Rac1 function during fucoid development

Morphogenesis in fucoid algae begins with adhesive secretion and rhizoid germination, developmental events that secure the alga within the intertidal zone. The importance of the actin cytoskeleton during these processes has been well established; but in general, little is known about actin regulation within the stramenopile lineage. Based on conserved strategies for regulation of actin in other lineages, co-localization of the Arp2/3 complex with actin structures that are essential for rhizoid formation may implicate members of the Rho family of small GTPases in the signaling pathway(s) regulating actin polymerization during fucoid development. Our lab recently demonstrated Rac1 dependent regulation of endomembrane polarization, polarization of adhesive secretion, germination and tip growth in the fucoid brown alga Silvetia compressa. We also present new evidence revealing Rac1 localization during germination in S. compressa, and show that membrane localization is essential for proper Rac1 function.Key words: actin, Arp2/3 complex, manumycin A, NSC23766, Rac1, Rho GTPase, Scar/WAVE, Silvetia compressa     

Establishment and expression of cellular polarity in fucoid zygotes.

Zygotes of fucoid algae have long been studied as a paradigm for cell polarity. Polarity is established early in the first cell cycle and is then expressed as localized growth and invariant cell division. The fertilized egg is a spherical cell and, by all accounts, bears little or no asymmetry. Polarity is acquired epigenetically a few hours later in the form of a rhizoid/thallus axis. The initial stage of polarization is axis selection, during which zygotes monitor environment gradients to determine the appropriate direction for rhizoid formation. In their natural setting in the intertidal zone, sunlight is probably the most important polarizing vector; rhizoids form away from the light. The mechanism by which zygotes perceive environmental gradients and transduce that information into an intracellular signal is unknown but may involve a phosphatidylinositol cycle. Once positional information has been recorded, the cytoplasm and membrane are reorganized in accordance with the vectorial information. The earliest detectable asymmetries in the polarizing zygote are localized secretion and generation of a transcellular electric current. Vesicle secretion and the inward limb of the current are localized at the presumptive rhizoid. The transcellular current may establish a cytoplasmic Ca2+ gradient constituting a morphogenetic field, but this remains controversial. Localized secretion and establishment of transcellular current are sensitive to treatment with cytochalasins, indicating that cytoplasmic reorganization is dependent on the actin cytoskeleton. The nascent axis at first is labile and susceptible to reorientation by subsequent environmental vectors but soon becomes irreversibly fixed in its orientation. Locking the axis in place requires both cell wall and F-actin and is postulated to involve an indirect transmembrane bridge linking cortical actin to cell wall. This bridge anchors relevant structures at the presumptive rhizoid and thereby stabilizes the axis. Approximately halfway through the first cell cycle, the latent polarity is expressed morphologically in the form of rhizoid growth. Elongation is by tip growth and does not appear to be fundamentally different from tip growth in other organisms. The zygote always divides perpendicular to the growth axis, and this is controlled by the microtubule cytoskeleton. Two microtubule-organizing centers on the nuclear envelope rotate such that they align with the growth axis. They then serve as spindle poles during mitosis. Cytokinesis bisects the axial spindle, resulting in a transverse crosswall. Although the chronology of cellular events associated with polarity is by now rather detailed, causal mechanisms remain obscure.     

The Rac1 inhibitor,NSC23766, depolarizes adhesive secretion,endomembrane cycling,and tip growth in the fucoid alga, <Emphasis Type="Italic">Silvetia compressa</Emphasis>

Proper cell morphogenesis is dependent on the establishment and expression of cellular polarity. In the fucoid zygote, cell shape is critical for establishing the developmental pattern of the adult, and is achieved by guiding insertion of new membrane and wall to the rhizoid tip. Selection and growth of the appropriate tip site are accompanied by formation of dynamic actin arrays associated with the actin-nucleating Arp2/3 complex. In eukaryotes, a major pathway for activation of the Arp2/3 complex is via the Rho family GTPase, Rac1, which stimulates the Scar/WAVE complex. To determine whether Rac1 controls actin nucleation in Silvetia compressa (J. Agardh) E. Serrao, T. O. Cho, S. M. Boo et Brawley, we tested the effects of the Rac1-specific inhibitory compound, NSC23766, on actin dependent processes and on actin arrays. We found that NSC23766 disrupted polar secretion of adhesive, polarization of endomembranes, and tip-focused growth in the rhizoid. Similarly, NSC23766 altered actin and Arp2 localization in the growing rhizoid. In contrast, NSC23766 had no effect on selection of the growth site or on cytokinesis. These data suggest that Rac1 participates in nucleation of specific actin arrays in the developing zygote.     

