1. Pinocytosis:

Using time-lapse photography to study tissue culture cells, W. H. Lewis in 1931 described what seemed to be a curious phenomenon in which small amounts of culture medium were trapped in invaginations of the plasma membrane and then pinched off to form small cytoplasmic vesicles.

Because the entire process ap­peared much like some form of organized cell drink­ing, Lewis termed the phenomenon pinocytosis (“pinos” means “I drink” in Greek).

Lewis’ observa­tions with tissue culture cells were confirmed in 1934 by S. O. Mast and W. L. Doyle studying amoebae in which pinocytosis is readily observed with the light microscope.

Using electron microscopy, it became clear in the 1950s that pinocytosis is a common phe­nomenon occurring at different times in many kinds of cells and tissues including leukocytes, kidney cells, intestinal epithelium, liver macrophages, and plant root cells.

Pinocytosis is induced by the presence of appropri­ate concentrations of proteins, amino acids, or certain ions in the medium surrounding the cell. The first step in the process (Fig. 15-43) involves the binding of the inducer substance to specific receptors in the plasma membrane. This is followed by invagination of the membrane to form either pinocytic vesicles or narrow channels (Fig. 15-43).

Stages of Pinocytosis

Although binding of the in­ducer is not inhibited by cyanide or low temperature, the formation of pinocytic vesicles is, and it is there­fore dependent on cell metabolism. Actin filaments are associated with the margins of pinocytic vesicles and are believed to play some role in the vesicle’s in­vagination. Pinocytic vesicles detach from the plasma membrane and migrate toward the interior of the cell, where they may fragment into smaller vesicles or coa­lesce to form larger ones. Unless the vesicles are “tagged” by inducing pinocytosis in the presence of radioactive tracers, they soon become indistinguish­able from other vacuoles in the cell.

Two levels of pinocytosis can be distinguished. In one of these, called micropinocytosis, the vesicles formed have a diameter of about 0.1 µm and are de­rived from small depressions in the cell surface (caveolae). In macropinocytosis, the vesicles are con­siderably larger, having diameters of 1-2 µm. The larger vesicles are formed either from larger invagi­nations of the plasma membrane or from surface ruf­fles. Although pinocytosis is induced by the presence of specific substances in the cell surroundings, other materials are also enclosed by the pinocytic vesicles, including water, salts, and so on.

In this regard, pinocytosis is partly (perhaps even predominantly) nonspecific and is to be contrasted with receptor-mediated endocytosis, which is almost entirely specific. Molecules that induce pinocytosis, together with water and other so­lutes entrapped by the infolding plasma membrane are presumed to enter the cytosol from the vesicles that are formed by diffusion, active transport, or re­lated transport mechanisms. Pinocytosis, like other forms of endocytosis, is carried out at some energy cost on the part of the cells engaged in this activity.

2. Receptor-Mediated Endocytosis:

In receptor-mediated endocytosis, binding of molecules such as hormones, antibodies and other proteins, and lipids to specific receptors in the plasma membrane is followed by their clustering (i.e., concentration) at specific membrane sites, which are then internalized by the cell. The process is depicted diagrammatically in Figure 15-44.

Concentrative, receptor Mediated Endocytosis

The receptors that mediate the endocytosis are integral proteins that span the membrane. Substances bound by these receptors, called ligands, are “plucked” from the extracellular milieu even when present in extremely low concentrations and surrounded by a large excess of other, unrelated solutes. Ligand-receptor complexes move laterally in the membrane toward coated pits in which the complexes are then concentrated. Because lateral movement through the membrane by the ligand-receptor complex is quite rapid, a coated pit is encountered within a few seconds.

Once a ligand-receptor complex enters a pit, its lateral movement is halted; as a result, many such complexes are quickly accumulated in a single pit. Uncomplexed receptors, which also move laterally in the plasma membrane, may also be concentrated in a coated pit. Coated pits are present in almost all animal cells and account for about 2% of the membrane’s total surface area.

The pits’ “coats” consist primarily of a layer of peripheral protein called clathrin tightly appressed to the cytosol-facing surface of each pit. The clathrin’s subunits (molecular weight about 180,000) occur in three-armed trimers called triskelions that interact with one another to form a cage like net around the pit.

