Blue-light-induced cortical fiber reticulation concomitant with chloroplast aggregation in the alga Vaucheria sessilis

Point illumination with low-intensity blue light induces the chloroplasts and other organelles, which normally stream in the cytoplasm of Vaucheria sessilis (Vauch.) D.C. (Xanthophyceae), to aggregate in the illuminated region of the cell. Aggregation is passive and results from the trapping of the organelles as they stream into the blue light. Prior to illumination, longitudinal fibers along which the organelles appear to move, can readily be seen through a light microscope fitted with differential interference contrast optics. Upon actinic irradiation, these fibers appear to become destabilized, branching and forming a cortical fiber reticulum in the light. The reticulation process always precedes chloroplast aggregation. Aggregation itself occurs after a lag period which is inversely related to fluence rate. The lag period at high fluence rates (>400 mW m^-2) may be as short as 20 s. Studies of wavelength dependence show that wavelengths near 480 nm are maximally effective while those longer than 530 nm are inactive.C.I.W.-D.P.B. Publication No. 642     

A light-dependent current associated with chloroplast aggregation in the alga Vaucheria sessilis

Local irradiation of the alga Vaucheria sessilis (Vauch.) D.C. with blue light, which stimulates cortical fiber reticulation and chloroplast aggregation (M.R. Blatt and W.R. Briggs, 1980, Planta 147, 355–362), also induces an outward-directed current from the irradiated region of the cell. This current appears in conjunction with cortical fiber reticulation and precedes chloroplast aggregation. The current is not photosynthetic in origin, as indicated by experiments with 3(3,4-dichlorophenyl)-1,1-dimethyl urea and carbonyl-cyanide-m-chlorophenylhydrazone (CCCP). It shows a wavelength-dependence similar to that of chloroplast aggregation and reaches a maximum of 500 nA cm^-2 with saturating light intensities. The current is not dependent upon the presence of Na⁺, K⁺, or Cl^- in a test medium containing only Na⁺, K⁺, Ca²⁺, and Cl^-, but is inhibited, apparently nonspecifically, in the absence of external calcium. Both the light-induced current and chloroplast aggregation are stimulated by increases in the external KCl concentration and are inhibited by sub-micromolar concentrations of CCCP or by external pHs below approximately 5.5. We suggest that blue light stimulates the local extrusion of cations, possibly of protons, at the plasma membrane, an event which may act to destabilize the cortical fibers in Vaucheria, disrupt cytoplasmic streaming, and eventually lead to organelle aggregation in the light.C.I.W.-D.P.B. Publication No. 712     

Regulation of aplanospore germination in Vaucheria

Germination of aplanospores in Vaucheria longicaulis Hoppaugh var. macounii Blum proceeds through three stages of development. Stage I begins with the initiation of germination and lasts approx. 2 h. During this stage germinating filaments grow at an accelerated rate (266 ± 12 m · h^–1). Stage II is characterized by a sharp decline in the growth rate of germinating filaments (96 ± 4 m · h^–1) and lasts 4 h. This is followed, during the next 4 h, by a recovery in the growth rate (168 ± 8 m · h^–1) of germinating filaments, stage III. Growth rates stabilize and remain unchanged during subsequent development (Oliveira and Fitch, 1988, J. Submicrosc. Cytol. Pathol. 20, 397–406). The Ca²⁺-influx modulators LaCl₃, nifedipine and methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4 (2-trifluoromethylphenyl)-pyridine-5-carboxylate (Bay K-8644), the ionophore calcimycin (A23187), the intracellular Ca²⁺-release antagonist 8-N-N'-(diethylamino)-octyl-3,4, 5-trimethoxybenzoate (TMB-8), the Ca²⁺-uptake inhibitor ruthenium red and the phosphoinositide-cycle modulators LiCl and myo-inositol show that the events required for the initiation are distinct from those required for the completion of each stage of germination. These studies in conjunction with microinjection of germinating filaments with inositol 1,4,5-trisphosphate, the natural ligand for Ca²⁺ release from Ca-storing organelles (endoplasmic reticulum, vacuole), and treatment with chlorotetracycline (CTC), to visualize the distribution of membrane-bound Ca²⁺ reveal that both the initiation and completion of each stage of germination are controlled by Ca²⁺ signals which are restricted to well-defined time intervals and are modulated by the origin (source) of Ca²⁺.Abbreviations BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid - Bay K-8644 methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4(2-trifluoromethylphenyl)-pyridine-5-carboxylate - CTC chlorotetracycline - InsP₃ inositol 1,4,5-trisphosphate - RR ruthenium red - TMB-8 8-N-N-(diethylamino)-octyl-3,4,5-trimethoxybenzoate The author wishes to express his gratitude to the technical group of the Immunocytochemistry Unit for their help with the microinjection studies. This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (grant A-7844).     

