The membrane fusion and cell swelling stages of Sendai virus-mediated cell-cell fusion have been studied by thin-section and freeze-fracture electron microscopy. Sites of membrane fusion have been detected in human erythrocytes arrested at the membrane fusion stage of cell fusion and in virtually all cases a fused viral envelope or envelope components has been identified thus providing further direct evidence that cell-viral envelope-cell bridge formation is the membrane fusion event in Sendai virus-induced cell fusion. Radial expansion of a single virus bridge connecting 2 cells is sufficient to produce a fused cell. Membrane redistribution which occurs during this cell swelling stage of the fusion process is often accompanied by the formation of a system of membrane tubules in the plane of expansion of the virus bridge. The tubules originate from points of fusion between the bridging virus envelope and the erythrocyte membrane and also expand radially as cells swell. Ultimately membrane rupture occurs and the tubules appear to break down as small vesicles. When previously observed in cross-sectioned cells these membrane tubules were interpreted as sites of direct membrane fusion. The present study indicates that this interpretation is incorrect and shows that the tubules are generated subsequent to membrane fusion when 2 cells connected by a virus bridge are induced to swell. A mechanism to explain the formation of this system of membrane tubules is proposed.

Three stages during Sendai virus-mediated cell fusion have been distinguished: (1) cell agglutination; (2) membrane fusion; (3) cell swelling (Knutton, 1978). Stage 1 involves the crosslinking of adjacent cells by virus particles (Bächi & Howe, 1972); stages 2 and 3 have only recently been distinguished as separate events (Knutton & Pasternak, 1979) and the mechanism of these 2 events is less well understood.

Two different mechanisms for the membrane fusion event have been proposed. The cell-viral envelope-cell bridge mechanism (Apostolov & Almeida, 1972), which implicates fusion of a viral envelope directly in the formation of a cytoplasmic connexion, has not been widely accepted because of the lack of direct morphological evidence although several examples showing involvement of a virus particle or viral envelope components at sites of fusion have recently been reported (Knutton, 1977, 1978). The alternative cell-cell bridge mechanism (Bächi & Howe, 1972; Toister & Loyter, 1973), which has been more widely accepted, proposes that a fused virus particle is not directly involved in membrane fusion between cells. In this mechanism fusion of virus particles with the cell plasma membrane is thought to modify the structure of the membrane making it susceptible to fusion. This mechanism was proposed to explain the finding that virus particles, virus fragments or viral antigens were not seen at cytoplasmic connexions between fused cells (Schneeberger & Harris, 1966; Cassone, Calio & Pesce, 1973; Bächi, Aguet & Howe, 1973; Hosaka & Shimizu, 1977)

The difficulty in determining which mechanism is correct stems from the fact that fusion of virus particles with the cell surface results in cell swelling (Knutton & Bächi, 1980). Sites of membrane fusion, therefore, only have a transient existence and since cell swelling leads to a dispersal of viral envelope components (Knutton & Bächi, 1980), ultrastructural studies have not yet provided conclusive evidence in favour of either mechanism. The recent introduction of a method for growing nonhaemolytic Sendai virus (Homma, Shimizu, Shimizu & Ishida, 1976) now makes it much easier to localize sites of fusion because this virus does not induce cell swelling and so cell-cell fusion is arrested at the membrane fusion stage (Knutton, 1979). The same situation can also be achieved with haemolytic virus if fusion is carried out in the presence of high concentrations of divalent cations since divalent cations inhibit virally-mediated cell swelling and haemolysis (Toister & Loyter, 1973).

Most ultrastructural studies of cell-cell fusion have concentrated on the problem of how membrane fusion occurs and little or no consideration has been given to the problem of how 2 cells joined at sites of membrane fusion expand to form a fused cell. The realization that Sendai virus induces cell swelling and that it is an event subsequent to membrane fusion had led to the proposal that osmotic cell swelling is the mechanism whereby cells connected at sites of membrane fusion expand such sites to form a fused cell (Knutton & Pasternak, 1979) and evidence to support this proposal has been presented (Knutton & Bächi, 1980). The process of membrane redistribution during cell swelling, however, has not been investigated in any detail.

