Organisation and regulation of the cytoskeleton in plant programmed cell death

Programmed cell death (PCD) involves precise integration of cellular responses to extracellular and intracellular signals during both stress and development. In recent years much progress in our understanding of the components involved in PCD in plants has been made. Signalling to PCD results in major reorganisation of cellular components. The plant cytoskeleton is known to play a major role in cellular organisation, and reorganization and alterations in its dynamics is a well known consequence of signalling. There are considerable data that the plant cytoskeleton is reorganised in response to PCD, with remodelling of both microtubules and microfilaments taking place. In the majority of cases, the microtubule network depolymerises, whereas remodelling of microfilaments can follow two scenarios, either being depolymerised and then forming stable foci, or forming distinct bundles and then depolymerising. Evidence is accumulating that demonstrate that these cytoskeletal alterations are not just a consequence of signals mediating PCD, but that they also may have an active role in the initiation and regulation of PCD. Here we review key data from higher plant model systems on the roles of the actin filaments and microtubules during PCD and discuss proteins potentially implicated in regulating these alterations.     

Actin depolymerization is sufficient to induce programmed cell death in self-incompatible pollen

Self-incompatibility (SI) prevents inbreeding through specific recognition and rejection of incompatible pollen. In incompatible Papaver rhoeas pollen, SI triggers a Ca2+ signaling cascade, resulting in the inhibition of tip growth, actin depolymerization, and programmed cell death (PCD). We investigated whether actin dynamics were implicated in regulating PCD. Using the actin-stabilizing and depolymerizing drugs jasplakinolide (Jasp) and latrunculin B, we demonstrate that changes in actin filament levels or dynamics play a functional role in initiating PCD in P. rhoeas pollen, triggering a caspase-3-like activity. Significantly, SI-induced PCD in incompatible pollen was alleviated by pretreatment with Jasp. This represents the first account of a specific causal link between actin polymerization status and initiation of PCD in a plant cell and significantly advances our understanding of the mechanisms involved in SI.     

Signal-mediated depolymerization of actin in pollen during the self-incompatibility response

Signal perception and the integration of signals into networks that effect cellular changes is essential for all cells. The self-incompatibility (SI) response in field poppy pollen triggers a Ca(2+)-dependent signaling cascade that results in the inhibition of incompatible pollen. SI also stimulates dramatic alterations in the actin cytoskeleton. By measuring the amount of filamentous (F-) actin in pollen before and during the SI response, we demonstrate that SI stimulates a rapid and large reduction in F-actin level that is sustained for at least 1 h. This represents quantitative evidence for stimulus-mediated depolymerization of F-actin in plant cells by a defined biological stimulus. Surprisingly, there are remarkably few examples of sustained reductions in F-actin levels stimulated by a biologically relevant ligand. Actin depolymerization also was achieved in pollen by treatments that increase cytosolic free Ca(2+) artificially, providing evidence that actin is a target for the Ca(2+) signals triggered by the SI response. By determining the cellular concentrations and binding constants for native profilin from poppy pollen, we show that profilin has Ca(2+)-dependent monomeric actin-sequestering activity. Although profilin is likely to contribute to stimulus-mediated actin depolymerization, our data suggest a role for additional actin binding proteins. We propose that Ca(2+)-mediated depolymerization of F-actin may be a mechanism whereby SI-induced tip growth inhibition is achieved.     

The actin cytoskeleton is a target of the self-incompatibility response in Papaver rhoeas

The integration of signals received by a cell, and their transduction to targets, is essential for all cellular responses. The cytoskeleton has been identified as a major target of signalling cascades in both animal and plant cells. Self-incompatibility (SI) in Papaver rhoeas involves an allele-specific recognition between stigmatic S-proteins and pollen, resulting in the inhibition of incompatible pollen. This highly specific response triggers a Ca(2+)-dependent signalling cascade in incompatible pollen when a stigmatic S-protein interacts with it. It has been demonstrated recently that SI induces dramatic alterations in the organization of the pollen actin cytoskeleton. This implicates the actin cytoskeleton as a key target for the SI-stimulated signals. The cytological alterations to the actin cytoskeleton that are triggered in response to SI are described here and there seem to be several stages that are distinguishable temporally. Evidence was obtained that F-actin depolymerization is also stimulated. The current understanding that the actin cytoskeleton is a target for the signals triggered by the SI response is discussed. It is suggested that these F-actin alterations may be Ca(2+)-mediated and that this could be a mechanism whereby SI-induced tip growth inhibition is achieved. The potential for actin-binding proteins to act as key mediators of this response is discussed and the mechanisms that may be responsible for effecting these changes are described. In particular, the parallels between sustained actin rearrangements during SI and in apoptosis of animal cells are considered.     

