Cytochalasin Rearranges Cortical Actin of the Alga Nitella into Short, Stable Rods

The cortical cytoplasm of the alga Nitella contains reticulateactin that does not survive perfusion fixation with glutaraldehydeunless prestabilized with the cross-linker 3-maleimidobenzoyl-N-hydroxysuccinimidester (MBS). Cytochalasin D remodels thiscortical actin into short rods which are more stable, survivingaldehyde fixation without MBS pre-treatment. The overall alignmentof these actin rods correlates with that of cortical microtubules(transverse in young cells, random in old cells) but probablydoes not involve one-to-one correspondence. The time course,dose dependence and reversibility of these structural changesbroadly resemble those for streaming inhibition by cytochalasinbut the cortical actin responds to concentrations that do notslow streaming. Because the structural changes concern the corticaland not the subcortical actin, they seem unlikely to directlyinhibit streaming. Formation of cortical rods is not a responseto streaming inhibition per se since it does not occur whentwo other inhibitors of streaming (2,4-dinitrophenol (DNP) andNethyl maleimide (NEM)) are used. NEM, however, resembles MBSin stabilizing the reticulate form of cortical actin even thoughit cannot cross link. ¹Address from July 1995; Department of Biology, Faculty of Science,Osaka University, Machikaneyama 1-1, Tayonaka, Osaka, 560 Japan.     

Cell Geometry Guides the Dynamic Targeting of Apoplastic GPI-Linked Lipid Transfer Protein to Cell Wall Elements and Cell Borders in Arabidopsis thaliana

During cellular morphogenesis, changes in cell shape and cell junction topology are fundamental to normal tissue and organ development. Here we show that apoplastic Glycophosphatidylinositol (GPI)-anchored Lipid Transfer Protein (LTPG) is excluded from cell junctions and flat wall regions, and passively accumulates around their borders in the epidermal cells of Arabidopsis thaliana. Beginning with intense accumulation beneath highly curved cell junction borders, this enrichment is gradually lost as cells become more bulbous during their differentiation. In fully mature epidermal cells, YFP-LTPG often shows a fibrous cellulose microfibril-like pattern within the bulging outer faces. Physical contact between a flat glass surface and bulbous cell surface induces rapid and reversible evacuation from contact sites and accumulation to the curved wall regions surrounding the contact borders. Thus, LTPG distribution is dynamic, responding to changes in cell shape and wall curvature during cell growth and differentiation. We hypothesize that this geometry-based mechanism guides wax-carrying LTPG to functional sites, where it may act to “seal” the vulnerable border surrounding cell-cell junctions and assist in cell wall fortification and cuticular wax deposition.     

Transient exposure to ethylene stimulates cell division and alters the fate and polarity of hypocotyl epidermal cells

After transient exposure to the gaseous hormone ethylene, dark-grown cucumber (Cucumis sativus) hypocotyls developed unusual features. Upon ethylene's removal, the developing epidermis showed significant increases in cell division rates, producing an abundance of guard cells and trichomes. These responses to ethylene depended on the stage of development at the time of ethylene exposure. In the upper region of the hypocotyl, where cells were least differentiated at the onset of ethylene treatment, complex, multicellular protuberances formed. Further down the hypocotyl, where stomata and trichomes were beginning to develop at the onset of ethylene exposure, an increase in the number of stomata and trichomes was observed. Stomatal complexes developing after the ethylene treatment had a significant increase in the number of stomatal subsidiary cells and the number of cells per trichome increased. Analysis of division patterns in stomatal complexes indicated that exposure to ethylene either suspended or altered cell fate. Ethylene also altered cell division polarity, resulting in aberrant stomatal complexes and branched trichomes. To our knowledge, the results of this study demonstrate for the first time that transient treatment with physiological concentrations of ethylene can alter cell fate and increase the propensity of cells to divide.     

The C-terminal dilysine motif confers endoplasmic reticulum localization to type I membrane proteins in plants

The tomato Cf-9 disease resistance gene encodes a type I membrane protein carrying a cytosolic dilysine motif. In mammals and yeast, this motif promotes the retrieval of type I membrane proteins from the Golgi apparatus to the endoplasmic reticulum (ER). To test whether the C-terminal KKXX signal of Cf-9 is functional as a retrieval motif and to investigate its role in plants, green fluorescent protein (GFP) was fused to the transmembrane domain of Cf-9 and expressed in yeast, Arabidopsis, and tobacco cells. The fusion protein was targeted to the ER in each of these expression systems, and mutation of the KKXX motif to NNXX led to secretion of the fusion protein. In yeast, the mutant protein reached the vacuole, but plants secreted it as a soluble protein after proteolytic removal of the transmembrane domain. Triple hemagglutinin (HA)-tagged full-length Cf-9 was also targeted to the ER in tobacco cells, and cleavage was also observed for the NNXX mutant protein, suggesting an endoprotease recognition site located within the Cf-9 lumenal sequence common to both the GFP- and the HA-tagged fusions. Our results indicate that the KKXX motif confers ER localization in plants as well as mammals and yeast and that Cf-9 is a resident protein of the ER.     

Gibberellin-induced changes in growth anisotropy precede gibberellin-dependent changes in cortical microtubule orientation in developing epidermal cells of barley leaves. Kinematic and cytological studies on a gibberellin-responsive dwarf mutant, M489

We conducted kinematic and cytological studies on "between vein" epidermal cells of the gibberellin (GA)-deficient M489 dwarf mutant of barley (Hordeum vulgare L. Himalaya). GAs affect radial and axial components of cell expansion and cortical microtubule orientation. Adaxial cells in particular expand radially after leaving the elongation zone (EZ), probably as part of leaf unrolling. Exogenous gibberellic acid corrects the mutant's short, wide blades, short EZ, and slow elongation rate. Cell production rates increase more on the adaxial than on the abaxial surface. Cells spend equal periods of time elongating in dwarf and tall plants, but relative elemental growth rates start to decline sooner in the dwarf. GA increased the rate at which longitudinal wall area increased because the increased axial growth more than compensated for reduced radial growth. In dwarf leaves, increased radial expansion was detected in basal parts of the EZ before cortical microtubules lost transverse orientation in the distal elongation zone. We conclude that loss of microtubule orientation is not required for low GA levels to reduce growth anisotropy.     

