Direct live imaging of cell–cell protein transfer by transient outer membrane fusion in Myxococcus xanthus
- Aix Marseille University-CNRS UMR7283, France
- Institut de Microbiologie de la Méditerranée, France
Figures
Figure 1 with 1 supplement
Transfer is a highly efficient OM-specific process.
(A) Percentage of mCherry+ recipient cells as a function of time. For each strain and time point, at least 3000 cells were analyzed in triplicate. Error bars = SD. (B) Fluorescence intensity of …
Figure 1—figure supplement 1
Subcellular localization of indicated fluorescent probes before and after a plasmolysis treatment.
(A and B) Sub-cellular localization of the OMmcherry, OMsfGFP, IMmcherry, PERImcherry fusions before (−) and after (+) plasmolysis treatment (0.5 M NaCl). For each fusion, cells were immobilized in …
Figure 2 with 1 supplement
Cell-contact-dependent transfer of OMsfGFP/DiO between single cells.
(A) sfGFP transfer from a donor OMsfGFP+ (white contour in lower panel) cell to a recipient OMsfGFP− IMmCherry+ cell (orange contour in upper panel). Scale bar=1 µm. (B) Kymographs of green …
Figure 2—figure supplement 1
Cell-contact-dependent transfer of OMsfGFP.
(A) OMsfGFP is transferred in a traA dependent-manner. The transfer kinetics of OMmcherry are also shown for comparison. The percentage of fluorescent recipient cells is plotted as a function of …
Figure 3 with 1 supplement
Lipid tubes are OM-derived and are observed when cells move apart.
(A) A lipid tubes formed between two cells expressing OMsfGFP. (B) Lipid tubes formed by OMsfGFP IMmCherry-expressing cells (white arrow). Scale bar = 1 µm. (C) TEM images of lipid tubes. Tubes …
Figure 3—figure supplement 1
The tubular extensions are not Type-IV pili.
(A) Measured diameters of the tubes observed by TEM. The diameters distribution is shown as a boxplot (n=100). (B) Polar Type-IV pili observed in wt cells by Transmission Electron Microscopy. (Scale …
Figure 4 with 2 supplements
Transfer is driven by transient OM fusion between donor and recipient cells.
(A and B) Fluorescence recovery after photobleaching (FRAP) experiments targeting a tube connected to a single DiO+ cell. Rapid DiO exchange is observed between the tube and the cell body. The cell …
Figure 4—figure supplement 1
The OMsfGFP or OMmCherry fluorescent probes are not significantly exchanged through the lipid tubes.
OMsfGFP-stained tubes formed between an OMsfGFP+ IMmcherry− cell and an OMsfGFP− IMmcherry+ recipient cell. No significant exchange of green fluorescence can be observed through the tubes. Scale bar …
Figure 4—figure supplement 2
Fluorescence Recovery After Photobleaching (FRAP) experiments targeting indicated fluorescent probes.
(A) Representative fluorescence recovery after FRAP on the cell body of a DiO-stained cell. (B) Comparative recovery kinetics of DiO and OMsfGFP after FRAP.
Figure 5 with 1 supplement
Lipid tubes and vesicles are deposited in slime trails.
(A) TEM images of lipid tubes deposited in the wake of a moving cell (left panel). A higher magnification view of lipid tubes/vesicles is shown in the right panel. Scale bars = 250 nm. (B) …
Figure 5—figure supplement 1
Lipid tubes and vesicles are deposited in slime trails.
(A) OM materials are deposited in the wake of motile cells and specifically associated with slime. A higher magnification view of lipid tubes/vesicles is shown in the panel (B). Scale bars = 500 nm.
Figure 6 with 2 supplements
Distribution of TraA is restricted to the deltaproteobacteria.
(A) TraA homologues in Myxococcus xanthus DK1622 (gi|108763680), Myxococcus stipitatus (gi|442324418), Corallococcus coralloides (gi|383459429), Myxococcus fulvus (gi|338532052), Stigmatella …
Figure 6—figure supplement 1
TraA homologues in deltaproteobacteria.
ClustalW alignment of TraA homolog: Myxococcus xanthus DK1622 (gi|108763680), Myxococcus stipitatus (gi|442324418), Corallococcus coralloides (gi|383459429), Myxococcus fulvus (gi|338532052), Stigmat…
Figure 6—figure supplement 2
ClustalW alignment of TraA in Myxococcus xanthus strains.
Note that most amino acids variations are localized in the first 300 N-terminal residues region encompassing the so-called PA14 domain.
Videos
Video 1
Live observations of cell–contact dependent transfer of OMsfGFP between single cells.
Corresponding green fluorescence and red fluorescence are shown. For details see Figure 2. Pictures were taken every 30 s.
Video 2
Live observations of cell–contact dependent transfer of OMsfGFP between single cells.
Corresponding phase contrast, green fluorescence and red fluorescence are shown. Pictures were taken every 30 s.
Video 3
Live observations of cell–contact dependent transfer of DiO between single cells.
Corresponding phase contrast and green fluorescence which are displayed in pseudo colors, are shown. For details see Figure 3A. Pictures were taken every 30 s.
Video 4
Formation of OMsfGFP tubes between two cells.
Corresponding phase contrast and green fluorescence are shown. Pictures were taken every 30 s.
Video 5
Live observations of DiO transfer and formation of DiO+ tubes between two cells.
For details see Figure 3A. Corresponding phase contrast and green fluorescence are shown. Pictures were taken every 30 s.
Video 6
Live observations of DiO transfer and formation of DiO+ tubes between two cells.
For details see Figure 3C. Corresponding phase contrast and green fluorescence are shown. Pictures were taken every 30 s.
Additional files
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Supplementary file 1
(A) Strains used in this study. (B) Primers used in this study. (C) Plasmids used in this study and their mode of construction.
- https://doi.org/10.7554/eLife.00868.023