Self-incompatibility in Papaver: signalling to trigger PCD in incompatible pollen

Sexual reproduction in higher plants uses pollination, involving interactions between pollen and pistil. Self-incompatibility (SI) prevents self-fertilization, providing an important mechanism to promote outbreeding. SI is controlled by the S-locus; discrimination occurs between incompatible pollen, which is rejected, while compatible pollen can achieve fertilization. In Papaver rhoeas, S proteins encoded by the pistil part of the S-locus interact with incompatible pollen to effect rapid inhibition of tip growth. This self-incompatible interaction triggers a Ca(2+)-dependent signalling cascade. SI-specific events triggered in incompatible pollen include rapid depolymerization of the actin cytoskeleton; phosphorylation of soluble inorganic pyrophosphatases, and activation of a MAPK. It has recently been shown that programmed cell death (PCD) is triggered by SI. This provides a precise mechanism for the specific destruction of 'self' pollen. Recent data providing evidence for SI-induced caspase-3-like protease activity, and the involvement of actin depolymerization and MAPK activation in SI-mediated PCD will be discussed. These studies not only significantly advance our understanding of the mechanisms involved in SI, but also contribute to our understanding of functional links between signalling components and initiation of PCD in a plant cell. Recent data demonstrating SI-mediated modification of soluble inorganic pyrophosphatases are also described.     

Hydrogen peroxide modulates the dynamic microtubule cytoskeleton during the defence responses to Verticillium dahliae toxins in Arabidopsis

The molecular mechanisms of signal transduction of plants in response to infection by Verticillium dahliae (VD) are not well understood. We previously showed that NO may act as an upstream signalling molecule to trigger the depolymerization of cortical microtubules in Arabidopsis. In the present study, we used the wild-type, and atrbohD and atrbohF mutants of Arabidopsis to explore the mechanisms of action of H(2)O(2) signals and the dynamic microtubule cytoskeleton in defence responses. We demonstrated that H(2)O(2) may also act as an upstream signalling molecule to regulate cortical microtubule depolymerization. The depolymerization of the cortical microtubules played a functional role in the signalling pathway to mediate the expression of defence genes. The results indicate that H(2)O(2) modulates the dynamic microtubule cytoskeleton to trigger the expression of defence genes against V. dahliae toxins (VD-toxins) in Arabidopsis.     

A cascade signal pathway occurs in self-incompatibility of Pyrus pyrifolia

Pear (Pyrus pyrifolia L.) possesses an S-RNase-based gametophytic self-incompatibility (GSI) system and S-RNase, the self-incompatibility (SI) determinant in the pistil, has also been implicated in the rejection of self-pollen and genetically identical pollen. We have demonstrated that S-RNase depolymerises actin cytoskeleton, triggers mitochondrial alteration and DNA degradation in the incompatible pollen tube, which indicates programmed cell death (PCD) may occur in SI response of Pyrus pyrifolia. Recently, we have identified that S-RNase specifically disrupted tip-localized reactive oxygen species (ROS) of incompatible pollen tube via arrest of ROS formation in mitochondria and cell walls in Pyrus pyrifolia. Furthermore, tip-localized ROS disruption not only decreased the Ca²⁺ current and depolymerised the actin cytoskeleton, but it also induced nuclear DNA degradation in the pollen tube. The results mentioned above indicate that a cascade signal pathway may occur in SI of Pyrus pyrifolia and PCD is used to terminate the incompatible pollen tubes growth. In this addendum, we review the cascade signal pathway of Pyrus pyrifolia SI.Key words: S-RNase, programmed cell death, reactive oxygen species, actin cytoskeleton, Ca²⁺ current, nuclear DNA     

Actin-microtubule interactions in the algaNitella: analysis of the mechanism by which microtubule depolymerization potentiates cytochalasin's effects on streaming

