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. 2015 May 4;33(3):285–298. doi: 10.1016/j.devcel.2015.02.017

Figure 4.

Figure 4

High-Curvature Membrane Induction by Overexpressed Nup60

(A) Fluorescent imaging of cells expressing Nup188-GFP and plasmid-borne Nup60-mCherry constructs (GAL promoter). Induced refers to 4 hr of protein overproduction in galactose-containing medium. NLSNup60 (1-47): black arrow indicates expansion membrane; white arrow indicates NPC clusters. For expression levels and toxicity, see Figure S5B and (C). Because Nup60 (48-162) was not imported into the nucleus, we added an SV40 NLS to all Nup60 constructs for comparability. Accelerated import also reduces targeting of Nup60 (1-47) to the plasma membrane (compare Figure 4A with Figure S5F left). Scale bar represents 2 μm.

(B) TEM analysis of cells overproducing Nup60-mCherry constructs. NLSNup60 (1-47): asterisk marks an intranuclear organelle of unknown origin, black arrow labels newly formed membrane sheet. NLSNup60 (1-162): black arrows label membrane tubules in longitudinal and cross-section. N, nucleus; C, cytoplasm; E, expansion membrane; V, vacuole. Scale bar represents 500 nm.

(C) Electron micrographs of NLSNup60 (1-162) overproducing cells. Left and right images are TEM samples optimized for staining proteins or membranes, respectively. Left image shows membrane tubules in cross-section with a surrounding electron-dense coat (white arrow). Right image shows that tubules are made of a single lipid bilayer (black arrow, compare to INM/ONM). INM, inner nuclear membrane; ONM, outer nuclear membrane. Scale bar represents 100 nm.