Actin redistribution in mosquito malpighian tubules after a blood meal and cyclic AMP stimulation

Fluid secretion by mosquito Malpighian tubules is critical to maintaining fluid and electrolyte balance after a blood meal. Endogenous cAMP levels increase in Malpighian tubules after a blood meal. Here, we determined if corresponding changes in intracellular actin distribution occur after a blood meal or dibutyryl-cAMP (db-cAMP) stimulation and whether altering actin turnover inhibits secretion. In untreated Malpighian tubules, beta-actin immunostaining was more intense in the apical region of adult Malpighian tubules than in the cytoplasm. Stimulation by a blood meal or db-cAMP significantly decreased beta-actin immunostaining in the non-apical region of the cell. Db-cAMP had similar effects in larvae and pupae Malpighian tubules. In contrast, no detectable shift in F-actin distribution was detected; however, F-actin bundles within the cytoplasm increased in size after treatment with db-cAMP. Pretreatment of Malpighian tubules with agents perturbing actin fiber assembly and disassembly decreased basal secretion rates and inhibited the stimulatory effects of db-cAMP. Our results show (1) beta-actin redistributes toward the apical membrane after a blood meal and this correlates temporally with increase urine flow rate and intracellular cAMP levels, (2) Malpighian tubules from all developmental stages exhibit this same response to db-cAMP-stimulation, and (3) dynamic assembly and disassembly of beta-actin is required for db-cAMP-stimulated secretion.     

Immobilisation of organelles and actin bundles in the cortical cytoplasm of the alga Chara corallina Klein ex. Wild

The mechanism by which sub-cortical actin bundles and membranous organelles are immobilised in the cortical cytoplasm of the alga Chara was studied by perfusing cells with a solution containing 1% Triton X-100. Light and scanning electron microscopy and the release of starch grains and chlorophyll-protein complexes indicated that the detergent extensively solubilised the chloroplasts. However, the sub-cortical actin bundles remained in situ even though they were originally separated from the plasma membrane by the chloroplasts. A fibrous layer between chloroplasts and plasma membrane became readily visible after detergent extraction of the cells and could be released by low-ionic-strength ethylenediaminetetraacetic acid, thioglycollate and trypsin. The same treatments applied to cells not subject to detergent extraction released the membrane-bound organelles and actin bundles and no fibrous meshwork was visible on subsequent extraction with Triton. It is, therefore, concluded that a detergent-insoluble cortical cytoskeleton exists and contributes to the immobility of the actin and cortical organelles in the cells.Abbreviation EDTA ethylenediaminetetraacetic acid     

Actin is present in a green alga that lacks cytoplasmic streaming

Summary Negative-staining of crude cytoplasmic extracts from cells of the green algaErnodesmis verticillata reveals the presence of numerous microfilaments. Rabbit skeletal muscle heavy-meromyosin binds to the microfilaments (in the absence of ATP) in typical arrowhead arrays. These results demonstrate that actin is present in this alga and it is suggested that actin may be involved in cytoplasmic contractions effecting wound healing, since cytoplasmic streaming does not occur in this organism.     