Summary In the characean algaNitella, depolymerization of microtubules potentiates the inhibitory effects of cytochalasins on cytoplasmic streaming. Microtubule depolymerization lowers the cytochalasin B and D concentrations required to inhibit streaming, accelerates inhibition and delays streaming recovery. Because microtubule depolymerization does not significantly alter³H-cytochalasin B uptake and release, elevated intracellular cytochalasin concentrations are not the basis for potentiation. Instead, microtubule depolymerization causes actin to become more sensitive to cytochalasin. This increased sensitivity of actin is unlikely to be due to direct stabilization of actin by microtubules, however, because very few microtubules colocalize with the subcortical actin bundles that generate streaming. Furthermore, microtubule reassembly, but not recovery of former transverse alignment, is sufficient for restoring the normal cellular responses to cytochalasin D. We hypothesize that either tubulin or microtubule-associated proteins, released when microtubules depolymerize, interact with the actin cytoskeleton and sensitize it to cytochalasin.Abbreviations APW artificial pond water - Ca_c cytoplasraic free calcium concentration - DMSO dimethyl sulfoxide - MT microtubule-minus - MT+ microtubule-plus.     

Arabidopsis AIP1-1 regulates the organization of apical actin filaments by promoting their turnover in pollen tubes

Apical actin filaments are highly dynamic structures that are crucial for rapid pollen tube growth, but the mechanisms regulating their dynamics and spatial organization remain incompletely understood. We here identify that AtAIP1-1 is important for regulating the turnover and organization of apical actin filaments in pollen tubes. AtAIP1-1 is distributed uniformly in the pollen tube and loss of function of AtAIP1-1 affects the organization of the actin cytoskeleton in the pollen tube. Specifically, actin filaments became disorganized within the apical region of aip1-1 pollen tubes. Consistent with the role of apical actin filaments in spatially restricting vesicles in pollen tubes, the apical region occupied by vesicles becomes enlarged in aip1-1 pollen tubes compared to WT. Using ADF1 as a representative actin-depolymerizing factor, we demonstrate that AtAIP1-1 enhances ADF1-mediated actin depolymerization and filament severing in vitro, although AtAIP1-1 alone does not have an obvious effect on actin assembly and disassembly. The dynamics of apical actin filaments are reduced in aip1-1 pollen tubes compared to WT. Our study suggests that AtAIP1-1 works together with ADF to act as a module in regulating the dynamics of apical actin filaments to facilitate the construction of the unique "apical actin structure" in the pollen tube.     

Alterations in the actin cytoskeleton of pollen tubes are induced by the self-incompatibility reaction in Papaver rhoeas

Self-incompatibility (SI) is a genetically controlled process used to prevent self-pollination. In Papaver rhoeas, the induction of SI is triggered by a Ca(2)+-dependent signaling pathway that results in the rapid and S allele-specific inhibition of pollen tube tip growth. Tip growth of cells is dependent on a functioning actin cytoskeleton. We have investigated the effect of self-incompatibility (S) proteins on the actin cytoskeleton in poppy pollen tubes. Here, we report that the actin cytoskeleton of incompatible pollen tubes is rapidly and dramatically rearranged during the SI response, not only in our in vitro SI system but also in vivo. We demonstrate that nonspecific inhibition of growth does not result in similar actin rearrangements. Because the SI-induced alterations are not observed if growth stops, this clearly demonstrates that these alterations are triggered by the SI signaling cascade rather than merely resulting from the consequent inhibition of growth. We establish a detailed time course of events and discuss the mechanisms that might be involved. Our data strongly implicate a role for the actin cytoskeleton as a target for signaling pathways involved in the SI response of P. rhoeas.     

nhibiting Self-Pollen： Self-Incompatibility in Papaver Involves Integration of Several Signaling Events

Cellular responses rely on signal perception and integration. A nice example of this is self incompatibility （SI）, which is an important mechanism to prevent inbreeding. It prevents self-fertilization by using a highly discriminatory cellular recognition and rejection mechanism. Most Sl systems are genetically specified by the S-locus, which has a pollen and a pistil S-component. A receptor-ligand interaction is used by Papaver rhoeas to control SI. S proteins encoded by the pistil part of the S-locus interact with incompatible pollen to achieve rapid inhibition of tip growth. The incompatible Sl interaction triggers a Ca^2＋-dependent signaling cascade. A number of Sl-specific events are triggered in incompatible pollen, including rapid depolymerization of the actin cytoskeleton; phosphorylation of soluble inorganic pyrophosphatases （SPPases）, Prp26.1; activation of a mitogen activated protein kinase, p56; programmed cell death （PCD） involving a caspase-3-1ike activity. These events contribute to prevent self-fertilizaUon. We are attempting to establish the functional significance of these events, and their possible involvement in integrating a coordinated signaling response. Here we describe the identification of these components shown to be involved in Sl, together with recent progress in identifying links between some of them. These data constitute the first steps in elucidating how SI signaling is integrated.     