I have now used thin-section and freeze-fracture electron microscopy to examine both the membrane fusion and cell swelling stages of cell-cell fusion and in this paper I present further direct evidence to show that virus bridge formation is the membrane fusion event in Sendai virus-mediated cell fusion and I also describe the often complex process of membrane redistribution which occurs when cells connected by a virus bridge swell to form a fused cell.

Cell fusion

A 1% suspension of 3-times washed human erythrocytes in phosphate-buffered saline (PBS) was used for fusion experiments.

Batches of haemolytic and nonhaemolytic Sendai virus were grown in 10- or 11-day-old chick embryos for 72 or 24 h respectively and clarified by centrifugation as previously described (Knutton & Bächi, 1980).

For fusion experiments equal volumes of virus (∼ 103 haemagglutinating units/ml) and the erythrocyte suspension were mixed at 4 °C. After 15 min the agglutinated cell suspension was transferred to a waterbath at 37 °C and incubated with gentle shaking for various time intervals depending on the nature of the experiment. The fusion process was arrested by the addition of an equal volume of buffered 6% glutaraldehyde. In some experiments 20 mM Mn2+ was present; for these experiments Tricine-buffered saline (TBS) was used.

Electron microscopy

For freeze fracture glutaraldehyde-fixed cells were washed with buffer, infiltrated with 25% glycerol and fractured at −11o °C in a Denton freeze-fracture device. Platinum carbon replicas were cleaned with 5% sodium hypochlorite.

For thin sections cells were fixed for 1 h with 3% glutaraldehyde in 0·1 M cacodylate buffer pH 7·4, washed in 02 M cacodylate buffer and postfixed with 1% osmium tetroxide in 0·1 M cacodylate buffer. Ruthenium red (0· 2%, final concentration) was present in both fixative and wash solutions (Luft, 1971). Following fixation cells were dehydrated through a graded series of ethanol solutions and embedded in Epon. Sections were cut with glass knives using a Tesla ultramicrotome and stained with uranyl acetate and lead. Thin-section and freeze-fracture replicas were examined in either a Siemens 101 or Philips 301 electron microscope.

Membrane fusion

Fused virus particles have a highly convoluted morphology due to the formation of linear invaginations of the viral envelope prior to fusion. These linear segments of the viral envelope have a very small radius of curvature and, in freeze-fracture replicas, appear as smooth linear grooves on P fracture faces (Fig. 1A) and smooth linear ridges on E faces (Fig. 1 B). Fused virus particles can also be recognized in conventional thin-section preparations but, because of their highly irregular morphology are more easily identified when the envelope has been labelled with an electron-dense tracer like ruthenium red (Fig. 1 C-E). Cross-sectioned linear invaginations can be recognized by the low radius of curvature of the membrane (Fig. 1c, arrowheads) whereas tangential sections reveal their linear structure (Fig. 1D).Fig. 1D, E, are consecutive serial sections and illustrate the complex relationship between fused virus particles and the erythrocyte membrane. These characteristic linear components provide a unique marker for fused virus particles in both thin-section and freeze-fracture preparations. The helical coiled viral nucleocapsid is an additional marker for the virus in thin sections (Fig. 1C-E).

Fig. 1.

Freeze-fracture (A, B) and thin-section images (C-E) showing Sendai virus particles (svf) which have fused with an erythrocyte membrane. Linear viral envelope invaginations appear as smooth linear ridges on E fracture faces (B) and smooth linear grooves on P faces (A) and as linear profiles in tangential sections (D). In cross-sections they can be identified by the low radius of curvature of the membrane (c, arrowheads). tv, unfused virus particles; ne, nucleocapsid; es, extracellular space, A, B, × 56000; c, ×-6OOOO; D, E, × 65000.

Fig. 1.

Freeze-fracture (A, B) and thin-section images (C-E) showing Sendai virus particles (svf) which have fused with an erythrocyte membrane. Linear viral envelope invaginations appear as smooth linear ridges on E fracture faces (B) and smooth linear grooves on P faces (A) and as linear profiles in tangential sections (D). In cross-sections they can be identified by the low radius of curvature of the membrane (c, arrowheads). tv, unfused virus particles; ne, nucleocapsid; es, extracellular space, A, B, × 56000; c, ×-6OOOO; D, E, × 65000.

