Darryl L. Kropf

CYTOSKELETAL CONTROL OF POLAR GROWTH IN PLANT CELLS

There are two quite different modes of polar cell expansion in plant cells, namely, diffuse growth and tip growth. The direction of diffuse growth is determined by the orientation of cellulose microfibrils in the cell wall, which in turn are aligned by microtubules in the cell cortex. The orientation of the cortical microtubule array changes in response to developmental and environmental signals, and recent evidence indicates that microtubule disassembly/reassembly and microtubule translocation participate in reorientation of the array. Tip growth, in contrast, is governed mainly by F-actin, which has several putative forms and functions in elongating cells. Longitudinal cables are involved in vesicle transport to the expanding apical dome and, in some tip growers, a subapical ring of F-actin may participate in wall-membrane adhesions. The structure and function of F-actin within the apical dome may be variable, ranging from a dense meshwork to sparse single filaments. The presence of multiple F-actin structures in elongating tips suggests extensive regulation of this cytoskeletal array.

Cytosolic pH Gradients Associated with Tip Growth

The presence of a cytosolic pH gradient and its relation to polar tip growth was investigated in rhizoid cells of Pelvetia embryos with the use of pH-sensitive microelectrodes and by ratio imaging. Growing rhizoid cells generated a longitudinal pH gradient in which the apical cytosol was 0.3 to 0.5 units more acidic than the cytosol at the base of the cell. Treatment with a membrane-permeant weak acid, propionic acid, dissipated the cytosolic pH gradient and inhibited growth. The magnitude of the pH gradient correlated well with the rate of tip elongation. The pH gradient spatially superimposed on the cytosolic calcium gradient, and inhibition of calcium fluxes by treatment with lanthanum abolished the pH gradient and inhibited growth.

The Microtubule Plus-End Binding Protein EB1 Functions in Root Responses to Touch and Gravity Signals in Arabidopsis

Microtubules function in concert with associated proteins that modify microtubule behavior and/or transmit signals that effect changes in growth. To better understand how microtubules and their associated proteins influence growth, we analyzed one family of microtubule-associated proteins, the END BINDING1 (EB1) proteins, in Arabidopsis thaliana (EB1a, EB1b, and EB1c). We find that antibodies directed against EB1 proteins colocalize with microtubules in roots, an observation that confirms previous reports using EB1-GFP fusions. We also find that T-DNA insertion mutants with reduced expression from EB1 genes have roots that deviate toward the left on vertical or inclined plates. Mutant roots also exhibit extended horizontal growth before they bend downward after tracking around an obstacle or after a 90° clockwise reorientation of the root. These observations suggest that leftward deviations in root growth may be the result of delayed responses to touch and/or gravity signals. Root lengths and widths are normal, indicating that the delay in bend formation is not due to changes in the overall rate of growth. In addition, the genotype with the most severe defects responds to low doses of microtubule inhibitors in a manner indistinguishable from the wild type, indicating that microtubule integrity is not a major contributor to the leftward deviations in mutant root growth.

Localized membrane-wall adhesions inPelvetia zygotes

SummaryMembrane-wall adhesions in zygotes of the brown algaPelvetia were visualized following plasmolysis. Strands of cytoplasm remained firmly attached to the cell wall at discrete adhesion sites during plasmolysis. Adhesion sites were uniformly distributed in ungerminated zygotes, but were concentrated in the apical 5 μm of the elongating rhizoid in germinated zygotes. Few adhesions were detected along the flanks of the rhizoid or in the thallus region of germinated zygotes. The structure, physiology and function of apical adhesions in the rhizoid were characterized. F-actin was found at adhesion sites in plasmolyzed zygotes labeled with rhodamine phalloidin, and disruption of cortical F-actin reduced the number of adhesions. Manipulation of cytosolic H+ and Ca2+ activities also disrupted adhesions. On the extracellular surface, the number of adhesions was reduced by inhibition of cellulose synthesis, protease cleavage of wall proteins, and changes in extracellular H+ and Ca2+ activities. Chronic treatment with the synthetic peptide RGDS, which prevents cell adhesion in fibroblasts, also reduced adhesion number. The number of adhesions per cell did not correlate with growth rate, but was inversely correlated with the ability to establish new rhizoid growth sites. The results indicate that membrane wall adhesions containing F-actin on the cytoplasmic face are localized in the growing rhizoid apex. The adhesions may be structurally related to focal adhesions in animal cells.

Establishing a Growth Axis in Fucoid Algae

Recent studies indicate that fucoid zygotes establish developmental polarity much earlier than previously thought. A growth axis is first set in place at fertilization, with the site of sperm entry defining the rhizoid pole of the axis. This initial axis is a default axis, which is only used as the final growth axis if the zygote fails to detect spatial cues (such as sunlight) in its intertidal environment. However, the zygote usually senses vectorial information; it then abandons the sperm-induced axis and assembles a new axis de novo in accordance with the perceived vector(s).

