Bacterial mesosomes: Method dependent artifacts

The occurrence of mesosomes was investigated during septum formation of vegetative and sporulating cells of Bacillus cereus. It has been demonstrated that bacterial mesosomes which are considered by numerous microbiologists as an integrated constituent of Gram positive bacteria, are in reality artifacts arising during the preparation for electron microscopy. The conventional fixation methods allowed enough time for the cytoplasmic membrane to react to the changed conditions and to form the typical pocket-like membrane invaginations. With cryofixation followed by freeze-substitution it was shown in ultrathin sections that mesosomes do not occur. The extremely rapid freezing and the substitution of the ice by an organic solvent containing the fixative prevented the formation of membraneous artifacts.Non-standard abbreviations OsO₄ osmium tetroxide - UO₂Ac uranylacetate - PHB poly--hydroxy-butyric acid - M mesosome - CW cell wall - CM cytoplasmic membrane - PF plasmatic fracture of the cytoplasmic membrane     

CYTOCHALASIN B INFLUENCES DICTYOSOMAL VESICLE PRODUCTION AND MORPHOGENESIS IN THE DESMID EUASTRUM1

Cytochalasin B (CB) applied to young developing cells of the desmid Euastrum oblongum Ralfs ex Ralfs, at concentrations that do not entirely inhibit cytoplasmic streaming, retarded cell growth and caused malformations of cell shape. While the basic symmetry of the cell was maintained, only the first indentations were formed and the cell body appeared to be swollen. Electron microscopic investigations revealed that vesicle production at the dictyosomes was disturbed by cytochalasin. In contrast to untreated control cells, where vesicles with electron-dense contents (“dark vesicles”) were formed during primary wall formation, vesicles pinched off by the dictyosomes during CB treatment exhibited an “empty” appearance. These vesicles, which correspond to the “dark vesicles” in size, were accumulated around the dictyosomes without being transported to the plasma membrane and were frequently connected to the trans-cisternae of the Golgi bodies. We speculate that CB may influence the transfer of products from the endoplasmic reticulum (ER) to the dictyosomes via transition vesicles, which results in a disturbed vesicle production at the Golgi bodies. CB also causes a shift in ER and dictyosome distribution. Moreover, a cortical actin system appears to be involved in the cell shaping of Euastrum. The arrangement of microtubules around the nucleus is not affected by the drug.     

Microinjection of profilins from different sources into the green algaMicrasterias causes transient inhibition of cell growth

Summary Recombinant profilins from different sources (Betula verrucosa, Schizosaccharomyces pombe, Acanthamoeba castellani, or man) cause marked effects on cell growth and morphogenesis when microinjected into growing cells of the green algaMicrasterias denticulata. Whereas control injections with -lactoglobulin only result in a slight delay of cell growth, when profilin is injected cell differentiation ceases and only resumes about 1 to 2 h after the injection, depending on the dose. The resulting cell does not show any malformations, but is reduced in size and retarded in differentiation compared to controls. As a consequence of the profilin microinjection the pattern of cytoplasmic streaming and cytoplasmic structure are also altered. Gelsolin, injected for comparison, leads to minor retardation of cell development but produces less marked effects than profilin. Microinjection of fluorescently labeled profilin shows even distribution throughout the cytoplasm and more intense fluorescence in the nucleus. Electron microscopical investigations of cells fixed immediately after profilin injection show a normal distribution of dictyosomes, ER cisternae, microtubules, and secretory vesicles compared to noninjected controls at the same developmental stage. Our results indicate that disturbance of the natural actin turnover by the injection of actin-binding proteins strongly affects development ofMicrasterias, corroborating a key role of actin in the morphogenetic process.     

