Journal: Science Advances
Article Title: A phosphoinositide switch from PI(4,5)P 2 to PI4P triggers endocytosis by inducing dynamin-mediated fission in secretory cells
doi: 10.1126/sciadv.ady8065
Figure Lengend Snippet: ( A and B ) STED XZ -plane images of P4M-mTFP1 (labelling PI4P), PH G [labelling PI(4,5)P 2 ], and A532 (bath) for pre-Ω with a visible pore (Ω p , A) or a nonvisible pore (Ω np , B). XY -plane images across Ω’s pore region (dotted line) showed both PI4P and PI(4,5)P 2 at the pore region. Similar observations were made in 29 cells (five cultures, each culture from two to four glands). ( C ) PI(4,5)P 2 at the pore region: confocal (upper) and MINFLUX (lower) images of PH-SNAP-A647 (PH-domain attached with Alexa 647–tagged SNAP, labeling PI(4,5)P 2 ) for a Ω p and a Ω np at the XY plane across the pore region. Imaging were performed on fixed cells. ( D ) PI4P at the pore region: similar to (C), except for P4M-SNAP-A647 (P4M attached with Alexa 647–tagged SNAP, labeling PI4P). ( E ) STED XZ -plane images of FFN206 (labeling vesicles) and P4M-mTFP1 (merged at bottom): Vesicles docked at PI4P clusters at PM (triangles). Similar observations were made in 26 cells (four cultures). ( F ) STED XZ -plane images of P4M-mTFP1, dynamin 2-mNeonGreen (Dyn), and A532 (merged at bottom): dynamin 2 and PI4P were at the pre-Ω’s pore region. Similar observations were made in 10 cells (two cultures). ( G ) STED XZ -plane images of FFN206, Dyn, and P4M-mTFP1 (merged at bottom): PI4P and dynamin 2 were colocalized at the vesicle docking site (triangles) ready for mediating fusion pore closure. Similar observations were made in 26 cells (four cultures). ( H ) Sampled negative stain images (upper) and the outer diameter (lower) of ∆PRD–dynamin 1 decorated 30% DOPS, 68% DOPC tubes (Dyn-tubes) in the presence of PI4P (2%, n = 50 tubes) or PI(4,5)P 2 (2%, n = 50 tubes). *** P < 0.001, t test. ( I ) Sampled cryo-EM images (left), 2D class images (middle), and inner diameter (right) of Dyn-tubes in the presence of PI4P (2%, n = 120 tubes) or PI(4,5)P 2 (2%, n = 120 tubes). *** P < 0.001, t test. ( J ) Distribution of Dyn-tube inner diameter in the presence of PI4P (2%, n = 120 tubes) or PI(4,5)P 2 (2%, n = 120 tubes). Curves: gaussian fits.
Article Snippet: Subsequently, cells were incubated at room temperature for 50 min with SNAP substrate dye solution containing 1 μM SNAP-Surface Alexa Fluor 647 (NEB, S9136S), 0.5% bovine serum albumin, and 1 mM dithiothreitol (DTT), resulting in the attachment of SNAP-surface Alexa Fluor 647 to PH-SNAP (PH-SNAP-A647) or P4M-SNAP (P4M-SNAP-A647), which were used for MINFLUX imaging.
Techniques: Labeling, Imaging, Staining, Cryo-EM Sample Prep
![( A and B ) STED XZ -plane images of P4M-mTFP1 (labelling PI4P), PH G [labelling PI(4,5)P 2 ], and A532 (bath) for pre-Ω with a visible pore (Ω p , A) or a nonvisible pore (Ω np , B). XY -plane images across Ω’s pore region (dotted line) showed both PI4P and PI(4,5)P 2 at the pore region. Similar observations were made in 29 cells (five cultures, each culture from two to four glands). ( C ) PI(4,5)P 2 at the pore region: confocal (upper) and MINFLUX (lower) images of <t>PH-SNAP-A647</t> (PH-domain attached <t>with</t> <t>Alexa</t> 647–tagged SNAP, labeling PI(4,5)P 2 ) for a Ω p and a Ω np at the XY plane across the pore region. Imaging were performed on fixed cells. ( D ) PI4P at the pore region: similar to (C), except for P4M-SNAP-A647 (P4M attached with Alexa 647–tagged SNAP, labeling PI4P). ( E ) STED XZ -plane images of FFN206 (labeling vesicles) and P4M-mTFP1 (merged at bottom): Vesicles docked at PI4P clusters at PM (triangles). Similar observations were made in 26 cells (four cultures). ( F ) STED XZ -plane images of P4M-mTFP1, dynamin 2-mNeonGreen (Dyn), and A532 (merged at bottom): dynamin 2 and PI4P were at the pre-Ω’s pore region. Similar observations were made in 10 cells (two cultures). ( G ) STED XZ -plane images of FFN206, Dyn, and P4M-mTFP1 (merged at bottom): PI4P and dynamin 2 were colocalized at the vesicle docking site (triangles) ready for mediating fusion pore closure. Similar observations were made in 26 cells (four cultures). ( H ) Sampled negative stain images (upper) and the outer diameter (lower) of ∆PRD–dynamin 1 decorated 30% DOPS, 68% DOPC tubes (Dyn-tubes) in the presence of PI4P (2%, n = 50 tubes) or PI(4,5)P 2 (2%, n = 50 tubes). *** P < 0.001, t test. ( I ) Sampled cryo-EM images (left), 2D class images (middle), and inner diameter (right) of Dyn-tubes in the presence of PI4P (2%, n = 120 tubes) or PI(4,5)P 2 (2%, n = 120 tubes). *** P < 0.001, t test. ( J ) Distribution of Dyn-tube inner diameter in the presence of PI4P (2%, n = 120 tubes) or PI(4,5)P 2 (2%, n = 120 tubes). Curves: gaussian fits.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6769/pmc12526769/pmc12526769__sciadv.ady8065-f5.jpg)