Fusogen promotes cell-to-cell fusion in our live-cell imaging system
We aimed to develop a system to study fertilisation molecules in animal cultured cells by live imaging. Various cultured animal cell lines, such as COS-7 and HEK293T, have been used for transfection and expression in previous fertilisation molecule studies; for example, molecular adhesiveness between IZUMO and JUNO was shown by mixing gametes and transfected cultured-cells3,26,35. In this study, because we focused on cell migration and morphology for frequent contact of cells under microscopy, we selected a line of motile BHK cells that did not spontaneously adhere or fuse with each other (Fig. S1). BHK cells were also used in the original fusion assay18. We investigated whether cell fusion activity could be detected in known fertilisation molecules using our live-cell imaging technique by transfecting BHK cells with RFPcyto as a negative control, and GCS1/HAP2 to evaluate multinucleation rates. Ectopic expression of fusogens sometimes indicates toxicity to cells or the whole organism16,18,21,36; therefore, tight regulation was required to ensure expression at the preferred times. For this purpose, we used a mifepristone-inducible system as used in the fusion assay18.
To capture cell-to-cell fusion by live imaging, we used a Nipkow disc scanning confocal system embedded within the incubator to monitor cells in 1 mm × 1 mm areas every 6 min under stable low-phototoxicity conditions (Fig. 1). In each captured video, 130-476 cells expressing RFPcyto were monitored. When the gametic fusogen GCS1 (Arabidopsis thaliana GCS1; AtGCS1) was expressed, cell-to-cell fusion was observed (Fig. 1A, arrows; Video 1). At 12 h after induction, the multinucleation rate increased significantly, to 2.2 ± 0.4% (n = 4; P < 0.05; Dunnett’s test; Fig. 1B). To confirm that cell-to-cell fusion depended on the fusogen activity of the expressed proteins, we transfected the loop-deletion GCS1 (GCS1∆loop). Amino acids 166–178 in AtGCS1 represent a hydrophobic loop structure, which is necessary for membrane fusion17. The multinucleation rate was 1.2 ± 0.2% (n = 3), which was not significantly different from the negative control (Fig. 1B). This result suggested that fusogen-dependent cell-to-cell fusion was visualised by live imaging.
Our live imaging system is useful to monitor expression of a fluorescent marker upon cell-to-cell fusion. The dependency of individual fusion on the expression of transfected genes from 4 to 12 h after expression induction is shown in Fig. 1C. In experiments for wild-type GCS1, a combination of two cells labelled with red fluorescent protein (RFP) was more likely to fuse than a combination of labelled and non-labelled cells. This is partly due to the frequent fusion of daughter cells after cell division. Our result is consistent with the previous report that GCS1, as well as EFF-1, bilaterally promoted cell-to-cell fusion in the fusion assay18. Together, these results suggest that our live imaging technique is compatible with a modified fusion assay for the study of fusogen functions in cultured cells.
Adhesion molecules accumulate at the contact interfaces of adjacent cells
The fusion assay has never previously been used to evaluate gametic adhesion molecule function; conventional methods include adhesion and aggregation assays37,38. To develop a simple method suitable for live imaging-based studies, we introduced adhesion molecule pairs into the modified fusion assay.
First, we transfected the somatic adhesion molecule E-cadherin into BHK cells. Cadherins have been shown to play a role in Ca2+-dependent cell–cell adhesion39. The transfection of a single cadherin gene can potentially induce interaction of cadherin expressed in different cells (homophilic adhesion), eliminating the necessity of introducing an adhesion molecule pair such as IZUMO and JUNO (heterophilic adhesion). Cadherin was visualised by translational fusion with GFP, and no toxicity was observed despite the lack of an inducible gene expression system for cadherin in this experiment. E-cadherin-expressing BHK cells in our system did not adhere to neighbouring cells to show aggregation. E-cadherin appeared to be localised at the cell membrane and secretory pathways of the cell, showing no polarisation (Fig. 2A). However, upon temporary contact between E-cadherin-expressing cells, E-cadherin tended to accumulate at the contact interface (Fig. 2A, 7:30; Video 2); fluorescent intensity of E-cadherin-GFP increased specifically at the interface of cell contact (Fig. 2B, 7:30). This accumulation disappeared as cells detached (Fig. 2B, 8:00).