The endomembrane system of differentiating carposporangia in the red algaChondria tenuissima: Occurrence and participation in secretion of polysaccharidic and proteinaceous substances

Summary The endomembrane system during carposporogenesis inChondria tenuissima was studied using electron microscopy and histochemistry. Profiles of the nucleus are convoluted, resulting in a highly increased surface area. Stacked cisternae are found within the peripheral part of the nucleus. Vesicles, tubules and membrane bound fibrillar bodies occur within the nucleoplasm. The endoplasmic reticulum surrounds the nuclear envelope.The endoplasmic reticulum and the Golgi apparatus, together with small transition vesicles, represent a functional unit. They form two different secretory substances during carposporogenesis. In young stages, carbohydrates are produced by normal dictyosomes within large, normal exocytotic Golgi vesicles. They do not react positively with PAS or Thiéry method and are believed to represent cell wall material. In later stages, the central area of the Golgi cisternae becomes filled with electron dense material. The individual cisternae are transformed into cored vesicles at the trans-face of the dictyosomes. The dense core of the vesicles is proteinaceous and stains with coomassie brilliant blue R. The peripheral fibrillar material is polysaccharidic and reacts positively using the Thiéry method. The contents of the cored vesicles are believed to participate in carpospore attachment. The ER gives rise to cytolysosomes in which starch grains are sequestrated and digested. Mucilaginous sacs seem to be similarly formed.     

Different extent of F-actin bundling in walled cells and in protoplasts ofMougeotia scalaris

Summary F-actin was localized inMougeotia interphase cells by rhodamine phalloidin (RLP) using an extended, formaldehyde-based fixation protocol, which included a minimal concentration of 0.05% (v/v) glutardialdehyde and stabilization of the calcium-binding vesicles by presaturation with neutral red. Staining revealed a low level of RLP-fluorescence throughout the cytoplasm. An enhanced level of RLP-fluorescence was found around the nucleus and in mostly two parallel fringes along each longitudinal chloroplast edge; also close to the chloroplast edge, quite regularly spaced patches of RLP-fluorescence were seen possibly associated with dictyosomes. The diffuse staining indicates lack of F-actin bundles inMougeotia filamentous cells, in contrast toSpirogyra interphase cells orMougeotia protoplasts. The observations upon staining with RLP confirm previous findings by electron microscopy and indicate seemingly single actin filaments throughout the entireMougeotia filamentous cell. Thus, a special F-actin organization is evident here which for the chloroplast movement is in support of the hypothesis of pigment regulated plasmalemma anchorage sites to actin filaments.Abbreviations CaBV calcium-binding vesicle - DIC differential interference contrast - EGTA ethyleneglycol-bis-(-aminoethyl ether) N, N, N, N tetraacetic acid - FA formaldehyde - GA glutardialdehyde - MFSB microfilament stabilizing buffer - PIPES piperazine-N, N-bis(2-ethanesulfonic acid) - RLP rhodamine (labeled) phalloidin Dedicated to the memory of Professor Oswald Kiermayer     

Involvement of actin filaments in chloroplast division of the algaClosterium ehrenbergii

Summary Studies have been made of whether actin filaments and microtubules are involved in the chloroplast division ofClosterium ehrenbergii (Conjugatae). Fluorostaining with rhodamine-phalloidin showed 5 types of localization of F-actin: (1) cables of actin filaments running in the cortical cytoplasm along the cell's long axis, (2) condensed actin filaments at the septum, (3) perinuclear distribution of actin filaments, (4) F-actins in a marking pin-like configuration adjacent to the nucleus of semicells just before completion of chloroplast kinesis, and (5) actin filaments girdling the isthmus of the constricted and dividing chloroplasts. Cytochalasin D (CD) at a concentration of 6 to 25 M caused significant disruption of actin filaments and the arrest of chloroplast kinesis, nuclear division, septum formation and cytoplasmic streaming within 3 to 6h. Chloroplast kinesis and cytoplasmic streaming recovered when cells were transferred to the medium without CD after CD treatment, or were subjected to prolonged contact with CD for more than 9h. In these cells there was a coincidental reappearance of actin filaments. A tubulin inhibitor, amiprophos-methyl at 330 M, did not inhibit chloroplast kinesis but did inhibit division and positioning of the nucleus. These results suggest that actin filaments do play a role in the mechanism of chloroplast kinesis but that microtubules do not appear to be involved in the process.Abbreviations APM amiprophos-methyl - CD cytochalasin D - DAPI 4,6-diamidino-2-phenylindole - DIC Nomarski differential interference contrast - DMSO dimethyl sulfoxide - Rh-Ph rhodamine-phalloidin     