Three different procedures have been used to arrest cell fusion at the membrane fusion stage. Membrane fusion occurs within 1-2 min of warming an agglutinated cell suspension to 37 °C. With haemolytic virus, therefore, fusion was arrested after 90 s. In the presence of 20 mM Mn2+ or with nonhaemolytic virus, where significant cell swelling does not occur, timing is not so critical and in these 2 situations fusion was arrested at various time intervals up to 15 min. Using these 3 different procedures approximately 50 sites of fusion between cells have now been detected and in all but 4 a fused virus envelope or envelope components, identified using the criteria already described, can be recognized at sites of cell-cell fusion. Selected examples of cell-virus envelope-cell bridge formation identified in both thin sections and freeze-fracture replicas are shown in Figs. 2 and 3. Fig. 4 illustrates 2 examples where a fused virus cannot definitely be identified although in Fig. 4 A the sharp curvature of the membrane at the small cytoplasmic connexions between the cells most likely represents cross-sections through pairs of the linear viral envelope invaginations.

Fig. 2.

Thin-section images showing virus particles (svb) which have fused with 2 erythrocytes (1, 2) to form a cell-virus-cell bridge. In each case a cytoplasmic continuity between the cells can be seen and the bridging virus particles can be identified by the characteristic appearance of the sectioned linear viral envelope invaginations. A, × 82000; B, × 52000; C, D, ×67000.

Fig. 2.

Thin-section images showing virus particles (svb) which have fused with 2 erythrocytes (1, 2) to form a cell-virus-cell bridge. In each case a cytoplasmic continuity between the cells can be seen and the bridging virus particles can be identified by the characteristic appearance of the sectioned linear viral envelope invaginations. A, × 82000; B, × 52000; C, D, ×67000.

Fig. 3.

Freeze-fracture replicas showing examples of cell-virus-cell bridge formation. In some cases the bridging virus envelope (svb) fused to 2 erythrocytes (1, 2) can be seen (A, B); in others, linear viral envelope components (C-E, arrowheads) can be identified at sites of fusion between 2 cells (1, 2). E and Fare complementary replicas, A, × 1OOOOO; B-F, × 62500

Fig. 3.

Freeze-fracture replicas showing examples of cell-virus-cell bridge formation. In some cases the bridging virus envelope (svb) fused to 2 erythrocytes (1, 2) can be seen (A, B); in others, linear viral envelope components (C-E, arrowheads) can be identified at sites of fusion between 2 cells (1, 2). E and Fare complementary replicas, A, × 1OOOOO; B-F, × 62500

Fig. 4.

Thin-section (A) and freeze-fracture (B) images showing small cytoplasmic connexions (cc) between 2 erythrocytes (1, 2) where a fused viral envelope cannot definitely be identified. The low radius of curvature of the membrane (A, arrowheads) does suggest the involvement of a virus particle in this case, A, × 80000; B, × 40000

Fig. 4.

Thin-section (A) and freeze-fracture (B) images showing small cytoplasmic connexions (cc) between 2 erythrocytes (1, 2) where a fused viral envelope cannot definitely be identified. The low radius of curvature of the membrane (A, arrowheads) does suggest the involvement of a virus particle in this case, A, × 80000; B, × 40000

Cell swelling

With preparations of haemolytic virus extensive cell-cell fusion is seen within 2–3 min of warming an agglutinated cell suspension to 37 °C. The cell swelling stage of cell fusion was studied by examining cells fixed between 90 s and 3 min. Cell swelling occurs to varying degrees with different batches of nonhaemolytic virus (Knutton & Bächi, 1980) and so intermediate stages during swelling were also studied with appropriate batches of non-haemolytic virus, cells being fixed after 30 min at 37 °C-.