Nucleotide exchange factor GEF-H1 mediates cross-talk between microtubules and the actin cytoskeleton

Regulation of the actin cytoskeleton by microtubules is mediated by the Rho family GTPases. However, the molecular mechanisms that link microtubule dynamics to Rho GTPases have not, as yet, been identified. Here we show that the Rho guanine nucleotide exchange factor (GEF)-H1 is regulated by an interaction with microtubules. GEF-H1 mutants that are deficient in microtubule binding have higher activity levels than microtubule-bound forms. These mutants also induce Rho-dependent changes in cell morphology and actin organization. Furthermore, drug-induced microtubule depolymerization induces changes in cell morphology and gene expression that are similar to the changes induced by the expression of active forms of GEF-H1. Furthermore, these effects are inhibited by dominant-negative versions of GEF-H1. Thus, GEF-H1 links changes in microtubule integrity to Rho-dependent regulation of the actin cytoskeleton.     

Latrunculin B has different effects on pollen germination and tube growth

The actin cytoskeleton is absolutely required for pollen germination and tube growth, but little is known about the regulation of actin polymer concentrations or dynamics in pollen. Here, we report that latrunculin B (LATB), a potent inhibitor of actin polymerization, had effects on pollen that were distinct from those of cytochalasin D. The equilibrium dissociation constant measured for LATB binding to maize pollen actin was determined to be 74 nM. This high affinity for pollen actin suggested that treatment of pollen with LATB would have marked effects on actin function. Indeed, LATB inhibited maize pollen germination half-maximally at 50 nM, yet it blocked pollen tube growth at one-tenth of that concentration. Low concentrations of LATB also caused partial disruption of the actin cytoskeleton in germinated maize pollen, as visualized by light microscopy and fluorescent-phalloidin staining. The amounts of filamentous actin (F-actin) in pollen were quantified by measuring phalloidin binding sites, a sensitive assay that had not been used previously for plant cells. The amount of F-actin in maize pollen increased slightly upon germination, whereas the total actin protein level did not change. LATB treatment caused a dose-dependent depolymerization of F-actin in populations of maize pollen grains and tubes. Moreover, the same concentrations of LATB caused similar depolymerization in pollen grains before germination and in pollen tubes. These data indicate that the increased sensitivity of pollen tube growth to LATB was not due to general destabilization of the actin cytoskeleton or to decreases in F-actin amounts after germination. We postulate that germination is less sensitive to LATB than tube extension because the presence of a small population of LATB-sensitive actin filaments is critical for maintenance of tip growth but not for germination of pollen, or because germination is less sensitive to partial depolymerization of the actin cytoskeleton.     

Actin-Binding Proteins Implicated in the Formation of the Punctate Actin Foci Stimulated by the Self-Incompatibility Response in Papaver