Coated vesicles and other cytoplasmic components of growing root hairs of radish

Summary Root tips of radish,Raphanus sativus, were fixed in glutaraldehyde followed by osmium tetroxide. The fine structure of young root hairs, not exceeding about 130, in length, was studied to relate their apical growth pattern to their cytoplasmic organization. The cytoplasm in the terminal 3–5 it of the root hair is characterized by an electron dense matrix in which lie numerous smooth-surfaced vesicles, large irregularly-shaped fibrous inclusions, and clusters of ribosomes. Other organelles are largely or entirely excluded from this region. Farther than about 5, from the tip, the hair cytoplasm is filled with plastids, rough endoplasmic reticulum, mitochondria, and dictyosomes. The latter produce smooth vesicles similar in size and morphology to those present in the apical dome. Vesicles of a different kind appear in the peripheral cytoplasm along the entire length of the hair. These vesicles possess an alveolate or chambered coat about 20 m thick and have a diameter of about 85 m, including coat. They originate by evagination from the large, smooth-surfaced vesicles in the vicinity of dictyosomes. It is suggested that proteins and carbohydrates are concentrated in the dictyosomes and then segregated in the smooth vesicles released from the dictyosome cisternae. The coated vesicles which bud from the smooth vesicles may serve to isolate the proteins and transport them to the hair surface for participation in wall synthesis. The smooth vesicles are believed to convey carbohydrates to the region of active wall extension at the hair apex.This work was supported in part by grant GM-10493 from the National Institutes of Health. United States Public Health Service, to Dr. H. T. Bonnett, Jr., and grant RG-628 from the National Science Foundation to Dr. E. H. Newcomb.     

Effects of methyl benzimidazoIe-2-yl carbamate on microtubule and actin cytoskeleton inAspergillus nidulans

Summary The effects of methyl benzimidazole-2-yl carbamate (MBC) on microtubule and actin cytoskeleton were analyzed by indirect immunofluorescence and transmission electron microscopy in a wild-type strain and a benomyl-resistant mutant (benA ₁₀) ofAspergillus nidulans. The treatment of the wild-type strain with sublethal doses of MBC not only caused depolymerization of cytoplasmic microtubules (MTs), but also changed the pattern of actin at the hyphal tips. In the MBC-treated hyphae, the actin fluorescence was concentrated at the very tip region of the hypha, whereas in the control hyphae, the actin fluorescence was weak at the very tip and strong below the tip. The dose of MBC used for the wild-type strain did not depolymerize the MTs or modify the actin organization at the apex in the mutant strain, which confirmed that the change in actin distribution in the wild-type strain was due to the disruption of MTs. In the mutant strain, a seven times higher concentration of MBC than in the wild-type strain was required to depolymerize MTs and to alter the actin organization at the apex. The ultrastructural study of the MBC-treated hyphae revealed that the area containing apical vesicles was larger and the number of microvesicles was higher than in control hyphae. These changes probably resulted from the disassembly of MTs and the reorientation of actin cytoskeleton in MBC-treated apexes and suggested that MTs would organize the actin at the apex, which in turn would restrict the vesicle fusion to a narrow area at the hyphal tip. In treated hyphae of both strains without cytoplasmic MTs, mitotic spindles were detected although in lower number and with slightly modified morphology.Abbreviations DAPI 4,6-diamidino-2-phenylindole - DMSO dimethyl sulfoxide - EM electron microscopy - ER endoplasmic reticulum - IIP indirect immunofluorescence - MBC methyl benzimidazole-2-yl carbamate - MTs microtubules     

Ultrastructure and cell wall composition in cell division cycle mutants ofSchizosaccharomyces pombe deficient in septum formation

A number of temperature-sensitive cdc^- mutants ofSchizosaccharomyces pombe that are affected in septum formation were analyzed with respect to their ultrastructure and the composition of their cell wall polymers. One mutant strain, cdc 16–116, has a cell wall composition similar to the wild type (strain 972 h^-). However two other mutants, cdc 4 and cdc 7, show a higher galactomannan content and a lower -glucan content. In all the mutants tested, total glucose incorporation, protein, RNA and DNA synthesis increased similarly to wild type over 3 1/2 h. After 2–3 h of incubation at the non permissive temperature-35°C-, cell numbers remained constant although, increases in optical densities at 600 nm were observed. According to scanning electron microscopy, the mutants had aberrant shapes after 5h of incubation at 35°C. Transmission electron microscopy showed that cdc 3 is unable to complete septum formation. cdc 4 showed the most varied morphological shapes and aberrant depositions of cell wall material. cdc 8 exhibited a deranged plasma membrane and cell wall regions near of cell poles; an abnormal septum and several nuclei. cdc 7 showed elongated cells with several nuclei and with an apparently normal cell wall completely lacking in septum and septal material. cdc 16 showed more than one septum per cell.     

