The putative Arabidopsis arp2/3 complex controls leaf cell morphogenesis

The evolutionarily conserved Arp2/3 complex has been shown to activate actin nucleation and branching in several eukaryotes, but its biological functions are not well understood in multicellular organisms. The model plant Arabidopsis provides many advantages for genetic dissection of the function of this conserved actin-nucleating machinery, yet the existence of this complex in plants has not been determined. We have identified Arabidopsis genes encoding homologs of all of the seven Arp2/3 subunits. The function of the putative Arabidopsis Arp2/3 complex has been studied using four homozygous T-DNA insertion mutants for ARP2, ARP3, and ARPC5/p16. All four mutants display identical defects in the development of jigsaw-shaped epidermal pavement cells and branched trichomes in the leaf. These loss-of-function mutations cause mislocalization of diffuse cortical F-actin to the neck region and inhibit lobe extension in pavement cells. The mutant trichomes resemble those treated with the actin-depolymerizing drug cytochalasin D, exhibiting stunted branches but dramatically enlarged stalks due to depolarized growth suggesting defects in the formation of a fine actin network. Our data demonstrate that the putative Arabidopsis Arp2/3 complex controls cell morphogenesis through its roles in cell polarity establishment and polar cell expansion. Furthermore, our data suggest a novel function for the putative Arp2/3 complex in the modulation of the spatial distribution of cortical F-actin and provide evidence that the putative Arp2/3 complex may activate the polymerization of some types of actin filaments in specific cell types.     

Microtubules and microfilaments in cell morphogenesis in higher plants

Microtubules and microfilaments play important roles in cell morphogenesis. The picture emerging from drug studies and molecular-genetic analyses of mutant higher plants defective in cell morphogenesis shows that the roles played by them remain the same in both tip-growing and diffuse-growing cells. Microtubules are important for establishing and maintaining growth polarity whereas actin microfilaments deliver the materials required for growth to specified sites. The recent cloning of several cell morphogenesis genes has revealed that conserved mechanisms as well as novel signal transduction pathways spatially organize the plant cytoskeleton.     

Three Brick genes have distinct functions in a common pathway promoting polarized cell division and cell morphogenesis in the maize leaf epidermis

We have taken a genetic approach to investigating cytoskeleton-dependent mechanisms governing cell morphogenesis in the maize leaf epidermis. Previously, we showed that the Brick1 (Brk1) gene is required for the formation of epidermal cell lobes as well as for properly polarized divisions of stomatal subsidiary mother cells, and encodes an 8 kDa protein highly conserved in plants and animals. Here, we show that two additional Brick genes, Brk2 and Brk3, are involved in the same aspects of epidermal cell morphogenesis and division. As shown previously for Brk1, analysis of the cytoskeleton shows that Brk2 and Brk3 are required for the formation of local F-actin enrichments associated with lobe outgrowth in wild-type cells. Analysis of brk1;brk2, brk1;brk3 and brk2;brk3 double mutants shows that their phenotypes are the same as those of brk single mutants. Mosaic analysis shows that Brk1 acts non cell-autonomously over a short distance. By contrast, Brk2 and Brk3 act cell-autonomously to promote pavement cell lobe formation, but Brk3 acts non cell-autonomously, and Brk2 partially non cell-autonomously, to promote polarized subsidiary mother cell divisions. Together, these observations indicate that all three Brk genes act in a common pathway in which each Brk gene has a distinct function. Recent work demonstrating a function for the mammalian homolog of BRK1 (HSPC300) in activation of Arp2/3-dependent actin polymerization implicates the Brk pathway in local regulation of actin polymerization in plant cells.     

Quantitative analysis of microtubule orientation in interdigitated leaf pavement cells

Leaf pavement cells are shaped like a jigsaw puzzle in most dicotyledon species. Molecular genetic studies have identified several genes required for pavement cells morphogenesis and proposed that microtubules play crucial roles in the interdigitation of pavement cells. In this study, we performed quantitative analysis of cortical microtubule orientation in leaf pavement cells in Arabidopsis thaliana. We captured confocal images of cortical microtubules in cotyledon leaf epidermis expressing GFP-tubulinβ and quantitatively evaluated the microtubule orientations relative to the pavement cell growth axis using original image processing techniques. Our results showed that microtubules kept parallel orientations to the growth axis during pavement cell growth. In addition, we showed that immersion treatment of seed cotyledons in solutions containing tubulin polymerization and depolymerization inhibitors decreased pavement cell complexity. Treatment with oryzalin and colchicine inhibited the symmetric division of guard mother cells.     

