Exocytosis and Endocytosis are the process which help cells to release or take up large molecules or material respectively in a controlled mechanism by forming membrane enclosed vacuoles or vesicles around them.

Factors that play role in exocytosis and endocytosis

• The flexibility, self sealing and the fusion of cell membranes mediated by specific proteins play an important role in exocytosis and endocytosis and viral invasion.
• Formation of vesicles and their transport within the cell plays on important role in these processes.
• Microtubules and associated ATP driven motors appear to have a role in the intracellular transport of vesicles as they guide targeting vesicles to different and specific destination.
• Molecular motor, kinesin and dynein, transport vesicles along microtubules in the axon of a nerve cell.

Table of Contents

• Factors that play role in exocytosis and endocytosis
  • Exocytosis
    • Steps for exocytosis
    • Types of exocytosis
      • Constitutive exocytosis
      • Regulated exocytosis
    • Proteins and complexes involved in exocytosis
  • Endocytosis
    • Phagocytosis
    • Clathrin-mediated endocytosis (Receptor mediated endocytosis)
    • Caveolae
    • Macropinocytosis
    • Transcytosis

Exocytosis

• Exocytosis is the process by which the cells direct the contents of secretory vesicles out of the cell membrane.
• These secretory vesicles contain soluble proteins which is to be secreted to the extracellular environment. Also they contan membrane proteins and lipids that are sent to become components of the plasma membrane.
• It is the final step in the secretory pathway that typically starts in the endoplasmic reticulum (ER), passes through the Golgi apparatus using motor proteins and a cytoskeletal track to get closer to cell membrane, and ends at the outside of the cell.
• Some of the examples include secretion of proteins like enzymes, peptide hormones and antibodies from cells and release of neurotransmitter from presynaptic neurons.

Steps for exocytosis

1. Once these vesicles carrying secretory proteins each their targets, they come into contact with tethering factors that can restrain them.
2. Then the process of vesicle tethering distinguishes between the initial, loose tethering of vesicles from the more stable, packing interactions.
3. Tethering involves links over distances of more than about half the diameter of a vesicle from a given membrane surface (>25 nm).
4. The process of holding two membranes within a bilayer’s distance of one another (<5-10 nm) is called vesicle docking.
5. Stable docking indicates the molecular interactions underlying the close and tight association of a vesicle with its target may include the molecular rearrangements and ATP-dependent protein and lipid modifications, needed to trigger bilayer fusion called vesicle priming.
6. It is mostly takes place before exocytosis and used in regulated secretion type of exocytosis but not used in constitutive secretion.
7. It is followed by vesicle fusion which includes merging of the vesicle membrane with the target and hence there is release of large biomolecules in the extracellular space with the help of some protein complex.

Types of exocytosis

Exocytosis are of two types. Constitutive exocytosis and Regulated exocytosis.

Constitutive exocytosis

• Secretory materials are continuously released without requirement of any specific kind of signal.

Regulated exocytosis

• Regulated exocytosis requires an external signal, a specific sorting signal on the vesicles for release of components.
• It contains a class of secretory vesicles that fuse with plasma membrane following cell activation in presence of signal.
• Examples of regulated exocytosis are secretion of neurotransmitter, hormones and many other molecules.
• It can also be explained as  the vesicle containing secretory proteins releases its contents or membrane receptors etc. to the outside of the cell or to the plasma membrane on receiving a signal such as Ca²⁺ increase or hormone etc.
• The regulated exocytosis includes:-
  • The transport of Glut–4 (Glucose Transporter–4) containing vesicles to plasma membrane of adipose and muscle cells when insulin binds to its receptor in these cells.
  • Secretion of digestive enzymes enclosed in vesicles by acinar cells of pancrease directly into the lumen of the intestines on receiving hormonal signal.
  • Release of acetylcholine from vesicles containing acetyl choline which fuse with cell membrane at synaptic when Ca²⁺ level rises as a result of the opening of voltage gated Ca²⁺ channels in neurons.
  • In the Ca²⁺ induced formation of a barrier to further sperm penetration after fertilization of an ovum.

