Figure 4.

Immunogold electron microscopy analysis of FP-17 and -22 distribution in the ER and ERES after the 10°C block. (a–f) HeLa cells transfected with Venus-17 (a and c) or -22 (b and d) and incubated for 3 h at 10°C were processed for cryosectioning as described in Materials and methods. FPs and the ERES marker Sec16 are labeled with 18- and 12-nm gold particles, respectively. Arrowheads in a and c indicate 12-nm gold particles, which are in close proximity to 18-nm gold particles in the case of cells expressing FP-22 (b) but not FP-17 (a). c–f show two examples of how image analysis was performed. Starting from the images in c and d, we manually reconstructed the drawings in e and f, attributing unequivocally identifiable membrane profiles to ER (black) and, among these, membranes within 60 nm of a 12-nm gold particle to ERES (in red). Other intracellular membranes (not considered) are represented in gray. The dashed line in f represents the plasma membrane. Note that, as expected, in these transfected cells FP-22 is present also at the plasma membrane. Gold particles falling within 20 nm of ER (or ERES) and considered in the analysis are color-coded as follows: 12-nm gold particles used to identify ERES, red; 18-nm gold particles attributed to ER or to ERES, black and cyan, respectively. Bars, 100 nm. (g) Relative labeling index for FP-17 and -22 at the ER (white) and ERES (black). The analysis, performed on 25 and 19 micrographs for FP-17 and -22, respectively, demonstrates that neither of the two FP constructs is randomly distributed between ER and ERES, but although FP-17 is excluded from ERES, FP-22 is concentrated therein. Statistical significance was assessed by a test for randomness, modified from Mayhew et al., (2003; *, P = 0.044; **, P = 0.0007). For details, see Materials and methods and Tables S1–3, available at http://www.jcb.org/cgi/content/full/jcb.200710093/DC1.