Morquio’s disease à keratan& chondroitin sulphate

Tay Sachs disease à GM2 ganglioside

Niemann-Pick à sphingomyelin

Farberdisease à ceremide

Peroxisomes or Microbodies

The features of peroxisomes are:

Membrane-bound spherical organelles

Have a diameter of 0.2-2.0mm

Contain a granular matrix and an electron-dense crystalline core

Contain the enzymes catalase and urate oxidase

Have the ability to break down H2O2

Have a pH optimum of 7.5

Oxidases produce H₂O₂

RH2 + O2 à R + H2O2

Catalase decomposes H₂O₂

- by conversion to water

2H2O2 à H2O

+ O2

- by oxidation of another organic compound

H2O2 + AH2 à 2H2O2 + A

H2O2 is both produced and degraded in peroxisomes.

H2O2 is harmful to cells and has to be degraded almost as fast as it is produced.

Many substances are broken down or metabolised by oxidative reactions in peroxisomes.

The most important ones are:

1. b-Oxidation of fatty acids

very long are chain fatty acids are broken down to acetyl CoA with the production of H2O2

This reaction also occurs in mitochondria.

2. Transfer of NH3 (amino) groups from amino acids to ketoacids

- requires the enzyme aminotransferase

3. Oxidation of uric acid or urates to allantoin

requires the enzyme urate oxidase

All are associated with the production of H2O2

Peroxisomes also have synthetic functions including:

- synthesis of cholesterol and dolicholol (also occurs in SER)

- synthesis of plasmalogens (membrane components in brain & heart)

- synthesis of bile acids

Some diseases are caused by lack of specific peroxisome enzymes:

· Adrenoleukodystrophy

- a progressive neurological disorder

- a defect in b-oxidation of long chain fatty acid,

- results in accumulation of long chain fatty acids in neurons and other cells

· Gout due to accumulation of uric acid

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·

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- failure of conversion of uric acid to allantoin

- causes deposition of uric acid in joints, and consequent painful arthritis

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· Zellweger syndrome caused by absence of peroxisomes

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- causes severe neurological disorder, metabolic defects, and early death

Melanosomes

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Melanosomes are the organelles in which melanin synthesis occurs.

They are :

- Oval-shaped , membrane-bound bodies

- 0.3-1.3 mm in diameter

- found in melanocytes (pigment-producing cells)

- contain enzymes for the biosynthetic pathway of melanin

- are identified by the DOPA reaction

Biosynthetic pathway of melanin formation

A multi-step pathway in which tyrosine is DOPA by a series of reactions involving the enzyme tyrosinase as well other enzymes. DOPA is then converted to melanin.

In the DOPA reaction, cells are exposed to an excess of DOPA. Melanin pigment will be formed in the melanosomes, the organelles that have the necessary enzymes for the biosynthesis of melanin.

Morphological pathway of melanosome formation

Melanosomes are formed from two sources:

From the SER – pre-melanosomes are formed by coalescence of vesicles, and appear to contain parallel filamentous contents.

In the RER and Golgi complex, tyrosinase and other enzymes are synthesised and packaged into vesicles. These fuse with the pre-melanosomes.

Melanosomes are stimulated by Melanocyte Stimulating Hormone (MSH)

The plasma membrane of melanocyte has receptors for MSH. The MSH enters the cells by receptor-mediated endocytosis and the MSH-containing vesicles fuse with the melanosomes.

Melanin can be transferred from melanocytes to keratinocytes in the skin.

Melanocytes have numerous dendritic processes.

Melanin-containing vesicles are pinched off from the tips of these processes.

They are endocytosed by the keratinocytes, degraded by lysosomes and the melanin become dispersed within these cells.