Cell Fusion, Cell Hybrids

 Fusions of both the external and intracellular membranes of cells are important for differentiation and development. Moreover, enveloped viruses infect cells via fusion of their envelopes with cell or endosome membranes.

 Cell fusion is the process of fusion of the membranes of two or more cells and results in the formation of cells with multiple nuclei. It occurs at various stages of the natural development of organisms, such as in the first step of fertilization of an oocyte with sperm and in myotube formation by fusion of myoblasts during differentiation of skeletal muscles. Artificial cell fusion can be induced by the addition of a high concentration of Sendai virus, an enveloped virus of the paramyxovirus group, as was demonstrated in 1957–63 (1-3). This finding coincided with cell biology's beginning focus on the culture of somatic cells of birds and mammals. In 1961, Barski et al. (4) reported the appearance of hybrid cells after a few months of mixed culture of two different mouse cancer cell lines. These hybrid cells had a single nucleus containing chromosomes from both parent cell lines, and their appearance was considered to be due to the spontaneous fusion of cells of the two cell lines.

 Sendai virus proved to be an important development in this field because it has some useful characteristics for the fusion of somatic cells:

1.Its targets are sialoglycoproteins and sialolipids, which are present in the cell membranes of almost all mammalian and fowl cells. Consequently, it can induce the fusion of a wide range of cells (3).

2. Its cell fusion activity is not affected by procedures that inactivate the viral genome, such as exposure to ultraviolet (UV) light. Thus, fused cells could be prepared under conditions inhibiting virus growth, using a UV-inactivated virus.

3. There is no species specificity in its fusion of cells, so interspecific heterokaryons can be induced easily (5, 6), unlike in the case of fertilization.

4. The frequency of virus-induced hybrid formation is at least 1000 times greater than that of spontaneous hybridization.

Littlefield reported in 1964 (7) further progress in methods for the selection of hybrid cell clones, by fusing two different mutants defective in the salvage pathway for nucleotide biosynthesis and culturing them in a medium containing aminopterin, which inhibits de novo nucleotide synthesis. One of the mutant cell lines that were fused lacked thymidine kinase, the other hypoxanthine-guanine phosphoribosyl transferase. The only cells that could grow from the mixed culture were fused cells that had acquired mutual complementation of the two mutant defects. Based on these new techniques, the field of somatic cell genetics was established in the 1960s.

 That the chemical fusogen polyethylene glycol is effective for the fusion of protoplasts of plant cells was first reported in 1974 (8). With this fusogen, hybrid plants, such as the “pomato,” can be prepared in cultures of somatic hybrid cells. Moreover, electroporation (see Transfection) was found to be useful as a physical method for cell fusion in the 1980s (10, 11). Cell fusion by these two methods does not require viral receptors, so they have made possible the fusion of cells from all kinds of organisms.

In the early stage of somatic cell genetics in the 1960s and 70s, many reports provided important information on some basic phenomena in cell biology, permitting a focus on molecular biology in the next stage of the 1980s and 90s. The main findings were as follows:

 1. Immediately after multinucleated cell formation by artificial cell fusion, some nuclear proteins derived from the parents are rapidly transferred and mixed between the various nuclei. This tends to induce synchronization of the stage of DNA replication of the nuclei after the fusion of randomly growing cells. The degree of this synchronization is greatest in cells with only two nuclei and decreases sharply with an increasing number of nuclei in polykaryocytes (12, 13). This may be why almost all randomly isolated hybrid cells have in their nucleus one set of chromosomes derived from each parent. As a special case, the fusion of cells in mitosis with cells in interphase causes the rapid dissolution of the nuclear membrane of the interphase cells, followed by the condensation of its chromosomes, a process named chromosomal pulverization (14) or premature chromosome condensation (15). Reactivation of dormant nuclei from chick erythrocytes was demonstrated upon their fusion with cultured mammalian cells (16, 17). In the case of cell neoplastic transformation by tumor viruses, induction of Simian Virus 40 (SV40(production was observed after the fusion of SV40-transformed nonproducer hamster cells with monkey cells, which are a permissive host for SV40 (18). Later, a similar observation was reported on the fusion of Rous sarcoma virus (RSV)-transformed rat cells with chicken cells (a permissive host of RSV) (19). In the case of cell differentiation, the distinction between luxury and household functions of cells was observed by the formation of hybrids of cells with different phenotypes (20). The findings were important for choosing the correct combination of cells for hybridization of differentiated cells. In 1974, Köhler and Milstein (21) reported that monoclonal antibodies could be prepared from hybrid cells (hybridomas) of B cells and myelomas (tumor cells obtained from B cells). This method of preparing monoclonal antibodies is now a major technique in molecular, cell, and developmental biology, and in medicine.