Hypaphorine, an indole-3-acetic acid antagonist delivered by the ectomycorrhizal fungus Pisolithus tinctorius, induces reorganisation of actin and the microtubule cytoskeleton in Eucalyptus globulus ssp bicostata root hairs

Hypaphorine, an indole alkaloid from the ectomycorrhizal fungus Pisolithus tinctorius Coker & Couch., counteracts indole-3-acetic acid (IAA) activity and controls the rate of root hair elongation in Eucalyptus globulus ssp. bicostata. The present investigation shows that hypaphorine changes cytoskeletal organisation in elongating root hairs of the host. The actin cytoskeleton was investigated by two different fixation and labelling procedures, which gave similar results. In control root hairs, actin organisation was characterised by (i) an actin cap at the very tip region, (ii) a subapical region with reduced labelling and containing fine actin filaments, and (iii) axial bundles of actin filaments running from the subapical part to the base of the root hair. In the hypaphorine-treated root hairs no actin cap was distinguished. The fine actin filaments occurring in the subapical region were replaced by a few thick actin filament bundles that extended from the subapical region toward the root hair tip. In the hypaphorine-treated hairs the total number of actin filament bundles along most of the root hair length was significantly reduced, presumably due to aggregation of pre-existing actin filaments. The first signs of alteration to the cytoskeleton could be detected as soon as 15 min after hypaphorine treatment. In hypaphorine-treated, but not in control root hairs, a patch of aggregated microtubules regularly occurred at a distance of approximately 10 m from the tip, possibly as a consequence of changes induced by hypaphorine in the actin cytoskeleton. The hypaphorine-induced aggregations in the actin and microtubule cytoskeletons could stabilise the structure of cytoskeletal elements, which in turn could hinder the vesicle delivery at the tip necessary for elongation. Such cytoskeletal alterations may be a consequence of the antagonism between IAA and hypaphorine. The latter view was supported by restoration of the actin cytoskeleton in hypaphorine-treated root hairs by IAA application.     

Localization to the rhizoid tip implicates a<Emphasis Type="Italic"> Fucus distichus</Emphasis> Rho family GTPase in a conserved cell polarity pathway

Generation and expression of cell polarity in brown algal zygotes of the Fucales involve regulation of the actin cytoskeleton and localized secretion. We used degenerate PCR to isolate cDNAs that encode two small GTPases, FdRac1 and FdRab8, from zygotes of Fucus distichus (L.) Powell. Sequence analysis placed FdRac1 in the Rho family, which regulates actin, and FdRab8 in the Rab family, which regulates vesicle transport. As expected, bacterially expressed forms of both proteins bound GTP in vitro. When expressed in budding yeast, FdRac1 showed some functional overlap with CDC42, the Saccharomyces cerevisiae Rho family gene required for yeast cell polarity. Immunolocalization revealed an asymmetric distribution of FdRac1 in polarized zygotes and embryos, with FdRac1 concentrated at or near the growing tip of the algal rhizoid. Our data support the hypothesis that FdRac1 regulates algal cell polarity, possibly via the actin cytoskeleton. Because brown algae belong to the heterokont group, which diverged from other groups early in eukaryotic evolution, we argue that the Rho family function of regulating cell polarity is ancient and may extend throughout the eukaryotes.Electronic Supplementary Material Supplementary material is available in the online version of this article at Abbreviations AF After fertilization - GST Glutathione-S-transferase - MBP Maltose-binding protein     

Micromanipulation of statoliths in gravity-sensing Chara rhizoids by optical tweezers

