Acyltransferase and acid hydrolase activities of the abalone photoreceptor cell

Summary In order to study the synthesis and degradation processes of the photoreceptor membranes in the abalone, Nordotis discus, the localization of acyltransferase and acid hydrolase activities, respectively, were determined at the electron-microscopic level. Acyltransferase activity was localized on the cytoplasmic sides of thick (>10 nm) membranes of the following organelles: a few cisternae at the trans (or concave) side of Golgi apparatus, Golgi and probably related vesicles, short tubules, curved pentalaminar disks and limiting membranes of the phagosomal multivesicular bodies; all organelles were scattered in the peri- to supranuclear cytoplasm. The phospholipids, which are major components of the photoreceptor membrane, are considered to be synthesized by these membranes. Acid phosphatase activity was localized in the lumina of Golgi cisternae and vesicles, lysosomes, and smaller multivesicular and related bodies, but not in multilamellar bodies. The matrices of the larger multivesicular bodies and of the pigment granule complexes showed arylsulfatase activity. Vesiculated and autophagocytosed photoreceptor microvilli seemed to be degraded by acid hydrolases, forming multivesicular and related bodies. Supporting cells also showed acyltransferase and acid hydrolase activities.Abbreviations used in this Paper AcP acid phosphatase - ArS arylsulfatase - AT acyltransferase - ER endoplasmic reticulum - GERL Golgi-endoplasmic reticulum-lysosomal complex - MEB meshwork body - MLB multilamellar body - MVB multivesicular body - VLB vesiculolamellar body     

Improved structural preservation in freeze-substituted sporidia ofUstilago avenae—a comparison with low-temperature embedding

Summary In the present study the cellular fine structure of freeze-substituted sporidia of the phytopathogenic fungusUstilago avenae is investigated by means of thin-section electron microscopy. A conventional embedding method using Spurr's low viscosity resin is compared with the recently developed methacrylate mixtures Lowicryl^® K 4 M and HM 20 resin. Generally, freeze-substitution yields improved preservation of fine structural details of the fungus compared to previously applied conventional fixation methods. Using double fixation during freeze-substitution prior to conventional embedding the fungal membrane system (plasmalemma, endoplasmic reticulum), organelles (mitochondria, nucleus etc.) and other cytoplasmic features (ribosomes, cytoskeleton) appear well resolved and smoothly contoured. Aldehyde fixed and Lowicryl embedded sporidia ofU. avenae resemble these double fixed fungal specimens fairly closely. The complete low-temperature preparation produces visualization of distinct cellular details although contrast reversal of cellular membranes (er, mitochondria etc.) is sometimes observed. In particular, fine structure resolution is enhanced in Lowicryl HM 20 embedded fungal cells. This is due also to significant improvement in staining of the cellular membranes, cytoskeleton (microfilaments and microtubules) and Golgi apparatus-like areas, using tannic acid. In case of the fungusU. avenae, freeze-substitution in combination with mild glutaraldehyde fixation and final low-temperature embedding in HM 20 resin prove suitable for improved preservation of cellular ultrastructure.Abbreviations cw cell wall - cy cytoplasm - FS freeze-substitution - FS-A GA/OsO₄ freeze-substitution and Spurr's LV-embedding - FS-B GA freeze-substitution and Lowicryl K 4 M LT-embedding - FS-C GA freeze-substitution and Lowicryl HM 20 LT-embedding - go Golgi apparatus-like body - GA glutaraldehyde - g glycogen deposit - l lipid droplet - LT low temperature - Lowicryl LT-embedding Lowicryl low-temperature embedding - Lowicryl LT-resin Lowicryl low-temperature resin - MeOH methanol - mf microfilament - mt microtubule - m mitochondrion - mvb multivesicular body - ne nuclear envelope - np nuclear pore - npl nucleoplasm - nu nucleolus - n nucleus - OsO₄ osmium tetroxide - pl plasmalemma - pr polyribosomes - Pb-citrate Reynolds' lead citrate - r ribosome - RT room temperature - rer rough endoplasmic reticulum - Spurr's LV-embedding Spurr's low viscosity embedding - Spurr's LV-resin Spurr's low viscosity resin - t tonoplast - Uac uranyl acetate - v vacuole     

Spore discharge in Cordana musae (Zimm.) H?hnel and Zygosporium oscheoides Mont.