2. Weiss and Green (22) observed that the chromosomal balance is unstable in interspecific man/mouse hybrids and human chromosomes disappear randomly during serial passage in culture. Based on this finding, mapping of the human chromosomes became possible (23) and was followed by the technique of direct in situ hybridization of the chromosomes with complementary DNA.

3. In 1972, Bootsma and colleagues (24) demonstrated that genetic complementation groups in a hereditary disease, xeroderma pigmentosum, could be classified by the cell fusion technique. This was the first successful genetic analysis of a human hereditary disease.

1. Mechanism of Cell Fusion

 Artificial cell fusion using the various fusogens mentioned above has been established as a routine laboratory method. These fusogens, viral, chemical, and physical, have different modes of action on cell membranes, but induce similar changes in the cell membrane for fusion of the lipid bilayers. In general, glycoproteins are distributed on cell surfaces, with their hydrophobic domains embedded in the lipid bilayer of the cell membrane, their hydrophilic domains exposed to the outside, while their intracytoplasmic domains are associated with the cytoskeletal system. Water molecules are also associated with the outside of cell membranes. These structures on the exteriors of cells inhibit the close contact of lipid bilayers of neighboring cell membranes, which is essential for cell fusion. Close contact of membranes requires on the cell membrane surface the transient appearance of areas from which glycoproteins are excluded. All the fusogens can induce such areas.

 Sendai virus has two glycoproteins: HN with receptor binding and destroying activities, and F with fusogenic activity that is essential for fusion. The first step in cell fusion is the attachment of the virus to cell surfaces and agglutination of the cells, which is produced by HN activity. The second step is insertion of the fusogenic domain of F into the lipid bilayer. This domain is located at the N-terminus of F and consists of 15 relatively hydrophobic residues (25); it has the unique characteristic of trapping cholesterol molecules in its tertiary structure at 37°C (26). The amino acid sequence of this domain is well conserved in paramyxoviruses.

Simultaneous removal of cholesterol molecules from multiple sites in the cell membrane by the attachment of several hundred virus particles perturbs the cell membrane and causes breakdown of the normal barrier to ions. At this stage, calcium ions from the medium promptly penetrate the cell and induce changes in cell structures, such as separating the connection between the cytoplasmic domains of membrane proteins and the cytoskeletal system. Macromolecules can also diffuse through the cell membrane, and membrane fluidity increases, so that membrane proteins can move more freely in the lipid bilayer. This results in clustering of intramembrane domains of the proteins (demonstrable as cold-induced clustering) and the appearance of areas with no membrane proteins. The appearance of these areas is followed by close attachment of the lipid bilayers of neighboring cells, due to strong hydrophobic interactions, and cell fusion (27).

 Polyethylene glycol is reported to induce clustering of membrane proteins similar to that induced by Sendai virus (28). Electroporation by electric pulses causes pores to be formed in cell membranes that may allow Ca²⁺ ions into the cytoplasm, as is also caused by Sendai virus. Thus, the fundamental mechanisms of these three fusogens appear to be similar.

It is known that calcium ions (29) and an energy (ATP) supply (2) are required for cell fusion. In the absence of either one, cells rapidly degenerate and fusion is greatly decreased. Evidence suggests that calcium ions associate directly with phospholipid molecules to normalize the perturbed membrane structure and promote a connection between the two lipid bilayers, in addition to acting in the cytoplasm as mentioned above. Why does cell fusion require an energy supply? In simple terms, cell fusion would be expected to produce a favorable decrease in free energy, by changing from a number of small vesicles to one large one, in which case an energy supply would not be necessary. Energy may be required for the removal of excess calcium ions introduced into the cytoplasm during cell fusion, but not for the membrane fusion itself. On culture of fused cells, the calcium ions are rapidly sequestered in organelles of the cells, and their level in the cytoplasm returns to normal. If the supply of energy is delayed, the cells degenerate.

