Endocytosis late endosome

Under some circumstances, lysosomal hydrolases may fail to be properly packaged in the TGN, so they enter the default pathway to the cell surface, where they are secreted. Although these hydrolases do little harm at the nearly neutral pH of most extracellular fluids, they can also be returned to lysosomes by a pathway known as receptor-mediated endocytosis. In this pathway, M6P receptors are sent to the plasma membrane, where they bind escaped lysosomal hydrolases and bring them back to lysosomes through the early and late endosomes. Receptor-mediated endocytosis is a major component of the endocytic pathways for trafficking of membrane proteins and merit more detailed consideration. 

In endocytosis, vesicles are formed at the plasma membrane and then transported to an endosome. (More precisely, endosomes should at least be classified into early endosomes and late endosomes, but this fact is ignored here.) The endocytic pathway also includes the following routes from the endosome to the lysosome, from the endosome to the plasma... 

Early endosomes are the main sorting station in the endocytic pathway. In their acidic interior (pH 5.9-6.0), the receptor and its ligand can be released. The receptor may be recycled to the surface by vesicles that fuse with the plasma membrane. Material that cannot escape from the early endosomes is further transported via multivesicular bodies to late endosomes and digesting lysosomes that contain a broad spectrum of peptidases and hydrolases in an acidic surrounding [for reviews on endocytosis see Refs. (10-12), for review on clathrin uptake see Refs. (9,13)]. 

Figure 1 The mode of action for bacterial AB-type exotoxins. AB-toxins are enzymes that modify specific substrate molecules in the cytosol of eukaryotic cells. Besides the enzyme domain (A-domain), AB-toxins have a binding/translocation domain (B-domain) that specifically interacts with a cell-surface receptor and facilitates internalization of the toxin into cellular transport vesicles, such as endosomes. In many cases, the B-domain mediates translocation of the A-domain into the cytosol by pore formation in cellular membranes. By following receptor-mediated endocytosis, AB-type toxins exploit normal vesicle traffic pathways into cells. One type of toxin escapes from early acidified endosomes (EE) into the cytosol, thus they are referred to as short-trip-toxins . In contrast, the long-trip-toxins take a retrograde route from early endosomes (EE) through late endosomes (LE), trans-Golgi network (TGN), and Golgi apparatus into the endoplasmic reticulum (ER) from where the A-domains translocate into the cytosol to modify specific substrates.

Figure 11.1 The intracellular trafficking pathway of plasmid DNA complexed by poly cationic lipid (lipoplex). Critical steps are indicated by numbers (1) endocytosis, sorting and recycling via vesicular compartments comprising the early (EE) and sorting endosomes (2) entrapment and degradation in the late-endosomes (LE) and lysosomes (3) destabilization of the endo-lysosomal membrane and release into the cytosol, (the precise location of this step is not known) (4) diffusion toward the nuclear pore complex (NPC) and degradation in the cytoplasm, and (5) nuclear translocation across the NPC.

In the confocal microscopy experiment, it is recommended to include a negative control. This could be done by incubating cells with phages at 4°C, which should minimize internalization and thus only result in cell surface localization. In addition, endocytosis inhibitors could be used to monitor this event. The subcellular localization could be assessed by co-staining with antibodies that are reactive with different intracellular compartments. For instance, early endosome can be visualized by an EEA1 antibody, whereas late endosomes can be stained by an antibody against the mannose-6-phosphate receptor. 

Gentamicin is more toxic to LLC-PKj monolayers when exposed at the apical side, indicating a preferential uptake from the luminal membrane [136]. The uptake mechanism is proposed to be via megalin mediated endocytosis, a protein which is abundantly expressed in the proximal tubule [137]. A pathway delineated in LLC-PKl cells is proposed, whereby internalized aminoglycosides and other small molecular weight cationic compounds are transported from the early and late endosomes, through the Golgi complex. 

Figure 9.1. Receptor-mediated endocytosis. Exogenous ligands (yellow ovals) bind specifically to their cell surface receptors. The plasma membrane invaginates around the ligand-receptor complexes to form an intracellular vesicle (endosome). As the lumen of the maturing endosome acidifies, the receptor often releases the ligand. Eventually, the fates of both the ligand and the receptor are determined during a sorting process within late endosomal compartments.

Most mammalian cells produce cell-surface receptors that specifically bind to apoB-100 and internalize LDL particles by receptor-mediated endocytosis. After endocytosis, the LDL particles are transported to lysosomes via the en-docytlc pathway and then are degraded by lysosomal hydrolases. LDL receptors, which dissociate from their ligands in the late endosome, recycle to the cell surface. 

The fate of lipoprotein cholesterol after internalization is a key issue in understanding the biology and pathology of lesional macrophages. After internalization by receptor-mediated endocytosis or phagocytosis, the lipoproteins are delivered to late endosomes or lysosomes, where hydrolysis of proteins and lipids occurs. Most importantly, the large lipoprotein-CE stores are hydrolyzed by a lysosomal enzyme called lysosomal acid lipase. The liberated FC then trafficks to the plasma membrane and other cellular sites [14]. 

Fig. 10.16. Lysosomes in receptor-mediated endocytosis via clathrin-coated pits. 1 Endo-cytotic vesicles fuse to form early endosomes. 2 Vesicle contents are sorted, and receptors, clathrin, and lipids are sent back to the plasma membrane. 3 Transport vesicles from the trans-Golgi carry lysosomal hydrolases to the late endosome. 4 Lysosomes containing concentrated hydrolases digest proteins and other components acquired from endocytotic vesicles.

Endolysosomal Escape. Polyplexes bind to the surface of cells via nonspecific electrostatic interactions and are internalized via adsorptive pinocyto-sis. Alternatively, polyplexes derivatized with targeting ligands may bind to specific cell-snrface receptors, in which case they are often internalized by receptor-mediated endocytosis. In either case, the polyplexes become localized within endocytic vesicles, which isolate the polyplex from the rest of the cell. The endocytic pathway represents a hostile environment for polyplexes. The first vesicle, termed the early endosome, fuses with sorting endosomes from which the internalized material may be transported back to the membrane and ont of the cell by exocytosis. More generally, however, polyplexes are believed to be trafficked into late endosomes, vesicles that rapidly acidify to pH 5-6 becanse of the action of an ATPase proton-pump enzyme in the vesicle membrane. Polyplexes may subsequently be trafficked into lysosomes, which further acidify to... 

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