From 2 cells connected by a virus bridge to a spherical fused cell, cells pass through intermediate dumbbell-shaped configurations as radial expansion of the cytoplasmic connexion occurs (Knutton & Bächi, 1980). In fact, many fused cells never seem to swell beyond a large dumbbell-shape and so this configuration is frequently seen (Fig. 5). As far as the problem of membrane redistribution during cell swelling is concerned, the interesting region of the cell surface is the plane of expansion of the virus bridge (Fig. 5, dotted lines). In this region of many large dumbbell-shaped cells only cytoplasm is seen (Fig. 5), whereas in many other cells a linear arrangement of vesicular or elongated membrane profiles of variable number can be seen (Fig. 6). Both freeze-fracture (Fig. 6A, B) and thin-section images (Fig. 6 c, D) indicate that these membrane profiles are, in fact, part of a system of membrane tubules. Oblique fractures clearly show their tubular structure (Figs. 6A, B, 9A) and the observation that these membrane profiles all become stained with ruthenium red shows that they are continuous with the external space (Fig. 6c, D). A fused virus envelope or envelope components associated with these membrane tubules is also frequently seen (Fig. 6).

Fig. 5.

A large dumbbell-shaped fused cell. The fracture through the centre of the dumbbell (dotted lines) reveals only cytoplasm, × 12000.

Fig. 5.

A large dumbbell-shaped fused cell. The fracture through the centre of the dumbbell (dotted lines) reveals only cytoplasm, × 12000.

Fig. 6.

Freeze-fracture (A, B) and thin-section images (c, D) showing cross-sections through large dumbbell-shaped fused cells in the region shown by the dotted line in Fig. 5. In many fused cells a variable number of tubular membrane profiles (t) can be seen. Fused viral envelopes (A, svf) or linear viral envelope components (B, C, arrowheads) can also frequently be seen, A, × 30000; B, × 40000; c, × 55000; D, × 48000.

Fig. 6.

Freeze-fracture (A, B) and thin-section images (c, D) showing cross-sections through large dumbbell-shaped fused cells in the region shown by the dotted line in Fig. 5. In many fused cells a variable number of tubular membrane profiles (t) can be seen. Fused viral envelopes (A, svf) or linear viral envelope components (B, C, arrowheads) can also frequently be seen, A, × 30000; B, × 40000; c, × 55000; D, × 48000.

In order to understand how these different dumbbell-shaped configurations and this system of tubules arise it was necessary to examine cells at earlier stages of swelling. Three examples showing the cytoplasmic connexion between fused cells at early stages during the expansion of a virus bridge are shown in Fig. 7. In some cases the bridging virus particle appears to become incorporated into the erythrocyte membrane as swelling proceeds (Fig. 7 A) whereas in other cases this does not appear to happen and most of the bridging virus envelope appears to remain anchored at the site of fusion as the cytoplasmic connexion expands (Fig. 7B). In neither of these 2 examples are membrane tubules apparent. Fig. 7 c, on the other hand, shows that when they do occur the tubules are present at early stages during swelling. Unfortunately, in this particular fracture the origin of the tubules and their relationship to the bridging virus particle is not apparent. Fractures through the plane of expansion at early stages of swelling showing both the bridging virus envelope and the system of tubules are rare but a few examples have now been obtained and 2 are shown in Fig. 8. In both examples the tubules can be seen to be continuous with linear elements of the bridging virus envelope (Fig. 8, arrowheads).

Fig. 7.

Freeze-fracture replicas showing cytoplasmic connexions between fused cells at early stages of swelling. In some cases the bridging virus particle appears to become incorporated into the erythrocyte membrane (A) whereas in others most of the bridging virus (B, re4) remains anchored at the site of fusion. Fractures through the plane of expansion of the virus bridge (c, dotted line) show that membrane tubules (t) are formed at very early stages of swelling. Linear vira) envelope components are indicated by arrowheads, A, × 60000; B, × 52000; c, × 50000.

Fig. 7.

Freeze-fracture replicas showing cytoplasmic connexions between fused cells at early stages of swelling. In some cases the bridging virus particle appears to become incorporated into the erythrocyte membrane (A) whereas in others most of the bridging virus (B, re4) remains anchored at the site of fusion. Fractures through the plane of expansion of the virus bridge (c, dotted line) show that membrane tubules (t) are formed at very early stages of swelling. Linear vira) envelope components are indicated by arrowheads, A, × 60000; B, × 52000; c, × 50000.