The morphogenesis of lobed plant cells in the mesophyll and epidermis: organization and distinct roles of cortical microtubules and actin filaments

The morphogenesis of lobed plant cells has been considered to be controlled by microtubule (MT) and/or actin filament (AF) organization. In this article, a comprehensive mechanism is proposed, in which distinct roles are played by these cytoskeletal components. First, cortical MT bundles and, in the case of pavement cells, radial MT arrays combined with MT bundles determine the deposition of local cell wall thickenings, the cellulose microfibrils of which copy the orientation of underlying MTs. Cell growth is thus locally prevented and, consequently, lobes and constrictions are formed. Arch-like tangential expansion is locally imposed at the external periclinal wall of pavement cells by the radial arrangement of cellulose microfibrils at every wall thickening. Whenever further elongation of the original cell lobes occurs, AF patches assemble at the tips of growing lobes. Intercellular space formation is promoted or prevented by the opposite or alternate, respectively, arrangement of cortical MT arrays between neighboring cells. The genes that are possibly involved in the molecular regulation of the above morphogenetic procedure by MT and AF array organization are reviewed.     

Effects of mechanical tension on protrusive activity and microfilament and intermediate filament organization in an epidermal epithelium moving in culture

Mechanical tension influences tissue morphogenesis and the synthetic, mitotic, and motile behavior of cells. To determine the effects of tension on epithelial motility and cytoskeletal organization, small, motile clusters of epidermal cells were artificially extended with a micromanipulated needle. Protrusive activity perpendicular to the axis of tension was dramatically suppressed. To determine the ultrastructural basis for this phenomenon, cells whose exact locomotive behavior was recorded cinemicrographically were examined by transmission electron microscopy. In untensed, forward-moving lamellar protrusions, microfilaments appear disorganized and anisotropically oriented. But in cytoplasm held under tension by micromanipulation or by the locomotive activity of other cells within the epithelium, microfilaments are aligned parallel to the tension. In non-spreading regions of the epithelial margin, microfilaments lie in tight bundles parallel to apparent lines of tension. Thus, it appears that tension causes alignment of microfilaments. In contrast, intermediate filaments are excluded from motile protrusions, being confined to the thicker, more central part of the cell. They roughly follow the contours of the cell, but are not aligned relative to tension even when microfilaments in the same cell are. This suggests that the organization of intermediate filaments is relatively resistant to physical distortion and the intermediate filaments may act as passive structural support within the cell. The alignment of microfilaments under tension suggests a mechanism by which tension suppresses protrusive activity: microfilaments aligned by forces exerted through filament-surface or filament-filament interconnections cannot reorient against such force and so cannot easily extend protrusions in directions not parallel to tension.     

Arabidopsis interdigitating cell growth requires two antagonistic pathways with opposing action on cell morphogenesis

Coordinating growth and communication between adjacent cells is a critical yet poorly understood aspect of tissue development and organ morphogenesis. We report a Rho GTPase signaling network underlying the jigsaw puzzle appearance of Arabidopsis leaf pavement cells, in which localized outgrowth in one cell is coordinated with localized inhibition of outgrowth of the adjacent cell to form interdigitating lobes and indentations. Locally activated ROP2, a Rho-related GTPase from plants, activates RIC4 to promote the assembly of cortical actin microfilaments required for localized outgrowth. Meanwhile, ROP2 inactivates another target RIC1, whose activity promotes well-ordered cortical microtubules. RIC1-dependent microtubule organization not only locally inhibits outgrowth but in turn suppresses ROP2 activation in the indentation zones. Thus, outgrowth-promoting ROP2 and outgrowth-inhibiting RIC1 pathways antagonize each other. We propose that the counteractivity of these two pathways demarcates outgrowing and indenting cortical domains, coordinating a process that gives rise to interdigitations between adjacent pavement cells.     

Calcium, microfilaments and morphogenesis

Morphogenesis, the generation of tissue form, is important not only in the embryogenesis of a new individual, but also because a change in morphogenesis may be involved in the establishment of differences between individuals during evolution. Morphogenetic movements are effected in part by coordinated changes in the shapes of individual cells and over the past decade the cellular organelles responsible for cell shape have been identified as microfilaments and microtubules. In non-embryonic systems the contraction of microfilaments is controlled by the level of intracellular free calcium, and so calcium is implicated as an intermediate control mechanism in morphogenisis. Through techniques which perturb the calcium balance of cells, or which measure calcium ion concentration directly, evidence is accumulating that calcium is involved in morphogenetic movements such as gastrulation and neurulation, and related phenomena such as wound healing. Thus fundamental questions about the control of morphogenesis in embryogenesis and evolution may now be couched in more precise terms of the control of intracellular calcium ion balance.     