Infrared laser traps (optical tweezers) were used to micromanipulate statoliths in gravity-sensing rhizoids of the green alga Chara vulgaris Vail. We were able to hold and move statoliths with high accuracy and to observe directly the effects of statolith position on cell growth in horizontally positioned rhizoids. The first step in gravitropism, namely the physical action of gravity on statoliths, can be simulated by optical tweezers. The direct laser microirradiation of the rhizoid apex did not cause any visible damage to the cells. Through lateral positioning of statoliths a differential growth of the opposite flank of the cell wall could be induced, corresponding to bending growth in gravitropism. The acropetal displacement of the statolith complex into the extreme apex of the rhizoid caused a temporary decrease in cell growth rate. The rhizoids regained normal growth after remigration of the statoliths to their initial position 10–30 m basal to the rhizoid apex. During basipetal displacement of statoliths, cell growth continued and the statoliths remigrated towards the rhizoid tip after release from the optical trap. The resistance to statolith displacement increased towards the nucleus. The basipetal displacement of the whole complex of statoliths for a long distance (>100 m) caused an increase in cell diameter and a subsequent regaining of normal growth after the statoliths reappeared in the rhizoid apex. We conclude that the statolith displacement interferes with the mechanism of tip growth, i.e. with the transport of Golgi vesicles, either directly by mechanically blocking their flow and/or, indirectly, by disturbing the actomyosin system. In the presence of the actin inhibitor cytochalasin B the optical forces required for acropetal and basipetal displacement of statoliths were significantly reduced to a similar level. The lateral displacement of statoliths was not changed by cytochalasin B. The results indicate: (i) the viscous resistance to optical displacement of statoliths depends mainly on actin, (ii) the lateral displacement of statoliths is not impeded by actin filaments, (iii) the axially directed actin-mediated forces against optical displacement of statoliths (for a distance of 10 m) are stronger in the basipetal than in the acropetal direction, (iv) the forces acting on single statoliths by axially oriented actin filaments are estimated to be in the range of 11–110 pN for acropetal and of 18–180 pN for basipetal statolith displacements.Abbreviation CB cytochalasin B This work was supported by the Bundesminister für Forschung und Technologie, and by Fonds der Chemischen Industrie. We thank Professor Dr. A. Sievers (Botanisches Institut, Universität Bonn, Germany) for helpful discussions.     

Poly(a)+ rna during vegetative development ofacetabularia peniculus

Summary In the juvenile stage, the diploid giant-celled green algae Acetabularia spp. are differentiated into an upright stalk and an irregularly branched rhizoid. Early amputation and grafting experiments as well as biochemical and molecular analyses have shown that mRNA (as poly(A)⁺ RNA) is continuously supplied from the primary nucleus in the rhizoid and accumulates in the stalk apex. In the present study, localization of poly(A)⁺ RNA in the juvenile stage of theAcetabularia peniculus was investigated by fluorescent in situ hybridization using oligo(dT) as a probe. The signal was localized in the apical cytoplasm and, in addition, multiple longitudinal striations throughout the stalk and rhizoid cytoplasm. A large portion of the poly(A)⁺ RNA striations exhibited structural polarity, broadened at one end and gradually thinned toward the other end. Some of the striations in the rhizoid cytoplasm were continuous with a zone of signal in the area of the perinuclear rim. The poly(A)⁺ RNA striations were associated with thick bands of longitudinal actin bundles which run through the entire length of the stalk. Cytochalasin D caused fragmentation of the actin bundles and irregular distribution of the fluorescent signal. We suggest that the poly(A)⁺ RNA striations constitute a hitherto unknown form of packaged mRNA that is transported over large distances along the actin cytoskeleton to be stored and expressed in the growing apex.     

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. Received July 15, 2002; accepted November 27, 2002; published online June 13, 2003 RID="*" ID="*" Correspondence and reprints: Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, UT 84112-0840, U.S.A.     

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.     

Pyrus pyrifolia stylar S-RNase induces alterations in the actin cytoskeleton in self-pollen and tubes in vitro

Summary. Pears (Pyrus pyrifolia L.) have an S-RNase-based gametophytic self-incompatibility system, and S-RNases have also been implicated in self-pollen or genetically identical pollen rejection. Tip growth of the pollen tube is dependent on a functioning actin cytoskeleton. In this study, configurations of the actin cytoskeleton in P. pyrifolia pollen and effects of stylar S-RNases on its dynamics were investigated by fluorescence and confocal microscopy. Results show that actin filaments in normal pollen grains exist in fusiform or circular structures. When the pollen germinates, actin filaments assembled around one of the germination pores, and then actin bundles oriented axially throughout the shank of the growing tube. There was a lack of actin filaments 5–15 μm from the tube tip. When self-stylar S-RNase was added to the basal medium, pollen germination and tube growth were inhibited. The configuration of the actin cytoskeleton changed throughout the culturing time: during the first 20 min, the actin configurations in the self-pollen and tube were similar to the control; after 20 min of treatment, the actin filaments in the pollen tube gradually moved into a network running from the shank to the tip; finally, there was punctate actin present throughout the whole tube. Although the actin filaments of the self-pollen grain also disintegrated into punctate foci, the change was slower than in the tube. Furthermore, the alterations to the actin cytoskeleton occurred prior to the arrest of pollen tube growth. These results suggest that P. pyrifolia stylar S-RNase induces alterations in the actin cytoskeleton in self-pollen grains and tubes. Correspondence: Shao-ling Zhang, College of Horticulture, Nanjing Agricultural University, Nanjing, Jiangsu 210095, People’s Republic of China.     

