Fluid-phase uptake by pinocytosis can be broadly categorised according to the size of the endocytic vesicle and this also relates to how the vesicle is coated (Figure 35). The rate of internalisation is directly proportional to (i) the concentration of extracellular molecules, (ii) the volume enclosed by the vesicle and (iii) the rate of vesicle formation. Greater efficiency of endocytosis can be achieved by binding of the extracellular molecules to the membrane. The most efficient uptake occurs when molecules from the extracellular environment bind to specific receptors, i.e. receptor-mediated endocytosis. In contrast, phagocytosis is concentration-independent, but as with pinocytosis, the entry into the cell is determined by the type of cargo and its receptor. Both processes also serve to internalise and recycle membrane proteins and lipids.
In many cell types, stimulation by growth factors often induces membrane disturbance, which ultimately leads to macropinocytosis. Macropinocytosis is a non-specific mechanism for internalisation, in which lamellipodia extend at a site of membrane ruffling to form irregular vesicles, containing large volumes of extracellular fluid. Macropinocytosis is often induced as part of the response to stimulation by growth factors. The extension of lamellipodia is driven by the extension of actin filaments, in a process controlled by small GTPases belonging to the Rho family. (Be careful not to confuse this family of molecules with the Rho protein involved in termination of transcription.) Rho was the first member of a large family of GTPases to be discovered. It is involved in numerous cellular events, including pinocytosis, cell signalling and cell migration. Many of these events involve the reorganisation of the cytoskeleton, for different purposes. Other members of the Rho family are Rac, Rap1 and Cdc42.
In contrast to macropinocytosis, the mechanism of clathrin-coated-vesicle dependent, clathrin-coated-vesicle independent and caveolin-mediated endocytosis proceeds by involution of selective plasma-membrane domains that give rise to smaller pinocytic vesicles (Figure 35). Assembly of endosomal vesicles is often preceded by the formation of domains within the membrane, consisting of specific lipids and proteins. For example, caveolae form from lipid rafts (cholesterol-rich domains within the membrane), which can selectively incorporate or exclude particular proteins. The cytoskeletal protein actin is thought to constrain the lateral mobility of rafts, increasing their stability in the membrane. Moreover, actin is involved in the initial intracellular movement of the caveolae.
What are the principal traffic pathways of material entering the cell by endocytosis?
Molecules pass from early endosomes to late endosomes, and from there may move to lysosomes or recycle to the plasma membrane or intersect with secretory vesicles from the Golgi. Some molecules such as the bacterial toxins (Box 4) move to the Golgi network and the ER.
What are the sorting signals that guide proteins through the endocytic maze? In some cases of receptor-mediated endocytosis, covalent attachment of ubiquitin can act as a signal for endocytosis – several proteins appear to control this complex process and some of them are distinguished by the fact that they become tagged with a single copy of the molecule ubiquitin. Monoubiquitination (the addition of one ubiquitin molecule) is well established as a signal for endocytosis in yeast, and is implicated in the regulated removal of cell surface receptors in animal cells. This tagging requires sequentially acting enzymes, the last one being a ubiquitin ligase that attaches ubiquitin to a lysine residue of the target protein. It is not known how the ligase distinguishes between proteins destined for degradation, which will be tagged with several molecules of ubiquitin, and proteins destined for endocytosis, which will be tagged with a single molecule of ubiquitin by the very same ligase. This process has been studied in relation to the protein eps15, a molecule that associates with clathrin-coated pits and which regulates endocytosis (Figure 36).
The signals that trigger internalisation vary according to the receptors. Some receptors are internalised continuously, but others remain exposed on the surface until ligand is bound to them, after which they become susceptible to endocytosis. In either case the receptors slide laterally into coated pits, and endocytosis starts by a common route that leads to several pathways in which receptors have different fates. It is not clear whether lateral diffusion can adequately explain receptor movement into coated pits or whether some additional forces are required.
Coated pits invaginate into the cytoplasm and pinch off to form coated vesicles. Clathrin forms an outer polyhedral layer on clathrin-coated vesicles, and the adaptins recognise the appropriate sequences in the cytoplasmic domains of receptors that are to be internalised, immobilising the receptor in the pit. As a result, the receptor is retained by the coated vesicle when it pinches off from the plasma membrane. These vesicles move to early endosomes, fuse with the target membrane and release their content. The immediate destination for endocytic clathrin-coated vesicles is the endosome, a rather heterogeneous structure consisting of membrane-bound tubules and vesicles. Early endosomes lie just beneath the plasma membrane and are reached by the internalised proteins within about a minute. By comparison, late endosomes are closer to the nucleus and are reached within 5–10 minutes.
The early endosomes provide the main location for sorting proteins on the endocytic pathway. The interior of the endosome is acidic (pH < 6), which is important in determining the fate of proteins taken up by endocytosis. For example, the fate of a receptor–ligand complex depends on its response to the acidic environment of the endosome. Exposure to a low pH environment changes the conformation of the external domain of receptors, causing the ligand to be released and/or changing the structure of the ligand (e.g. transferrin). But the receptor must not become irreversibly denatured by the acidic environment, and the presence of multiple disulfide bridges in the external domain may play an important role in maintaining stability.
Transport to the lysosomes is the default pathway and applies to any material that does not possess a signal specifically directing it elsewhere. The lysosomes contain the cellular supply of hydrolytic enzymes, which are responsible for degradation of the macromolecules. Like the endosome, the lysosomal lumen is acidic (pH ~ 5). There are two routes to the lysosomes. Proteins internalised from the plasma membrane may be directed via the early endosome to the late endosome. Newly synthesised proteins may be directed from the trans Golgi via the late endosome, as already described. The relationship between the various types of endosome and lysosome is not clear. Vesicles may be used to transport proteins along the pathways from one structure to the next, or early endosomes may mature into late endosomes, which in turn mature into lysosomes. Regardless of the sequence, the pathway is unidirectional and many proteins that have left the early endosome for the late endosome will end up in the lysosomes.