Proteins and complexes involved in exocytosis

• As already discussed, exocytosis is thus a general term used to denote vesicle transport and fusion at plasma membrane and the release of its contents.
• We know that exocytosis is the final step in the secretory pathway that begins at endoplasmic reticulum, passes through the Golgi apparatus and ends outside the cell.
• Sorting of contents in the vesicles occurs in the endoplasmic reticulum, golgi or post golgi compartments and are modified enzymatically into their mature form ready for delivery.
• Vesicle formation for transport between golgi involves:-
  • Vesicle formation for transport between golgi involves a mechanism in which GTP–protein complexes bind to the vesicle membrane which results in binding of COP (for coat protein) molecules and four other proteins to form a complex called Coatamer around the vesicle.
  • Budding of the vesicle at the binding site leads to the formation of a coated (non is clathrin coated) vesicle which play a role in intra golgi transport.
  • These coated vesicles uncoat at the target membrane as a result of GTP hydrolysis to GDP by Rab proteins in mammals, which are GTPases.
  • The Rab proteins play a crucial role where GTP hydrolysis is the signal for uncoating, docking and fusion of vesicles to its target membrane.
• Recognition of the vesicle for the target membrane sites is via special recognition proteins such as:
  • v–SNARE (where v is for vesicle and SNARE, soluble NSF attachment protein receptor, located in the external surface of the vesicle).
  • t–SNARE (t is for target vesicle) is the partner protein located on the cytosolie face of the target membrane which is plasma membrane in case of exocytosis.
  • Two other proteins, NSF, N–ethyl maleimide sensitive factor) and SNAP (a soluble NSF attachment Protein) besides other proteins are important in docking and fusion of vesicle in animals and yeast cells.
  • NSF may act to prime SNAREs before docking and to recycle SNAREs after fusion. Specific v–and t– SNARE are asociated with exocytosis.
  • Different combinations of SNAREs and other proteins are characteristic of other docking events. (Docking is the process by which the vesicle is fixed beneath the target membrane or plasma membrane on the cytosolic side before fusion requiring molecular recognition between vesicle and the target membrane).
• The basic model predicts that while docking a dimer or more likely multimeric complexes are formed with one or more SNAREs from each of the two fusing membrane.
• The fusion of the vesicles at the plasma membrane leads to the formation of fusion pore which is a channel that passes through the vesicle and the plasma membrane and allows the content of the vesicle to move out to the extracellualr milieu or compartment.
• After the transient fusion pore opening, as the contents of the vesicle are released, there is a rapid closure of the pore.
• Alternatively, the pore formation is followed by full pore opening with incorporation of vesicle membrane into the plasma membrane.
• Both transient and permanent fusion can be found in the same animal.
• In presynaphic nerve terminal when there is an increase in Ca²⁺, signals the release of acetylcholine, the core complex which is trimeric, comprising of two t–SNAREs Syntaxin and SNAP–25, (synaptosome associated protein of 25 KD) and synaptobrevim, a v–SNARE is formed which is a bundle of four .
• This pulls the two membranes together and the bilayer is disrupted at that point which leads to membrane fusion, fusion pore formation, and the release of the neurotransmitter at a synapse.
• The complex of SNAREs and SNAPs is digested by the Clostridium botulinum toxin which is a protease and thus blocks neurotransmission causing death of the organism.

Endocytosis

1. Endocytosis is the process by which cells absorb larger molecules and particles from the surrounding by engulfing them.
2. It is used by most of the cells because large and polar molecules cannot cross the plasma membrane.
3. The material to be internalized is surrounded by plasma membrane, which then buds off inside the cell to form vesicles containing ingested material.
4. Also, membrane proteins, receptors and transporters are endocytosed e.g. GLUT–4, a glucose transporter in muscle and adipose cells is endocytosed when glucose concentration in blood falls and insulin dissociates from its receptor.
5. The endocytosis pathway is divided into five categories:-
   1. Phagocytosis
   2. Clathrin-mediated endocytosis (Receptor mediated endocytosis)
   3. Caveolae
   4. Macropinocytosis
   5. Transcytosis

Phagocytosis

• Phagocytosis is the process by which certain living cells called phagocytes engulf larger solid particles such as bacteria, debris or intact cells.
• Certain unicellular organisms, such as the protists, use this particular process as means of feeding. It provides them part or all of their nourishment. This mode of nutrition is known as phagotrophic nutrition.
• In ciliates, a specialized groove or chamber, known as the cytostome, is present, where the process takes place.
• In amoeba, phagocytosis takes place by engulfing the nutrient with the help of pseudopods, that are present all over the cell. The various phases of phagocytosis in amoeba for food capturing are
  1. Adherence of the macromolecules to the receptor on the phagocytic cell
  2. Extension of pseudopodia and ingestion of microbe by phagocytic cell
  3. Formation of phagosome by the fusion of surrounding membrane
  4. Fusion of phagosome and lysosome to form phagolysosome
  5. Digestion of the ingested macromolecules by the acid hydrolytic enzymes in the lysosome
  6. Formation of residual body coating indigestible material
  7. Discharge of waste materials.
• Other examples of phagocytosis include some
  • immune system cells, that engulf and kill certain harmful. There are two types of phagocytes (WBC) in mammals:-
    • Macrophages and Neutrophils-In these cells, the engulfment of foreign material is facilitated by actin-myosin contractile system. It allows the cell membrane to expand in order to engulf the particle and then contract immediately, ingesting it.
    • Macrophages also remove dead cells
  • infectious micro-organisms and
  • other unwanted foreign materials which in turn provides defence against invading micro-organism and eliminate damaged cells from the body.