Next, we introduced mouse IZUMO and JUNO to determine whether a similar temporal accumulation at the contact interface of adjacent cells might occur even in heterophilic adhesion molecules. We developed a mixing assay, in which cells expressing IZUMO and JUNO were mixed at 4 hours after independent transfection and were incubated for 20 hours. IZUMO and JUNO were designed as translational fusion with fluorescent proteins. At 4 to 12 hours after the induction of IZUMO and JUNO expression by mifepristone, we found that IZUMO temporally accumulated at the contact interfaces of JUNO-expressing cells (47% of contacting cells, n = 53; Fig. 2C-E; Video 3).
We examined whether IZUMO accumulation is dependent on the interaction of IZUMO and JUNO expressed in adjacent cells. When IZUMO and JUNO were expressed simultaneously in the same cell by co-transfection, neither molecule accumulated on the cell surface (0%, n = 63; Fig. 2E). Next, we constructed a mutant IZUMO lacking the β-hairpin region (IZUMO∆) and examined the interaction between IZUMO∆ and JUNO. The central β-hairpin region of IZUMO is required for JUNO binding, as has been shown by co-crystal structure analysis4,5. When IZUMO∆- and JUNO-expressing cells were mixed, accumulation of IZUMO was rarely observed (7% of cases, n = 59; Fig. 2E). We also co-transfected JUNO with CD9, an egg tetraspanin required for gamete fusion2, to determine whether CD9 modulates IZUMO accumulation. When IZUMO- and JUNO/CD9-expressing cells were mixed, IZUMO accumulation and frequency were unchanged, consistent with the finding that CD9 is independent of gamete adhesion2,40 (50%, n = 42; Fig. 2E). These results demonstrate the usefulness of this assay for visualising the dependence of adhesion molecule interactions on the affinity of introduced adhesion molecule pairs. We named this assay system the live imaging-based adhesion molecule (LIAM) assay, which we propose for the examination of transfected adhesion molecules at cell contact sites.
Accumulation and translocation of adhesion molecules in the LIAM assay depended on critical amino acids
Subsequent experiments were conducted using high-throughput analysis in a non-inducible gene expression system (cytomegalovirus promoter), because we found that the inducible gene expression system was unnecessary for cadherin, IZUMO, and JUNO (translational fusion with fluorescent proteins). Prior to contact between mouse IZUMO- and JUNO-expressing cells, these adhesion molecules were distributed evenly throughout the cell, as also occurs in the inducible gene expression system. Accumulation was observed upon cell contact; however, both IZUMO and JUNO showed accumulation (Fig. 3A; Video 4). IZUMO accumulation was observed in 72% of contacting cells (39/54; Fig. 3B), whereas JUNO accumulation was observed in only 37% (20/54; Fig. 3B). IZUMO and JUNO accumulation were always simultaneously observed in the same cell pairs (opposite sides of a single contact site) when JUNO accumulation was observed.
To determine whether accumulation was dependent on the amino acid residues of IZUMO and JUNO for molecular interaction, we performed a LIAM assay using mutant IZUMO and JUNO, according to previous crystal structure analysis results4,5 (Fig. 3A). When mouse mutant IZUMO W148A-expressing cells were mixed with mouse JUNO-expressing cells, accumulation was not observed (0% of 45 contacting cells; Fig. 3B). When mouse IZUMO-expressing cells and mouse mutant JUNO W62A-expressing cells were mixed, accumulation was again not observed (0% of 67 contacting cells; Fig. 3B). Prior to cell contact, IZUMO and JUNO expression and localisation were unchanged by these point mutations (Fig. 3A). Next, we tested the sperm transmembrane protein SPACA6, which is required for fertilisation6,7, instead of IZUMO, which shows structural similarity with SPACA66 (Fig. S2), and found that neither SPACA6 nor JUNO accumulated (Fig. 3A). These results indicate that IZUMO and JUNO accumulation depends on amino acids critical for their interaction, according to the LIAM assay.
