Clathrin-dependent endocytosis

The 80 kDa glycoprotein transferrin (Tf) is responsible for intracellular iron transport via the transferrin receptor (TfR), using a clathrin-dependent endocytosis process. Tumor tissues frequently overexpress the TfR. While natural Tf recycles to... 

Figure 2 Proposed pathways for liposomal entry into the cell enhanced by peptides. These include direct cell entry suggested as the mechanism of entry by cell-penetrating peptides and receptor-mediated endocytosis by caveolae- and clathrin-dependent endocytosis.

Richard IP, Melikov K, Brooks H, et al. Cellular uptake of unconjugated TAT peptide involves clathrin-dependent endocytosis and heparan sulfate receptors. J Biol Chem 2005 280(15) 15300-15306. 

Clathrin-mediated (or clathrin-dependent) endocytosis normally occurs at specialized sites, where complex structures called coated pits are assembled in order to concentrate surface proteins for internalization. The coat consists of many different proteins that are needed for stabilization of both the pit and the forming of the clathrin-coated vesicle. The two most abundant proteins found within these structures are clathrin and the adaptor protein AP-2 (9). 

Mousavi SA, Malerod L, Berg T, Kjeken R. Clathrin-dependent endocytosis. Biochem J 2004 377(Pt 1) 1-16. 

According to the classical view, synaptic vesicles completely flatten during exo-cytosis, which is followed by retrieval of the membrane components by clathrin-dependent endocytosis. Evidence for direct retrieval (kiss-and-run) was provided... 

Follow the procedure in Subheading 3.2. However, in step 4 just prior to the addition of the ligand solution, add 100 ig/mL transferrin-AlexaFluor-647 or 300 (rg/mL dextran-Alexa Fluor 647 to the peptide/DNA mixture to visualize clathrin-dependent endocytosis or macropinocytosis, respectively. Analyze the cells either live or fixed and counter-stained. If the cells are fixed and counter-stained, omit adding TO-PRO-3 under step 18, as the fluorescence of TO-PRO-3 is in the far-red spectrum just as Alexa Fluor 647. 

Marchetti M, Monier MN, Fradagrada A, Mitchell K, Bayche- 126. tier F, Bid P, Johannes L, Lamaze C. Stat-mediated signaling induced by type I and type II interferons (IFNs) is differentially controlled through lipid microdomain association and clathrin-dependent endocytosis of IFN receptors. Mol. Biol. Cell 2006 17 2896-2909. 

Clathrin One of the main scaffolding proteins mediating clathrin-dependent endocytosis. 

It has been reported that NP internalised via clathrin-dependent endocytosis were generally entrapped within the endosomes and then fused with lysosomes, resulting in lysosomal degradation of the load [74]. On the other hand, NP internalised by caveolin-mediated endocytosis could normally bypass lysosomal degradation [75]. It was also shown that polymeric micelles incorporating nile red (M-NR) were capable of colocalisation with tubulin, an important protein that shapes the microtubule network of cells this colocalisation might be advantageous for M-NR uptake via dynamin and caveolin-mediated endocytosis [76, 77]. 

Lukacs, G. L., G. Segal, N. Kartner, S. Grinstein, and F. Zhang. (1997). Constitutive internalization of cystic fibrosis transmembrane conductance regulator occurs via clathrin-dependent endocytosis and is regulated by protein phosphorylation. Biochem ], Vol.328, pp.353-361. 

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]. 

Fig. 3. Effects of AMAP2 overexpression on receptor internalizations in HeLa cells. Clathrin-independent endocytosis of Tac molecules (A and C) and clathrin-dependent endocytosis of Tfn molecules (B and D) were analyzed. See text for details., P<0.01 against values of the EGFP control in (A) and (B). In (C), cells marked by asterisks expressed detectable levels of EGFP-AMAP2 or dynamin2 K44A, but exhibited more than 20% of Tac uptake as compared to those of the untransfected cells in the same field. These cells were hence judged as cells in which Tac uptake was not blocked. Panels A an B are reproduced from Hashimoto et al. (2004b).

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