Clathrin pathway

Some inhibitors for this pathway, often described in the literature, do not directly affect the clathrin pathway but rather affect features involved with other pathways. For example, the acidification of endosomes is employed by the other types of endocytosis as well—therefore, these inhibitors are less specific and are described in the section Intracellular Trafficking The same occurs with dynamin dependence or metabolic activity (section Metabolic Activity ). 

Either fluorescent EGF or its fluorescent receptor (GFP-EGFR) can be used to track the clathrin pathway (110). Following internalization, EGF is mainly targeted to early endosomes, then to lysosomes, and following this is subsequently degraded. After a 10-minute incubation period with EGF, the polypeptide hormone accumulates in early endosomes, and after 60 to 90 minutes, EGF... 

Endocytosis is essential for uptake of macromolecules and nanoparticles. Thereby, the physical and chemical characteristics of the cargo and transporter complex determine the nature of the cell uptake mechanism. Particles larger than 500 nm are typically phagocytosed or macropinocytosed, whereas smaller hydrophilic molecules are internalized by any of the various endocytotic processes [2,8]. Often, nanometersized transporter molecules or particles accomplish cell uptake simultaneously by several endocytotic mechanisms [9]. Clathrin-dependent endocytosis is the most widely understood pathway, which is present in nearly aU mammalian cells. The clathrin pathway is considered the most important uptake mechanism for several polymers such as poly(ethylene glycol)-polylactides, poly(lactide-c )-glycolide) (PLGA), silica-based nanoparticles, and chitosan nanoparticles [8, 10-12]. 

Clathrin-coated vesicles mediate transport within the late secretory and the endocytic pathways. Their major coat constituents are clathrin and various adaptor complexes. 

Along their route through the Golgi, secretory and membrane proteins destined for the various post-Golgi pathways are intermixed. Thus, proteins of distinct routes, i.e. the endosomal and the secretory route, are sorted into individual types of transport vesicles at the TGN. Among the best characterized types of TGN-derived vesicles are clathrin-coated vesicles. In addition, several types of non-clathrin-coated vesicles have been identified but their specific functions remain to be characterized. 

Since endocytosis ofLDH was confirmed by TEM images (Figure 13.9), forthe next step, its specific endocytic pathway for membrane entry was determined by immunofluorescence and confocal microscopy. Cells were incubated with LDH-FITC, fixed with 3.7% freshly made formaldehyde, and then stained with either anti-clathrin antibody or anti-caveolin-1 antibody both conjugated to the red fluorescent dye Texas Red (TR). The confocal microscopic images showed that green fluorescent... 

Other systems like electroporation have no lipids that might help in membrane sealing or fusion for direct transfer of the nucleic acid across membranes they have to generate transient pores, a process where efficiency is usually directly correlated with membrane destruction and cytotoxicity. Alternatively, like for the majority of polymer-based polyplexes, cellular uptake proceeds by clathrin- or caveolin-dependent and related endocytic pathways [152-156]. The polyplexes end up inside endosomes, and the membrane disruption happens in intracellular vesicles. It is noteworthy that several observed uptake processes may not be functional in delivery of bioactive material. Subsequent intracellular obstacles may render a specific pathway into a dead end [151, 154, 156]. With time, endosomal vesicles become slightly acidic (pH 5-6) and finally fuse with and mature into lysosomes. Therefore, polyplexes have to escape into the cytosol to avoid the nucleic acid-degrading lysosomal environment, and to deliver the therapeutic nucleic acid to the active site. Either the carrier polymer or a conjugated endosomolytic domain has to mediate this process [157], which involves local lipid membrane perturbation. Such a lipid membrane interaction could be a toxic event if occurring at the cell surface or mitochondrial membrane. Thus, polymers that show an endosome-specific membrane activity are favorable. 

Rejman J, Oberle V, Zuhom IS, Hoekstra D (2004) Size-dependent internalization of particles via the pathways of clathrin- and caveolae-mediated endocytosis. Biochem J 377 159-169... 

The bulk of pinocytosis in the nervous system is mediated by clathrin-mediated endocytosis (CME) [55] and this is the best-characterized pathway. More detail about clathrin-mediated pathways will be given when receptor-mediated endocytosis and the synaptic vesicle cycle pathways are considered. Pinocytosis through CME is responsible for uptake of essential nutrients such as cholesterol bound to low density lipoprotein (LDL) and transferring, but also plays a role in regulating the levels of membrane pumps and channels in neurons. Finally, CME is critical for normal synaptic vesicle recycling. 