Fig. 8.

Dumbbell-shaped fused cells at early stages during swelling showing fractures through the plane of expansion of the virus bridge (dotted line). Linear components of the bridging virus particle arrowheads) can be seen to be continuous with a system of erythrocyte membrane tubules (t); sv, unfused virus particles; svf, fused virus particles, A, × 50000; B, ×45000.

Fig. 8.

Dumbbell-shaped fused cells at early stages during swelling showing fractures through the plane of expansion of the virus bridge (dotted line). Linear components of the bridging virus particle arrowheads) can be seen to be continuous with a system of erythrocyte membrane tubules (t); sv, unfused virus particles; svf, fused virus particles, A, × 50000; B, ×45000.

Oblique fractures through large dumbbell-shaped cells at late stages of swelling suggest that, once generated, the tubules expand radially as cells swell (Fig. 9A, B) but ultimately become unstable and breakdown to form small vesicles (Fig. 9 c).

Fig. 9.

Freeze-fracture replicas showing the central region of large dumbbell-shaped fused cells. Obliquely (A) and cross-fractured (B) tubules (t) appear to radiate from a point at the centre of the plane of expansion. In some highly swollen cells (c) the tubules (t) appear to breakdown to small vesicles (v). A, × 33000; B, c, × 30000.

Fig. 9.

Freeze-fracture replicas showing the central region of large dumbbell-shaped fused cells. Obliquely (A) and cross-fractured (B) tubules (t) appear to radiate from a point at the centre of the plane of expansion. In some highly swollen cells (c) the tubules (t) appear to breakdown to small vesicles (v). A, × 33000; B, c, × 30000.

Membrane fusion

Two basic problems are involved in distinguishing between the cell-cell bridge and the cell-virus-cell bridge mechanisms of cell fusion: (1) identifying sites of membrane fusion, and (2) identifying the presence or absence of a fused viral envelope at sites of fusion. The linear viral envelope invaginations provide a unique morphological marker for identifying fused virus particles and the possibility of arresting cell-cell fusion at the membrane fusion stage has now allowed many sites of membrane fusion to be localized. In virtually all instances a fused virus envelope or envelope components has been identified at sites of cell-cell fusion thus confirming previous conclusions that cell-virus-cell bridge formation is the membrane fusion event in Sendai virus-induced cell fusion (Knutton, 1977, 1978; Knutton & Pasternak, 1979). Many of the observations of cells at early stages during the subsequent cell swelling stage of fusion also reveal a bridging virus particle.

Since considerable evidence has previously been presented in support of the alternative direct cell-cell fusion mechanism, one must consider seriously whether direct fusion of erythrocyte membranes also occurs. In the early studies, before it was realized that virus particles undergo a dramatic change in structure prior to fusion (Knutton, 1976), there was no reliable morphological criterion for identifying a fused virus (Schneeberger & Harris, 1966; Okada, Murayama & Yamada, 1966; Hosaka & Koshi, 1968; Bächi & Howe, 1972). More recent studies have used ferritin-conjugated antibody labelling techniques to identify viral envelope components (Bächi et al. 1973; Shimizu, Shimizu, Ishida & Homma, 1976). In these studies, however, cells were examined at late stages of fusion when cell swelling had produced large dumbbell-shaped cells and so, I believe, an incorrect assignment of fusion sites has been made. The observations presented in this paper illustrate how this occurred. The inset to Fig. 9B illustrates the sort of image one would observe if a section were cut through a large dumbbell-shaped fused cell similar to the ones shown in Fig. 9 A, B. It is thin-section images of fused cells such as this which have previously been interpreted as being multiple sites of fusion between 2 cells and the absence of viral antigens at such sites has been some of the main evidence used in support of the direct cell-cell fusion mechanism of cell fusion (Hosaka & Shimizu, 1977; Poste & Pasternak, 1978; Knutton, 1978). The present observations show that this interpretation is incorrect and that such images do not represent sites of membrane fusion but instead represent cross-sections through a system of membrane tubules which are generated subsequent to membrane fusion during the cell swelling stage of cell fusion (see below).