In Cordana musae and Zygosporium oscheovides violent spore dischargeoccurs under conditions of rapidly decreasing vapour-pressure;discharge is not dependent on light. Mechanisms of dischargeare postulated. In C. musae, on drying, water evaporates frominside the conidium and conidiophore, causing a negative pressureto develop in the solutions inside these structures and thewalls to be drawn inwards under tension. The sudden appearanceof a gas bubble in one or both of these structures releasesthe tension, and the resultant jolt causes the conidium to beshot away. In Z. oscheoides similar tension and wall distortiondevelop in a specialized conidiophore cell, the vesicle, onwhich the conidia are poised. On appearance of a gas phase,the vesicle wall springs back to its original form and the conidiaare shot away. The vesicle is likened to a single annulus cellin the fern sporangium. The similarity between discharge inC. musae, Z. oscheoides, and Deightoniella torulosa is brieflydiscussed.     

Multivesicular body formation and function in the light-adapted crayfish retina: a new interpretation

Summary Quantitative TEM refutes the hypothesis of multivesicular body formation at dawn via the degradation of photoreceptor. No significant relationship exists between multivesicular bodies and rhabdom diameter or between multivesicular bodies and coated vesicles. The density of multivesicular bodies is related to the vesicle-producing Golgi bodies. A new theory concerning the formation and possible function of multivesicular bodies is discussed.     

Consecutive monitoring of lifelong production of conidia by individual conidiophores of Blumeria graminis f. sp. hordei on barley leaves by digital microscopic techniques with electrostatic micromanipulation

Conidial formation and secession by living conidiophores of Blumeria graminis f. sp. hordei on barley leaves were consecutively monitored using a high-fidelity digital microscopic technique combined with electrostatic micromanipulation to trap the released conidia. Conidial chains formed on conidiophores through a series of septum-mediated division and growth of generative cells. Apical conidial cells on the conidiophores were abstricted after the conidial chains developed ten conidial cells. The conidia were electrically conductive, and a positive charge was induced in the cells by a negatively polarized insulator probe (ebonite). The electrostatic force between the conidia and the insulator was used to attract the abstricted conidia from the conidiophores on leaves. This conidium movement from the targeted conidiophore to the rod was directly viewed under the digital microscope, and the length of the interval between conidial septation and secession, the total number of the conidia produced by a single conidiophore, and the modes of conidiogenesis were clarified. During the stage of conidial secession, the generative cells pushed new conidial cells upwards by repeated division and growth. The successive release of two apical conidia was synchronized with the successive septation and growth of a generative cell. The release ceased after 4-5 conidia were released without division and growth of the generative cell. Thus, the life of an individual conidiophore (from the erection of the conidiophore to the release of the final conidium) was shown to be 107 h and to produce an average of 33 conidia. To our knowledge, this is the first report on the direct estimation of life-long conidial production by a powdery mildew on host leaves.     

Symbiotic vesicle ultrastructure in high pressure-frozen,freeze-substituted actinorhizae

