JCS01579 Supplementary Material

Files in this Data Supplement:

• Movie 1A
• Movie 1B
• Movie 1C
• Movie 1D
• Movie 1E
• Movie 1F -

  Fig. S1. Localisation of PH-domain probes during phagocytosis. Time series of phagocytosis of yeast cells by Dictyostelium cells expressing different PH-domain GFP fusion proteins.
  (A) Cell expressing the PI(3,4,5)P3 specific GRP1-PH-GFP during phagocytosis of a yeast cell, fluorescence and corresponding brightfield images, interval between subsequent images: 5s.
  (B) Cell expressing the PI(3,4)P2 specific TAPP1-PH-GFP during phagocytosis of a yeast cell, fluorescence and corresponding brightfield images, interval between subsequent images: 5s.
  (C) Cell expressing the PI(3,4,5)P3/PI(3,4)P2 specific CRAC-PH-GFP during phagocytosis of a yeast cell, fluorescence and corresponding brightfield images, interval between subsequent images: 5s.
  (D) Cell expressing the PI(3,4,5)P3/PI(3,4)P2 specific DAPP1-PH-GFP during phagocytosis of a yeast cell, fluorescence and corresponding brightfield images, interval between subsequent images: 5s.
  (E) Cell expressing the PI(3,4)P2 specific mutated DAPP1^G176A-PH fused to GFP during phagocytosis of a yeast cell, fluorescence and corresponding brightfield images, interval between subsequent images: 5s.
  (F) Montage of the fluorescence images of the various PH domain expressing cell lines during phagocytosis; interval between subsequent images: 5s

• Movie 2 -

  Fig. S2. Localisation of Coronin-GFP during phagocytosis. Sequence showing fluorescence and corresponding brightfield images of two cells expressing Coronin-GFP during the uptake and internalization of yeast cells. Interval between subsequent images: 5s. Note the rapid redistribution of Coronin-GFP around the phagosome and the propulsion of the ingested yeast towards the cell interior

• Movie 3 -

  Fig. S3. Localisation of PTEN-GFP during phagocytosis. PTEN^null cells expressing PTEN-GFP during phagocytosis of yeast cells. Time interval between subsequent images: 5s. Note the localisation of the PTEN-GFP fusion protein at the plasma membrane, but it is not detectable on the inner membrane of the phagocytic cup.

• Movie 4 -

  Fig. S4. Cellular localisation of PI(4)P during phagocytosis. Cell expressing the PI(4)P specific FAPP1-PH-GFP during the phagocytosis of a yeast cell. Time interval between subsequent images: 5s. Note the association of FAPP1-PH with the Golgi and small dynamic bright spots/vesicles. However, there is no localization to the plasma membrane or the phagosomal membrane.

• Movie 5 -

  Fig. S5. Changes in 3-phosphoinositide levels during macropinocytosis. Time series showing the differences in membrane binding during macropinocytosis by the different strains. Time interval between subsequent images: 5s. Note the rapid disappearance of GRP1-PH from the macropinosomes while the PI(3,4)P2 specific probes show a strong increase in fluorescence around the macropinosomes following fluid uptake.

• Movie 6 -

  Fig. S6. Effect of cAMP stimulation on PH-domain localisation. Time series showing the transient translocation of PI(3,4,5)P3 specific PH-domain probes to the plasma membrane in response to a pulse of cAMP  (final concentration 5µM) while no translocation is observed in the cell lines expressing the PI(3,4)P2 specific probes TAPP1-PH and DAPP1^G176A-PH. Time interval between subsequent images: 2s.

• Movie 7A
• Movie 7B
• Movie 7C -

  Fig. S7. 3-phosphoinositide distribution during aggregation of Dictyostelium cells.
  (A) Time series showing the localization of GRP1-PH-GFP localisation in aggregation stream cells mowing towards an aggregation centre on the right hand side. Note the small periodic changes in membrane localization in response to the propagated extracellular cAMP waves. Time interval between subsequent images: 10s.
  (B) Time series showing the localization of CRAC-PH -GFP localisation in aggregation stream cells mowing towards an aggregation centre on the right hand side. Time interval between subsequent images: 10s.
  (C) Time series showing the localization of DAPP1^G176A -GFP localisation in aggregation stream cells mowing towards an aggregation centre on the right hand side. Time interval between subsequent images: 10s. Note the engulfment of another Dictyostelium cell, which is accompanied by a transient rise of PI(3,4)P2 levels at the phagosome. Time interval between subsequent images: 10s.