2. Supplement

 1. In the case of Sendai virus, cell-to-cell fusion is also possible as a result of viral envelope fusion itself, if one virus envelope fuses with the membranes of two different cells. But envelope fusion is slower than that induced by a high concentration of the virus and is observed to occur after completion of cell-to-cell fusion. Finally, as a result of viral envelope fusion, many viral glycoproteins are integrated into the fused cell surfaces, which are excluded from the cell surface by internalization by coated vesicles in culture (30) .

 Polykaryocytes (syncytia) are often observed in pathological tissues infected by an enveloped virus, such as paramyxoviruses, retroviruses and the Herpes virus. This polykaryocyte formation may occur by cell fusion via viral envelope fusion. Newly synthesized viral glycoproteins are distributed massively on the surfaces of infected cells, which consequently are quite similar to the viral envelope, and they may fuse with neighboring noninfected cells, as in viral envelope fusion.

2. Enveloped viruses cause infection by fusion of their envelopes either (a) with cell membranes at neutral pH or (b) with endosome membranes at acidic pH after their internalization from the cell surface. Infections by paramyxoviruses, retroviruses, and the Herpes virus are of the first type and induce syncytia formation in vivo and in vitro. Influenza virus is of the second type and does not induce syncytia in vivo, but the viral envelope can fuse with cell membranes under acidic conditions in vitro. This is because the influenza fusogenic glycoprotein (HA, or hemagglutinin) is not functional at neutral pH, but becomes functional under acidic conditions by a conformational change, forming trimers (31).

The fusogenic glycoproteins of various enveloped viruses differ, but all of them contain a hydrophobic domain that can interact with the lipid bilayer of cellular membranes. In some cases, the glycoproteins of the viruses are known to be synthesized in an inactive form in which their hydrophobic domain is hidden and then activated by proteolytic cleavage exposing this domain.

This was first demonstrated by Homma (32) with the Sendai virus. In this virus, the F glycoprotein is synthesized as an inactive form F0 and is then cleaved to F1 and F2, with the fusogenic domain being exposed at the N-terminus of F1. The glycoprotein (gp160) of human immunodeficiency virus (HIV(a retrovirus, is cleaved to gp120 and gp41, and the fusogenic domain is exposed at the N-terminus of gp41 (33). The HA glycoprotein of influenza virus is also activated by the cleavage of inactive HA0 to HA1 and HA2, with the hydrophobic domain being exposed at the N-terminus of HA2 (34) and becoming functional by trimer formation under acidic conditions.

 3. Application of Fusogenic Reactions to Cell Engineering

Various biotechniques for the reconstitution of cells or introduction of macromolecules into cells have been developed using the unique characteristics of the interactions of fusogens with cell membranes. Introduction of macromolecules such as DNA, RNA, and proteins from the medium into the cytoplasm has become possible by perturbation of the cell membrane. Electroporation is usually used for that purpose (35), but treatment of the cells with Sendai virus can also be used (36).

Another technique for the introduction of macromolecules is based on the mechanism of infection of Sendai virus with cell membranes. In 1979, Uchida et al. (37) reported on the reassembly of viral envelopes as artificial vesicles, with HN and F glycoproteins embedded in their surface and any macromolecules present trapped inside. These pseudovirus particles containing the macromolecules instead of viral nucleocapsids will introduce those macromolecules into a cell that they “infect.” This technique is especially useful in vivo. Subsequently, modifications have been made to simplify the preparation procedure, using spontaneous fusion of the UV-inactivated virus with simple, artificial liposomes containing the desired macromolecules. Using such a preparation, the human hepatitis B virus genome has been introduced into rat liver cells in vivo to induce hepatitis (38). This technique is useful for drug delivery in vivo and gene therapy (39).

 Reconstruction of cells was first reported by Veomett et al. in 1974 (40). On incubation with cytochalasin, cells could be separated into nucleoplasts (enclosed by cell membranes but lacking cytoplasm) and cytoplasts (without a nucleus). Viable cells could be reconstituted by the fusion of nucleoplasts with cytoplasts. Heterologous combinations could also be constituted. This technique has been expanded to the preparation of cloned animals. Introduction of nuclei from somatic cells at the early stages after cleavage of fertilized eggs into enucleated eggs, by Sendai virus-mediated fusion, is reported to result in a high frequency of development of animals (41).

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