In 38% (15/39) cells showing IZUMO accumulation, IZUMO was found to translocate to the plasma membrane of the opposite cell (Fig. 3C). Several fluorescent foci of IZUMO or JUNO on the opposite cell were observed upon the detachment of contacting cells (Video 5). IZUMO was translocated to JUNO-expressing cells in 27% of contacting cells (15/54; Fig. 3D) and JUNO was translocated to IZUMO-expressing cells in 7.4% of contacting cells (4/54; Fig. 3D). This translocation of IZUMO and JUNO was unilateral, not bilateral, in each cell pair, and translocation was selectively observed for either IZUMO or JUNO in each cell pair. In addition, IZUMO or JUNO translocation was always observed after IZUMO accumulation, but not necessarily after JUNO accumulation, perhaps due to the difficulty associated with visualising JUNO accumulation on the cell membrane.
This translocation was dependent on the amino acid sequence of IZUMO and JUNO required for the interaction (Fig. 3D). When mutant IZUMO W148A or mutant JUNO W62A was used, neither translocation not accumulation was observed (Fig. 3D). SPACA6 and JUNO also did not induce translocation (Fig. 3D). In subsequent experiments, this translocation of adhesion molecules was also monitored in the LIAM assay.
Species specificity in JUNO and IZUMO interactions suggested by the LIAM assay
Using the LIAM assay, we investigated the species specificity of IZUMO and JUNO from mouse, human, hamster, and pig (Fig. 4; Tables 1–4). Among these four mammalian species, mice are most closely related to hamsters, and humans and pigs are phylogenetically close to each other41. Molecular phylogenetic analysis results for IZUMO and JUNO proteins were consistent with the phylogenetic relationships among these four species (Figs. S3–5). The mouse IZUMO protein shares 61%, 52%, and 50% homology (identity) with hamster, human, and pig IZUMO proteins, respectively (Figs. S3, S5). In contrast, the mouse JUNO protein shares 74%, 68%, and 65% homology with hamster, human, and pig JUNO proteins, respectively (Figs. S4, S5). These four species were selected because species specificity has been observed biochemically in their IZUMO and JUNO interactions, in some but not all combinations, using the AVEXIS assay3. In the AVEXIS assay, IZUMO and JUNO interaction was shown in all conspecific combinations, hamster JUNO and all other three species, and mouse JUNO and human IZUMO but not in human JUNO and mouse IZUMO.
Table 1
IZUMO accumulation in conspecific and heterospecific cell combinations. Numbers in brackets are the numbers of contacted cells for each combination.
|
IZUMO
|
JUNO
|
Mouse
|
Human
|
Hamster
|
Pig
|
Mouse
|
72% (54)
|
0.0% (50)
|
31% (36)
|
0.0% (64)
|
Human
|
50% (38)
|
28% (51)
|
0.0% (58)
|
48% (63)
|
Hamster
|
78% (45)
|
0.0% (46)
|
77% (57)
|
13% (84)
|
Pig
|
0.0% (25)
|
0.0% (38)
|
0.0% (37)
|
24% (41)
|
We examined all combinations of IZUMO and JUNO from mouse, human, hamster, and pig cells using the LIAM assay. We began with conspecific combinations. Mouse IZUMO and JUNO showed accumulation in 72% (39/54; Fig. 3; Table 1) and 37% (20/54; Fig. 3; Table 2) of contacting cells, respectively. Translocation of mouse IZUMO and JUNO was also observed, in 27% (15/54; Table 3; Fig. 3C) and 7.4% (4/54; Table 4) of contacting cells, respectively. This finding is consistent with previous AVEXIS assay results3, which showed that mouse IZUMO and JUNO show relatively strong binding. In this study, we observed IZUMO accumulation as often as JUNO accumulation in human IZUMO × human JUNO interactions (IZUMO accumulation: 28%, 14/51, Table 1; JUNO accumulation: 29%, 15/51, Table 2). In this combination, IZUMO translocation was not observed (Table 3), whereas JUNO translocation was frequently observed (64%, 33/51, Table 4, Fig. 4A). In hamster IZUMO × hamster JUNO, IZUMO and JUNO accumulation (IZUMO: 77%, 41/57, Table 1; JUNO: 28%, 16/57, Table 2) and translocation (IZUMO: 31%, 18/57, Table 3; JUNO: 28%, 16/57, Table 4, Fig. 4B) were observed, consistent with mouse IZUMO and JUNO. In pig IZUMO × pig JUNO interactions, we observed IZUMO accumulation at a low rate (24%, 10/41, Table 1, Fig. 4C). Conspecific interaction of IZUMO and JUNO was confirmed for all combinations, and the frequencies of IZUMO and JUNO accumulation and translocation differed among species.
