Stages 4 and 5. Co-expression of DMP1-eGFP (a), TPK1-mRFP (b) and YFP-HDEL (C, false-colored) shows colocalization of DMP1-eGFP and TPK1-mRFP and dissociation from the ER (d). The ER is compressed into interstices and junctions formed by foamy membrane structures (c and g, arrows and arrowheads respectively). On single plane images (e-h1) but not maximum projection (a-d), the colocalization between DMP1-eGFP (e) and TPK1-mRFP (f) appears incomplete (h1). In the majority of membrane segments analysed the fluorescence signal peaks are shifted between 200 nm and 300 nm (h
, panels 1–3), suggesting a double membrane structure, one membrane being labeled by DMP1-eGFP and the other with TPK1-mRFP. In some membrane segments the fluorescence peaks match perfectly, indicating colocalization (h1 and h2, segment and panel 4 respectively). Vacuolar sheets and foamy membrane formations have double membranes (i and k). Interstices and membrane junctions contain cytoplasm and trapped organelles (i, arrow and arrowhead). Crystalloid ER in a late stage 4 cell (j1 and j2). Cells displaying foamy vacuolar membrane structures (l) and labeling of the whole tonoplast (p) enter cell death by vesiculation of the entire ER network (m, arrows) except for the nuclear envelope (m, arrowhead). DMP1-eGFP (l) and RFP-p24 (m) were fully dissociated (n, magnified in o) as in stage 4. The Golgi vesicles remain unaffected in these cells (q and r). This process was visualized by EM (s). Scale bar, A-H1 and S, 10 μm; I, J1 and K, 0,5 μm; L-N and P-R, 20 μm; J2, 0,1 μm.