The Golgi apparatus in developing endosperm of wheat (Triticum aestivum L.)

The ultrastructure and distribution of the Golgi apparatus in developing wheat endosperm was investigated using a zinc iodide-osmium tetroxide staining complex in conjunction with low and high voltage electron microscopy. Dictyosomes were numerous in starchy endosperm and aleurone at 15 days after anthesis, and during the period of rapid storage protein deposition 25 d after anthesis. Fewer dictyosomes were seen in maturing endosperm. Two types of vesicles were associated with the dictyosomes; small, heavily-stained vesicles were sited at the ends of fine tubules which extend from the cisternae, and larger less-stained vesicles were associated with the periphery of the cisternae. Stereo-pairs of micrographs up to 1 m thick were taken to demonstrate the interconnections between cisternal and tubular endoplasmic reticulum. Elements of tubular ER were closely associated with dictyosomes, but connections were not observed. These results are discussed in relation to the transport of endosperm storage proteins from their site of synthesis on the cisternal ER to their site of storage, the protein bodies.     

Development of the pellicle and thecal plates following ecdysis in the dinoflagellateGlenodinium foliaceum

Summary The ultrastructure and development of the amphiesma of the dinoflagellateGlenodinium foliaceum was studied using conventional electron microscopy and immunocytochemistry. Ecdysis (shedding of the flagella, the outer two membranes of the cell, and the thecal plates) was induced by centrifugation. The cells were resuspended and the thickening of the pellicle and the development of the new thecal vesicles and plates was studied over a 9 h period. After ecdysis, the thin pellicle which underlay the thecal plates in the motile cells thickens to form a complex structure of four distinct layers: an outer layer of randomly oriented fibrils, a 50 nm layer of fibrils oriented perpendicular to the dense layer, the dense layer which has a trilaminate structure, and a wide inner homogeneous layer. The new thecal vesicles form in these pelliculate cells by the migration of electron translucent amphisomal vesicles over the layer of peripheral microtubules to a position directly under the plasmalemma. The thecal vesicles then flatten and elongate. A discontinuous pellicular layer appears within them. Subsequently, the thecal vesicles widen and are filled with a fibrillogranular substance overlying the pelliculate layer. The thecal plates form on top of this fibrillogranular material. By this time, most cells have escaped from the pellicle and are motile. At first, the outer thecal vesicle membrane is continuous with the inner thecal vesicle membrane at the sutures, but when this connection is broken, the dense pelliculate layers become continuous across the suture as does the inner thecal vesicle membrane. At ecdysis, this membrane becomes the new plasmalemma of the cell. Cells at each stage of pellicle thickening and thecal development were labelled with a polydonal antiserum raised against the 70 kDa epiplasmic protein ofEuglena acus. This antiserum labelled both the thecal plates of the motile cells and the inner homogeneous layer of the pellicle of ecdysed non-motile cells. No other amphiesmal structure was labelled, nor was any intracellular compartment.Abbreviations PBS phosphate-buffered saline - PIPES piperazine-N,N-bis[2-ethane sulfonic acid]     

Ultrastructure of mitosis and cytokinesis inZygnema sp. (Zygnematales,Chlorophyta)