Localization of Actin mRNA during the Establishment of Cell Polarity and Early Cell Divisions in Fucus Embryos

Localization of mRNA is a well-described mechanism to account for the asymmetric distribution of proteins in polarized somatic cells and embryos of animals. In zygotes of the brown alga Fucus, F-actin is localized at the site of polar growth and accumulates at the cell plates of the first two divisions of the embryo. We used a nonradioactive, whole-mount in situ hybridization protocol to show the pattern of actin mRNA localization. Until the first cell division, the pattern of actin mRNA localization is identical to that of total poly(A)+ RNA, that is, a symmetrical distribution in the zygote followed by an actin-dependent accumulation at the thallus pole at the time of polar axis fixation. At the end of the first division, actin mRNA specifically is redistributed from the thallus pole to the cell plates of the first two divisions in the rhizoid. This specific pattern of localization in the zygote and embryo involves the redistribution of previously synthesized actin mRNA. The initial asymmetry of actin mRNA at the thallus pole of the zygote requires polar axis fixation and microfilaments but not microtubules, cell division, or polar growth. However, redistribution of actin mRNA from the thallus pole to the first cell plate is insensitive to cytoskeletal inhibitors but is dependent on cell plate formation. The F-actin that accumulates at the rhizoid tip is not accompanied by the localization of actin mRNA. However, maintenance of an accumulation of actin protein at the cell plates of the rhizoid could be explained, at least partially, by a mechanism involving localization of actin mRNA at these sites. The pattern and requirements for actin mRNA localization in the Fucus embryo may be relevant to polarization of the embryo and asymmetric cell divisions in higher plants as well as in other tip-growing plant cells.     

Association of spectrin-like proteins with the actin-organized aggregate of endoplasmic reticulum in the Spitzenkörper of gravitropically tip-growing plant cells

Spectrin-like epitopes were immunochemically detected and immunofluorescently localized in gravitropically tip-growing rhizoids and protonemata of characean algae. Antiserum against spectrin from chicken erythrocytes showed cross-reactivity with rhizoid proteins at molecular masses of about 170 and 195 kD. Confocal microscopy revealed a distinct spherical labeling of spectrin-like proteins in the apices of both cell types tightly associated with an apical actin array and a specific subdomain of endoplasmic reticulum (ER), the ER aggregate. The presence of spectrin-like epitopes, the ER aggregate, and the actin cytoskeleton are strictly correlated with active tip growth. Application of cytochalasin D and A23187 has shown that interfering with actin or with the calcium gradient, which cause the disintegration of the ER aggregate and abolish tip growth, inhibits labeling of spectrin-like proteins. At the beginning of the graviresponse in rhizoids the labeling of spectrin-like proteins remained in its symmetrical position at the cell tip, but was clearly displaced to the upper flank in gravistimulated protonemata. These findings support the hypothesis that a displacement of the Spitzenk?rper is required for the negative gravitropic response in protonemata, but not for the positive gravitropic response in rhizoids. It is evident that the actin/spectrin system plays a role in maintaining the organization of the ER aggregate and represents an essential part in the mechanism of gravitropic tip growth.     

QUANTITATIVE STUDY OF DEVELOPMENT OF THE GIANT COENOCYTE,CAULERPA PROLIFERA

The lengths of rhizomes, rhizoid clusters, and leaves of many plants of Caulerpa prolifera cultured under controlled conditions were measured daily. Their rates and patterns of growth were analyzed mathematically with the aid of an electronic computer. All organs elongated at the rate of ca. 4.4 mm per day. Rhizomes elongated linearly for more than 2 weeks, and curvilinear regression usually did not improve the fit significantly. Rhizome elongation is apical. Every 1.18 days, on the average, a rhizoid cluster developed on the lower surface of each rhizome. The length of the younger rhizoid clusters shows a linear relationship to their distance from the rhizome tip. Little or no significant improvement of fit resulted from fitting polynomials of the 2nd through 4th degree. Accordingly, by extrapolating the straight line to zero rhizoid length, we calculated the presumptive site of rhizoid initiation to be 0.84 mm from the rhizome tip. The rate and distribution of growth were compared to those of other coenocytes and of multicellular plants.     