The lack of evidence for a fused virus particle at small cytoplasmic connexions between cells (e.g. Fig. 4B) does not, in itself, prove that direct membrane fusion occurred since any one section or fracture plane reveals only part of the membrane at the cytoplasmic bridge. It seems quite reasonable that, in a small number of cases, a virus particle would not be detected using the linear viral envelope structures as sole morphological marker. Taking the data as a whole it seems most likely that cell-virus-cell bridge formation is the only membrane fusion event in Sendai-virus mediated cell fusion and that direct cell-cell fusion does not occur. This conclusion means that in order to understand the mechanism of membrane fusion in Sendai virus-induced cell fusion we only need to study the mechanism of viral envelope-cell fusion since virus bridge formation is simply a special case of virus-cell fusion.

Cell swelling

Previous morphological and biochemical studies have shown that virally-induced cell swelling is a distinct stage in cell-cell fusion, that it occurs subsequent to the membrane fusion event and it has been proposed that cell swelling is the mechanism whereby cells which have established sites of membrane fusion expand such sites to form polykaryons (Knutton, 1977, 1978; Knutton & Pasternak, 1979; Knutton & Bächi, 1980). The observations presented in this paper showing intermediate stages during swelling are consistent with these general conclusions. In addition, new data on the actual process of membrane redistribution during cell swelling have been presented. Although this is clearly a complex topological problem and apparently differs for each fused cell, some insights into how membrane redistribution occurs and the factors affecting its outcome can be deduced from the data presented.

The system of membrane tubules seen in many dumbbell-shaped fused cells has previously been seen in both thin-section and freeze-fracture studies of cell fusion. When first described they were interpreted as multiple sites of membrane fusion (Bächi et al. 1973) (see above). I previously suggested that they might represent membrane vesicles which become internalized during cell swelling although no mechanism was proposed to explain how or why this might occur (Knutton, 1977, 1978). What this study has now shown is that the linear arrangement of membrane profiles seen in large dumbbell-shaped fused cells is a system of erythrocyte membrane tubules which originate from the bridging virus particle. In favourable fractures (e.g. Fig. 8) the tubules are seen to be continuous with linear segments of viral envelope suggesting that each tubule originates from a different point of fusion between the erythrocyte membrane and the bridging virus particle; viral envelope-erythrocyte membrane fusion is known to involve the linear viral envelope invaginations (Knutton,. 1977)-

I suggest that tubules are generated at points of viral envelope-erythrocyte membrane fusion because at such points the erythrocyte membrane is restricted. Most of the erythrocyte surface is free to redistribute itself as cells swell except where it is. fused with the bridging virus particle. To use an analogy, as cells swell it is as though the membrane of both cells is ‘pulling’ at the same virus particle and the effect of this is to distort the erythrocyte membrane at points where it is fused with the bridging virus. This distortion of the erythrocyte membrane results in the formation of the tubules. In such a scheme the variable number of tubules seen in different fused cells can be explained on the basis of a variable number of fusion points between the bridging virus particle and the erythrocyte membrane. For example, in the case where only 2 fusion points occur, the minimum necessary to form a virus bridge, no tubules would be formed and during the expansion of such a cylindrical virus bridge the fused viral envelope would become integrated into the surface of the expanding fused cell. Fig. 7 A probably represents such an example. Alternatively, large virus particles with many linear invaginations (e.g.Fig. 1B) should be capable of multiple fusion events and thus inducing the formation of more tubules than smaller virus particles. This also appears to be the case (cf. Fig. 8 A, B). The one problem with this scheme is that in a case where numerous tubules are formed (e.g. Fig. 9) there is no evidence to suggest that a virus particle fuses at many points when the virus bridge is formed. Rather, studies of virus-cell fusion have shown that a virus particle usually fuses with a cell at a single point (Knutton, 1977). In order to explain how several tubules could be formed, therefore, we have to consider what happens to a virus particle following this initial fusion event. In the case of virus—cell fusion, during the subsequent cell swelling stage, the entire viral envelope becomes incorporated into the erythrocyte membrane and it is during this stage that multiple fusion points between the viral envelope and erythrocyte membrane appear to form. In Fig. 1A one can see several such points of fusion (arrows) separated by regions of extracellular space (es). Distortion of the erythrocyte membrane where it is fused to the virus would generate a series of membrane tubules and this is what one might expect to happen in the case of cell-virus-cell fusion for the reasons already discussed.