Summary Mitosis and cytokinesis have been studied in the green algaZygnema C. A. Agardh using interference-contrast light and transmission electron microscopy. At prophase, the nucleolus disintegrates and numerous extranuclear microtubules near the nuclear periphery penetrate into the nucleoplasm. When aligned in the equatorial plane of the open metaphase spindle the chromosomes are coated with persistent nucleolar fragments. At anaphase, vacuoles intrude into the interzonal spindle region and seemingly contribute to the anaphase movement of the chromosomes. At telophase, the spindle is persistent and the reforming nuclei are separated by cytoplasmic strands containing microtubules, interspersed with vacuoles. Extensive bundles of microtubules, dictyosomes and parallel, slightly inflated ER-profiles extend from the poles of the telophase nucleus along the longitudinal side of the chloroplast. Conceivably, these microtubules guide the nucleus during its post-mitotic migration towards its central interphase position between the two halves of the dividing chloroplast. Throughout the mitotic cycle, ubiquitous dictyosomes, positioned near the chloroplast core, seem very active. Arrays of microtubules run towards these dictyosomes and may conduct the dictyosome-vesicles to the cleavage plane. At metaphase, septum growth becomes visible as an annular ingrowth of the plasmalemma. At late telophase or at entering interphase, an extensive clump of vesicles, associated with longitudinal bundles of microtubules, appears between the leading edges of the advanced furrow. Apparent fusion of these vesicles with the head of the centripetally-growing furrow results in its completion. The pattern of mitosis and cytokinesis inZygnema is compared with that of closely related green algae.     

Hyphal fusion during initial stages of trap formation in Arthrobotrys oligospora

Hyphal fusion during initial stages of trap formation by Arthrobotrys oligospora was studied by video-enhanced contrast and electron microscopy. Trap initials grew perpendicularly to the parent hypha, then curved around and anastomosed with a peg that developed on the hypha. Trap initials usually developed 40–140 m apart while the anastomosis occurred 20–25 m from the initial. Vigorous cytoplasmic movements in trap initials and developed traps corresponded to intense staining with fluorescein diacetate (FDA) of these cells. In addition, bundles of microfilaments were seen in developing loops of traps. On fusion organelle migration took place from the tip cell of the trap into the peg. Later on a septum was formed at the site of fusion.     

Relationship of actin organization to growth in the two forms of the dimorphic yeastCandida tropicalis

Summary Candida tropicalis is a dimorphic yeast capable of growing both as a budding yeast and as filamentous hyphae depending upon the source of the carbon used in the culture medium. The organization of F-actin during growth of the yeast form (Y-form) and the hyphal form (H-form) was visualized by rhodamine-conjugated phalloidin by using a conventional fluorescence microscope as well as a laser scanning confocal fluorescence microscope. In single cells without a bud or non-growing hyphae, actin dots were evenly distributed throughout the cytoplasm. Before the growth of the bud or hypha, the actin dots were concentrated at one site. During bud growth, actin dots were located solely in the bud. They filled the small bud and then filled the apical two-thirds of the cytoplasm of the middlesized bud. During growth of the large bud, actin dots which had filled the apical half of the cytoplasm gradually moved to the tip of the bud. In the formation of the septum, actin dots were arranged in two lines at the conjunction of the bud and the mother cell. During hyphal growth, the majority of actin dots were concentrated at the hyphal apex. A line of clustered spots or a band of actin was observed only at the site where the formation of a new septum was imminent. This spatial and temporal organization of actin in both categories of cells was demonstrated to be closely related to the growth and local deposition of new cell wall material by monitoring the mode of growth with Calcofluor staining. Treatment of both forms of cells with cytochalasin A (CA) confirmed the close relationship between actin and new cell wall deposition. CA treatment revealed lightly stained unlocalized actin which was associated with abnormal cell wall deposition as well as changes in morphology. These results suggest that actin is required for proper growth and proper deposition of cell wall material and also for maintaining the morphology of both forms of cells.Abbrevations FM fluorescence microscopy - EM electron microscopy - rh rhodamine - CA cytochalasin A - CD cytochalasin D - PBS phosphate-buffered saline - DMSO dimethylsulfoxide - GA glutaraldehyde     

Actin cytoskeleton is responsible for the change of cytoplasmic organization in root hair cells induced by a protein phosphatase inhibitor, calyculin A