Mitosis and mitotic wave propagation in the coenocytic alga,<Emphasis Type="Italic"> Vaucheria terrestris</Emphasis> sensu Goetz

Mitosis and cytoplasmic microtubule (MT) dynamics were observed for the first time in Vaucheria terrestris sensu Goetz. Mitosis could occasionally be seen in part of the cylindrical coenocytic cell. The frequency of encountering cells with dividing nuclei was highest (ca 12%) 4 h after the onset of light in 12 h light/12 h dark regimes; it decreased thereafter and approached zero during the dark period. From the anterior end of every interphase nucleus a unique, long MT bundle extended. Differential-interference optics reveals that there is a filamentous structure in front of the moving nucleus. In prophase, the interphase bundle disappeared and shorter MT bundles emanated from both ends of the nucleus. In metaphase, the cytoplasmic MTs completely disappeared, probably being recycled to spindles. Continuous MTs elongated in anaphase and developed into an interzonal spindle in telophase; this elongated up to as much as 10 m. The daughter nuclei were pushed away from each other by the interzonal spindle. Mitosis started synchronously in a relatively narrow region, and the mitotic stage propagated as a mitotic wave to adjacent regions, most frequently from tip to base. The role of the mitotic wave in tip growth and morphogenesis of a coenocytic cell is discussed.This paper is dedicated to the memory of Dr. Eiji Kamitsubo who passed away on 25 April 2003.     

Identification of calmodulin in the green alga Mougeotia and its possible function in chloroplast reorientational movement

A soluble protein was isolated from Mougeotia by chloropromazine-sepharose 4 B affinity chromatography. The protein matches the properties of calmodulin in terms of heat stability, Ca²⁺-dependent electrophoretic mobility in sodium-dodecyl-sulfate polyacrylamide gels, and its ability to activate cyclic nucleotide phosphodiesterase in a Ca²⁺-dependent manner. Phytochrome-mediated chloroplast reorientational movement in Mougeotia was inhibited by the calmodulin antagonist trifluoperazine, a hydrophobic compound, or N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a hydrophilic compound; 50% inhibition (IC₅₀) of chloroplast movement is caused by 20–50 mol l^-1 trifluoperazine or 100 mol l^-1 W-7. The Ca²⁺-calmodulin may act as an intermediate in the chloroplast reorientational response in Mougeotia governed by phytochrome.Abbreviations EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - SDS sodium dodecyl sulfate - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide     

The involvement of F-actin inUromyces cell differentiation: The effects of cytochalasin E and phalloidin

Summary The role of F-actin in cell differentiation ofUromyces appendiculatus (bean rust fungus) germlings was examined by treating differentiating and nondifferentiating germlings with the actin-binding drugs cytochalasin E (CE) and phalloidin. Prolonged exposure of urediospores to 5×10^–3–5 × 10^–5 M CE induced nuclear division in up to 28–45% of the resulting germlings, whereas the rate of mitosis in established germlings exposed to these concentrations of CE was significantly lower (4–11%). Germlings treated with CE shifted from polarized apical growth to spherical expansion, cytoplasmic microfilaments were depolymerized, and nuclear inclusions became enlarged. Differentiating germlings exposed to a 10 minute pulse of 5×10^–6M CE before the initiation of septum formation prevented the establishment of the F-actin septal ring and growth of the crosswall delimiting the appressorium. Although these CE treatments resulted in morphological and nuclear events similar to those occurring during normal appressorium formation, transient microfilament depolymerization was not sufficient to induce differentiation. Phalloidin stabilized cytoplasmic microfilaments, especially posteriorly-located microfilaments, but did not affect differentiation, nor did it significantly inhibit the effects of CE.Abbrevations CE cytochalasin E - DAPI 4,6-diamidino-2-phenylindole - DMSO dimethyl sulfoxide - F-actin filamentous actin     