Cell swelling is a consequence of the fusion of virus particles having ‘permeable’ envelopes with the erythrocyte membrane (Shimizu et al. 1976; Poste & Pasternak, 1978) and during cell-cell fusion many virus particles will fuse with each cell. Since viral envelope-cell fusion does not occur synchronously the relationship between fusion of the bridging virus and the onset of cell swelling will vary in each fused cell. Such variations, in addition to variations in the size of the bridging virus, could account for the different modes of membrane redistribution seen in different fused cells.

Erythrocytes have only a limited capacity to swell before membrane rupture and haemolysis occurs (Knutton et al. 1976). Similarly, with a fused cell there is a limit to the radial expansion of the tubules before the membrane ruptures. In this case, the tubules appear to breakdown into small vesicles which become internalized along with, presumably, the bridging virus particle. Previous observations showing that some membrane profiles seen inside large dumbbell-shaped fused cells did not stain with ruthenium red also confirm their vesicular nature at late stages of swelling (Knutton, 1978).

A diagrammatic representation of the proposed mechanism of Sendai virus-induced cell fusion illustrating how the membrane tubules are generated during the cell swelling stage is shown in Fig. 10. This illustrates a simple case involving the fusion of 2 cells joined by a single virus bridge. In practise, many cells fuse together and it is common to see more than 1 virus bridge between the same 2 cells. In such cases, membrane redistribution during cell swelling is even more complex.

Fig. 10.

Diagrammatic representation of the process of Sendai virus-induced cell fusion. Stage 1: Cell agglutination involves the crosslinking of adjacent cells by virus particles. Stage 2: Prior to fusion virus particles undergo a cell-mediated change in envelope structure which involves the formation of linear invaginations pairs of which fuse with the cell membrane (A). Cell-cell fusion occurs when a virus particle fuses with two crosslinked cells to form a cell-virus-cell bridge (B). Stage 3: Virus-induced cell swelling expands cells connected by a virus bridge to form a spherical fused cell (A-D). During cell swelling a system of membrane tubules originating from points of fusion between the viral envelope and the cell membrane (A) are sometimes generated in the plane of expansion (dotted line). The tubules expand radially as cells swell (B, C) but eventually break down into small vesicles (D).

Fig. 10.

Diagrammatic representation of the process of Sendai virus-induced cell fusion. Stage 1: Cell agglutination involves the crosslinking of adjacent cells by virus particles. Stage 2: Prior to fusion virus particles undergo a cell-mediated change in envelope structure which involves the formation of linear invaginations pairs of which fuse with the cell membrane (A). Cell-cell fusion occurs when a virus particle fuses with two crosslinked cells to form a cell-virus-cell bridge (B). Stage 3: Virus-induced cell swelling expands cells connected by a virus bridge to form a spherical fused cell (A-D). During cell swelling a system of membrane tubules originating from points of fusion between the viral envelope and the cell membrane (A) are sometimes generated in the plane of expansion (dotted line). The tubules expand radially as cells swell (B, C) but eventually break down into small vesicles (D).

I am grateful to Miss Susan O’Niel for excellent technical assistance and the Cancer Research Campaign for financial support.

