Endocytosis early endosome

Microtubules are used to cover relatively long distances in the cell. Therefore, the short distance between the cell surface and the early endosomes does not require microtubules (94,136). They are involved in the later steps in endocytosis early endosomes accumulate endocytic material for about 10 minutes and then generate transport vesicles (0.5 pm in diameter), which will be taken to the cell center with a relatively high traveling speed of Ipm/sec (with velocities of up to 2.5pm/sec) (95,137). These transport vesicle move on microtubule-tracks to the cell center (and to the ly sosome) (94). When performing live cell imaging studies, it should be kept in mind that microtubules are extremely sensitive to ultraviolet light, which causes their polymerization. 

Figure 13.9 represents the TEM image of LDH particles and their cellular internalization. As expected, LDH particles are internalized by endocytosis. Figure 13.9(A) shows the cellular uptake process of LDHs after 3h of treatment, and demonstrates a successive entry of LDH by endocytosis first the LDH particles were located around the cell membrane due to their positive charge ( ), then they migrate to the membrane ruffles which are considered as endocytic bodies ( ), finally the coated intracellular vesicles were formed as early endosomes ( ). Figure 13.9(B)...

However, not all proteins proceed directly to their eventual destination. Some proteins relocate from one plasma membrane compartment to another by means of trans-cytosis. Transcytosis involves endocytosis of selected proteins in one membrane compartment, followed by subsequent transport through early endosomes to recycling endosomes and finally translocation to a different membrane compartment, for example from the apical to the basolateral surfaces. Sorting at the TGN and endo-some recycling steps appear to have a primary role in the steady state distribution of proteins in different plasma membrane domains [47], However, selective retention of proteins at the plasma membrane by scaffolding proteins or selective removal may also contribute to normal distributions. Finally, microtubule-motor regulatory mechanisms have been discovered that might explain the specific delivery of membrane proteins to discrete plasma membrane domains [48]. 

Synncs, M., Prydz, K., Lovdal, T., Brech, A. and Berg, T. (1999). Fluid phase endocytosis and galactosyl receptor-mediated endocytosis employ different early endosomes, Biochim. Biophys. Acta-Biomembranes, 1421, 317-328. 

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)]. 

As described above (section Clathrin-Mediated Uptake ), several ligands for clathrin-mediated endocytosis (see section Clathrin-Mediated Endocytosis EGF, Tfn, LDL) can be used to highlight early endosomes or lysosomes, depending on different incubation times. 

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 8 Ubiquitin and endocytosis. Receptors on the plasma membrane undergo monoubiquitination as a result of ligand (e.g., neurotransmitter). Ubiquitinated receptors bind to proteins called epsins, which in turn interact with adaptor proteins (adaptin) bound to clathrin-coated pits. Ubiquitination also functions to sort the internalized membrane protein into early endosomes, which directs them to degradation by lysosome through the multivesicular body. If ubiquitin from the endocytosed receptors is removed by an UBP, the receptor recycles back to the membrane. Proteasome inhibitors block endocytotic degradation of some proteins such as glutamate receptor subunits indicating a possible role for the proteasome.

Fig. 5 Synaptic vesicle recycling in the synapse. For synaptic vesicle recycling, several endocytic mechanisms appear to co-exist in synaptic nerve terminals. In the case of fast kiss-and-ran exo-cytosis/endocytosis, the fused vesicle does not collapse into the membrane but is retrieved directly by a fast process. The molecular machinery underlying this pathway is unknown. Vesicles that have fully collapsed into the membrane are recycled by clathrin-mediated endocytosis. Clathrin, along with other proteins, is involved in membrane invagination (see figure and text) and leads finally to the formation of a constricted pit. The GTPase dynamin (black ring) mediates membrane scission of the constricted pit. After removal of the clathrin coat, two pathways are possible (direct recycling and recycling via the early endosome). In all cases, before fusion the recycled vesicles have to be loaded with neurotransmitters (NT).

Fig. 3.3 Intracellular sorting in epithelial cells after clathrin-mediated endocytosis. AEE apical early endosome ARE apical recycling endosome CE common endosome ...

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. 

Phosphatidyl-inositol-3-OH kinase (PI(3)kinase) plays an important role in the fusion of endosomes. Phosphatidyl-inositol-3-phosphate (PI(3)P), a product of this enzyme, is enriched in early endosomes, and blocking PI(3)kinase activity with the small molecule wortmannin prevents endosome fusion. This fungal natural product has been shown to inhibit the endocytosis of transferrin, horseradish peroxidase, and albumin (44, 45). 

Figure 1 Overview of the synaptic vesicle cycle, (a) Within the presynaptic terminal, synaptic vesicles are filled with neurotransmitter by the action of specific vesicular neurotransmitter transporters, (b) Neurotransmitter-filled vesicles translocate to the active-zone membrane where they undergo docking, (c) Docked vesicles transition to a release-competent state through a series of priming or prefusion reactions, (d) Invasion of an action potential into the presynaptic terminal and subsequent calcium influx induces rapid fusion of the synaptic vesicle membrane with the terminal membrane, which thereby releases the neurotransmitter into the synaptic cleft, (e) Spent vesicles are internalized by clathrin-mediated endocytosis and are recycled for reuse, which thus completes the synaptic vesicle cycle. SV, synaptic vesicle CCV, clathrin-coated vesicle EE, early endosome. NOTE The use of arrows indicates a temporal sequence of events. Physical translocation of synaptic vesicles is unlikely to occur between the docking and fusion steps.

Receptors may also be located in the plasma membrane of the endothelial cells of the BBB, but are not restricted to this location. They can be internalized and transported via the early endosome to the ly sosomes or even transcytosed and shuttled back to the plasma membrane again. Internalization occurs via an endocy totic process. Endocytosis is used in this context to indicate vesicular transport pathways in eukaryotic cells to internalize extracellular fluid and particles (<500 ran) as well as plasma membrane molecules (34). Endocytosis may be very fast in some cultured mammalian cells, 50% of the entire cell surface may be internalized every hour (35). 

Concerning endocytosis, the internalization and degradation of EGF receptors is inhibited by primary, but not secondary alcohols [157]. Overexpression of PLDl and PLD2 reduces surface levels of the EGF receptor, but this is not observed with catalytically inactive mutants [157]. The activation of MAP kinase by EGF depends on the internalization of the EGF receptor [158], and there is evidence that this is inhibited by butan-l-ol, but not butan-2-ol [157]. The fusion of early endosomes may also involve PLD since this is stimulated by exogenous PLD and blocked by butan-l-ol, but not butan-2-ol [159]. A role for the phospholipase in the assembly of clafhrin coats on lysosomes has also been reported [160]. 

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