Clathrin-mediated endocytosis (Receptor mediated endocytosis)

• Clathrin-mediated endocytosis is also known as receptor mediated endocytosis.
• It is the process of internalizing molecules into the cell by the inward budding of plasma membrane vesicles containing proteins with receptor sites specific to the molecules being internalized.
• Phases of clathrin-mediated endocytosis:-
  1. Macromolecules (as ligands) bind to the specific cell surface receptors.
  2. Then the receptors are concentrated in specialized regions of plasma membrane and clathrin and adaptor protein binds to these receptor forming clathrin-coated pits.
  3. These pits bud from the membrane and form clathrin-coated vesicles containing receptors, proteins and ligands.
  4. Then these vesicles fuse with early endosomes, in which the contents are sorted for the transport to lysosomes and receptors and proteins are recycled to plasma membrane.
• The example for clathrin mediated endocytosis is uptake of cholesterol by the mammalian cells. Here cholesterol is transported through the blood stream in the form of lipoprotein or LDL.
  • LDL–receptor is a glycoprotein. It specifically binds apolipoprotein B–100 and apoE. The receptor synthesized in the endoplasmic reticulum, matured in the golgi complex, migrates to plasma membrane by exocytosis. It is located in an invagination pit or coated pit where the most abundant protein is clathrin. Clathrin is made up of triskelion units which are self interacting and form a cage like structure.
  • Phases for receptor mediated endocytosis for cholesterol uptake involves:-
  1. Receptor Binding & its activation: Here LDL receptor binds to Apo-B protein on the LDL particle.
  2. Coated Pit Formation: Clathrin forms cage around forming endosome.
  3. Clathrin-Coated Vesicle Budding.
  4. Uncoating of the Vesicle.
  5. Early Endosome associates with other vesicles.
  6. Formation of CURL (Compartment for Uncoupling of Ligand and Receptor) or Late Endosome.
  7. Recycling of the Receptor to the cell surface.
  8. Fusion of Transport Vesicle with Lysosome.
  9. Digestion of the LDL to Release Cholesterol.
• Hypercholesterolemia
  • In patients suffering from familial hypercholesterolemia, having high levels of cholesterol in serum and hence suffer from heart attacks early in life. Because these patients are unable to internalize LDL from the extracellular fluids, result in high accumulation of cholesterol.
  • Normal individual possess LDL for transport of cholesterol but familial hypercholesterolemia results from inherited mutation in LDL receptor.
  • These mutations can happen in two ways.
    • Either the patients simply fail to bind with LDL, demonstrating that a specific cell surface receptor is required for uptake of cholesterol.
    • The patients are able to bind with LDL but are unable to internalize it. Because they are unable to concentrate in coated pits, demonstrating that coated pits in receptor plays an important role for cholesterol uptake.
    • This mutation lies in the cytoplasmic tail of the receptor and can be subtle as the change of a single tyrosine to cysteine.
  • Other example for receptor mediated endocytosis is for iron uptake

Caveolae

• Caveolae is a pathway which is independent of clathrin- endocytosis process and involves in the uptake of molecules in small invaginations of the plasma membrane (50 to 80 mm diameter).
• These are enriched in lipid rafts of cholesterol, phospholipid and sphingolipids and possess a distinct coat formed by a protein called caveolin (cholesterol binding protein).
• It is abundant in smooth muscle, type I pneumocytes, fibroblasts, adipocytes, and endothelial cells.
• Cells mostly use caveolae for the selective uptake of molecules as small as folate to full size proteins such as albumin and alkaline phosphatase.
• Many studies have shown that caveolae-mediated uptake of materials is not limited to macromolecules.
• In certain cell-types, viruses as simian virus 40 and even entire bacteria as some specific strains of E. Coli are engulfed and transferred to intracellular compartments in a caveolae-dependant fashion.

Macropinocytosis

• The process of uptake of fluids in large vesicles (0.15 to 5 μm in diameter) is called macropinocytosis.

Transcytosis

• The process of transfer of internalized receptor across the cell to opposite domain of the plasma membrane is called transcytosis.
• It occurs in polarized cells or epithelial cells mostly.
• It is used for protein sorting and also a mean for transport of macromolecules across the epithelial cell sheets.
• Example:-
  • Transport of Abs from blood to other secreted fluids. The Abs bind to the receptor on basolateral surface and then transcytosed along with their receptors to apical surface. The receptors are then cleaved and release Abs into extracellular secretions.