Summary In root hair cells ofLimnobium stoloniferum, a protein phosphatase inhibitor, calyculin A (CA), at concentrations higher than 50 nM inhibits cytoplasmic streaming and induces remarkable morphological changes in the cytoplasm: the transvacuolar strands disperse and spherical cytoplasmic bodies emerge. The mechanism of the morphological changes of the cytoplasm induced by CA was studied by pharmacological analyses. The formation of spherical bodies in cells treated with CA was suppressed by the actin-depolymerizing and -fragmenting drugs latrunculin B and cytochalasin D at concentrations higher than 100 nM and 5 M, respectively. In contrast, 100 M propyzamide, a microtubule-depolymerizing drug, did not affect the formation of spherical bodies by CA. Interestingly, 60 mM 2,3-butanedione monoxime, an inhibitor of myosin, also suppressed the CA-induced formation of cytoplasmic spherical bodies. These results indicate that the actin cytoskeleton is intimately involved in the morphological changes of the cytoplasm induced by CA.Abbreviations APW artificial pond water - BDM 2,3-butanedione monoxime - CD cytochalasin D - DMSO dimethylsulfoxide - LB latrunculin B - Pro propyzamide     

Numerical and structural changes in dictyosomes during zygospore germination ofClosterium ehrenbergii

Summary Numerical and structural changes in dictyosomes during the germination of zygospores inClosterium ehrenbergii were examined by electron microscopy. In the dormant mature zygospores, two parallel cisternac were seen which were derived from the disorganization of dictyosomes during the maturation of zygospores. After the induction of germination, the two parallel cisternae developed into dictyosomes with ten or eleven cisternae. The dictyosomes doubled in number by division every day for four days and reached, at the time of germination, a density of distribution similar to that found in the youngest zygospore. On the 4th day after the induction of germination, dictyosomes produced two kinds of vesicles which appear to be involved in the formation of new cell wall layers. The germination of the zygospore was effected by the escape of the cell covered with the new cell wall layers through the broken old cell wall layers.     

The electron microscopic and classic Golgi apparatus

The relationship of the membrane structure, designated in electron microscopy as the Golgi apparatus, to the classic Golgi apparatus in the light microscope were studied withFagopyrum. Comparison of these structures in plant cells with the same or similar structures in animal cells led to the following conclusions: there exist two groups of formations, impregnable with osmium or silver, considered as the classic Golgi apparatus. The first group contains the active membrane structures. These are the dictyosomes and the anastomosing form of the electron microscopic Golgi apparatus. To this group belongs also the endoplasmatic reticulum, which in plant cells forms dense vacuoles, having the appearance of the classic Golgi apparatus, and in animal cells occasionally has a similar arrangement as the anastomosing form of the Golgi apparatus. The second group comprises formation containing reserve and secretion material, i.e. predominantly products of the activity of the electron microscopic Golgi apparatus and of the endoplasmic reticulum (matter of the dense vacuoles, lipochondria, secretory granula etc.). In the plant cells, especially ofFygopyrum, the dictyosomes contained in the structures of the first group are separated from the formations of a reserve character in the second group, formed in the lumen of the endoplasmic reticulum (dense vacuoles). The identity of the dictyosomes with the osmiophilic platelets, considered by some authors in the light microscope as the classic Golgi apparatus, has not been proved up to present, because of the one-sidedness of the methods used nowadays. WithFagopyrum no foundation has been observed for the assumed formation of net-form structures by grouping of the dictyosomes. Structures similar to the net-form of the classic Golgi apparatus in the animal cell form only dense vacuoles. On the basis of the differentiation of both types of formations in the plant cell, the foundations were laid for the characterization of the classic Golgi apparatus in the animal cell. The net-form of the classic Golgi apparatus in the animal cell is obviously not artificial, but reflects the ultrastructural arrangement of the electron microscopic Golgi apparatus or of the endoplasmic reticulum. The problem of the suitability and specification of the name Golgi apparatus in the animal and plant cell was also discussed. In contrast to the opinion of some authors, it does not appear useful to remove the name golgi apparatus, designating the dictyosomes and the anastomosing forms of the smooth membranes.     

