Supplementary Materials

Golgi-derived PI(4)P-containing vesicles drive late steps of mitochondrial division

Shun Nagashima, Luis-Carlos Tábara, Lisa Tilokani, Vincent Paupe, Hanish Anand, Joe H. Pogson, Rodolfo Zunino, Heidi M. McBride, Julien Prudent

Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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  • Materials and Methods
  • Figs. S1 to S15
  • Captions for Movies S1 to S16
  • References
Table S1

Images, Video, and Other Media

Movie S1
Arf1-GFP is enriched at mitochondrial constriction sites before division. HeLa cells transfected for 24 hr with Arf1-GFP (green) and TOM20-mCherry (white) were observed under ANDOR Dragonfly spinning disk confocal microscope using 100X objective and an sCMOS (Zyla) camera. 3 stacks of 0.3 μm each were acquired every 5 seconds. Images were then compiled by "max projection". Time-lapse images are shown in Figure 3C.
Movie S2
Additional example showing the Arf1-GFP accumulation at mitochondrial constriction sites before division. HeLa cells transfected for 24 hr with Arf1-GFP (green) and TOM20-mCherry (white) were observed under ANDOR Dragonfly spinning disk confocal microscope using 100X objective and an EMCCD (iXon Ultra 888) camera. Images were acquired every 5 seconds.
Movie S3
Arf1-GFP accumulates at mitochondrial fission sites after Drp1-scarlet recruitment. HeLa cells transfected for 24 hr with Arf1-GFP (green) and Drp1-scarlet (purple) and with mitochondria labelled with MitoTracker deep red FM (white) were observed under ANDOR Dragonfly spinning disk confocal microscope using 100X objective and an EMCCD (iXon Ultra 888) camera. Images were acquired every 2 seconds. Time-lapse images are shown in Figure 3E.
Movie S4
Arf1-GFP is enriched at mitochondria-ER contact sites before mitochondrial division. HeLa cells transfected for 24 hr with Arf1-GFP (green) and ER-dsRED2 (purple) and with mitochondria labelled with MitoTracker deep red FM (white) were observed under ANDOR Dragonfly spinning disk confocal microscope using 100X objective and an EMCCD (iXon Ultra 888) camera. Images were acquired every 5 seconds. Time-lapse images are shown in Fig. S11D.
Movie S5
Additional example showing the Arf1-GFP localization at mitochondria-ER contacts before mitochondrial division. HeLa cells transfected for 24 hr with Arf1-GFP (green) and ER-dsRED2 (purple) and with mitochondria labelled with MitoTracker deep red FM (white) were observed under ANDOR Dragonfly spinning disk confocal microscope using 100X objective and an EMCCD (iXon Ultra 888) camera. Images were acquired every 5 seconds.
Movie S6
Arf1-GFP is recruited at mitochondrial fission sites independently of LAMP1- mCherry. HeLa cells transfected for 24 hr with Arf1-GFP (green) and LAMP1-mCherry (purple) and with mitochondria labelled with MitoTracker deep red FM (white) were observed under ANDOR Dragonfly spinning disk confocal microscope using 100X objective and an EMCCD (iXon Ultra 888) camera. Images were acquired every 2 seconds. Time-lapse images are shown in Figure 3G.
Movie S7
Arf1-GFP is recruited at mitochondrial fission sites with TGN46-mCherry. HeLa cells transfected for 24 hr with Arf1-GFP (green) and TGN46-mCherry (purple) and with mitochondria labelled with MitoTracker deep red FM (white) were observed under ANDOR Dragonfly spinning disk confocal microscope using 100X objective and an EMCCD (iXon Ultra 888) camera. Images were acquired every 2 seconds. Time-lapse images are shown in Figure 3I.
Movie S8
