Macropinocytosis

Previous work has shown that the majority of cells internalize liposomes through an endocytic pathway (4,5). There are multiple pathways for internalization involving vesicles of 50 300 nm in diameter. These include clathrin-mediated endocytosis, caveolae-mediated endocytosis, phagocytosis, macropinocytosis, and nonclathrin- noncaveolae-dependent endocytosis (6). 

Figure 1 Endocytosis of liposomes five different routes into the cell. Multiple pathways can be used by the cell to internalize liposomes. Besides the well-characterized clathrin-mediated endocytosis, other pathways can be applied by the cell. Possible alternative pathways include phagocytosis or macropinocytosis—two pathways that internalize by an actin-driven protuberance of the plasma membrane. Other routes include the involvement of caveolae where substances are taken up into the cell bypass the traditional endosome/lysosome system (particles might escape from being degraded in lysosomes). Finally there exists an ill-defined mechanism that is neither mediated by caveolae nor by clathrin. In a single cell type, two or more of these mechanisms can coexist. Source Adapted from Ref 8.

Phagocytosis and macropinocytosis are actin-dependent and clathrin-independent processes that lead to the uptake of particles into large vesicles. 

In addition to the well characterized roles of clathrin-caveolae-mediated endocytosis and macropinocytosis/phagocytosis, an ill-defined route of nonclathrin-noncaveolae mediated endocytosis still exists (31,32). It seems that all of the until now poorly understood mechanisms of internalization can be summarized in this topic. 

Nystatin Bind cholesterol inhibitor for caveolae, but also stimulate macropinocytosis Block cholesterol-sensitive 60 ... 

Phorbol esters are described as being protein kinase C activators and should be selective inhibitors for caveolae uptake [1 pM Phorbol 12-myristate 13-acetate (PMA) for 30 minutes] (48,68). The inhibition occurs as quickly as five minutes after adding PMA to cell culture medium and is not reversible within five hours (MA104 cells). However, PMA has several other effects on several other pathways. An example of this is that it has been described to stimulate macropinocytosis and transcytosis (see the following). 

Because of the strict requirement for actin, the most commonly used inhibitors of macropinocytosis are the cytochalasins, especially cytochalasin D or toxin C. These substances also block phagocytosis and intracellular trafficking along actin filaments. Therefore, the results from these experiments are described in the trafficking section Actin Dependence on Liposome Uptake. ... 

As phagocytosis occurs only in specialized cells, for the purposes of this review, we do not differentiate macropinocytosis from phagocytosis. 

The uptake via macropinocytosis is described as being blocked by amiloride and its derivatives. These all inhibit the Na" 7H exchange and EGF-R tyrosine kinase (70,71). For inhibition, cells are incubated with amiloride (3mM, five minutes), hexamethylamiloride, (100 pM), or dimethylamiloride (0.1 mM, 5 minutes to 10 pM, 60 minutes) (72). 

No specific cargo molecules have been identified to track macropinocytosis, which makes it difficult to follow the intracellular fate of macropinosomes. However, one substance that is applied to study macropinocytosis and to track macropinocytic vesicles is labeled high-molecular-weight dextran (molecular weight 70,000) (77). 

Araki N, Johnson MT, Swanson JA. A role for phosphoinositide 3-kinase in the completion of macropinocytosis and phagocytosis by macrophages. J Cell Biol 1996 135(5) 1249-1260. 

Zenni MK, Giardina PC, Harvey HA, et al. Macropinocytosis as a mechanism of entry into primary human urethral epithelial cells by Neisseria gonorrhoeae. Infect Immiin 2000 68(3) 1696-1699. 

Lee E, Knecht DA. Visualization of actin dynamics during macropinocytosis and exocytosis. Traffic 2002 3(3) 186-192. 

Sallusto, F., Celia, M., Danieli, C. and Lanzavecchia, A. (1995) Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment downregulation by cytokines and bacterial products. J. Exp. Med., 182, 389M00. 

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. 

Wadia, J. S., Stan, R. V, and Dowdy, S. F. (2004) Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat Med 10,310-315. 

New, E.J. and Parker, D. (2009) The mechanism of cell uptake for luminescent lanthanide optical probes the role of macropinocytosis and the effect of enhanced membrane permeability on compartmentalization. Organic... 

Cell-surface receptors are involved in both phagocytosis and pinocytosis. At least four distinct mechanisms of pinocytosis have been characterized macropinocytosis, clathrin-mediated endocytosis, raft/caveolae-mediated endocytosis, and clathrin-and caveolae-independent endocytosis (1). Selected receptor-mediated aspects of these mechanisms are outlined below. 

---