- Review Article
- Open Access
Cellular Endocytosis and Gene Delivery
© The Feinstein Institute for Medical Research 2010
- Received: 30 July 2009
- Accepted: 2 February 2010
- Published: 3 February 2010
Endocytosis is the process by which cells take up macromolecules from the surrounding medium. The best-characterized process is the so-called clathrin-dependent endocytosis, although much is also currently known about clathrin-independent endocytic processes such as those involving caveolae and lipid rafts. An understanding of endocytosis and the cellular trafficking that occurs thereafter has a great deal of relevance to current molecular medicine. Gene therapy, which is presently being investigated for its therapeutic potential in treating immunodeficiency and metabolic diseases, cancer and heart disease, employs a variety of viral and nonviral vectors, which can be delivered to the target cells of the body and are subsequently endocytosed and dissembled. A variety of vectors can be used to deliver genes to organs invivo or cells exvivo. Various routes of vector delivery have been investigated. The mechanisms by which vectors such as adenoviruses, adeno-associated viruses, retroviruses and liposomes enter the cell are increasingly being investigated as the effort to increase the efficiency of gene therapy continues. This review focuses on mechanisms of endocytosis and how they relate to the internal trafficking of viral and nonviral vectors in gene therapy.
Cells are able to take up macromolecules from the surrounding medium by endocytosis. In this process, the material to be internalized is surrounded by an area of plasma membrane, which then buds off inside the cell to form a vesicle containing the ingested material. The best-characterized form of this process is receptor-mediated endocytosis, which provides a mechanism for the selective uptake of specific macromolecules. The macromolecules to be internalized first bind to specific cell surface receptors. These receptors are concentrated in specialized regions of the plasma membrane, called clathrin-coated pits (1,2), which can be internalized to form clathrin-coated vesicles (CCV). There is evidence that cells also possess clathrin-independent endocytosis pathways. One pathway of clathrin-independent endocytosis involves the uptake of molecules in small invaginations of the plasma membrane called caveolae, which have been implicated in cell signaling and a variety of transport processes, including endocytosis (3).
The CCV have been extensively studied and their characteristics well described. They comprise a clathrin coat that is linked to cargo-bearing receptors via a heterotetrameric adaptin complex. The adaptin complex comprises HA2 and AP-2 subunits, which in turn are made up of 100-kDa a- and b2 adaptins together with smaller µ2 (50 kDa) and s2 (17 kDa) subunits. a-Adaptin and b-adaptin both contain clathrin-binding sites.
Signals for the incorporation of cargo receptors into plasma membrane-derived CCV have been defined based on examination of their protein sequences. The best-characterized portion of the protein is the YXXØ motif, in which Ø represents a bulky hydrophobic amino acid (4). Such signals are found in both constitutively endocytosed receptors (for example, transferrin receptor) and those such as the epidermal growth factor (EGF) receptor, which are endocytosed only after lig-and binding. In the latter case, ligand binding causes phosphorylation of the receptor and a conformational change that results in exposure of the internalization signal. The µ2 adaptin subunit has been shown to interact with these tyrosine-based internalization signals. An alternative internalization signal, dileucine, does not interact with the µ2 chain in vitro, although there is evidence indicating an interaction with AP-2 (5). An exception to the model of AP-2-mediated recruitment of receptors to CCV is provided by G-protein-coupled receptors. In this instance the activated proteins are recruited by b-arrestin, which is believed to function as a specialized adaptor protein because it also binds to clathrin (6).
