Endocytosis inhibition

LDL (apo B-lOO, E) receptors occur on the cell surface in pits that are coated on the cytosolic side of the cell membrane with a protein called clathrin. The glycoprotein receptor spans the membrane, the B-lOO binding region being at the exposed amino terminal end. After binding, LDL is taken up intact by endocytosis. The apoprotein and cholesteryl ester are then hydrolyzed in the lysosomes, and cholesterol is translocated into the cell. The receptors are recycled to the cell surface. This influx of cholesterol inhibits in a coordinated manner HMG-CoA synthase, HMG-CoA reductase, and, therefore, cholesterol synthesis stimulates ACAT activ-... 

Akagi et al. demonstrated the use of nanoparticles composed of amphiphilic poly (amino acid) derivatives as vaccine delivery and adjuvants [62, 102-104]. To evaluate the uptake of OVA encapsulated within y-PGA-Phe nanoparticles (OVA-NPs) by DCs, murine bone marrow-derived DCs were incubated with 250 nm-sized OVA-NPs for 30 min at 37 °C. The cells were then analyzed by flow cytometry (FCM) and confocal laser scanning microscopy (CLSM). OVA-NPs were efficiently taken up into DCs, whereas the uptake of OVA alone was barely detectable at the same concentration of OVA (Fig. 13). OVA-NPs were more efficiently taken up than OVA alone by DCs, and the uptake of OVA-NPs was inhibited at 4 °C. These results suggest that OVA-NPs were phagocytosed mainly via endocytosis by the DCs. In the case of OVA alone, an approximately 30-fold... 

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]. 

E. B. Malarkey, R. C. Reyes, B. Zhao, R. C. Haddon, V. Parpura, Water soluble single-walled carbon nanotubes inhibit stimulated endocytosis in neurons, Nano Letters, vol. 8, pp. 3538-3542, 2008. 

It has also been proposed that CPP are taken up by clathrin-independent mechanisms such as caveolin-mediated endocytosis (100). TAT-containing liposomes were colocalized with caveolin 1, a marker for caveolar endocytosis, but not with markers for clathrin (101). An inhibitor of caveolin and nystatin reduced TAT peptide reporter in HepG2 and CHO cells by 50% (80). However, it was pointed out by Brooks et al. that HepG2 cell lines do not contain caveolin 1 (102,103). The caveolin pathways relevance to CPP uptake may depend on the cell type because nystatin inhibits... 

Acidification of the cytoplasm has been described as blocking clathrin-mediated endocytosis, but might affect cell morphology and viability (43,44). The mechanism of inhibition is still ill defined but it is clear that the pH of the medium has no effect on the intralysosomal pH (45). It should be noted that cytosolic acidification shows cell-type variations and might not be highly specific. 

As described above, mpCD is a commonly used inhibitor for both the clathrin and the caveolae pathway. See the section on Inhibiting Clathrin-Mediated Endocytosis for details. 

At 4°C, endocytosis is inhibited, but binding to cell surface receptors and passive fusion processes still can occur (82,93,101). Often cells are incubated for 60 minutes at 4°C. When applying this technique, it should be made sure that all buffers and substances are not warmer than 4°C because recovery occurs within seconds. 

Doxsey SJ, Brodsky FM, Blank GS, Helenius A. Inhibition of endocytosis by anti-clathrin antibodies. Cell 1987 50(3) 453-463. 

Zhao X, Greener T, Al-Hasani H, Cushman SW, Eisenberg E, Greene LE. Expression of auxilin or AP180 inhibits endocytosis by mislocalizing clathrin evidence for formation of nascent pits containing API or AP2 but not clathrin. J Cell Sci 2001 114(Pt 2) 353-365. 

Acetylcholine release is inhibited by one of the most potent toxins, the botulims toxin produced by the anaerobic bacterium Clostridium botulinum. The toxin, lethal at 1 ng/kg in humans, enters the synapse by endocytosis at nonmyelinated synaptic membranes and produces muscle paralysis by blocking the active zone of the presynaptic membrane... 

VLDL in the plasma is converted to LDL—a much smaller, denser particle. Apo CM and apo E are returned to HDLs, but the LDL retains apo B-100, which is recognized by receptors on peripheral tissues and the liver. LDLs undergo receptor-mediated endocytosis, and their contents are degraded in the lysosomes. A deficiency of functional LDL receptors causes type II hyperlipidemia (familial hypercholesterolemia). The endocytosed cholesterol inhibits HMG CoA reductase and decreases synthesis of LDL receptors. Some of it can also be esterified by acyl CoAxholesterol acyltransferase and stored. 

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