TIPs and Microtubule Regulation. The Beginning of the Plus End in Plants

Plants have evolved novel microtubule (MT) arrays to regulate cell division and cell expansion. How these MT arrangements are managed has been a question of long-standing interest to plant cell biologists. Do plants have unique ways of regulating MTs or have they co-opted mechanisms that are

Polarity establishment requires dynamic actin in fucoid zygotes.

Summary. Previous work has demonstrated that actin plays important roles in axis establishment and polar growth in fucoid zygotes. Distinct actin arrays are associated with fertilization, polarization, growth, and division, and agents that depolymerize actin filaments (cytochalasins, latrunculin B) perturb these stages of the first cell cycle. Rearrangements of actin arrays could be accomplished by transport of intact filaments and/or by actin dynamics involving depolymerization of the old array and polymerization of a new array. To investigate the requirement for dynamic actin during early development, we utilized the actin-stabilizing agent jasplakinolide. Immunofluorescence of actin arrays showed that treatment with 1–10 μM jasplakinolide stabilized existing arrays and induced polymerization of new filaments. In young zygotes, a cortical actin patch at the rhizoid pole was stabilized, and in some cells supernumerary patches were formed. In older zygotes that had initiated tip growth, massive filament assembly occurred in the rhizoid apex, and to a lesser degree in the perinuclear region. Treatment disrupted polarity establishment, polar secretion, tip growth, spindle alignment, and cytokinesis but did not affect the maintenance of an established axis, mitosis, or cell cycle progression. This study suggests that dynamic actin is required for polarization, growth, and division. Rearrangements in actin structures during the first cell cycle are likely mediated by actin depolymerization within old arrays and polymerization of new arrays.

Cell wall deposition during morphogenesis in fucoid algae

Abstract. Cell wall deposition was investigated during morphogenesis in zygotes of Pelvetia compressa (J. Agardh) De Toni. Young zygotes are spherical and wall is deposited uniformly, but at germination (about 10 h after fertilization) wall deposition becomes localized to the apex of the tip-growing rhizoid. Wall deposition was investigated before and after the initiation of tip growth by disrupting cytoskeleton, secretion or cellulose deposition; effects on wall strength and structure were examined. All three were involved in generating wall strength in both spherical and tip-growing zygotes, but their relative importance were different at the two developmental stages. Much of the wall strength in young zygotes was dependent on F-actin, whereas cellulose and a sulfated component, probably a fucan (F2), were most important in tip growing zygotes. Some treatments had contrasting effects at the two developmental stages; for example, disruption of F-actin or inhibition of secretion weakened walls in spherical zygotes but strengthened those in tip-growing zygotes. Transmission electron microscopic analysis showed that most treatments that altered wall strength induced modifications of internal wall structure.

Asymmetric Division in Fucoid Zygotes Is Positioned by Telophase Nuclei

The relative contributions of cell polarity and nuclear position in specifying the plane of asymmetric division in fucoid zygotes were investigated. In zygotes developing normally, telophase nuclei were positioned parallel to the polar growth axis, and the division plane bisected both axes. To assess division plane specification, the colinearity of the nuclear and growth axes was uncoupled by treatment with pharmacological agents. Spatial correlations between the growth axis, telophase nuclei, and the division plane were analyzed in the treated zygotes. In all cases, cytokinesis was oriented transverse to the telophase mitotic array and was less well aligned with the growth axis. Telophase nuclei also played a predominant role in positioning the division plane in polyspermic zygotes. Microtubules from the telophase nuclei interdigitated throughout the plane of subsequent cytokinesis, and we speculate that they specify the division plane. Morphological markers of the division plane were not observed before telophase; the earliest division marker detected was a plate of actin that assembled in the zone of microtubule overlap late in telophase. These findings are consistent with division plane specification at cytoplast boundaries.

Polarization of the endomembrane system is an early event in fucoid zygote development

BackgroundFucoid zygotes are excellent experimental organisms for investigating mechanisms that establish cell polarity and determine the site of tip growth. A common feature of polarity establishment is targeting endocytosis and exocytosis (secretion) to localized cortical domains. We have investigated the spatiotemporal development of endomembrane asymmetry in photopolarizing zygotes, and examined the underlying cellular physiology.ResultsThe vital dye FM4-64 was used to visualize endomembranes. The endomembrane system preferentially accumulated at the rhizoid (growth) pole within 4 h of fertilization. The polarized endomembrane array was initially labile and reoriented when the developmental axis changed direction in response to changing light cues. Pharmacological studies indicated that vesicle trafficking, actin and microtubules were needed to maintain endomembrane polarity. In addition, endocytosis required a functional cortical actin cytoskeleton.ConclusionEndomembrane polarization is an early event in polarity establishment, beginning very soon after photolocalization of cortical actin to the presumptive rhizoid site. Targeting of endocytosis and secretion to the rhizoid cortex contributes to membrane asymmetry. We suggest that microtubule-actin interactions, possibly involving microtubule capture and stabilization at actin-rich sites in the rhizoid, may organize the endomembrane array.