Figure 2 : Visualizing membrane extrusions at the abscission sites.

From: Resolving new ultrastructural features of cytokinetic abscission with soft-X-ray cryo-tomography

Figure 2

(a) Reconstructed tomographic Z sections (70 nm steps) of the intercellular bridge shown in Fig. 1, centered on membrane vesicles protruding on both sides of the dark zone (scheme of intercellular bridge stage, left; cyan arrows, membrane extrusions). (b) Representative intercellular bridge at stage 3. First panel (left to right): fluorescence image of the intercellular bridge acquired on an SD (scale bar, 10 μm), fluorescence image acquired at BESSY II, a scheme indicating the stage of abscission. Reconstructed tomographic Z sections of the fluorescent intercellular bridge are shown in panels 2–4. Second panel: representative Z sections spanning the entire intercellular bridge (350 nm steps). Third panel: subsets of the tomogram (70 nm steps) centered on membrane protrusions observed at the abscission site. Fourth panel: subsets of the tomographic Z sections (70 nm steps) centered on the dark zone. Cyan arrow, membrane extrusion at the abscission site; blue arrow, dark zone membrane extrusion; yellow arrow, cortical filaments at dark zone. (c) 3D rendering of tomographic Z sections shown in (a) (left) and in (b) (right). Red, intercellular bridge membrane; yellow, dark zone; green, microtubules; cyan, abscission site membrane extrusions; blue, dark zone membrane extrusions. n (extrusion at abscission site) = 6/13; stage 1, 2/5; stage 2, 2/5; stage 3, 1/2; stage 4, 1/1). n (extrusions at dark zone) = 12/13. Scale bars: (a–b) 1 μm. (d) MDCK cells expressing CAAX-GFP were synchronized, fixed, stained with anti-tubulin antibodies and imaged by SIM. Shown are 3D rendered images of a representative cell undergoing abscission (Left: tubulin alone; Right: tubulin and CAAX). Arrows indicates points of membrane extrusions from constriction sites. Individual Z stacks are shown in Figure S1. n = 5. Scale bar = 2 μm.