Endocytosis

Endocytosis is the process by which cells absorb molecules (such as proteins) from outside the cell by engulfing it with their cell membrane. It is used by all cells of the body because most substances important to them are large polar molecules that cannot pass through the hydrophobic plasma membrane or cell membrane. The process opposite to endocytosis is exocytosis.

Types
There are three main types of endocytosis that are distinguished by the size of the vesicle formed and the cellular machinery involved.
 * Phagocytosis (literally, cell-eating) is the process by which cells ingest large objects, such as bacteria, viruses, or the remnants of cells which have undergone apoptosis. The membrane invaginates enclosing the wanted particles in a pocket, then engulfs the object by pinching it off, and the object is sealed off into a large vacuole known as a phagosome.


 * Pinocytosis (literally, cell-drinking). This process is concerned with the uptake of solutes and single molecules such as proteins. Both phagocytosis and pinocytosis are non-receptor-mediated forms of endocytosis, and may result in the cell engulfing non-specific or unwanted particles.


 * Receptor-mediated endocytosis is a more specific active event where the cytoplasm membrane folds inward to form coated pits. In this case, proteins or other trigger particles lock into receptors/ ligands in the cell’s plasma membrane. It is then, and only then that the particles are engulfed. These inward budding vesicles bud to form cytoplasmic vesicles. This process may also result in engulfing of unwanted particles, however not to the extent of pino/phagocytosis.

Function of endocytosis
Endocytosis is required for a vast number of functions that are essential for the well being of cell. It intimately regulates many processes, including nutrient uptake, cell adhesion and migration, receptor signaling, pathogen entry, neurotransmission, receptor downregulation, antigen presentation, cell polarity, mitosis, growth and differentiation, and drug delivery.

Endocytosis pathways
Endocytosis pathways could be subdivided into four categories: namely, clathrin-mediated endocytosis, caveolae, macropinocytosis, and phagocytosis.


 * Clathrin-mediated endocytosis is mediated by small (approx. 100 nm in diameter) vesicles that have a morphologically characteristic crystalline coat made up of a complex of proteins that mainly associated with the cytosolic protein clathrin. Clathrin-coated vesicles (CCVs) are found in virtually all cells and from domains of the plasma membrane termed clathrin-coated pits. Coated pits can concentrate a large extracellular molecules that have different receptors responsible for the receptor-mediated endocytosis of ligands, e.g. low density lipoprotein, transferrin, growth factors, antibodies and many others.


 * Caveolae are the most common reported non-clathrin coated plasma membrane buds, which exist on the surface of many, but not all cell types. They consist of the cholesterol-binding protein caveolin (Vip21) with a bilayer enriched in cholesterol and glycolipids. Caveolae are small (approx. 50 nm in diameter) flask-shape pits in the membrane that resemble the shape of a cave (hence the name caveolae). They can constitute up to a third of the plasma membrane area of the cells of some tissues, being especially abundant in smooth muscle, type I pneumocytes, fibroblasts, adipocytes, and endothelial cells. Uptake of extracellular molecules is also believed to be specifically mediated via receptors in caveolae.


 * Macropinocytosis, which usually occurs from highly ruffled regions of the plasma membrane, is the invagination of the cell membrane to form a pocket, which then pinches off into the cell to form a vesicle (0.5-5µm in diameter) filled with large volume of extracellular fluid and molecules within it (equivalent to 103 to 106 CCVs). The filling of the pocket occurs in a non-specific manner. The vesicle then travels into the cytosol and fuses with other vesicles such as endosomes and lysosomes.


 * Phagocytosis is the process by which cells bind and internalize particulate matter larger than around 0.75 µm in diameter, such as small-sized dust particles, cell debris, micro-organisms and even apoptotic cells, which only occurs in specialized cells. These processes involve the uptake of larger membrane areas than clathrin-mediated endocytosis and caveolae pathway.

More recent experiments have suggested that these morphological descriptions of endocytic events may be inadequate, and a more appropriate method of classification may be based upon the clathrin-dependence of particular pathways, with multiple subtypes of clathrin-dependent and clathrin-independent endocytosis. Mechanistic insight into non-phagocytic, clathrin-independent endocytosis has been lacking, but a recent study has shown how Graf1 regulates a highly prevalent clathrin-independent endocytic pathway known as the CLIC/GEEC pathway.