Actin in living and fixed characean internodal cells: identification of a cortical array of fine actin strands and chloroplast actin rings

Summary We report on the novel features of the actin cytoskeleton and its development in characean internodal cells. Images obtained by confocal laser scanning microscopy after microinjection of living cells with fluorescent derivatives of F-actin-specific phallotoxins, and by modified immunofluorescence methods using fixed cells, were mutually confirmatory at all stages of internodal cell growth. The microinjection method allowed capture of 3-dimensional images of high quality even though photobleaching and apparent loss of the probes through degradation and uptake into the vacuole made it difficult to record phallotoxin-labelled actin over long periods of time. When injected at appropriate concentrations, phallotoxins affected neither the rate of cytoplasmic streaming nor the long-term viability of cells. Recently formed internodal cells have relatively disorganized actin bundles that become oriented in the subcortical cytoplasm approximately parallel to the newly established long axis and traverse the cell through transvacuolar strands. In older cells with central vacuoles not traversed by cytoplasmic strands, subcortical bundles are organized in parallel groups that associate closely with stationary chloroplasts, now in files. The parallel arrangement and continuity of actin bundles is maintained where they pass round nodal regions of the cell, even in the absence of chloroplast files. This study reports on two novel structural features of the characean internodal actin cytoskeleton: a distinct array of actin strands near the plasma membrane that is oriented transversely during cell growth and rings of actin around the chloroplasts bordering the neutral line, the zone that separates opposing flows of endoplasm.     

Actin cytoskeletons regulate the stretch-induced increase of Ca2+ current in human gastric myocytes

Using the whole-cell and single channel recording techniques, the influence of actin cytoskeletons on L-type Ca2+ current was investigated in human gastric smooth muscle cells. In isotonic condition, an actin depolymerizer cytochalasin D (Cyt-D) markedly decreased the whole-cell current (I(Ba)) without changing steady-state voltage dependency and single channel conductance. Intracellular dialysis of phalloidin, an actin polymerizer, significantly increased the I(Ba). Hypotonic stretch (222 mOsm/L) of the myocytes increased the I(Ba), and Cyt-D significantly inhibited the I(Ba) increase by the stretch. Phalloidin was without effect on the I(Ba) increase by the stretch. Phalloidin antagonized the Cyt-D inhibition of the stretch-induced I(Ba) increase. Neither heterotrimeric G protein modifiers (GTPgammaS and GDPbetaS) nor rho GTPase inhibitor (C3 exoenzyme) influenced the stretch-induced responses. These results reveal that the integrity of the actin cytoskeleton is an important factor which determines the activity of L-type Ca2+ channels and a response to stretch.     

Actin isoforms in non-infected roots and symbiotic root nodules of Phaseolus vulgaris L.

The present report describes an initial characterization of actin from non-infected roots and symbiotic nodules of Phaseolus vulgaris L. cv. Negro damapa. Using anti-actin monoclonal antibodies, a 42-kDa polypeptide was identified in plant extracts. After two-dimensional SDS-PAGE analysis and Western blotting of actin fractions enriched using diethylaminoethyl-resin, the presence of one major isoform of actin in symbiotic nodules and two main isoforms in non-infected roots was revealed. Possible implications of this finding are discussed.Abbreviations MAB(s) monoclonal antibody(ies) - IEF isoelectric focusing We thank Jose Luis Zitlalpopoca for technical assistance. H.P. gratefully acknowledges financial support from CONACYT (Mexico) and the National Institute of Public Health of Mexico. This work was partially supported by grants DGAPA-UNAM IN208489 and CEE. C11 06228-M.     