The process of CCV formation is generally agreed to be dependent on cytosol and ATP, with the additional requirement for multiple GTP-binding proteins of which the best characterized is dynamin (7). As well as binding directly to a-adaptin, dynamin binds to proteins that contain Src homology 3 (SH3) domains through interactions with its proline-rich C terminus (8). The growth-factor receptor-binding protein Grb2 also binds dynamin via an SH3 domain and activates its GTPase activity in a synergistic manner with phosphoinositides (9). This requirement for proteins such as dynamin is apparent on disruption of EGF-receptor recruitment of Grb2. Indeed, on inhibition of Grb2 binding via microinjection of a peptide corresponding to its SH2 domain, receptor endocytosis is impaired (10). Recent work has also shown that the F-actin cytoskeleton is required for the formation and internalization of clathrin-coated pits. Reflectively, via fluorescence microscopy, disruption of F-actin dynamics has been shown to significantly impede clathrin-coated mediated endocytosis (11).
In contrast, caveolae typically appear as rounded plasma membrane invaginations of 50 to 80 nm in diameter on electron microscopy sections. Their composition, appearance and function are cell-type dependent. In endothelial cells, caveolae can be more constricted at the mouth, or they may contain a diaphragm that restricts diffusion (12). In muscle cells, caveolae are often observed in the form of composite clusters or linear rows of multiple flask-shaped units involved in the formation of T-tubules (13). Although caveolae do not show an electron-dense layer on their cytosolic surface in thin-section electron micrographs, they do have a protein coat composed primarily of a protein called caveolin-1 (or caveolin-3 in muscle cells) (14).
Caveolins are integral membrane proteins of 21 kDa. They have an unusual topology in that the cytosolic N- and C-terminal domains are connected by a hydrophobic sequence that is buried in the membrane but does not span the bilayer (15). Caveolins are palmitoylated in the C-terminal segment; they can be phosphorylated on tyrosine residues, bind cholesterol, and form dimers and higher oligomers (15–17).
Caveolins are essential for the formation and stability of caveolae. In the absence of caveolins no caveolae are observed, and when caveolins are expressed in cells that lack caveolae, they induce caveolar formation (18). During endocytosis of caveolae, caveolin-1 moves along with the vesicles into the cytosol with no visible remnant left in the plasma membrane (19). Recent data indicate that the presence of caveolin-1 may actually slow down the endocytic process via caveolae (20). These data suggest that the role of caveolin may be to stabilize caveolae and thus to counteract an underlying raft-dependent endocytic process. The finding that certain ligands internalize through a lipid-raft-dependent but clathrin-independent mechanism in cells that lack caveolae has led to the postulation that lipid rafts can internalize independently of caveolae (21). In this context, it is significant that caveolin knockout mice survive relatively well, although their cells do not have detectable caveolae (3). More recently, caveolin has been shown to be involved not only in endocytosis, but also in cellular processes such as calcium signaling, homeostasis and cholesterol transport. Hence, these domains may actually have a multitude of significant functions within the cell and may serve as key regulatory domains (22,23).
In addition to caveolins, caveolae are known to contain dynamin (24), the same GTPase that is involved in the formation of CCV described previously. Dynamin has been localized to the neck of flask-shaped caveolar indentations and is therefore likely involved in pinching off the caveolar vesicle through a mechanism similar to its role in coated vesicle fission (25).
In recent years evidence has been accumulating that in addition to caveolae, several other clathrin-independent endocytosis pathways exist and that they may have a more important role in cell function than previously suspected. In particular, much effort is focused on understanding membrane lipid rafts and microdomains (26). Rafts have been defined as dynamic assemblies of cholesterol and glycosphingolipids that are present in the lateral plane of membranes. The hypothesis that lipid rafts harbor a functional role in membrane transport was initially based on two observations. First, glycosphingolipids were found to be preferentially targeted to the apical pole of polarized cells (27). Furthermore, glycosphingolipids were shown to be part of detergent-resistant membranes that also contained apically targeted glycosylphosphatidyl inositol-anchored proteins (28). Thus, the hallmarks of the raft hypothesis are the spontaneous partitioning of lipids and proteins in discrete membrane domains (microdomains), a behavior based on their intrinsic physicochemical characteristics, and the experimental recovery by biochemical flotation of these microdomains, along with their associated proteins, as detergent-resistant complexes. Recently, two membrane proteins, flotillin 1 and flotillin 2, have been found to coassemble to form microdomains with many of the predicted properties of lipid rafts (29). Although much remains unknown about the role of these proteins in endocytosis, the microdomains formed during their interaction have been shown to invaginate and form vesicles through mechanisms that are independent of clathrin and caveolae (30,31).