The actin cytoskeleton is a key target for signaling networks and plays a central role in translating signals into cellular responses in eukaryotic cells. Self-incompatibility (SI) is an important mechanism responsible for preventing self-fertilization. The SI system of Papaver rhoeas pollen involves a Ca²⁺-dependent signaling network, including massive actin depolymerization as one of the earliest cellular responses, followed by the formation of large actin foci. However, no analysis of these structures, which appear to be aggregates of filamentous (F-)actin based on phalloidin staining, has been carried out to date. Here, we characterize and quantify the formation of F-actin foci in incompatible Papaver pollen tubes over time. The F-actin foci increase in size over time, and we provide evidence that their formation requires actin polymerization. Once formed, these SI-induced structures are unusually stable, being resistant to treatments with latrunculin B. Furthermore, their formation is associated with changes in the intracellular localization of two actin-binding proteins, cyclase-associated protein and actin-depolymerizing factor. Two other regulators of actin dynamics, profilin and fimbrin, do not associate with the F-actin foci. This study provides, to our knowledge, the first insights into the actin-binding proteins and mechanisms involved in the formation of these intriguing structures, which appear to be actively formed during the SI response.The ability to perceive and integrate signals into networks is essential for all eukaryotic cells. The actin cytoskeleton is a major target and integrator of signaling networks in eukaryotic cells. In plants, many extracellular stimuli lead to rapid structural changes in the actin cytoskeleton (Staiger, 2000; Hussey et al., 2006). Although many of the signaling intermediates that regulate actin dynamics are well defined in animal cells and yeast (Iden and Collard, 2008; Thomas et al., 2009), considerably less is known for plants. However, it is generally accepted that actin-binding proteins (ABPs) function as transducers of cellular stimuli into changes in cellular architecture (Hussey et al., 2006; Staiger and Blanchoin, 2006; Thomas et al., 2009). This includes three abundant monomer-binding proteins, profilin, actin-depolymerizing factor (ADF), and cyclase-associated protein (CAP), which function synergistically to stimulate actin turnover in vitro (Chaudhry et al., 2007). Bundling and cross-linking proteins, such as fimbrin, function to stabilize actin filaments into higher order structures (Kovar et al., 2000b; Thomas et al., 2009). These and other regulators of actin turnover are likely targets for signal-mediated changes in actin architecture in response to biotic and abiotic stresses.Self-incompatibility (SI) is a genetically controlled system to prevent self-fertilization in flowering plants. SI is controlled by a multiallelic S-locus; S-specific pollen rejection results from the interaction of pollen S- and pistil S-determinants that have matching alleles (Franklin-Tong, 2008). In Papaver rhoeas, the pistil S-determinants (previously called S proteins, recently renamed PrsS; Foote et al., 1994; Wheeler et al., 2009) act as ligands, interacting with the pollen S-determinant PrpS (Wheeler et al., 2009), triggering increases in calcium influx and increases in cytosol-free calcium in incompatible pollen (Franklin-Tong et al., 1993, 1997, 2002). The Ca²⁺-mediated signaling network results in rapid inhibition of incompatible pollen tube growth and triggers programmed cell death (PCD) involving several caspase-like activities (Thomas and Franklin-Tong, 2004; Bosch and Franklin-Tong, 2007).The SI response and the Ca²⁺-signaling pathway in Papaver stimulate rapid reorganization and massive depolymerization of actin filaments in incompatible pollen tubes (Geitmann et al., 2000; Snowman et al., 2002). Moreover, it has been demonstrated that changes in actin dynamics are necessary and sufficient for PCD initiation (Thomas et al., 2006). Intriguingly, there seems to be cross talk between actin and microtubule cytoskeletons in mediating PCD in pollen (Poulter et al., 2008). Thus, there is compelling evidence for signaling to the actin cytoskeleton in mediating PCD during SI (for a recent review, see Bosch et al., 2008). SI also triggers further changes to the actin cytoskeleton. Small F-actin foci are formed, and these increase in size within the first hour after SI stimulus and remain observable for at least 3 h (Geitmann et al., 2000; Snowman et al., 2002). These aggregates contain F-actin, as they stain with rhodamine phalloidin. The formation of the small actin foci and the larger F-actin structures occurs after cessation of pollen tube growth, so they are unlikely to play a role in pollen inhibition.Punctate F-actin foci are unusual structures, and there appears to be a paucity of examples of their formation in any eukaryotic cell type. Actin patches are associated with endocytosis in normally growing yeast (Pelham and Chang, 2001; Kaksonen et al., 2003; Ayscough, 2004; Young et al., 2004), and “actin nodules” are formed during filipodia formation in platelets (Calaminus et al., 2008). Actin bodies are formed when yeast cells enter the quiescent cycle (Sagot et al., 2006), and Hirano bodies are observed in animal cells and Dictyostelium undergoing stress or in the disease state (Hirano, 1994; Maselli et al., 2002). Large star-shaped actin arrays have been observed in pollen tubes growing in vivo (Lord, 1992), but their nature and function are unknown. When we first described the SI-induced structures, we avoided the terminology of patches or bodies, as it was not known whether they were either structurally or functionally comparable to any of these previously characterized actin-based structures.Studies on the SI-mediated actin responses to date have focused on the initial phase of depolymerization, and no analysis of what is involved in the formation of the large punctate actin foci has been made. Here, we show that the formation of punctate actin foci requires actin polymerization, but once formed they are unusually stable. Moreover, we find that their formation correlates with changes in the intracellular localization of two ABPs, CAP and ADF, but not with two other key regulators of actin dynamics, profilin and fimbrin.     

