cytochalasin-d and amiprophos-methyl

Arabinogalactan proteins (AGPs), a superfamily of plant hydroxyproline-rich glycoproteins, are present at cell surfaces. Although precise functions of AGPs remain elusive, they are widely implicated in plant growth and development. A well-characterized classical tomato (Lycopersicon esculentum) AGP containing a glycosylphosphatidylinositol plasma membrane anchor sequence was used here to elucidate functional roles of AGPs. Transgenic tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells stably expressing green fluorescent protein (GFP)-LeAGP-1 were plasmolysed and used to localize LeAGP-1 on the plasma membrane and in Hechtian strands. Cytoskeleton disruptors and beta-Yariv reagent (which binds and perturbs AGPs) were used to examine the role of LeAGP-1 as a candidate linker protein between the plasma membrane and cytoskeleton. This study used a two-pronged approach. First, BY-2 cells, either wild type or expressing GFP-microtubule (MT)-binding domain, were treated with beta-Yariv reagent, and effects on MTs and F-actin were observed. Second, BY-2 cells expressing GFP-LeAGP-1 were treated with amiprophosmethyl and cytochalasin-D to disrupt MTs and F-actin, and effects on LeAGP-1 localization were observed. beta-Yariv treatment resulted in terminal cell bulging, puncta formation, and depolymerization/disorganization of MTs, indicating a likely role for AGPs in cortical MT organization. beta-Yariv treatment also resulted in the formation of thicker actin filaments, indicating a role for AGPs in actin polymerization. Similarly, amiprophosmethyl and cytochalasin-D treatments resulted in relocalization of LeAGP-1 on Hechtian strands and indicate roles for MTs and F-actin in AGP organization at the cell surface and in Hechtian strands. Collectively, these studies indicate that glycosylphosphatidylinositol-anchored AGPs function to link the plasma membrane to the cytoskeleton.

Topics: Actins; Cell Membrane; Cells, Cultured; Cytochalasin D; Glucosides; Green Fluorescent Proteins; Microtubules; Mucoproteins; Nicotiana; Nitrobenzenes; Organothiophosphorus Compounds; Phloroglucinol; Plant Proteins; Plants, Genetically Modified; Recombinant Fusion Proteins; Solanum lycopersicum; Tubulin Modulators

Plastid stromules are stroma-filled tubular extensions of the plastid envelope membrane. These structures, which have been observed in a number of species, allow transfer of proteins between interconnected plastids. The dramatic shape of stromules and their dynamic movement within the cell provide an opportunity to study the control of morphology and motion of plastids. Using inhibitors of actin and tubulin, we found that both microfilaments and microtubules affect the shape and motility of non-green plastids. Actin and tubulin control plastid and stromule structure by independent mechanisms, while plastid movement is promoted by microfilaments but inhibited by microtubules. The presence or absence of stromules does not affect the motility of plastids. Photobleaching experiments indicate that actin and tubulin are not necessary for the bulk of green fluorescent protein (GFP) movement between plastids via stromules.

Topics: Actin Cytoskeleton; Cytochalasin D; Microtubules; Nicotiana; Nitrobenzenes; Organothiophosphorus Compounds; Plastids

Apical cells of protonemata of the moss Ceratodon purpureus are unusual among plant cells with sedimentation in that only some amyloplasts sediment and these do not fall completely to the bottom of vertical cells. To determine whether the cytoskeleton restricts plastid sedimentation, the effects of amiprophos-methyl (APM) and cytochalasin D (CD) on plastid position were quantified. APM treatments of 30-60 min increased the plastid sedimentation that is normally seen along the length of untreated or control cells. Longer APM treatments often resulted in more dramatic plastid sedimentation, and in some cases almost all plastids sedimented to the lowermost point in the cell. In contrast, the microfilament inhibitor CD did not affect longitudinal plastid sedimentation compared to untreated cells, although it did disturb or eliminate plastid zonation in the tip. These data suggest that microtubules restrict the sedimentation of plastids along the length of the cell and that microtubules are load-bearing for all the plastids in the apical cell. This demonstrates the importance of the cytoskeleton in maintaining organelle position and cell organization against the force of gravity.

Topics: Actin Cytoskeleton; Cytochalasin D; Cytoskeleton; Gravitation; Microscopy, Fluorescence; Microtubules; Nitrobenzenes; Organothiophosphorus Compounds; Plants; Plastids