PMC

Exit of a small vesicle through a transient pore. The small vesicle (marked by an arrow) of radius R_cargo ≈ 0.6 μm is initially inside a larger one, of radius R ≈ 16 μm (first frame, t = 0 s). When a pore opens (second frame, t = 0.76 s), the small vesicle starts moving, dragged by the leaking fluid. It passes through the pore (fourth frame, t = 3.93 s) just before the pore reseals (sixth frame, t = 5.24 s). The position of the small vesicle and the pore radius are plotted as a function of time in . The vesicles are labeled using the dye RH237 (; also see Materials and Methods).

Translocation of Myo1b–GFP and Myo1c–GFP to fused secretory vesicles. (A) ATII cells were co-transfected with Myo1c–GFP and actin–DsRed and stained with LTB. Myo1c–GFP translocated to secretory vesicles after fusion. Arrowheads indicate fusing vesicles and time=0 indicates the last frame before fusion. Scale bar: 10 µm. The time of fusion pore opening was determined by LTB fluorescence decrease (upper row). Insets show an enlarged view of the fused vesicle. Scale bar: 1 µm. (B) Mean±s.e.m. of the Myo1c–GFP and actin–DsRed fluorescence change on fusing lamellar bodies (n=31). Myo1c–GFP translocated to fused vesicles before actin polymerization. The dashed line indicates the last frame before the fusion pore opening, determined by LTB fluorescence decrease. (C) ATII cells were co-transfected with Myo1b–GFP and actin–DsRed and stained with LTB. Myo1b–GFP translocated to secretory vesicles after fusion. Arrowheads point at fusing lamellar bodies and time=0 indicates the last frame before the fusion pore opening. Scale bar: 10 µm. The time of fusion pore opening was determined by LTB fluorescence decrease (upper row). Insets show an enlarged view of one fusing vesicle. Scale bar: 1 µm. (D) Mean±s.e.m. of the Myo1b–GFP and actin–DsRed fluorescence change on the fusing lamellar bodies (n=42). The kinetics of Myo1b–GFP translocation was slower than actin polymerization and also slower than Myo1c translocation (B). The dashed line indicates the last frame before the fusion pore opening.

A. Fluorescent images of ‘fast’ MDA-MB-231 cell nucleus shown migrating towards and through a 50 × 6 µm pore. White arrows point to nucleus as cell approaches pore (10 min – 120 min), contracts into pore (125 min – 130 min), migrates through the pore and exits from the opposite side (135 min – 245 min). B. Fluorescent images of ‘slow’ MDA-MB-231 cell nucleus transmigration through a 50 × 6 µm pore. White arrows indicate nucleus as cell migrates towards the pore opening (370 min – 620 min), contracts its cell body into the pore (625 min – 720 min), migrates through pore and exits from the opposite side (720 min –930 min). Arrows indicate position of nucleus, time shown in white at bottom of each frame. Scale bars = 20 µm.

(A) Membrane insertion outline of the closed discontinuous state of E put TM with pore water (in red and white spheres) inside the pore structure and packed by lipid-like pseudo atoms (green spheres). Only pore water molecules remain after pore water generation through high-temperature dynamics. (B) The closed discontinuous state of E put TM inserted in a 1.5 lipid bilayer (POPC, POPE, POPI, POPS, Cholesterol, and SM) having 194 lipid components (94 in the upper leaflet and 100 in the lower leaflet) and modified with a TIP3P water model. The water molecules are seen in the cytoplasmic and the ERGIC luminal side. (C) A magnified side view of the model water molecules in the pore structure of the closed discontinuous state of the E put TM inserted in the ERGIC membrane mimic. (D) Top view of the model water molecules in the pore structure of the E put TM inserted in the closed discontinuous state within the ERGIC membrane mimic. (E) Membrane insertion outline of the continuous channel state 1 of E put TM with pore water (in red and white spheres) inside the pore structure and packed by lipid-like pseudo atoms (green spheres). Only pore water molecules remain after pore water generation through high-temperature dynamics. (F) The continuous channel state 1 of E put TM inserted in a 1.5 lipid bilayer (POPC, POPE, POPI, POPS, Cholesterol, and SM) having 194 lipid components (94 in the upper leaflet and 100 in the lower leaflet) and modified with a TIP3P water model. The water molecules are seen in the cytoplasmic and the ERGIC luminal side. (G) A magnified side view of the model water molecules in the pore structure of the continuous channel state 1 of the E put TM inserted in the ERGIC membrane mimic. (H) Top view of the model water molecules in the pore structure of the E put TM inserted in the continuous channel state 1 within the ERGIC membrane mimic. (I) Snapshots of the morphed protein models between open and closed state intermediate of the E put at a 10-frame interval (from frame 1–61). (J) Cross-sectional pore size profile of the membrane-inserted E put TM along the Z-axis in the closed discontinuous state (black) and continuous channel state 1 (red). (K) Cross-sectional pore area profile of the membrane-inserted E put TM along the Z-axis in the closed discontinuous state (close in black) and continuous channel state 1 (open in red).