Fig. 5. Subcellular immunolocalisation of KNOLLE protein in root cells of 35S::KN seedlings. (A-C) Confocal laser-scanning microscopy of dividing cells in wild-type root tip stained for KN (Cy3, red) and DAPI (false colour, green) to show the temporal and spatial dynamics of KN relocalisation during cytokinesis; cells are delineated by broken lines. (A) Prophase: KN patches, presumably Golgi. (B) Telophase: KN in plane of cell division and nearby patches. (C) Late phase of cytokinesis: KN-positive cell plate extends to the parental cell wall, only few patches remain. (D-L) 35S::KN transgenic root cells. (D-F) Semi-thick cryo-sections through the central cylinder of root. (D,E) Cross section: (D) KN immunofluorescence; (E) phase contrast of D; cells close to the phloem show strong signals (D) at the cell surface and inside the cells (asterisks, D; arrowheads, E) point to the xylem. (F) KN immunofluorescence of longitudinal section: the plasma membrane (arrowhead, pm) and Golgi-like intracellular structures (arrow, g) are labelled. (G-I,L) Ultrathin cryo sections of root (G-I) and root hair (L) labelled with anti-KN immunogold. Cryo sections were required because the anti-KNOLLE antiserum does not detect the integral membrane protein KNOLLE on conventional EM sections after chemical fixation (Lauber et al., 1997). Cryosections do not preserve membrane structures very well. (G) Root cell adjacent to xylem cell (overview). Gold labelling within areas delineated by dashed or dotted lines is highlighted by yellow (internal staining) or red (plasma membrane) asterisks to visualise gold label that is not readily detectable at this low magnification. g, Golgi; m, mitochondrion; pm, plasma membrane. (H) Higher magnification of upper boxed area in G rotated 90° clockwise showing KN immunogold label at the plasma membrane (pm), Golgi (g) and post-Golgi area. m, mitochondrion. (I) KN immunogold labelling of Golgi (g) and trans-Golgi (t) compartments. (J,K) Ultrathin section of chemically fixed and embedded root hair. (K) Higher magnification of boxed area in J: root hair tip filled with rough endoplasmic reticulum (er) and numerous electron-dense vesicles (v). (L) Strongly KN-labelled vesicles (v) and trans-Golgi network (t) from root hair tip. Scale bar: 20 µm in D-F; 250 nm in H,I,K,L.