Generally speaking, normal endocytosis can be divided into three stages: (1) bud formation; (2) membrane bending and vesicle maturation; and (3) membrane rupture and release into the cytoplasm. Multiple endocytosis pathways overlap, so the general process of endocytosis is highly flexible and complex.
Clathrin-mediated endocytosis
Clathrin-mediated endocytosis (CME) is conceptually a simple process that involves several consecutive and partially overlapping steps. CME can be initiated structurally by some receptors on the plasma membrane or after binding with ligands and/or antibodies. CME begins when the endocapsid proteins in the cytoplasm begin to accumulate on the inner lobules of the plasma membrane. Capsid proteins continue to assemble and grow by recruiting from the cytoplasm and interacting with additional protein adapters. The key binding protein bends the membrane, thus concentrating the internalized receptors and ligands into a “clathrin-coated pit” (CCP). Due to the increase in CCP invagination, when the neck of the CCP narrows, it is separated from the plasma membrane through a fracture process. Actin polymerization helps to pull CCP inward into the cytoplasm until the breakage is complete and CCP is released and becomes a clathrin-coated vesicle (CCV). Finally, the CCV shell is broken down and the CCV fuses with the endosome to be transported to a specific subcellular location, or it can be recycled back to the cell surface.
Clathrin is the key component of CME, which is composed of heavy chain and light chain. The heavy and light chains of three clathrins form a trimer, which interacts with other trimers and forms a polygonal lattice around the emerging CCP. Connexin 2 (AP-2) is a heterotetramer complex that mediates neck contraction in CCP. Dynamin is a GTP enzyme that forms a spiral polymer in the neck of mature vesicles. After hydrolysis of GTP, dynamin-induced vesicles divide from the plasma membrane.
Caveolae-mediated endocytosis
Clathrin-independent endocytosis includes caveolae-mediated endocytosis, caveolae-independent carrier protein/GPI-enriched early endocytic compartments, and macropinocytosis.
The caveolae is a small bottle-shaped invagination of the plasma membrane, characterized by high levels of cholesterol and glycosphingolipids, which mediates endocytosis through a clathrin-independent pathway and exists in most cell types. The main scaffold protein of the caveolae is caveolin, which is an integral membrane protein of 20mur24 kDa that forms oligomers. Caveolins share common scaffold domains that mediate interactions with themselves and other proteins containing caveolin binding domains.
Although the caveolae have an invagination shape similar to that of CCPs, they are different. Simply put, the density of CCP is constant, while the density of caveolae varies greatly from cell type to cell type. CCPs increase with the maturation of the germinated endosome, while the vesicular remains the same size. Once inside the cell, the caveolae form a higher-order structure rather than a simple spherical endosome formed by CCPs.
Another unique aspect of caveolin-mediated endocytosis is that only about 1% of the caveolae germinate from the plasma membrane. In a small part of the internalized caveolae, it seems to follow a circulatory pathway co-located with Rab5 (a marker of early endoderm). This may pose a challenge to ADC targeting receptors that use caveolin-mediated endocytosis.
CLIC/GEEC endocytosis
CLIC/GEEC is an endocytosis chamber that occurs mainly in ligand-activated cells, which may be caused by growth factors, antibody receptor cross-links, bacterial toxins, or viruses. In addition, the cell membrane must be in a state of high fluidity because CLIC/GEEC does not work when it is lower than the physiological temperature or when the membrane is under higher tension.
CLIC increased at the frontal edge of the migrating cells. Other relevant parameters for identifying the CLIC/GEEC pathway include dynamic independent membrane breakage, sensitivity to cholesterol consumption, acquisition of Rab5/ fusion with early endosomes, placental alkaline phosphatase (PLAP), and GTPase regulatory associated with FAK (GRAF1).
Macropinocytosis
Macropinocytosis is a larger form of endocytosis that usually involves highly wrinkled areas/processes of the plasma membrane, which then fuse with each other or with the plasma membrane. The wrinkled membrane is the morphological feature of macropinocytosis.
Macropinocytosis depends on actin polymerization, Rac1 protein, and p21-activated kinase 1 (PAK1). PAK1 is a key regulator because it interacts with Rac1. Rac1 activates phosphatidylinositol-3-kinase (PI3K), Ras, Src, and Hsp90 to promote macropinocytosis. Macropinocytosis is also cholesterol-dependent, which is necessary to recruit Rac1. These components eventually lead to endocytosis with a larger absorption area than CME and caveolins.