PI(4)P builds up on mitochondrial constriction sites preceding mitochondrial division. HeLa cells transfected for 24 hr with GFP-PHFAPP1 (green) and TOM20-mCherry (white) were observed under ANDOR Dragonfly spinning disk confocal microscope using 100X objective and an sCMOS (Zyla) camera. 3 stacks of 0.3 μm each were acquired every 5 seconds. Images were then compiled by "max projection". Time-lapse images are shown in Figure 4B.
Movie S9
Additional example showing the PI(4)P builds up on mitochondrial constriction sites preceding mitochondrial division. HeLa cells transfected for 24 hr with GFP-PHFAPP1 (green) and TOM20-mCherry (white) were observed under ANDOR Dragonfly spinning disk confocal microscope using 100X objective and an sCMOS (Zyla) camera. 3 stacks of 0.3 μm each were acquired every 5 seconds. Images were then compiled by "max projection".
Movie S10
PI(4)P builds up on mitochondria-ER contacts preceding mitochondrial division. HeLa cells transfected for 24 hr with GFP-PHFAPP1 (green) and ER-dsRed2 (purple) with mitochondria labelled with MitoTracker deep red FM (white) were observed under ANDOR Dragonfly spinning disk confocal microscope using 100X objective and an EMCCD (iXon Ultra 888) camera. Images were acquired every 5 seconds. Time-lapse images are shown in Fig. S13.
Movie S11
GFP-PHFAPP1 accumulates at mitochondrial fission sites after Drp1-scarlet recruitment. HeLa cells transfected for 24 hr with GFP-PHFAPP1 (green) and Drp1-scarlet (purple) and with mitochondria labelled with MitoTracker deep red FM (white) were observed under ANDOR Dragonfly spinning disk confocal microscope using 100X objective and an EMCCD (iXon Ultra 888) camera. Images were acquired every 2 seconds. Time-lapse images are shown in Figure 4D.
Movie S12
GFP-PHFAPP1 is recruited at mitochondrial fission sites with TGN46-mCherry. HeLa cells transfected for 24 hr with GFP-PHFAPP1 (green) and TGN46-mCherry (purple) and with mitochondria labelled with MitoTracker deep red FM (white) were observed under ANDOR Dragonfly spinning disk confocal microscope using 100X objective and an EMCCD (iXon Ultra 888) camera. Images were acquired every 2 seconds. Time-lapse images are shown in Figure 4F.
Movie S13
GFP-PHFAPP1 is recruited at mitochondrial fission sites independently of LAMP1-mCherry. HeLa cells transfected for 24 hr with GFP-PHFAPP1 (green) and LAMP1-mCherry (purple) and with mitochondria labelled with MitoTracker deep red FM (white) were observed under ANDOR Dragonfly spinning disk confocal microscope using 100X objective and an EMCCD (iXon Ultra 888) camera. Images were acquired every 2 seconds. Time-lapse images are shown in Figure 4H.
Movie S14
PI(4)P builds up on mitochondrial constriction sites preceding mitochondrial division. HeLa cells transfected for 24 hr with mCherry-P4M (green) and TOM20-mEmerald (white) were observed under ANDOR Dragonfly spinning disk confocal microscope using 100X objective and an EMCCD (iXon Ultra 888) camera. Images were acquired every 5 seconds. Time-lapse images are shown in Fig. S14A..
Movie S15
GFP-P4M is recruited to mitochondrial fission sites with TGN46-mCherry. HeLa cells transfected for 24 hr with GFP-P4M (green) and TGN46-mCherry (purple) and with mitochondria labelled with MitoTracker deep red FM (white) were observed under ANDOR Dragonfly spinning disk confocal microscope using 100X objective and an EMCCD (iXon Ultra 888) camera. Images were acquired every 2 seconds. Time-lapse images are shown in Fig. S14C.
Movie S16
GFP-P4M accumulates at mitochondrial fission sites after Drp1-scarlet recruitment. HeLa cells transfected for 24 hr with GFP-P4M (green) and Drp1-scarlet (purple) and with mitochondria labelled with MitoTracker deep red FM (white) were observed under ANDOR Dragonfly spinning disk confocal microscope using 100X objective and an EMCCD (iXon Ultra 888) camera. Images were acquired every 2 seconds. Time-lapse images are shown in Fig. S14E.