ROP GTPase-dependent actin microfilaments promote PIN1 polarization by localized inhibition of clathrin-dependent endocytosis

Cell polarization via asymmetrical distribution of structures or molecules is essential for diverse cellular functions and development of organisms, but how polarity is developmentally controlled has been poorly understood. In plants, the asymmetrical distribution of the PIN-FORMED (PIN) proteins involved in the cellular efflux of the quintessential phytohormone auxin plays a central role in developmental patterning, morphogenesis, and differential growth. Recently we showed that auxin promotes cell interdigitation by activating the Rho family ROP GTPases in leaf epidermal pavement cells. Here we found that auxin activation of the ROP2 signaling pathway regulates the asymmetric distribution of PIN1 by inhibiting its endocytosis. ROP2 inhibits PIN1 endocytosis via the accumulation of cortical actin microfilaments induced by the ROP2 effector protein RIC4. Our findings suggest a link between the developmental auxin signal and polar PIN1 distribution via Rho-dependent cytoskeletal reorganization and reveal the conservation of a design principle for cell polarization that is based on Rho GTPase-mediated inhibition of endocytosis.     

Spatial gene expression in the T-stage mouse metanephros

The E11.5 mouse metanephros is comprised of a T-stage ureteric epithelial tubule sub-divided into tip and trunk cells surrounded by metanephric mesenchyme (MM). Tip cells are induced to undergo branching morphogenesis by the MM. In contrast, signals within the mesenchyme surrounding the trunk prevent ectopic branching of this region. In order to identify novel genes involved in the molecular regulation of branching morphogenesis we compared the gene expression profiles of isolated tip, trunk and MM cells using Compugen mouse long oligo microarrays. We identified genes enriched in the tip epithelium, sim-1, Arg2, Tacstd1, Crlf-1 and BMP7; genes enriched in the trunk epithelium, Innp1, Itm2b, Mkrn1, SPARC, Emu2 and Gsta3 and genes spatially restricted to the mesenchyme surrounding the trunk, CSPG2 and CV-2, with overlapping and complimentary expression to BMP4, respectively. This study has identified genes spatially expressed in regions of the developing kidney involved in branching morphogenesis, nephrogenesis and the development of the collecting duct system, calyces, renal pelvis and ureter.     

Polar lobe formation and cytokinesis in fertilized eggs of Ilyanassa obsoleta: I. Ultrastructure and effects of cytochalasin B and colchicine

Formation of polar lobe constrictions and cleavage furrows in fertilized eggs of the marine mudsnail, Ilyanassa obsoleta, is associated with localized concentrations of microfilaments in the cortical cytoplasm. These microfilaments disappear after treatment with cytochalasin B, with concomitant regression of polar lobe constrictions and cleavage furrows. Microtubules are present in the noncortical cytoplasm of the polar lobe and disappear after treatment with colchicine. Colchicine application early in lobe development inhibits both polar lobe formation and cytokinesis; however, drug treatment later in lobe development, although still inhibiting cytokinesis, fails to inhibit the continued constriction of the polar lobe neck and subsequent lobe resorption. The data therefore suggest that separate colchicine-sensitive steps are required for the complete constriction of the polar lobe neck and for initiation of cytokinesis. Polar lobe necks constrict at two markedly different rates in contrast to the smooth, single-phase progression of cleavage furrows. As with cytokinesis, polar lobe formation may occur by the contraction of a microfilament ring whose polymerization or activity is regulated by microtubules.     

Mutations in actin-related proteins 2 and 3 affect cell shape development in Arabidopsis

ACTIN-RELATED PROTEINS 2 and 3 form the major subunits of the ARP2/3 complex, which is known as an important regulator of actin organization in diverse organisms. Here, we report that two genes, WURM and DISTORTED1, which are important for cell shape control in Arabidopsis, encode the plant ARP2 and ARP3 orthologs, respectively. Mutations in these genes result in misdirected expansion of various cell types: trichome expansion is randomized, pavement cells fail to produce lobes, hypocotyl cells curl out of the normal epidermal plane, and root hairs are sinuous. At the subcellular level, cell shape changes are linked to severe filamentous actin aggregation and compromised vacuole fusion. Because all seven subunits of the ARP2/3 complex are present in plants, our data indicate that this complex may play a pivotal role during plant cell morphogenesis.     