Apostolov
,
K.
&
Almeida
,
J. D.
(
1972
).
Interaction of Sendai virus (HVJ) with human erythrocytes: a morphological study of haemolysis and cell fusion
.
J, gen. Virol
.
15
,
227
234
.
Bächi
,
T.
,
Aguet
,
M.
&
Howe
,
C.
(
1973
).
Fusion of erythrocytes by Sendai virus studied by immuno-freeze-etching
.
J. Virol
,
11
,
1004
1012
.
Bächi
,
T.
&
Howe
,
C.
(
1972
).
Fusion of erythrocytes by Sendai virus studied by electron microscopy
.
Proc. Soc. exp. Biol. Med
.
141
,
141
149
.
Cassone
,
A.
,
Calio
,
R.
&
Pesce
,
C. D.
(
1973
).
Interaction of Sendai virus with human erythrocytes. II. The fusion reactions
.
Boll. Inst. Sieroter, Milan
52
,
218
223
.
Homma
,
M.
,
Shimizu
,
K.
,
Shimizu
,
Y. K.
&
Ishida
,
N.
(
1976
).
On the study of Sendai virus haemolysis. I. Complete Sendai virus lacking haemolytic activity
.
Virology
71
,
41
47
.
Hosaka
,
Y.
&
Koshi
,
Y.
(
1968
).
Electron microscopic study of cell fusion by HVJ virions
.
Virology
34
,
419
434
.
Hosaka
,
Y.
&
Shimizu
,
K.
(
1977
).
Cell fusion by Sendai virus
.
In Cell Surface Reviews
. vol.
2
,
Virus Infection and the Cell Surface
, (ed.
G.
Poste
&
G. L.
Nicolson
), pp.
129
155
. Amsterdam: North-Holland.
Knutton
,
S.
(
1976
).
Changes in viral envelope structure preceding infection
.
Nature, Lond
,
264
,
672
673
.
Knutton
,
S.
(
1977
).
Studies of membrane fusion. II. Fusion of human erythrocytes by Sendai virus
.
J. Cell Sci
.
28
,
189
210
.
Knutton
,
S.
(
1978
).
The mechanism of virus-induced cell fusion
.
Micron
9
,
133
154
.
Knutton
,
S.
(
1979
).
Studies of membrane fusion. V. Fusion of human erythrocytes with non-haemolytic Sendai virus
.
J. Cell Sci
.
36
,
85
96
.
Knutton
,
S.
&
Bächi
,
T.
(
1980
).
The role of cell swelling and haemolysis in Sendai virus-induced cell fusion and in the diffusion of incorporated viral antigens. J
.
Cell Sci
.
42
,
153
167
.
Knutton
,
S.
,
Jackson
,
D.
,
Graham
,
J. M.
,
Micklem
,
K. J.
&
Pasternak
,
C. A.
(
1976
).
Microvilli and cell swelling
.
Nature, Lond
.
262
,
52
54
.
Knutton
,
S.
&
Pasternak
,
C. A.
(
1979
).
The mechanism of cell-cell Fusion
.
Trends in biochem. Sci
.
4
,
220
223
.
Luft
,
J. H.
(
1971
).
Ruthenium red and violet. I. Chemistry, purification, methods of use for electron microscopy and mechanism of action
.
Anat. Rec
.
171
,
347
368
.
Okada
,
Y.
,
Murayama
,
F.
&
Yamada
,
K.
(
1966
).
Requirement of energy for the cell fusion reaction of Ehrlich ascites tumour cells by HVJ
.
Virology
24
,
115
130
.
Poste
,
G.
&
Pasternak
,
C. A.
(
1978
).
Virus-induced cell fusion
.
In Cell Surface Reviews
, vol.
5
, Membrane Fusion (ed.
G.
Poste
&
G. L.
Nicolson
), pp.
306
367
.
Amsterdam
:
North-Holland
.
Schneeberger
,
E. E.
&
Harris
,
H.
(
1966
).
An ultrastructural study of interspecific cell fusion induced by inactivated Sendai virus
.
J. Cell Sci
.
1
,
401
406
.
Shimizu
,
Y. K.
,
Shimizu
,
K.
,
Ishida
,
N.
&
Homma
,
M.
(
1976
).
On the study of Sendai virus haemolysis. II. Morphological study of envelope fusion and haemolysis
.
Virology
71
,
48
60
.
Toister
,
Z.
&
Loyter
,
A.
(
1973
).
The mechanism of cell fusion. II. Formation of chicken erythrocyte polykaryons. ? ?ol. Chem
.
248
,
422
432
.