Endocytotic pathways

C1C-3, -4 and -5 form the second branch of the CLC gene family. These proteins are 80% identical, and with the exception of C1C-5, which is most highly expressed in kidney and intestine, show a broad expression pattern. C1C-3 to C1C-5 reside in intracellular membranes of the endocytotic pathway [4]. Disruption of C1C-5 leads to a defect in endocytosis in mouse... 

EJ Hughson, CR Hopkins. Endocytotic pathways in polarized Caco-2 cells Identifi-... 

The abundant expression of a variety of transporters in Caco-2 cells also makes it attractive to apply functional genomics tools, such as cDNA arrays in order to map the expression [31] and relative abundance of these transporters [32], Also, genomic mapping of surface receptors on Caco-2 cells can be performed to study receptor-mediated endocytosis and other endocytotic pathways for larger molecules in enterocytes [33-36]. 

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...

This receptor-mediated endocytotic pathway has been especially well studied in the uptake of iron from blood plasma. Iron, because of its very low-solubility product (< 1(T17 at pH 7.4), is transported in plasma bound to the iron-binding protein transferrin. Two Fe3+ ions bind to each transferrin molecule. Entry into... 

West MA, Bretscher MS, Watts C. Distinct endocytotic pathways in epidermal growth factor-stimulated human carcinoma A431 cells. J Cell Biol 1989 109(6 Pt l) 2731-2739. 

Based on the knowledge of the endocytotic pathway, it is reasonable to assume that uhiquitination is reversible until the endocytosed membrane proteins such as neurotransmitter receptors are routed to the multivesicular body for lysosomal degradation. 

Weiner et al. (173) showed that the uptake of a folate-conjugate dendrimer into tumor cells overexpressing high affinity Folate Receptor (hFR), occurs through this type of endocytotic pathway. 

The viability and function tests described above are used to evaluate the hepatocytes within the slice. Up to now, tests to measure the viability of the non-parenchymal cells have not been reported. The presence of the latter cell types is one of the conceptual advantages of slices as compared to isolated hepatocytes. As some drug targeting devices are designed to target non-parenchymal cells in the liver, the development of tests for the sinusoidal cell types deserves more attention. For example, the uptake of substrates such as succinylated human serum albumin (Suc-HSA,which is specifically endocytosed by endothelial cells [79]), or hyaluronic acid [80], can be used to assess the functionality of endocytotic pathways in the endothelial cells in the liver [81]. Other modified proteins that are specifically taken up by Kupffer cells such as mannosylated HSA, may be used to assess the functionality of the endocytotic pathway in Kupffer cells [79]. Another parameter which can be used to assess the functionality of these non-parenchymal liver cells, is the excretion of cytokines in response to pro-inflammatory stimuli. Non-parench5mal cell function in liver slices will be described in more detail in the Section 12.7. 

Already in 1965, Ryser and Hancock provided evidence that histones and polyamino acids could greatly enhance albumin uptake by cultured tumor cells (6). More recently, several polybasic peptides (so-called protein transduction domains, PTDs or cell-penetrating peptides, CPPs) have been shown to efficiently mediate uptake of nucleic acids, bioactive peptides, phage particles, and liposomes into a wide variety of mammalian cells. The initially proposed ability of CPPs to penetrate plasma membranes via a temperature-independent, non-endocytotic pathway was later shown to be a fixation artifact, and it is currently widely accepted that CPP-mediated macromolecular delivery follows energy-dependent endocytotic pathways that in most cases depend on the expression of cell-surface heparan sulfate proteoglycans (HSPGs) (7). 

Wittrup and Belting have in several studies on CPP-mediated DNA delivery established protocols for fluorescence assisted cell sorting (FACS) analysis to obtain reliable, quantitative data on CPP-DNA complex uptake in cultured cancer cells (9). Also, procedures for co-localization studies with known markers of various endocytotic pathways using confocal microscopy are described as well as the expression of dominant negative dynamin (GTPase deficient dynamin-2) to evaluate the dynamin dependence of the uptake mechanism. The use of various drugs commonly used to disrupt endocytosis is discussed, especially with regard to their limited specificity (9). 

Arf Activates the ADP-ribosyltransferase of the cholera toxin A subunit regulates vesicular trafficking pathways activates phospholipase D Rab Plays a key role in secretory and endocytotic pathways... 

The molecular defect in most cases of familial hypercholesterolemia is an absence or deficiency of functional receptors for LDL. Receptor mutations that disrupt each of the stages in the endocytotic pathway have been identified. Homozygotes have almost no functional receptors for LDL, whereas heterozygotes have about half the normal number. Consequently, the entry of LDL into liver and other cells is impaired, leading to an increased plasma level of LDL. Furthermore, less IDL enters liver cells because IDL entry, too, is mediated by the LDL receptor. Consequently, IDL stays in the blood longer in familial hypercholesterolemia, and more of it is converted into LDL than in normal people. All deleterious consequences of an absence or deficiency of the LDL receptor can be attributed to the ensuing elevated level of LDL cholesterol in the blood. 

In all these different endocytotic pathways the role of lipid-lipid and lipid-protein interactions with their associated signal transduction pathways need additional studies to further elucidate the importance of lipid membrane microdomains in these endocytotic processes and their relation to diseases. 

Size can also be a factor determining which endocytotic pathways are involved in ligand absorption. For example, in nonphagocytotic cells, latex beads with diameters less than 200 nm were taken up by the clathrin-dependent pathway, and latex beads with diameters between 200 and 500 nm were taken up by a caveolin-dependent process. Similarly, absorption of DNA-polycation complexes showed clear size dependence sizes greater than 200 nm were internalized by macropinocytosis. Intermediate size... 

Rab Targeting of vesicles involved in secretory and endocytotic pathways and formation of v-SNARE-t-SNARE complexes Dynamin, Rab (11-33) Anchored to lipid membranes with geranylgeranyl (C20 isoprenoid) groups and other lipids... 

Figure 5.18 Receptor-mediated endocytosis. (a) Schematic diagram of the main steps in the endocytotic pathway. Binding at the cell surface induces clustering of receptor-ligand complexes in specialized regions of the membrane. Endocytotic vesicles form in these regions separation of receptor and ligand occurs as the endosome matures. Separated receptors can recycle to the surface, while remaining material is degraded within lysosomes. (b) Typical time course of internalization and degradation.

Several lipoprotein formulations have been tested for delivery of photosensitizers to tumor tissue [54-56] and it has been shown that LDL is a particularly suitable delivery vehicle for different porphyrins. Uptake of LDL complex is mainly mediated via an active endocytotic pathway, involving LDL receptors on both tumor cells and endothelial cells [57-60]. Depending on the time after administration, the LDL complexed photosensitizers can be found either in the vasculature [61], or in the tumor cells themselves [57,62]. Covalent linkage of LDL to chlorin e6 resulted in conjugates which were specifically taken up by LDL-receptor carrying fibroblasts, and a retinoblastoma cell line, indicating its potential use in ocular tumors [54]. 

Several mechanisms of endocytotic pathways exist. These include clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis, and non-clathrin receptor-mediated endocytosis (Table 4). All of these pathways can be exploited for drug trafficking into resistant cancer cells. For example, the... 

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