Principal components of endocytic pathway
The endocytic pathway of mammalian cells consists of distinct membrane compartments that internalize molecules from the plasma membrane and recycle them back to the surface (early endosomes and recycling endosomes) or sort them to degradation (late endosomes and lysosomes). The principle components of endocytic pathway are:
 * Early endosomes are the first station on the endocytic pathway. Early endosomes are often located in the periphery of the cell and receive most of types of vesicles coming from the cell surface. They have a characteristic tubulo-vesicular morphology (vesicles up to 1µm in diameter with connected tubules of approx. 50nm diameter) and a mildly acid pH. They are principally sorting organelles where many ligands dissociate from their receptors in the acid pH of the lumen and from which many of the receptors recycle to the cell surface (via tubules). It is also the site of sorting into transcytotic pathway to late components (via vesicular component which can form multivesicular bodies (MVB) or endosomal carrier vesicles (ECVs)).
 * Late endosomes receive internalized material en route to lysosomes, usually from early endosomes in the endocytic pathway, from trans-Golgi network (TGN) in the biosynthetic pathway, and from phagosomes in the phagocytic pathway. Late endosomes often contain many membrane vesicles or membrane lamellae and proteins characteristic of lysosomes, including lysosomal membrane glycoproteins and acid hydrolases. They are acidic (approx. pH 5.5), and are part of the trafficking itinerary of mannose-6-phosphate receptors. Late endosomes are thought to mediate a final set of sorting events prior to delivery of material to lysosomes.
 * Lysosomes are the last compartment of the endocytic pathway. They are acidic (approx. pH 4.8) and by EM usually appear as large vacuoles (1-2 µm in diameter) containing electron dense material. They have a high content of lososomal membrane proteins and active lysosomal hydrolases, but no mannose-6-phosphate receptor. They are generally regarded as the principle hydrolytic compartment of the cell.

Clathrin-mediated endocytosis
The major route for endocytosis in most cells, and the best-understood, is that mediated by the molecule clathrin. This large protein assists in the formation of a coated pit on the inner surface of the plasma membrane of the cell. This pit then buds into the cell to form a coated vesicle in the cytoplasm of the cell. In so doing, it brings into the cell not only a small area of the surface of the cell but also a small volume of fluid from outside the cell.

Coats function to deform the donor membrane to produce a vesicle, and they also function in the selection of the vesicle cargo. Coat complexes have been well characterized so far including: coat protein-I (COP-I), COP-II, and clathrin. Clathrin coats are involved in two crucial transport steps: (i) receptor-mediated and fluid-phase endocytosis from the plasma membrane to early endosome and (ii) transport from the TGN to endosomes. In endosytosis, the clathrin coat is assembled on the cytoplasmic face of the plasma membrane, forming pits that invaginate to pinch off (scission) and become free CCVs. In cultured cells, the assembly of a CCV takes ~ 1min, and several hundred to a thousand or more can form every minute. The main scaffold component of clathrin coat is the 190 kD protein called clathrin heavy chain (CHC) and the 25 kD protein called clathrin light chain (CLC), which form three-legged trimers, called triskelions.

Vesicles selectively concentrate and exclude certain proteins during formation and are not representative of the membrane as a whole. AP2 adaptors are multisubunit complexes that perform this function at the plasma membrane. The best-understood receptors that are found concentrated in coated vesicles of mammalian cells are the LDL receptor (which removes LDL from circulating blood), the transferrin receptor (which brings ferric ions bound by transferrin into the cell) and certain hormone receptors (such as that for EGF).

At any one moment, about 25% of the plasma membrane of a fibroblast is made up of coated pits. As a coated pit has a life of about a minute before it buds into the cell, a fibroblast takes up its surface by this route about once every 16 minutes. Coated vesicles formed from the plasma membrane have a diameter of about 36 nm and a lifetime measured in a few seconds. Once the coat has been shed, the remaining vesicle fuses with endosomes and proceeds down the endocytic pathway. The actual budding-in process, whereby a pit is converted to a vesicle, is carried out by clathrin assisted by a set of cytoplasmic proteins, which includes dynamin and adaptors such as adaptin.

Coated pits and vesicles were first seen in thin sections of tissue in the electron microscope by Matt Lions and Parker George. The importance of them for the clearance of LDL from blood was discovered by R. G Anderson, Michael S. Brown and Joseph L. Goldstein in 1976. Coated vesicles were first purified by Barbara Pearse, who discovered the clathrin coat molecule.