The Polar Organization of the Growing Chara Rhizoid and the Transport of Statoliths are Actin-Dependent*

Horizontally positioned Chara rhizoids continue growth without gravitropic bending when the statoliths are removed from the apex by basipetal centrifugation. The transport of statoliths in centrifuged rhizoids is bidirectional: 50–60 % of the statoliths are re-transported on a straight course to the apex at velocities from 1 to 14 μm . min^?1 increasing towards the rhizoid tip. The centrifuged statoliths which are located closest to the nucleus are basipetally transported and caught up in the cytoplasmic streaming of the cell. Those statoliths which are located near the apical side of the nucleus are transported either apically or basally. A de-novo-formation of statoliths was not observed. After retransport to the apex some statoliths transiently sediment, a process which can induce a local inhibition of cell wall growth. The rhizoid bends again gravitropically only if a few statoliths finally sediment in the apex; the more statoliths that sediment in the apex the shorter the radius of bending becomes. The transport of statoliths is mediated by actin filaments which form a network of thin filaments in the apical and subapical zone of the rhizoid, and thicker parallel bundles in the basal zone where cytoplasmic streaming occurs. Both subpopulations of actin filaments overlap in the nucleus zone.     

Mannose,but not glucose or sucrose,disturbs actin cytoskeleton in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> leaves

A dynamic cytoskeleton able to recognize and respond to both abiotic and biotic stimuli is necessary for the proper functioning of a living cell. The cytoskeleton is involved in cell growth and division, maintenance of cell shape, cytoplasmic streaming, and organelle movements. Numerous studies have focused on the relationships between sugar metabolism, sugar signaling, and the cytoskeleton in yeast and animal cells. Data on such connections in plants are scarce. In the present study we investigated the effects of exogenously delivered sugars on the plant actin cytoskeleton. Detached Arabidopsis thaliana leaves were incubated with sugars for 2 days and the cytoskeleton was visualized using fluorescent-labeled phalloidin. Glucose and sucrose did not influence the pattern of the actin cytoskeleton. In contrast, mannose caused the disappearance of filamentous structures and generated actin foci. The symptoms started to be visible after 24 h of the exposure to mannose. The effect did not occur in Nicotiana tabacum mesophyll cells. This insensitivity was probably due to the presence of phosphomannose isomerase in tobacco cells. Mannose is commonly used as a selection marker for the transformation of plants lacking the enzymes responsible for the metabolism of mannose-6-phosphate. Exposure to this hexose has been linked with DNA fragmentation and a release of cytochrome c from mitochondria. Both responses are treated as features of programmed cell death. However, in our experiments no DNA laddering was observed in mannose-treated Arabidopsis leaves.     

Rhodamine-phalloidin staining of F-actin in pollen after dimethylsulphoxide permeabilization

Summary Bundles of actin filaments were observed in in vitro cultured pollen of Lilium longiflorum and Nicotiana tabacum which had been permeabilized in a buffered medium containing 5% dimethylsulphoxide (DMSO), EGTA, rhodaminephalloidin for F-actin staining, and sucrose as an osmoticum. In imbibed pollen grains, especially those of lily, numerous short bundles and small foci of F-actin were clearly visible. In germinated pollen grains, fine bundles of F-actin could be seen to converge at the aperture of the pollen grain. Along the entire length of the pollen tube, including the extreme tip, a dense three-dimensional netaxial distribution of actin filaments was observed. The F-actin patterns visualized after permeabilization with DMSO are much finer and more detailed than those observed after conventional fixation with formaldehyde.     

The Arp2/3 complex nucleates actin arrays during zygote polarity establishment and growth

Previous work has demonstrated that dynamic actin arrays are important for axis establishment and polar growth in the fucoid zygote, Silvetia compressa. Transitions between these arrays are mediated by depolymerization of an existing array and polymerization of a new array. To begin to understand how polymerization of new arrays might be regulated, we investigated the role of the highly conserved, actin-nucleating, Actin-related protein 2/3 (Arp2/3) complex. Arp2, a subunit of the complex, was cloned and peptide antibodies were raised to the C-terminal domain. In immunolocalization studies of polarizing zygotes, actin and Arp2 colocalized around the nucleus and in a patch at the rhizoid pole. In germinated zygotes, a cone of Arp2 and actin extended from the nucleus to the subapex. Within the rhizoid tip, three structural zones were observed in the majority of zygotes: the extreme apex was devoid of label, the subapex was enriched for Arp2, and further back both actin and Arp2 were present. This zonation suggests that actin nucleation occurs at the leading edge of the cone, in the Arp2-enriched region. In two sets of experiments, we showed that tip zonation is important for growth. First, pharmacological treatments that disrupted Arp2/actin zonation arrested tip growth. Second, changes in the direction of tip growth during negative phototropism were preceded by a reorientation of the zonation in accordance with the new growth direction. This work represents the first investigation of Arp2/3 complex localization in tip-growing algal cells.     

