Fig. 5: TMEM16K at ER-endosome membrane contact sites.
From: TMEM16K is an interorganelle regulator of endosomal sorting
a Immunocytochemistry to visualize TMEM16K proximity labeling of endosomes. COS-7 cells were transfected with TMEM16K tagged with proximity biotinylation enzyme, incubated with biotin for 6 h, and immunostained with fluorescently conjugated Streptavidin and antibody against endogenous Rab7. a. Row 1, Left: View of the entire cell expressing myc-BioID-TMEM16K. Scale bar 10 µm. a. Row 1, Right: Magnified region of the cell showing myc-BioID-TMEM16K, its pattern of proximity biotinylation and endogenous Rab7. Scale bar 5 µm. a. Row 2: High magnification insets with line scan quantification of the three channels marked in overlay. Scale bar 1 µm. b Live imaging of the U-2OS cells transfected with TMEM16K-V5-mNeonGreen (TMEM16K-V5-mNG), tdTomato-Rab7, and EGF-Alexa647, pulse chased for 45 min, imaged with spinning disk confocal microscope. See Supplementary Movie 3. b Row 1: Snapshots of the live imaging showing TMEM16K, Rab7, EGF and their overlay. Scale bar 10 µm. b. Row 2, High magnification insets with line scan quantification of the three channels marked in overlay. Scale bar 0.5 µm c Widefield image to view entire cell expressing TMEM16K-V5. Scale bar 10 µm. Inset marks cell region imaged with structured illumination microscopy (SIM). d. Single plane structured illumination microscopy of U-2OS cells transfected with TMEM16K-V5 and immunolabelled for endogenous Rab7. Scale bar 2 µm. e High magnification insets 1 and 2 from SIM images with corresponding line scan quantification of the two channels marked in overlay. Scale bar 1 µm. f Split-GFP assay positive control with cells transfected with TMEM16K-V5-GFP and GFP(1–10)-HA-TMEM16K, as TMEM16K acts as dimer. Scale bar 10 µm. g Scheme of the split-GFP experiment where molecule of the GFP can be reconstituted only if the two proteins contact. h Split GFP reconstitution assay between TMEM16K-V5-GFP and GFP(1-10)-HA-Rab7. Scale bar 10 µm, inset 2 µm. i Split-GFP reconstitution assay betweenTMEM16K-V5-GFP11 and constitutively active mutant of Rab7 Q67L tagged with GFP(1–10) Scale bar 10 µm, inset 2 µm. j Split-GFP reconstitution assay between TMEM16K-V5-GFP11 and inactive mutant of Rab7 T22N tagged with GFP(1–10) Scale bar 10 µm.