Actin in penetration pegs of the fungal rice blast pathogen,Magnaporthe grisea

Summary The penetration peg is the structure used byMagnaporthe grisea to pierce the surface of rice leaves or very hard nonbiodegradable substrates. Penetration pegs produced by appressoria in vitro were examined by electron microscopy and immunofluorescence microscopy using various fluorophore labeled anti-actins. Freeze-substitution preparation of appressoria at early stages of substrate penetration showed that peg cytoplasm consisted primarily of a zone of exclusion, excluding even ribosomes, and was continuous with a similar region in the appressorium. Apical vesicles were, however, observed in short, presumably elongating pegs. Immunofluorescence microscopy was used to demonstrate binding of a monoclonal anti-actin to penetration peg cytoplasm, following permeabilization of appressoria by means of a brief sonication. Occasional filaments and ca. 300 nm diameter plaques were labeled in appressorial cytoplasm. Western blot analysis of germ tube extracts showed that the monoclonal probe bound predominantly to a single band with a molecular weight similar to that of rabbit muscle actin. Preincubation of the antibody with actin virtually eliminated peg labeling. We conclude that the penetration peg contains actin which may play a role in the formation of the zone of exclusion.Abbreviations PE polyethylene - Tris tris(hydroxymethyl)-amino-methane - TBS Tris-buffered saline - TBS-B Tris buffered saline plus 3% bovine serum albumin     

Dynamic changes in the actin cytoskeleton during the high-fluence rate response of theMougeotia chloroplast

Summary Since photo-induced orientation movement of a single, ribbon-shaped chloroplast in each cell of the filamentous green algaMougeotia is inhibited in the presence of cytochalasin B, actin is thought to be involved in the process of chloroplast movements. However, this possibility remains to be proved. A specific class of cytoplasmic filaments, which emerge from the advancing front of the moving chloroplast, can be seen by differential interference contrast (DIC) microscopy. However, no one has yet succeeded in defining the nature of these filaments. We have been able to stain the actin filaments (AFs) associated with the moving chloroplast with fluorescein-conjugated phalloidin (FP) after pre-treatment withm-maleimidobenzoyl N-hydroxysuccinimide ester (MBS). No filamentous structures were observed in cells that had been pre-irradiated with low-fluence rate red light. However, transversely oriented fluorescent filaments appeared at the front edge of the moving chloroplast when it began to rotate under irradiation with high-fluence rate white light. These filaments disappeared after completion of the orientation movement, suggesting the simultaneous appearance of AFs and the orientation movement of the chloroplast. Thick cytoplasmic strands connecting the edge of the chloroplast with the parietal cytoplasm were often seen by DIC microscopy before and after completion of the high-fluence rate orientation movement. These thick cytoplasmic strands could not be stained by FP, but were often stained by 3,3-dihexyloxacarbocyanine iodide (DiOC₆(3)), suggesting that they are transvacuolar strands that include endoplasmic reticulum.     

Electron microscopy of gametangial interaction and oospore development in Phytophthora capsici

Gametangial development and oospore formation were studied, with emphasis on cell wall morphogenesis, on mated cultures (A¹xA²) of Phytophthora capsici. In this species, the oogonial and antheridial hyphae interact to produce a typical amphigynous antheridium. The following developmental steps were recognized: 1) contact between oogonial and antheridial initials; 2) penetration of the antheridial initial by the oogonial initial; 3) reemergence of the oogonial initial; 4) oogonial expansion; 5) gametangial delimitation and oogonial wall thickening; 6) penetration of the oogonium by the antheridial fertilization tube; 7) oosphere formation; 8) periplasm degeneration and outer oospore wall formation; and 9) inner oospore wall formation. Electron micrographs were obtained of steps 3–9. Steps 1 and 2 were reconstructed from subsequent events. Steps 3–6 are stages of active wall formation with clear indication of intensive dictyosome activity leading to the formation of numerous wall-destined vesicles of two different sizes and electron densities. No vesicles were seen associated with the development of the inner oospore wall; however, by this stage of development the oosphere cytoplasm exhibited an overall intense electron density that obscured fine detail. Cytoplasmic appearance changed enormously during differentiation, from a developing oogonium rich in mitochondria, ribosomes, rough endoplasmic reticulum, dictyosomes and their vesicles, through an oosphere filled with large finger-print vacuoles and lipid-like bodies, to a mature oospore with a large central vacuole (ooplast) surrounded by a cortex of numerous lipid-like bodies; other organelles are confined to the interstitial space between these storage bodies.