Furthermore, Cheng et al. demonstrated that several different clathrin-independent mechanisms of endocytosis occur in Chinese hamster ovary cells and that each has a distinct need for sphingolipids. Indeed, depletion of glycosphingolipids, a subgroup of sphin-golipids, was found to differentially affect and decrease endocytosis dependent on caveola, RhoA and Cdc42 (4,32). Despite the fact that by some estimates as much as 50% of the overall endocytic activity of the cell (33) occurs via such clathrin-independent mechanisms, the lack of well-defined systems has prevented the detailed identification and understanding of the precise function of these alternate pathways despite the surge in interest in identifying the roles of such raft constituents in endocytic platforms (34).
Phagocytosis and macropinocytosis are forms of endocytosis in which larger molecules are engulfed into the cell. Large molecules such as pathogens, dust particles and cell debris can be ingested by specialized immune cells and then encapsulated by a vesicle termed a phagosome. Phagosomes mature to fuse with lysosomes, ultimately becoming vacuoles within which the engulfed pathogen is degraded. Single proteins and fluid particles can be taken up via macropinocytosis, which involves the invagination of the plasma membrane and trafficking to the endosomes. Ultimately, these processes are more nonspecific and involve larger particles than the aforementioned clathrin- and caveolae-dependent endocytosis pathways (35).
After their internalization, endocytosis vesicles rapidly fuse with endosomes. Once again, the best-studied pathway is that of the CCV. The delineation of the various components of the pathway in the cytosol and the regulation of the steering of different molecules to their respective targets is not well understood (1). The most accepted model is internalization followed by the formation of early endosomes, late endosomes and lysosomes (36). This classification reflects a definition of endocytic compartments based on pulse or pulse-chase protocols and on the presence of marker proteins associated with these respective compartments.
The early endosome is the major sorting station on the endocytic pathway. From this organelle, material can be directed toward the pathway of recycling to the plasma membrane (for example, transferrin), to later endocytic compartments and to regulated secretory vesicles (37). Studies on transferrin-receptor recycling have defined a tubular “recycling endosome” into which transferrin receptors enter after sorting from late-endosome-directed material (12). This recycling endosome is typically less acidic (pH 6.4–6.5) than the sorting endosome (pH 6.0) and exhibits a pericentriolar localization (38).
Late endosomes are the point on the endocytic pathway where the cation-independent mannose 6-phosphate receptor (M6PR) is most concentrated. Degradative enzymes are active in this compartment but are more concentrated in lysosomes (39). The M6PR protein has been found to be concentrated on the internal vesicles of late endosomes, whereas lysosomal membrane proteins are on the external membrane (40). Transfer of material between late endosomes and lysosomes appears to be a direct fusion event that results in a transient hybrid organelle (41). Lysosomes are also multivesicular but completely lack M6PR.
Among the proteins that are present in the various compartments of the endocytic pathway are several small GTPases. These proteins have been particularly scrutinized because of their usefulness as molecular markers for various types of endosomes. Rab5, one such GTPase, regulates the rate of clathrin-dependent endocytosis at the plasma membrane (42) and could be used as a marker for early sorting endosomes in addition or in conjunction with the transferring receptor. Rab11 localizes to a pericentriolar subpopulation of transferrin-labeled endosomes (recycling endosomes) (43).
Viral vectors and endocytic pathways.