Las1 Is an Essential Nuclear Protein Involved in Cell Morphogenesis and Cell Surface Growth

Saccharomyces cerevisiae mutations that cause a requirement for SSD1-v for viability were isolated, yielding one new gene, LAS1, and three previously identified genes, SIT4, BCK1/SLK1, and SMP3. Three of these genes, LAS1, SIT4, and BCK1/SLK1, encode proteins that have roles in bud formation or morphogenesis. LAS1 is essential and loss of LAS1 function causes the cells to arrest as 80% unbudded cells and 20% large budded cells that accumulate many vesicles at the mother-daughter neck. Overexpression of LAS1 results in extra cell surface projections in the mother cell, alterations in actin and SPA2 localization, and the accumulation of electron-dense structures along the periphery of both the mother cell and the bud. The nuclear localization of LAS1 suggests a role of LAS1 for regulating bud formation and morphogenesis via the expression of components that function directly in these processes.     

IQ67 DOMAIN protein 21 is critical for indentation formation in pavement cell morphogenesis

In plants, cortical microtubules anchor to the plasma membrane in arrays and play important roles in cell shape. However, the molecular mechanism of microtubule binding proteins, which connect the plasma membrane and cortical microtubules in cell morphology remains largely unknown. Here, we report that a plasma membrane and microtubule duallocalized IQ67 domain protein, IQD21, is critical for cotyledon pavement cell(PC) morphogenesis in Arabidopsis. iqd21 mutation caused increased indentation wi...     

The Yck2 yeast casein kinase 1 isoform shows cell cycle-specific localization to sites of polarized growth and is required for proper septin organization

Casein kinase 1 protein kinases are ubiquitous and abundant Ser/Thr-specific protein kinases with activity on acidic substrates. In yeast, the products of the redundant YCK1 and YCK2 genes are together essential for cell viability. Mutants deficient for these proteins display defects in cellular morphogenesis, cytokinesis, and endocytosis. Yck1p and Yck2p are peripheral plasma membrane proteins, and we report here that the localization of Yck2p within the membrane is dynamic through the cell cycle. Using a functional green fluorescent protein (GFP) fusion, we have observed that Yck2p is concentrated at sites of polarized growth during bud morphogenesis. At cytokinesis, GFP-Yck2p becomes associated with a ring at the bud neck and then appears as a patch of fluorescence, apparently coincident with the dividing membranes. The bud neck association of Yck2p at cytokinesis does not require an intact septin ring, and septin assembly is altered in a Yck-deficient mutant. The sites of GFP-Yck2p concentration and the defects observed for Yck-deficient cells together suggest that Yck plays distinct roles in morphogenesis and cytokinesis that are effected by differential localization.     

Four-dimensional imaging with virtual reality to quantitatively explore jigsaw puzzle-like morphogenesis of Arabidopsis cotyledon pavement cells

In most dicotyledonous plants, leaf pavement cells exhibit complex jigsaw puzzle-like cell morphogenesis during leaf expansion. Although detailed molecular biological information and mathematical modeling of this jigsaw puzzle-like cell morphogenesis are now available, a full understanding of this process remains elusive. Recent reports have highlighted the importance of three-dimensional (3D) structures (i.e., anticlinal and periclinal cell wall) in understanding the mechanical models that describe this morphogenetic process. We believe that it is important to acquire 3D shapes of pavement cells over time, i.e., acquire and analyze four-dimensional (4D) information when studying the relationship between mechanical modeling and simulations and the actual cell shape. In this report, we have developed a framework to capture and analyze 4D morphological information of Arabidopsis thaliana cotyledon pavement cells by using both direct water immersion observations and computational image analyses, including segmentation, surface modeling, virtual reality and morphometry. The 4D cell models allowed us to perform time-lapse 3D morphometrical analysis, providing detailed quantitative information about changes in cell growth rate and shape, with cellular complexity observed to increase during cell growth. The framework should enable analysis of various phenotypes (e.g., mutants) in greater detail, especially in the 3D deformation of the cotyledon surface, and evaluation of theoretical models that describe pavement cell morphogenesis using computational simulations. Additionally, our accurate and high-throughput acquisition of growing cell structures should be suitable for use in generating in silico model cell structures.