Developmental transitions and dynamics of the cortical ER of Arabidopsis cells seen with green fluorescent protein.

Arabidopsis thaliana plants were stably transformed with DNA encoding green fluorescent protein and with sequences ensuring retention in the endoplasmic reticulum (ER). Confocal laser scanning microscopy shows fluorescent ER in many cells of seedlings so allowing developmental changes to be documented. The arrangement of the cortical ER changes as cells mature in the hypocotyl and root epidermis. In the root, cells that have completed expansion have reticulate cortical ER resembling the ER described in many previous studies. Expanding cells, however, show extensive perforated sheets of cortical ER which transform quite abruptly into a loose reticulum at the basipetal end of the elongation zone. The reticulum compacts in trichoblasts beginning at sites where root hairs are about to emerge. The compacted form is maintained throughout the hair until growth ceases and the open reticulate form returns. All forms of cortical ER are dynamic and we use a color overlay method to distinguish stable and moving structures in a single composite image. Reticulate ER continuously rearranges its polygonal layout and perforations move and change their shape in the ER sheets of younger cells. ER deeper in the cell (i.e. not close to the plasma membrane) moves more actively so that almost no tubules remain stable even over short periods of less than one minute. The function of the perforated sheets of cortical ER present in growing cells is unknown.

Actin of chara giant internodal cells: a single isoform in the subcortical filament bundles and a larger, immunologically related protein in the chloroplasts.

Internodal cells of Chara corallina Klein ex. Wild have been studied to determine the number of actin isoforms they contain and whether actin occurs at locations in the cortical cytoplasm outside the filament bundles. A monoclonal antibody to chicken actin is specific for actin in numerous animal cells but binds to two Chara proteins after their separation by two-dimensional polyacrylamide gel electrophoresis. One protein resembles known actins in relative molecular mass (43,000-M(r)) and isoelectric point (5.5) while the other is distinctly different (58,000-M(r), isoelectric point = 4.8). Because it is indetectable in cells whose actin bundles have been extracted, the 43,000-M(r) protein is assigned to the bundles and concluded to be rare or absent in the remaining cortical cytoplasm. The 58,000-M(r) protein, in contrast, does not extract with the actin bundles. It was localized within the chloroplasts by immunofluorescence and by the dependence of proteolysis on the permeabilization of the chloroplast envelope.

Growth and regrowth of actin bundles in Chara: bundle assembly by mechanisms differing in sensitivity to cytochalasin.

Cytochalasin is known to inhibit cytoplasmic streaming rapidly in characean cells without disassembling their actin bundles. Lower cytochalasin concentrations than those needed for streaming inhibition are now shown to disrupt bundle assembly and, over longer periods, assembled bundles. After local wounding, cytochalasin limited bundle regeneration to the production of polygons and straight, discontinuous bundles that rarely connected to bundles outside the wound. The regenerated bundles supported only scattered organelle movements, whereas long, oriented bundles of control cells were connected to those outside the wound and supported bulk endoplasmic streaming. Unwounded Chara plants cultured for up to 2 weeks in 1 microM-cytochalasin maintained normal bundle orientation and rapid cytoplasmic streaming, but the mean number of bundles per file of chloroplasts fell from 5.2 in controls to 2.0 in growing cells and 3.4 in nongrowing cells. These structural effects seem more likely than the streaming inhibition to reflect cytochalasin's in vitro effect of blocking extension at the barbed but not the pointed end of F-actin. In particular, cytochalasin inhibited the extension into the wound of bundles in which only the barbed ends of filaments would be exposed. However, short lengths of isolated bundles grew within the wound and bundle growth in the intact cell continued, albeit in modified form. It is suggested that these examples of continuing bundle growth involve cytochalasin-resistant mechanisms that are not wholly dependent on barbed-end filament growth.

Selective extraction of Chara actin bundles: identification of actin and two coextracting proteins.

The sub-cortical actin bundles of the alga Chara corallina can be selectively extracted with a low salt solution except when cytochalasin B is present. Proteins with molecular weights of 160000, 43000 and 37000 share this extraction behaviour. While chemical cleavage of the 43000 band indicates that it is actin, the nature of the other proteins is unknown. Although the 37000 protein resembles tropomyosin in molecular weight it lacks tropomyosin's distinctively large change in electrophoretic mobility in the presence of urea.

Regeneration of actin bundles in Chara: polarized growth and orientation by endoplasmic flow.

Strong irradiation of localized areas of the alga Chara produces chloroplast damage and extensive loss of the actin bundles responsible for cytoplasmic streaming. Immunofluorescence using a monoclonal antibody binding to the actin bundles has been used to follow their regrowth. Bundle regeneration is polarized so that new bundles develop from the ends of the actin bundles delivering endoplasm to the damaged area and not from bundles removing endoplasm. According to the previously established polarity of the actin filaments this growth is occurring from the "barbed" but not the "pointed" ends of the component filaments. The frequently irregular orientation of the regenerated bundles contrasts with the straight, parallel arrangement of the bundles before destruction. The arrangement of the regenerated bundles is suggested to depend on orientation by passive endoplasmic flow rather than a cortical template. As a result, bundles follow sweeping curves and can form a U-turn connecting oppositely polarized bundles normally separated by the neutral line. In addition to development in continuity with the free ends of pre-existing bundles, visualization of small, discrete fluorescent structures suggests that bundles can begin to form in isolation within the damaged areas. The results are discussed in terms of the polarized actin polymerisation seen in vitro, additional controls which may operate on bundle growth in vivo, and the ability of flow to orient F-actin. The relevance of the findings to normal cell ontogeny is assessed.