Other pinocytotic pathways also exist that do not depend on either caveolae or clathrin, although these are not as well defined [55]. Specific receptors continue to be internalized in the absence of clathrin or caveolin and these pathways can be monitored by following glycosyl phos-phatidylinositol (GPI (-anchored proteins. Nonclathrin, noncaveolin pathways may also be responsible for the reuptake of membrane in neuroendocrine cells after stimulated secretion. Some, but not all, of these pathways appear to require dynamin. 

The constitutive pathway has not been studied as intensively as regulated secretion [54]. In particular, relatively little is known about targeting and regulatory mechanisms for these transport vesicles. Clathrin seems not to be directly involved in the constitutive secretory pathway. Antibodies that disrupt clathrin assembly in vitro inhibit endocytosis, but constitutive exocytosis is not affected [63]. 

Retrieval of membrane components in the secretory pathway through receptor-mediated endocytosis (RME) is a clathrin-coat-dependent process [5]. The clathrin coat provides stability to the vesicle core and allows uptake of specific membrane proteins for reuse or degradation. RME shows a remarkable degree of specificity, allowing cells to internalize with astonishing efficiency only those selected molecules independent of their extracellular concentration. 

As noted above, synaptic vesicles are not typically generated at the level of the TGN. Instead, they are assembled from endocytosed material retrieved from the synaptic plasma membrane. Synaptic vesicle and plasma membrane lipids and proteins are synthesized in the endoplasmic reticulum and modified in the Golgi apparatus, where they are then packaged in secretory vesicles. These synaptic precursors are delivered to the plasma membrane from the cell body by the constitutive secretory pathway. Synaptic vesicle proteins must be retrieved by clathrin-mediated synaptic vesicle endocytosis, a variant of RME with some neuron-specific components. Once the vesicle sheds its clathrin coat, the uncoated vesicle fuses with a... 

In the classic model of synaptic vesicle recycling in nerve terminals, synaptic vesicles fuse completely with the plasma membrane and the integrated vesicle proteins move away from the active zone to adjacent membrane regions (Fig. 9-9A). In these regions, clathrin-mediated synaptic vesicle endocytosis takes place rapidly after neurotransmitter release (within seconds) [64]. The process starts with the formation of a clathrin-coated pit that invaginates toward the interior of the cell and pinches off to form a clathrin-coated vesicle [83]. Coated vesicles are transient organelles that rapidly shed their coats in an ATP/chaperone dependent process. Once uncoated, the recycled vesicle fuses with a local EE for reconstitution as a synaptic vesicle. Subsequently, the recycled synaptic vesicle is filled with neurotransmitter and it returns to the release site ready for use. This may be the normal pathway when neurotransmitter release rates are modest. Clathrin/ EE-based pathways become essential when synaptic proteins have been incorporated into the presynaptic plasma membrane. 

However, an alternative pathway that bypasses clathrin-mediated endocytosis and EEs appears to be available as well. This model of endocytosis known as kiss and run or its variant kiss and stay have attracted increasing interest in recent years [74] (Fig. 9-9B). Kiss and run has been directly demonstrated with dense-core granules in neuroendocrine cells [84, 85], and this model would explain some observations that are not readily accommodated by the classical pathway. The kiss and run model proposes that neurotransmitters are released by a transient fusion pore, rather than by a complete fusion with integration of the synaptic vesicle components into the plasma membrane. Synaptic membrane proteins never lose their association and the vesicle reforms when the pore closes. As a result, the empty vesicle can be refilled and reused without going through clathrin-mediated endocytosis and sorting in the EEs. 

Figure 4. A schematic of endocytotic pathways in a cell. P = pinocytosis Ex = Exocytosis R = receptor C = clathrin CP, CV = coated pit and coated vesicle E = endosome L = lysosome. Open arrow indicates recycling of clathrin and receptors. Solid arrows indicate pathways. See the text for discussion...

Many of the investigations into endosomal pathways have concentrated on receptor-mediated endocytosis, as in the iron-transferrin-receptor complex, and it is not clear how the systems vary depending on whether or not the pathway is clathrin-dependent or clathrin-independent [54],... 

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