End destination in cytoplasm
Target cellular organelle
Coxsackievirus-adenovirus receptor cell surface protein
Heparan sulfate proteoglycan, αVβ5, fibroblast growth factor receptor
Nuclear pores (?)/endosome
Transmembrane (TVA950) or glycophosphatidylinositol-anchored (TVA800)
Lipid-raft-dependent pathway, clathrin-coated pits
Clathrin-coated pits/membrane fusion
The endocytic route by which a molecule, virus or drug complex enters the cell is highly dependent on its size, charge and composition, as well as on the cell type into which it is entering. For example, although specialized immune cells may nonspecifically take up molecules such as the remnants of other cells that have undergone apoptosis via phagocytosis, specific complementary sequences are needed by hormones to enter the target epithelial cells via receptor-mediated endocytosis. Furthermore, although approximately 100 nm is the optimal size for the uptake of a molecule via the numerous types of clathrin-mediated endocytosis, particles of 500 nm in size are typically endocytosed via caveolae-dependent endocytosis (44).
Because the endocytic route by which a drug is able to be taken up can influence which cell type it targets, its levels of degradation after cell entry and the efficiency by which it can enter the cell, suitable vectors must be found depending on the purpose of the medication being administered. For example, drugs can be encapsulated in large lipsomes and coated with opsins to target them for phagocytic uptake, and others can be complexed with a peptide ligand for receptors overexpressed by tumor cells. Such processes allow for manipulation of the various endocytic pathways to achieve specificity and efficiency.
Adenoviruses carry double-stranded DNA and at the time of infection introduce their DNA into the host cell. Unlike adeno-associated viruses (AAV), the genetic material of adenoviruses is not incorporated into the genome of the target cell. Thus, although the viral genes are transcribed, they are not replicated on cell division and they will not be passed on to daughter cells. Despite the fact that any adenoviral gene therapy treatment would be transient, such viruses are nevertheless being considered for their role as vectors for therapeutic genes to help treat infarcted areas of the heart, tumors and other diseased target tissues.
Adenoviruses enter target cells via receptor-mediated endocytosis. Penton and fiber proteins of the caspid of the virus interact with the coxsackievirus-adenovirus receptor (CAR) cell-surface protein to initiate cell binding (45,46). These receptors are then localized to clathrin-coated pits via an NPXY motif of β3 and β5 integrin subunits of the cell. Dynamin, a cystolic GTPase, mediates the endocytosis of the virus into the cell by facilitating the constriction and budding off of the clathrin-coated pits. Preceding endocytosis, the proteins of the viral capsid are shed and the pH of the viral endosome decreases as a result of proton pumps. At pH 6.0, the virus is able to escape from the vesicle and enter the cytosol. The caspid proteins of the virus are trafficked to the nucleus by microtubules and further dismantled. Although the majority of the proteins of the caspid remain at the periphery of the nucleus, the viral DNA is extruded through the nuclear pores (47).
AAV are also being considered in the search for efficient gene therapy vectors because of their nonpathogenic properties. In contrast to adenoviruses, AAV carry single-stranded DNA, which is inserted into the genome of the host cell. These viruses enter host cells via a mechanism very similar to that of adenoviruses. Heparan sulfate proteoglycan is the primary receptor to which AAV attach, and fibroblast growth factor receptor 1 and αvβ5 integrin coreceptors facilitate the entry of the virus into the cell. AAV enter the cell through the internalization of clathrin-coated pits and, like adenoviruses, escape endosomal degradation via acidification of the late endosome. AAV are trafficked through the cytoplasm and uncoated in the nucleus. Their single-stranded genome is converted to double-stranded DNA and subsequently inserted into chromosome 19 (48). The clinical relevance of such work was demonstrated by Spencer et al., who successfully delivered a lysosome enzyme across the blood-brain barrier of mice via a lentivirus delivery system targeting the receptors of the central nervous system. Such work is extremely significant in that this area is difficult to target under normal circumstances because proteins are unable to cross this physiological barrier, yet the delivery of such macromolecules may be necessary for the treatment of many neurological disorders (49).