Novel changes in the plasma membrane and cortical cytoplasm during wound-induced contraction in a giant-celled green alga

Summary Changes in the plasma membrane surface and in the cortical cytoplasm during wound healing in giant green algal cells ofErnodesmis verticillata (Kützing) Brgesen were followed using scanning and transmission electron microscopy. Microvillus-like structures that contain cytoplasmic and cytoskeletal constituents were observed emanating from the surface of the plasma membrane at the retracting/cut end of wounded cells. These delicate structures seem to be remnants of cell wall-plasmalemma connections that draw out the plasma membrane and cortical components from the contracting cytoplasm as it pulls away from the cell wall. Most of these connections break during wound healing and, when contraction stops, the microvillus-like protrusions become progressively shorter. In cells treated with a calmodulin antagonist (W-7), a number of distinctive bodies accumulate that are of unknown composition, are oblong in shape, and have a diameter slightly smaller than the protoplasmic protrusions. Ultrastructural and other data indicate that these bodies result from retrieved constituents of the plasma-membrane protrusions, as they do not accumulate in unwounded drugtreated cells or in cells treated in W-5. These findings suggest that the protoplasmic protrusions accumulate membrane and cytoplasmic components that are retrieved and recycled during wound healing inErnodesmis by a novel mechanism. The combined plasma membrane surfaces of the microvillus-like protrusions may help to account for the drastic decrease in surface area that occurs during wound healing.Abbreviations SEM scanning electron microscopy - TEM transmission electron microscopy - W-7 N-[6-aminohexyl]-5-chloro-1-naph-thalenesulfonamide - W-5 N-[6-aminohexyl]-1-naphthalenesulfonamide     

Structure and assembly of the cortical microtubule cytoskeleton in the green alga,Boodlea coacta

Summary Examination was made of the structure and assembly of the cortical microtubule (MT) cytoskeleton in the coenocytic green algaBoodlea coacta (Dickie) Murray et De Toni by immunofluorescence microscopy. Cortical MTs inBoodlea protoplasts are arranged randomly but some show a meridional arrangement within 6 h after protoplast formation. At 6–9 h such MTs become highly concentrated and parallel to each other in certain areas. At 12 h the concentration is uniformly high throughout the cell, indicating the completion of high density meridional arrangement of cortical MTs. Cortical MTs exhibiting a high density, meridional arrangement show characteristic disassembly by treatment with 10 M amiprophos-methyl (APM) or cold treatment (0 °C). Disassembly occurs by each MT unit at positions skipping 30–40 m in the transverse direction, and neighboring MTs subsequently disassemble to form MT groups. Each group becomes slender and then disappears completely within the following 24 h. The meridional arrangement of cortical MTs is disrupted by N-ethylmaleimide (NEM) accompanied by a remarkable reduction in density. The remaining MTs form groups at 30–40 m intervals from each other, as also occurs with drug or cold treatment, but disruption and density return to normal levels following removal of NEM. It appears that there are meridionally oriented channels, anchor-rich and anchor-poor, in the plasma membrane. The channels could be distributed alternately and anchors could be deposited in a cross-linking manner with cortical MTs to form a stable cortical MT-cytoskeleton. MTs comprising the cortical MT cytoskeleton could be oriented by meridionally oriented channels of anchors which are distributed following establishment of cell polarity.Abbreviations APM amiprophos-methyl - MT microtubule - MTOC microtubule organizing center - NEM N-ethylrnaleimide     

Actin cytoskeleton and calcium-ATPase in the process of abomasal mucus secretion in cattle

Summary The distribution of actin filaments in pyloric gland cells of cattle was studied with respect to their functional significance in the process of exocrine secretion by use of rhodamine-phalloidin labelling and immunogold-electron microscopy based on the biotinstreptavidin bridge technique. Actin concentrates on the filamentous network of the luminal cell cortex. Membranes of secretory vesicles accumulating in the cell cortex are also labelled for actin. The present results support the concept of a barrier function of cortical microfilaments entrapping vesicles and linking them to the cytoskeleton. In addition, intracellular localization of calcium-ATPase activity was determined. Enzyme activity associated with the microfilamentous cortical matrix is supposed to be of cytoskeletal nature indicating participation of myosin (-like) structures in the dynamic secretion event. Deposition on the interior aspect of secretory vesicle membranes points to an ATPase transporting calcium into these organelles and enabling them to participate via storage of the cation in intracellular calcium homeostasis, thereby influencing the functional architecture of the cortical cytoskeleton.     