SB401, a pollen-specific protein from Solanum berthaultii, binds to and bundles microtubules and F-actin

We characterize a novel, pollen-specific, microtubule-associated protein, SB401, found in Solanum berthaultii. This protein binds to and bundles taxol-stabilized microtubules and enhances tubulin polymerization in a concentration-dependent manner, particularly at lower temperatures. Electron microscopy revealed that the protein decorates the entire length of microtubules. Cross-linking and electrophoresis studies showed that SB401 protein forms dimers, and suggest that dimerization could account for bundling. Double immunofluorescent staining of pollen tubes of S. berthaultii showed that SB401 protein co-localized with cortical microtubule bundles. SB401 protein also binds to and bundles actin filaments, and could connect actin filaments to microtubules. SB401 protein had a much higher affinity for microtubules than for actin filaments. In the presence of both cytoskeletal elements, the protein preferentially bound microtubules to form bundles. These results demonstrate that SB401 protein may have important roles in organizing the cytoskeleton in pollen tubes.     

Regulation of the pollen-specific actin-depolymerizing factor LlADF1

Pollen tube growth is dependent on a dynamic actin cytoskeleton, suggesting that actin-regulating proteins are involved. We have examined the regulation of the lily pollen-specific actin-depolymerizing factor (ADF) LlADF1. Its actin binding and depolymerizing activity is pH sensitive, inhibited by certain phosphoinositides, but not controlled by phosphorylation. Compared with its F-actin binding properties, its low activity in depolymerization assays has been used to explain why pollen ADF decorates F-actin in pollen grains. This low activity is incompatible with a role in increasing actin dynamics necessary to promote pollen tube growth. We have identified a plant homolog of actin-interacting protein, AIP1, which enhances the depolymerization of F-actin in the presence of LlADF1 by approximately 60%. Both pollen ADF and pollen AIP1 bind F-actin in pollen grains but are mainly cytoplasmic in pollen tubes. Our results suggest that together these proteins remodel actin filaments as pollen grains enter and exit dormancy.     

Distribution of callose synthase, cellulose synthase, and sucrose synthase in tobacco pollen tube is controlled in dissimilar ways by actin filaments and microtubules

Callose and cellulose are fundamental components of the cell wall of pollen tubes and are probably synthesized by distinct enzymes, callose synthase and cellulose synthase, respectively. We examined the distribution of callose synthase and cellulose synthase in tobacco (Nicotiana tabacum) pollen tubes in relation to the dynamics of actin filaments, microtubules, and the endomembrane system using specific antibodies to highly conserved peptide sequences. The role of the cytoskeleton and membrane flow was investigated using specific inhibitors (latrunculin B, 2,3-butanedione monoxime, taxol, oryzalin, and brefeldin A). Both enzymes are associated with the plasma membrane, but cellulose synthase is present along the entire length of pollen tubes (with a higher concentration at the apex) while callose synthase is located in the apex and in distal regions. In longer pollen tubes, callose synthase accumulates consistently around callose plugs, indicating its involvement in plug synthesis. Actin filaments and endomembrane dynamics are critical for the distribution of callose synthase and cellulose synthase, showing that enzymes are transported through Golgi bodies and/or vesicles moving along actin filaments. Conversely, microtubules appear to be critical in the positioning of callose synthase in distal regions and around callose plugs. In contrast, cellulose synthases are only partially coaligned with cortical microtubules and unrelated to callose plugs. Callose synthase also comigrates with tubulin by Blue Native-polyacrylamide gel electrophoresis. Membrane sucrose synthase, which expectedly provides UDP-glucose to callose synthase and cellulose synthase, binds to actin filaments depending on sucrose concentration; its distribution is dependent on the actin cytoskeleton and the endomembrane system but not on microtubules.     