Summary Using tissue stained en bloc with chromic acid or tissue prepared by high pressure-freezing and freeze-substitution, it was possible to analyze quantitatively the ultrastructure of symbiotic vesicle envelopes (SVE) inAlnus serrulata, Ceanothus americanus, Elaeagnus umbellata, andMyrica cerifera. The lamina measured about 4.7 nm in thickness in thin section. Despite diverse symbiotic vesicle morphology, the SVE thickness was similar in all of these symbioses: 36–71 nm, which corresponded to 6–15 laminae based on counts of chromic acid-stained SVEs. This similarity in structure suggests that a similar environmental signal regulates envelope thickness in the different root nodules. Based on previous studies, this is likely to be pO₂. Three types of envelope morphologies were distinguished: (1) theAlnus-type (as inAlnus andElaeagnus), which had localized thickenings around the vesicle and had thickest dimensions over the stalk; (2) theCeanothus-type. characterized as a relatively uniform envelope over both vesicle and attached hypha, and (3) theMyrica-type, which had no stalk region and a basal SVE thickness of about six laminae throughout except where localized thickening occurred. Localized thickening of the SVE resulted from extra numbers of laminae being deposited, generally over regions where septa contacted the edge of the vesicle. Freeze-substituted symbiotic vesicles had a variety of novel structures that are poorly preserved in chemically-fixed tissue. A paracrystalline body inAlnus symbiotic vesicles may be composed of particles that also exist free in the symbiotic vesicle cytoplasm. In addition, a previously unknown complex at the base of theAlnus-type symbiotic vesicle and within its stalk was evident in freeze-substituted tissues.Abbreviations HPF/FS high pressure-frozen/freeze-substituted - SV symbiotic vesicle - SVE symbiotic vesicle envelope Dedicated to the memory of Professor John G. Torrey     

The conserved oligomeric Golgi complex is involved in penetration resistance of barley to the barley powdery mildew fungus

Membrane trafficking is vital to plant development and adaptation to the environment. It is suggested that post‐Golgi vesicles and multivesicular bodies are essential for plant defence against directly penetrating fungal parasites at the cell wall. However, the actual plant proteins involved in membrane transport for defence are largely unidentified. We applied a candidate gene approach and single cell transient‐induced gene silencing for the identification of membrane trafficking proteins of barley involved in the response to the fungal pathogen Blumeria graminis f.sp. hordei. This revealed potential components of vesicle tethering complexes [putative exocyst subunit HvEXO70F‐like and subunits of the conserved oligomeric Golgi (COG) complex] and Golgi membrane trafficking (COPIγ coatomer and HvYPT1‐like RAB GTPase) as essential for resistance to fungal penetration into the host cell.     

Ultrastructural aspects of the hyphal tip ofSclerotium rolfsii preserved by freeze substitution

Summary The hyphal tip ofSclerotium rolfsii was examined after fixation by freeze substitution. The Spitzenkörper consisted of a dense mass of apical vesicles and microvesicles surrounding a vesicle-free zone. Linear arrangements of microvesicles were occasionally observed within the Spitzenkörper. Abundant microfilaments were seen within the Spitzenkörper region, often in close association with apical vesicles and microvesicles. Microtubules passed through the Spitzenkörper and terminated at the plasmalemma at the extreme hyphal apex. Filasomes were mostly observed within the apical region and were in close proximity to the plasmalemma. Rough ER, mitochondria, microtubules, and vacuoles were abundant in the subapical region and were usually oriented parallel to the long axis of the hypha. Ribosomes were aligned on the outer surfaces of mitochondria. Golgi body equivalents were observed throughout the subapical region and appeared as inflated cisternae of varying shapes and electron opacities. Relationships to other basidiomycetous hyphal tip cells are discussed.Abbreviations AV apical vesicle - C Celsius - diam diameter - f filasome - G Golgi body equivalent - h hour - nm nanometer - M mitochondria - ME membranous elements; min minute - MV microvesicle - MVB multivesicular body - N nucleus - OsO₄ osmium tetroxide - R ribosome - ER endoplasmic reticulum - S Spitzenkörper - Va vacuole - m micrometer     

Association of polyphosphate with protein in freeze-substituted sclerotia ofSclerotinia minor