Table 2
JUNO accumulation in conspecific and heterospecific cell combinations. Numbers in brackets are the numbers of contacted cells for each combination.
|
IZUMO
|
JUNO
|
Mouse
|
Human
|
Hamster
|
Pig
|
Mouse
|
37% (54)
|
2.0% (50)
|
25% (36)
|
0.0% (64)
|
Human
|
0.0% (38)
|
29% (51)
|
4.1% (58)
|
49% (63)
|
Hamster
|
0.0% (45)
|
0.0% (46)
|
28% (57)
|
7.1% (84)
|
Pig
|
0.0% (25)
|
0.0% (38)
|
0.0% (37)
|
0.0% (41)
|
Table 3
IZUMO translocation in conspecific and heterospecific cell combinations. Numbers in brackets are the numbers of contacted cells for each combination.
|
IZUMO
|
JUNO
|
Mouse
|
Human
|
Hamster
|
Pig
|
Mouse
|
28% (54)
|
0.0% (50)
|
0.0% (36)
|
0.0% (64)
|
Human
|
0.0% (38)
|
0.0% (51)
|
0.0% (58)
|
3.2% (63)
|
Hamster
|
0.0% (45)
|
0.0% (46)
|
32% (57)
|
0.0% (84)
|
Pig
|
0.0% (25)
|
0.0% (38)
|
0.0% (37)
|
0.0% (41)
|
Next, we examined heterospecific interaction between mouse IZUMO and human, hamster, and pig JUNO. When mouse IZUMO-expressing cells and human JUNO-expressing cells were mixed, mouse IZUMO accumulated at the cell interface (50%, 19/38; Table 1), but neither JUNO accumulation nor IZUMO/JUNO translocation was observed (Tables 2–4). When mouse IZUMO-expressing cells and hamster JUNO-expressing cells were mixed, only mouse IZUMO accumulated (78%, 35/45, Table 1), consistent with mouse IZUMO and human JUNO. When mouse IZUMO-expressing cells and pig JUNO-expressing cells were mixed, we observed neither accumulation nor transference. These results indicate that the translocation of adhesion molecules to opposite cells correlated well with conspecific interactions.
Table 4
JUNO translocation in conspecific and heterospecific cell combinations. Numbers in brackets are the numbers of contacted cells for each combination.
|
IZUMO
|
JUNO
|
Mouse
|
Human
|
Hamster
|
Pig
|
Mouse
|
7.4% (54)
|
0.0% (50)
|
0.0% (36)
|
0.0% (64)
|
Human
|
0.0% (38)
|
65% (51)
|
0.0% (58)
|
32% (63)
|
Hamster
|
0.0% (45)
|
0.0% (46)
|
28% (57)
|
0.0% (84)
|
Pig
|
0.0% (25)
|
0.0% (38)
|
0.0% (37)
|
0.0% (41)
|
Next, we examined heterospecific interaction between human IZUMO and mouse, hamster, and pig JUNO. Neither accumulation nor translocation was detected in any combination (Tables 1–4). When hamster IZUMO-expressing cells and mouse JUNO-expressing cells were mixed, IZUMO and JUNO accumulation were detected, although at lower rates than for conspecific combinations (mouse IZUMO × mouse JUNO, hamster IZUMO × hamster JUNO) (Tables 1 and 2). This result is consistent with the crossing results reported by Yanagimachi et al.27 and those of a previous AVEXIS assay3. No translocation was observed (Tables 3 and 4). Finally, we combined pig IZUMO-expressing cells with mouse, human, and hamster JUNO-expressing cells. When pig IZUMO-expressing cells and mouse JUNO-expressing cells were mixed, neither accumulation nor translocation was observed (Tables 1–4). However, when pig IZUMO-expressing cells and human JUNO-expressing cells were mixed, IZUMO and JUNO accumulation was detected. Unexpectedly, translocation of both IZUMO and JUNO was detected in this heterospecific combination of pig IZUMO and human JUNO (Tables 1–4, Fig. 4D). This strongly positive result was obtained using our LIAM assay, but has not been tested previously using other assays.