Figure 1.

OSER membrane biogenesis is sustained by monomeric fluorescent protein fusion expression and does not involve Climp63 and SUN proteins. A. HEK293 cells grown on coverslips were transfected with calnexin-mCherry (CNX-mCherry) construct and observed by confocal microscopy 24 hours later, as described under "Materials and methods". OSER membrane expansion was observed in almost every expressing cell (n = 14). B. HEK293 cells were co-transfected with calnexin-CFP (CNX-CFP) and Climp63-GFP constructs and processed as in A. OSER structures positive for calnexin and negative for Climp63-GFP are indicated by an arrow (number of cells showing Climp63-GFP excluded from the OSER membranes, n = 23). C-E. HEK293 cells grown on glass coverslips transfected with calnexin-CFP were cultured for 24 h and labelled with an antibody for endogenous SUN1 (C), SUN2 (D) or Nesprin-1 (E), stained with a Texas Red-conjugated secondary antibody and mounted on glass slides for confocal microscopy. All but one cell stained for SUN1 (n = 56) or SUN2 (n = 35), showed exclusion of the co-stained SUN proteins from the OSER; the calnexin/SUN colocalization observed in a single cell of each set was presumably due to fortuitous targeting of SUN proteins into the OSER structures (some of which may originate from the NE [10]). All Nesprin-1-stained cells cells showed its exclusion from the OSER (n = 34). Multilamellar OSER structures positive for calnexin-CFP but negative for the co-stained proteins are indicated by open arrows; solid arrows indicate SUN1-, SUN2- or Nesprin-1-stained NE (SUN1 and Nesprin-1 labelling produced intra-nuclear staining, in addition to the NE staining, under various experimental conditions). Scale bar in each image is 10 μm.

Korkhov and Zuber BMC Cell Biology 2009 10:59   doi:10.1186/1471-2121-10-59
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