Supplementary Materials

Lipid-gated monovalent ion fluxes regulate endocytic traffic and support immune surveillancem

Spencer A. Freeman, Stefan Uderhardt, Amra Saric, Richard F. Collins, Catherine M. Buckley, Sivakami Mylvaganam, Parastoo Boroumand, Jonathan Plumb, Ronald N. Germain, Dejian Ren, Sergio Grinstein

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

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  • Materials and Methods
  • Figs. S1 to S10
  • Captions for Movies S1 to S8
  • References

Images, Video, and Other Media

Movie S1
Resident tissue macrophages (LysM-tomato) of the peritoneal serosa continuously probe their local microenvironment. Intravital microscopy. Scale in lower right indicates time in minutes.
Movie S2
The stimulation of resident tissue macrophages (LysM-tomato) with M-CSF causes the formation of large macropinosomes as the cells continue to probe. Intravital microscopy. Scale in lower right indicates time in minutes.
Movie S3
Na+ efflux is required for macropinosome shrinkage. BMDM were stimulated with M-CSF in isosmotic medium containing either 150 mM NaCl or N-methylglucamine chloride as primary solutes, as indicated. Solutions also contained 1 mM CaCl2, 1 mM MgCl2, 5 mM KCl, 20 mM HEPES, plus 70 kDa tetramethylrhodamine dextran to identify the newly formed vacuoles. Fields of 5-10 cells were imaged every 30 s. Scale in lower right indicates time in minutes.
Movie S4
Tetrandrine, a cation channel inhibitor, blocks vacuole shrinkage. BMDM treated with vehicle control or tetrandrine (1 μM) were simultaneously stimulated with M-CSF in medium containing 70 kDa tetramethylrhodamine-dextran to identify the newly formed vacuoles. Fields of 5-10 cells were imaged every 30 s. Scale in lower left indicates time in minutes.
Movie S5
Osmotically-induced shrinkage of macropinosomes promptly causes tubulation. Macropinosomes loaded with sulforhodamine B and N-methylglucamine chloride and imaging started 10 min after their initial formation. After acquisition of the first frame, cells were exposed to a medium made hypertonic (500 mOsm final) by addition of sucrose. Images were acquired every 10 s.
Movie S6
Preventing vacuole resolution by inhibition of PIKfyve abrogates tissue surveillance. Intravital microscopy. Scale in lower right indicates time in minutes.
Movie S7
Lysosome swelling upon inhibition of PIKfyve is reversed by exposure to hyperosmotic solution. HeLa cells expressing LAMP1-RFP were treated with 500 nM of the PIKfyve inhibitor YM201636 for 1 h, causing swelling of the vesicles to the extent that their lumen could be resolved by confocal microscopy. The vesicles were monitored at 1 frame/10 s for the indicated times before and after addition of sucrose (second frame) to bring the medium osmolarity to 500 mOsm.
Movie S8
TPC2 expression level controls lysosome tubulation in a PtdIns(3,5)P2-dependent manner. HeLa cells expressing TPC2wt-GFP or TPC2K321A-GFP were recorded at 10 Hz for 30 s.