Pollen tube growth: coping with mechanical obstacles involves the cytoskeleton

Cellular growth and movement require both the control of direction and the physical capacity to generate forces. In animal cells directional control and growth forces are generated by the polymerization of and traction between the elements of the cytoskeleton. Whether actual forces generated by the cytoskeleton play a role in plant cell growth is largely unknown as the interplay between turgor and cell wall is considered to be the predominant structural feature in plant cell morphogenesis. We investigated the mechano-structural role of the cytoskeleton in the invasive growth of pollen tubes. These cells elongate rapidly by tip growth and have the ability to penetrate the stigmatic and stylar tissues in order to drill their way to the ovule. We used agents interfering with cytoskeletal functioning, latrunculin B and oryzalin, in combination with mechanical in vitro assays. While microtubule degradation had no significant effect on the pollen tubes’ capacity to invade a mechanical obstacle, latrunculin B decreased the pollen tubes’ ability to elongate in stiffened growth medium and to penetrate an obstacle. On the other hand, the ability to maintain a certain growth direction in vitro was affected by the degradation of microtubules but not actin filaments. To find out whether both cytoskeletal elements share functions or interact we used both drugs in combination resulting in a dramatic synergistic response. Fluorescent labeling revealed that the integrity of the microtubule cytoskeleton depends on the presence of actin filaments. In contrast, actin filaments seemed independent of the configuration of microtubules.     

A microtubule-binding Rho-GEF controls cell morphology during convergent extension of Xenopus laevis

During Xenopus development, convergent extension movements mediated by cell intercalation drive axial elongation. While many genes required for convergent extension have been identified, little is known of regulation of the cytoskeleton during these cell movements. Although microtubules are required for convergent extension, this applies only to initial stages of gastrulation, between stages 10 and 10.5. To examine the cytoskeleton more directly during convergent extension, we visualized actin and microtubules simultaneously in live explants using spinning disk confocal fluorescence microscopy. Microtubule depolymerization by nocodazole inhibits lamellipodial protrusions and cell-cell contact, thereby inhibiting convergent extension. However, neither taxol nor vinblastine, both of which block microtubule dynamics while stabilizing a polymer form of tubulin, inhibits lamellipodia or convergent extension. This suggests an unusual explanation: the mass of polymerized tubulin, not dynamics of the microtubule cytoskeleton, is crucial for convergent extension. Because microtubule depolymerization elicits striking effects on actin-based protrusions, the role of Rho-family GTPases was tested. The effects of nocodazole are partially rescued using dominant negative Rho, Rho-kinase inhibitor, or constitutively active Rac, suggesting that microtubules regulate small GTPases, possibly via a guanine-nucleotide exchange factor. We cloned full-length XLfc, a microtubule-binding Rho-GEF. Nucleotide exchange activity of XLfc is required for nocodazole-mediated inhibition of convergent extension; constitutively active XLfc recapitulates the effects of microtubule depolymerization. Morpholino knockdown of XLfc abrogates the ability of nocodazole to inhibit convergent extension. Therefore, we believe that XLfc is a crucial regulator of cell morphology during convergent extension, and microtubules limit its activity through binding to the lattice.     

Re-organisation of the cytoskeleton during developmental programmed cell death in Picea abies embryos

Cell and tissue patterning in plant embryo development is well documented. Moreover, it has recently been shown that successful embryogenesis is reliant on programmed cell death (PCD). The cytoskeleton governs cell morphogenesis. However, surprisingly little is known about the role of the cytoskeleton in plant embryogenesis and associated PCD. We have used the gymnosperm, Picea abies, somatic embryogenesis model system to address this question. Formation of the apical-basal embryonic pattern in P. abies proceeds through the establishment of three major cell types: the meristematic cells of the embryonal mass on one pole and the terminally differentiated suspensor cells on the other, separated by the embryonal tube cells. The organisation of microtubules and F-actin changes successively from the embryonal mass towards the distal end of the embryo suspensor. The microtubule arrays appear normal in the embryonal mass cells, but the microtubule network is partially disorganised in the embryonal tube cells and the microtubules disrupted in the suspensor cells. In the same embryos, the microtubule-associated protein, MAP-65, is bound only to organised microtubules. In contrast, in a developmentally arrested cell line, which is incapable of normal embryonic pattern formation, MAP-65 does not bind the cortical microtubules and we suggest that this is a criterion for proembryogenic masses (PEMs) to passage into early embryogeny. In embryos, the organisation of F-actin gradually changes from a fine network in the embryonal mass cells to thick cables in the suspensor cells in which the microtubule network is completely degraded. F-actin de-polymerisation drugs abolish normal embryonic pattern formation and associated PCD in the suspensor, strongly suggesting that the actin network is vital in this PCD pathway.