Apical myosin XI anticipates F‐actin during polarized growth of Physcomitrella patens cells

Tip growth is essential for land colonization by bryophytes, plant sexual reproduction and water and nutrient uptake. Because this specialized form of polarized cell growth requires both a dynamic actin cytoskeleton and active secretion, it has been proposed that the F‐actin‐associated motor myosin XI is essential for this process. Nevertheless, a spatial and temporal relationship between myosin XI and F‐actin during tip growth is not known in any plant cell. Here, we use the highly polarized cells of the moss Physcomitrella patens to show that myosin XI and F‐actin localize, in vivo, at the same apical domain and that both signals fluctuate. Surprisingly, phase analysis shows that increase in myosin XI anticipates that of F‐actin; in contrast, myosin XI levels at the tip fluctuate in identical phase with a vesicle marker. Pharmacological analysis using a low concentration of the actin polymerization inhibitor latrunculin B showed that the F‐actin at the tip can be significantly diminished while myosin XI remains elevated in this region, suggesting that a mechanism exists to cluster myosin XI‐associated structures at the cell's apex. In addition, this approach uncovered a mechanism for actin polymerization‐dependent motility in the moss cytoplasm, where myosin XI‐associated structures seem to anticipate and organize the actin polymerization machinery. From our results, we inferred a model where the interaction between myosin XI‐associated vesicular structures and F‐actin polymerization‐driven motility function at the cell's apex to maintain polarized cell growth. We hypothesize this is a general mechanism for the participation of myosin XI and F‐actin in tip growing cells.     

Actin dynamics in Phytophthora infestans; rapidly reorganizing cables and immobile,long‐lived plaques

The actin cytoskeleton is a dynamic but well‐organized intracellular framework that is essential for proper functioning of eukaryotic cells. Here, we use the actin binding peptide Lifeact to investigate the in vivo actin cytoskeleton dynamics in the oomycete plant pathogen Phytophthora infestans. Lifeact–eGFP labelled thick and thin actin bundles and actin filament plaques allowing visualization of actin dynamics. All actin structures in the hyphae were cortically localized. In growing hyphae actin filament cables were axially oriented in the sub‐apical region whereas in the extreme apex in growing hyphae, waves of fine F‐actin polymerization were observed. Upon growth termination, actin filament plaques appeared in the hyphal tip. The distance between a hyphal tip and the first actin filament plaque correlated strongly with hyphal growth velocity. The actin filament plaques were nearly immobile with average lifetimes exceeding 1 h, relatively long when compared to the lifetime of actin patches known in other eukaryotes. Plaque assembly required ～30 s while disassembly was accomplished in ～10 s. Remarkably, plaque disassembly was not accompanied with internalization and the formation of endocytic vesicles. These findings suggest that the functions of actin plaques in oomycetes differ from those of actin patches present in other organisms.     

Feeling for Filaments: Quantification of the Cortical Actin Web in Live Vascular Endothelium

Contact-mode atomic force microscopy (AFM) has been shown to reveal cortical actin structures. Using live endothelial cells, we visualized cortical actin dynamics simultaneously by AFM and confocal fluorescence microscopy. We present a method that quantifies dynamic changes in the mechanical ultrastructure of the cortical actin web. We argue that the commonly used, so-called error signal imaging in AFM allows a qualitative, but not quantitative, analysis of cortical actin dynamics. The approach we used comprises fast force-curve-based topography imaging and subsequent image processing that enhances local height differences. Dynamic changes in the organization of the cytoskeleton network can be observed and quantified by surface roughness calculations and automated morphometrics. Upon treatment with low concentrations of the actin-destabilizing agent cytochalasin D, the cortical cytoskeleton network is thinned out and the average mesh size increases. In contrast, jasplakinolide, a drug that enhances actin polymerization, consolidates the cytoskeleton network and reduces the average mesh area. In conclusion, cortical actin dynamics can be quantified in live cells. To our knowledge, this opens a new pathway for conducting quantitative structure-function analyses of the endothelial actin web just beneath the apical plasma membrane.