These two virus types remain among the most commonly used and studied gene therapy vectors. Although issues with these vectors remain, namely the immune response against the viruses, transient expression and suboptimal gene delivery efficiency due to lack of the respective primary receptors on target cells, some success has been observed in clinical and preclinical trials in which adenoviruses and AAV have been used. For example, Wohlfahrt et al. demonstrated that glioblastoma tumors could be effectively killed in mice via injection of the tumor cells with an adenovirus vector expressing TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) (50). Furthermore, adenoviral vectors harboring genes for vascular endothelial growth factor and fibroblast growth factor are currently being used in clinical trials aimed at treating ischemia (51,52). However, such trials have had limited success thus far, possibly owing to the brief expression of the encoded angiogenic genes after delivery of the virus. In clinical trials in varying stages, adenoviral vectors are also being used to treat cystic fibrosis, hemophilia, arthritis and heart disease (53,54). In several studies, cystic fibrosis transmembrane regulator cDNA was delivered to the epithelial cells of the lumen of the airways leading to the lungs. These trials served as a significant base that aided in the establishment of optimal dosages of the vector, and further trials similar in nature are now being conducted (54).
Retroviruses are another category of viruses that are used as vectors in gene therapy, and several features of retroviral gene therapy such as long-term expression and absence of immunogenicity are very attractive (55). However, there have been reports of leukemia associated with treatment in clinical trials of gene therapy for X-linked severe combined immunodeficiency (56–58), which has led many researchers to look for alternatives. Lentiviruses are another category of viral vectors that are related to retroviruses and are gaining increased attention because of potential better safety and efficiency (55,59).
The natural specificity of ligands and antibodies can be harnessed and used via their conjugation to synthetic compounds. Such specific targeting of diseased cells is crucial to the safety and efficiency of gene therapy, and remains the first of several major obstacles impeding the use of many of the currently designed vectors in a clinical setting. Furthermore, because cargo must escape from the endocytic vesicles and proceed to the nucleus after cellular uptake, synthetic compounds must harbor a mechanism that mimics that used by viral particles to facilitate release into the cytosol. Nuclear entry of nonviral vectors is also a significant barrier in nonmitotic cells. Although viral vectors can actively transfer their genetic material through the pores of the nucleus, synthetic vectors are currently unable to efficiently deliver their cargo through an intact nuclear envelope.
The efficiency of viral vectors makes them attractive models on which to base the synthesis of future compounds or, in contrast, foundations to modify such that toxicity and specificity can be optimized. For example, shielding, or coating, of viral surfaces with polyethylene glycol, a hydrophilic polymer, has been found to reduce the immunogenicity of such vectors (78). Furthermore, retargeting of viral vectors via conjugation to synthetic ligands after such shielding could induce novel and targeted specificities. In addition, it has been demonstrated that endosomal release of synthetic compounds such as DNA polyplexes can be facilitated via the covalent linkage of polyethylenimines to melittin analogs (79).
The delivery of nucleic acids (as well as proteins and drugs) to damaged or diseased cells has significant therapeutic implications, assuming efficient and practical vectors can be found. Hence, the mechanisms by which these molecules are taken up and processed by cells is becoming increasingly important. Despite our progress in understanding its various components, endocytosis is a complex process, much of which remains to be clarified. The need to elucidate the various pathways and steps of endocytosis increases in significance with continuing efforts to efficiently deliver and to prolong the expression of various genes. Ultimately, although a variety of cellular uptake and endocytic strategies can be manipulated in the effort to efficiently deliver genetic material to targeted cells, clinical use of any one of the existing viral or synthetic vectors remains limited. Fusions of the existing vector types to incorporate the desirable attributes of each may provide the most efficient means to obtain optimized vectors that successfully and specifically traverse the endocytic pathways of human cells.
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
IS Jovin’s work was supported by NIH Grant 5F32HL076016.
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