Cytoplasmic streaming in the cell model ofNitella

Summary The plasmalemma ofNitella internode was made freely permeable to solutes by treating the cell with detergent and EGTA under plasmolysis. After the treatment, the cytoplasmic streaming was stopped by bathing the cell in a medium lacking ATP. The streaming was reactivated by perfusing the exterior of the permeabilized cell with a medium containing both Mg²⁺ and ATP. The reactivated streaming could be reversibly stopped by depletion of ATP. However, depletion of Mg²⁺ irreversibly inhibited the streaming.Cytochalasin B at 5 g/ml irreversibly inhibited the reactivated streaming within a minute, showing that microfilaments are involved in the streaming.Abbreviations ATP adenosine-5-triphosphoric acid - CB cytochalasin B - CyDTA cyclohexanediamine-N,N-tetraacetic acid - DMSO dimethylsulfooxide - DTT dithiothreitol - EGTA ethyleneglycol-bis(-aminoethylether)-N,N tetraacetic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - PMSF phenylmethyl-sulfonylfluoride     

Assembly of cortical microtubules during cold treatment of the coenocytic green alga,Chaetomorpha moniligera

The effects of cold treatment on the cortical microtubules (MTs) of Chaetomorpha moniligera Kjellman were investigated by immunofluorescence microscopy. Cortical MTs in Chaetomorpha thallus are arranged longitudinally. In this study, 70–75% of MTs disassembled within 4 h on ice while the others remained stable under these conditions. Reticulate background immunofluorescence, assumed to indicate the presence of a tubulin monomer, was distributed about the stable MTs. Immunofluorescence was prominent in only 50% of the cells. Tubulin polymerization was noted where the background and MT immunofluorescence was strong. New MTs grew transversely as single strings or clusters from the sides of MTs after cold treatment for 4 h and elongated with time to take on a reticulate form at 24 h. The significance of this tubulin polymerization under cold treatment is discussed.Abbreviations MT microtubule - MTOC microtubule-organizing center     

Changes of the actin filament system in the green algaMicrasterias denticulata induced by different cytoskeleton inhibitors

Summary Two different techniques have been adapted forMicrasterias denticulata to depict the actin cytoskeleton of both untreated and inhibitor-treated developing cells: the quickstaining method, where the cells are fixed in a mixture of glutaraldehyde and formaldehyde followed by staining with phalloidin without embedding, and the methacrylate method, where the cells are also fixed by aldehydes and where the embedding medium is removed prior to incubation with an actin antibody. Both methods produce sufficient preservation and visualization of actin microfilaments (MFs) and confirm earlier observations on the presence of a cortical actin MF network in both the growing and the nongrowing semicell as well as of a basketlike MF arrangement around the migrating nucleus. The results show that a network of actin MFs is essential for the proper development of the young lobes ofM. denticulata. Early developmental stages expanding uniformly at the beginning of growth lack any netlike actin MF arrangement. The actin cytoskeleton in developing cells treated with the actin-targeting agents cytochalasin D and latrunculin B is markedly influenced. Cytochalasin D, which produces the most pronounced effects, causes a breakdown of the network of actin MFs, resulting in bright actin clusters as well as in short and abnormally thick actin fragments particularly in cortical cell regions. In latrunculin B-treated cells remnants of the former actin MF network are still visible, yet most of the actin cytoskeleton appears collapsed and is reduced to short filament pieces. The disturbance of the actin MF system visualized in the present study correlates with the severe morphological and ultrastructural changes occurring in desmid cells as a consequence of both drugs. The dinitroanilin herbicide oryzalin, known to deploymerize cytoplasmic microtubules, causes also an impairment of the actin cytoskeleton inM. denticulata though not sufficient to influence normal cell growth and differentiation.Abbreviations CB cytochalasin B - CD cytochalasin D - DMSO dimethyl sulfoxide - FA formaldehyde - GA glutaraldehyde - LAT-A latrunculin A - LAT-B latrunculin B - MFs microfilaments - MT microtubule Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday     

