Uptake-mechanisms in eukaryotic cells

The cellular membrane separating the cytosol from the extracellular environment has the function of a highly selective, but active barrier, which is able to transmit mechanical, electrical as well as biochemical cues. (Zellner et al., 2013) The plasma membrane is build by an asymmetric lipid bilayer composed of glycerophospholipids, sphingolipids, cholesterol and embedded or associated proteins, which serve for signal transduction or also as selective gates. For example, some ions are allowed to cross

the membrane via specialized ion channels, which is expressed in the membrane potential of living cells, and small molecules like saccharides are often internalized through mediated transport by transporter proteins. Additionally, steroid hormones, which mainly bind to intracellular receptors, are able to penetrate into the cell due to their hydrophobicity. However, uptake of larger objects like nanoparticles requires specialized routes of entry, which are summarized under the term “endocytosis”.

Endocytosis encompasses deformation of the membrane and wrapping of the material to be internalized by the membrane. Finally, a vesicle is pinched off from the plasma membrane. Endocytosis also plays an important role in the control of membrane compositions, which has an impact on the long-term sensitivity of a cell to external stimuli as receptors are often removed from the cell surface.(Doherty and McMahon, 2009) Despite many differences, all endocytotic pathways rely on active, energy consuming processes, which for example facilitate fission of the endocytotic vesicle from the plasma membrane or drive the formation of membrane protrusions or membrane ruffles. The main routes of endocytosis are summarized in Figure 2.8 and are discussed briefly in the next subchapters. A detailed description of endocytotic routes can be found in the review by Doherty and McMahon. (Doherty and McMahon, 2009) Furthermore, endocytosis in the context of bio-nano interactions has been summarized by Zellner et al..(Zellner et al., 2013) Endocytosis of Janus nanoparticles will be discussed in chapter 4.1.

Figure 2.8: Main routes of endocytosis. Phagocytosis is used by cells to engulf large solid objects and relies like macropinocytosis on protrusive forces generated by the actin cytoskeleton. Macropinocytosis enables the cell to take up large volumes of extracellular fluid

by the formation of actin-supported membrane ruffles. Small volumes can be taken up by the cell through clathrin-mediated and calveolar-type endocytosis. Dynamin facilitates the fission of

the vesicle from the plasma membrane. Size of the vesicles formed by the distinct endocytotic pathways can be found in brackets.(Pollard et al., 2008)(modified from (Conner and Schmid,

2003))

2.1.4.1 Clathrin-mediated endocytosis

Clathrin-mediated endocytosis (CME) is the best understood among the different endocytotic pathways and occurs in nearly all cell types. Many cargoes including nutrients, antigens, growths factors, pathogens and recycling receptors are taken up via this route. It plays also an important role in neurons, where CME is responsible for the regeneration of synaptic vesicles. CME is characterized by the presence of clathrin lattices that coat the membrane invaginations and freshly formed endocytotic vesicles at the cytosolic side. Clathrin itself exhibits the structure of a triskelion and is composed of three heavy chains and three light chains. CME is initiated by the recruitment of adaptor proteins to the membrane, e.g. adaptor protein-2, which serves as a ligand for clathrin. Upon binding, clathrin triskelions form highly organized cages, so called clathrin-coated pits that support the required curved membrane. The formed membrane invaginations are eventually pinched off from the plasma membrane under assistance of the GTPase dynamin. The actin cytoskeleton is responsible for the transport of the vesicle. The clathrin coat is removed from the endocytosed vesicle by the chaperone hsp70 (heat shock protein 70) under ATP-hydrolysis.(Takei and Haucke, 2001)

2.1.4.2 Caveolar-type endocytosis

Caveolae represent another endocytosis route, which facilitates the uptake of rather small cargoes. They have flask-like shape and their assembly relies on the protein caveolin. Hundred to two hundred calveolin molecules can be found per caveola. There are three isoforms of the protein, among whom calveolin1 has been shown to be the only one necessary for the formation of caveolae. It seems to be enriched in membrane domains with high curvature. Caveolin possesses a hairpin structure and binds to the inner leaflet of the plasma membrane, where it serves for the formation of cholesterol-rich microdomains. Depletion of cholesterol has been shown to flatten the caveolae and increase the lateral mobility of caveolin in the membrane. Caveolar-type endocytosis appears to be mainly involved in transcytosis, in which, for example, nutrients are transported from one side of the cell to another.(Doherty and McMahon, 2009)

2.1.4.3 Macropinocytosis

In contrast to the routes of endocytosis discussed previously, macropinocytosis allows the uptake of larger volumes. This pathway has been linked to the presence of membrane ruffles, which are produced by actin-polymerization under the co-assistance of rac1, a small GTPase of the Rho-family. These membrane ruffles engulf extracellular components and form endocytotic vesicles by fusing with themselves. It has been shown, that membrane ruffles are enriched in cholesterol. Cholesterol is important for the recruitment of rac1 to the plasma membrane. Other markers of lipid rafts have also been found in membrane ruffles suggesting a close interplay between inhomogeneity of the membrane and dramatic changes in the intracellular organization at these sides.(Doherty and McMahon, 2009) The uptake of objects by this pathway is rather unspecific but facilitates for example continuous nutrient uptake.(Pollard et al., 2008)

2.1.4.4 Phagocytosis

Like macropinocytosis, phagocytosis relies on protrusive forces acting on the membrane, which are generated by actin polymerization. The actin polymerization at the phagocytic membranes is achieved by recruitment of different regulating proteins to the membrane by the Rho-family protein cdc42. Specialized cells like macrophages, monocytes and neutrophils mainly use phagocytosis to take up pathogens or other opsonized particles.(Doherty and McMahon, 2009)