Indeed, polymerization of the triskelions is believed to form the pit, deepening it and eventually freeing it from the membrane as a small, coated vesicle. (Isolated and purified triskelions have been shown to spontaneously form hemispherical and spherical cage like structures in vitro.) Coated vesicles that have just detached from the plasma membrane move deeper into the cytoplasm, progressively shed their clathrin coats, and fuse with one another to form larger, smooth-surfaced vesicles called endosomes (sometimes receptosomes) (Fig. 15-44). The clathrin released by coated vesicles is believed to be recycled to the plasma membrane, where it gives rise to new coated pits.

As endosomes move even deeper into the cytoplasm, there is a progressive release of ligands from their receptors into the lumen of the vesicle. The endosome develops a tubular portion in which the membrane-bound but ligand-free receptors are concentrated and a vesicular portion containing free ligand. At this stage the endosome is sometimes referred to as a CURL (“compartment of uncoupling of receptor and ligand”). The tubular por­tion of the endosome is subsequently dispatched to the plasma membrane, thereby recycling its ligand recep­tors, and the vesicular portion continues its journey deeper into the cell.

If the endosome contains pro­teins, other macromolecules, or particles, it may fuse with a lysosome so that enzymatic digestion of the en­trained material can ensue. Figure 15-45 summarizes the overall process of receptor-mediated endocytosis, including the initial synthesis of membrane receptors by EF-bound ribosomes.

Synthesis and Recycling of Plasma Membrane Receptors

Sorting of Ligands and Receptors within a CURL Separation of ligand and receptor within a CURL is believed to be prompted by the low pH that exists within the compartment. Still uncertain is the mecha­nism by which receptors and ligands are sorted. How the receptors are moved into the lipid bilayer of the tu­bular extensions of the CURL prior to recycling to the plasma membrane, while the ligands remain in the fluid-filled vesicular body of the organelle, is unclear.

Two domains may be identified in a CURL: a membrane-rich (fluid-poor) domain consisting of the CURL’s narrow, tubular extensions, and a fluid-rich (membrane-poor) vesicular domain. It has been esti­mated that 90% of a CURL’s membrane is in the CURL’s tubular extensions, whereas only 10% is in the fluid-rich domain. L. H. Rome has suggested that such an asymmetric distribution of membrane could result in an automatic localization of receptors in the membrane-rich domain (so long as the receptors are free to migrate within the lipid bilayer). In other words, the receptors accumulate in the tubular (membrane-rich) extensions of a CURL by mass action.

Fragments of plasma membranes, membranes of the endocytic compartment, and membranes of the Golgi apparatus can be isolated from disrupted cells using various forms of density gradient centrifugation. The isolated fractions may then be analyzed for their contents of specific proteins (e.g., clathrin, re­ceptors, marker enzymes, etc.), lipids, and ligands.

Not surprisingly, receptors, ligands, and clathrin are differentially distributed among these membrane fractions. However, W. H. Evans and others have shown that in fractionated liver cells the cholesterol, sphingomyelin, and marker enzyme contents of the separated membrane components are unexpectedly similar and have suggested that an anatomical conti­nuity may exist between the plasma membrane and the membranes of the endocytic compartment.

3. Phagocytosis:

Phagocytosis, which was first described by E. Metchnikoff in the late nineteenth century, involves the endocytosis of much larger quantities of material than either pinocytosis or receptor-mediated endocytosis. For example, entire ciliates, rotifers, or other micro­scopic organisms may be phagocytosed by an amoeba and enclosed within one or more vacuoles called phagosomes, food vacuoles, or food cups (see Fig. 15-46).

Phagocytosis

During phagocytosis, the “prey” may be tem­porarily immobilized by secretions from the phagocytic cell. The phagocytosis of ciliates is characterized by the flowing of the amoeba’s cytoplasm into foot like projections (pseudopodia) that gradually encircle and fully encapsulate the ciliate.

Using a similar mecha­nism, certain white blood cells phagocytose hundreds of bacteria. The removal and destruction of old red blood cells in the liver, spleen, and bone marrow by reticuloendothelial cells in these organs also occurs by phagocytosis. Following phagocytosis, the phago­somes fuse with primary lysosomes in the cell. The hydrolytic enzymes from these lysosomes digest the en­gulfed material, converting it to a form that may be transported across the vacuolar membranes and into the cytosol.

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