Actin cytoskeleton in intact and wounded coenocytic green algae

Summary The subcellular distribution of actin was investigated in two related species of coenocytic green algae, with immunofluorescence microscopy. Either no, or fine punctate fluorescence was detected in intact cells of Ernodesmis verticillata (Kützing) Børgesen and Boergesenia forbesii (Harvey) Feldmann. A reticulate pattern of fluorescence appears throughout the cortical cytoplasm of Ernodesmis cells shortly after wounding; this silhouettes chloroplasts and small vacuoles. Slender, longitudinal bundles of actin become evident in contracting regions of the cell, superimposed over the reticulum. Thicker portions of the bundles were observed in well-contracted regions, and the highly-convoluted appearance of nearby cortical microtubules indicates contraction of the bundles in these thicker areas. Bundles are no longer evident after healing; only the reticulum remains. In Boergesenia, a wider-mesh reticulum of actin develops in the cortex of wounded cells, which widens further as contractions continue. Cells wounded in Ca²⁺-free medium do not contract, and although the actin reticulum is apparent, no actin bundles were ever observed in these cells. Exogenously applied cytochalasins have no effect on contractions of cut cells or extruded cytoplasm, and normal actin-bundle formation occurs in treated cells. In contrast, erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA) completely inhibits longitudinal contractions in wounded cells, and few uniformly slender actin bundles develop in inhibited cells. These results indicate that wounding stimulates a Ca²⁺-dependent, hierarchical assembly of actin into bundles, whose assembly and functioning are inhibited by EHNA. Contraction of the bundles and concomitant wound healing are followed by cessation of motility and disassembly of the bundles. The spatial and temporal association of the bundles with regions of cytoplasmic contraction, indicates that the actin bundles are directly involved in wound-induced cytoplasmic motility in these algae.Abbreviations EHNA erythro-9-[3-(2-hydroxynonyl)]adenine - MT(s) microtubule(s)     

Actin filaments predominate in morphogenic cell stages, whereas plaques predominate in non-morphogenic cell stages in Peronosporomycetes

We investigated the structural distribution of both types of actin arrays, filaments and plaques, in a soil-borne phytopathogenic peronosporomycete (oomycete), Aphanomyces cochlioides, under standardized host-free bioassays. The phenomenon was monitored during progression through all the asexual developmental processes of the organism. It was noted that the filamentous-form of actin was predominant during the morphogenic (morphologically active) stages of development. Conversely, during non-morphogenic (morphologically quiescent) stages, plaques dominated. From these analyses, we proposed a criterion that predominance of an actin form relates to, and precedes the morphological behaviour of a cellular stage in Peronosporomycetes. A decrease in the quantity of plaques in the encysted zoospore (non-morphogenic stage) during its developmental progression into morphogenic stages, both in germination and regeneration processes, asserted the notion that plaques function as the organization centres and are related to the reorganization of cell structure and the transition of the cell into a new stage. Furthermore, polymerization of filamentous-form during emergence stages in zoospore regeneration process revealed that filaments render motility to a developing zoospore. This unprecedented function of filaments in the developing zoospores was demonstrated using nicotinamide (0.8 × 10⁻⁶ m), which did not cause actin disruption, but could induce zoospore encystment, and its further replacement with water triggered the zoospore emergence process. Additionally, by using latrunculin B, an actin polymerization inhibitor, we also demonstrated the functional necessity of actin during various developmental processes in Aphanomyces.