Nuclear crystals, lampbrush-chromosome-like structures, and perinuclear cytoskeletal elements associated with nuclear fragmentation in characean internodal cells

Summary This article gives a survey of nucleus-associated structures and inclusions in a diverse range of characean algae includingChara braunii Gm.,Chara corallina Klein ex Willd., em. R.D.W.,Nitella cristata A.Br., em. R.D.W.,Nitella flexilis (L.) Ag.,Nitella furcata (Roxb. ex Bruz.) Ag. em. R.D.W.,Nitella hyalina (DC.) Ag.,Nitella pseudoflabellata A.Br., em. R.D.W.,Nitella pseudoflabellata var.imperialis T.F.A.,Nitella translucens var.axillaris (A.Br.) R.D.W. andNitellopsis obtusa (Desv. in Lois.) J.Gr. Lampbrushchromosome-like structures were found in nuclei ofNitella flexilis andNitellopsis obtusa and seem to be involved in the distribution of genetic material during nuclear fragmentation. Intranuclear tubular crystals of unknown protein composition were present in all species, especially in young, elongating cells, and could be important for establishing the main axis of the nuclei. Spindle-shaped protein crystals that originate in the nucleus and are released into the cytoplasm upon nuclear degeneration were observed in branchlet internodal cells of one population ofNitella flexilis. Perinuclear microtubules were present in all species, but perinuclear actin fibrils were hitherto only found in mostNitella species and inNitellopsis obtusa. None of these nucleus-associated structures seems to be responsible for the formation of constrictions leading to nuclear fragmentation. These constrictions were perpendicular to the main axis of the nucleus and symmetrical in theNitella species but asymmetric inC. braunii, C. corallina, and inNitellopsis obtusa. Statistical analysis of nuclear size, number and constriction sites indicate that fragmentation is a nonsynchronous process independent of the light-dark cycle.Abbreviations CLSM confocal laser scanning microscopy - DAPI 4,6-diamidino-2-phenylindole - DIC Nomarski differential interference contrast - LCLS lampbrush chromosome-like structure(s) Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday     

Mechanisms of Self-Organization of Cortical Microtubules in Plants Revealed by Computational Simulations

Microtubules confined to the two-dimensional cortex of elongating plant cells must form a parallel yet dispersed array transverse to the elongation axis for proper cell wall expansion. Some of these microtubules exhibit free minus-ends, leading to migration at the cortex by hybrid treadmilling. Collisions between microtubules can result in plus-end entrainment (“zippering”) or rapid depolymerization. Here, we present a computational model of cortical microtubule organization. We find that plus-end entrainment leads to self-organization of microtubules into parallel arrays, whereas catastrophe-inducing collisions do not. Catastrophe-inducing boundaries (e.g., upper and lower cross-walls) can tune the orientation of an ordered array to a direction transverse to elongation. We also find that changes in dynamic instability parameters, such as in mor1-1 mutants, can impede self-organization, in agreement with experimental data. Increased entrainment, as seen in clasp-1 mutants, conserves self-organization, but delays its onset and fails to demonstrate increased ordering. We find that branched nucleation at acute angles off existing microtubules results in distinctive sparse arrays and infer either that microtubule-independent or coparallel nucleation must dominate. Our simulations lead to several testable predictions, including the effects of reduced microtubule severing in katanin mutants.     

MICROTUBULE ORGANIZATION 1 regulates structure and function of microtubule arrays during mitosis and cytokinesis in the Arabidopsis root

MICROTUBULE ORGANIZATION 1 (MOR1) is a plant member of the highly conserved MAP215/Dis1 family of microtubule-associated proteins. Prior studies with the temperature-sensitive mor1 mutants of Arabidopsis (Arabidopsis thaliana), which harbor single amino acid substitutions in an N-terminal HEAT repeat, proved that MOR1 regulates cortical microtubule organization and function. Here we demonstrate by use of live cell imaging and immunolabeling that the mor1-1 mutation generates specific defects in the microtubule arrays of dividing vegetative cells. Unlike the universal cortical microtubule disorganization in elongating mor1-1 cells, disruption of mitotic and cytokinetic microtubule arrays was not detected in all dividing cells. Nevertheless, quantitative analysis identified distinct defects in preprophase bands (PPBs), spindles, and phragmoplasts. In nearly one-half of dividing cells at the restrictive temperature of 30 degrees C, PPBs were not detected prior to spindle formation, and those that did form were often disrupted. mor1-1 spindles and phragmoplasts were short and abnormally organized and persisted for longer times than in wild-type cells. The reduced length of these arrays predicts that the component microtubule lengths are also reduced, suggesting that microtubule length is a critical determinant of spindle and phragmoplast structure, orientation, and function. Microtubule organizational defects led to aberrant chromosomal arrangements, misaligned or incomplete cell plates, and multinucleate cells. Antiserum raised against an N-terminal MOR1 sequence labeled the full length of microtubules in interphase arrays, PPBs, spindles, and phragmoplasts. Continued immunolabeling of the disorganized and short microtubules of mor1-1 at the restrictive temperature demonstrated that the mutant mor1-1(L174F) protein loses function without dissociating from microtubules, providing important insight into the mechanism by which MOR1 may regulate microtubule length.