Summary In freeze-substituted sclerotia stained with aqueous toluidine blue O, metachromatic material was found throughout the cytoplasm in discrete granules. It was also distributed evenly throughout spherical and elongate protein bodies. This material stained at low pH and was extracted by cold acid, indicating that it was polyphosphate. Retention of metachromatic material was much greater than previously reported in chemically fixed, conventionally processed sclerotia. X-ray microanalysis of dry-cut, unstained sections of freeze-substituted sclerotia confirmed that phosphorus was distributed evenly throughout the protein bodies and was not localised in discrete granules but phosphorus levels in the cytoplasm were very low. It is concluded that polyphosphate is lost during conventional preparation procedures but retained in dry-cut, unstained sections of freeze-substituted material. However, when freeze-substituted sections were stained with toluidine blue O, water soluble polyphosphate was extracted and subsequently precipitated in the cytoplasm as polyphosphate granules. Therefore it is considered that polyphosphate granules are an artefact, and that protein bodies are the major site for storage of phosphorus in this fungus.Abbreviations STEM scanning transmission electron microscope - ER endoplasmic reticulum     

FUNCTIONS OF COATED VESICLES DURING PROTEIN ABSORPTION IN THE RAT VAS DEFERENS

The role of coated vesicles during the absorption of horseradish peroxidase was investigated in the epithelium of the rat vas deferens by electron microscopy and cytochemistry. Peroxidase was introduced into the vas lumen in vivo. Tissue was excised at selected intervals, fixed in formaldehyde-glutaraldehyde, sectioned without freezing, incubated in Karnovsky's medium, postfixed in OsO₄, and processed for electron microscopy. Some controls and peroxidase-perfused specimens were incubated with TPP,¹ GP, and CMP. Attention was focused on the Golgi complex, apical multivesicular bodies, and two populations of coated vesicles; large (> 1000 A) ones concentrated in the apical cytoplasm and small (<750 A) ones found primarily in the Golgi region. 10 min after peroxidase injection, the tracer is found adhering to the surface plasmalemma, concentrated in bristle-coated invaginations, and within large coated vesicles. After 20–45 min, it is present in large smooth vesicles, apical multivesicular bodies, and dense bodies. Peroxidase is not seen in small coated vesicles at any interval. Counts of small coated vesicles reveal that during peroxidase absorption they first increase in number in the Golgi region and later, in the apical cytoplasm. In both control and peroxidase-perfused specimens incubated with TPP, reaction product is seen in several Golgi cisternae and in small coated vesicles in the Golgi region. With GP, reaction product is seen in one to two Golgi cisternae, multivesicular bodies, dense bodies, and small coated vesicles present in the Golgi region or near multivesicular bodies. The results demonstrate that (a) this epithelium functions in the absorption of protein from the duct lumen, (b) large coated vesicles serve as heterophagosomes to transport absorbed protein to lysosomes, and (c) some small coated vesicles serve as primary lysosomes to transport hydrolytic enzymes from the Golgi complex to multivesicular bodies.     

MUCILAGE PROCESSING AND SECRETION IN THE GREEN ALGA CLOSTERIUM. II. ULTRASTRUCTURE AND IMMUNOCYTOCHEMISTRY1

We conducted an ultrastructural and immunocytochemical analysis of the subcellular components involved in mucilage secretion in Closterium. In conventionally fixed cells, the mucilage vesicle appears dense-cored with an electron-dense center surrounded by radiating fibrils. In freeze-substituted cells, the vesicles are highly osmiophilic. These mucilage vesicles are produced from peripheral swellings of the trans face cisternae of the Golgi apparatus (GA). The vesicles apparently move from the GA, found in cytoplasmic depressions between lobes of the plastid, to the sub-plasma membrane peripheral cytoplasm. Here, they become associated with components of the peripheral cytoskeletal network. The mucilage is ultimately released through flask-shaped pores in the cell wall.     

Anatomy and ultrastructure of the male reproductive system in Pleioplana atomata (Platyhelminthes: Polycladida)

Abstract. The ultrastructure of the male reproductive system in the polyclad flatworm Pleioplana atomata is described. Numerous testes are scattered throughout the entire body but are heavily concentrated on the ventral side. All stages of differentiating sperm cells are present in all testes follicles. Intercellular bridges connect spermatocytes and spermatids derived from a single spermatogonium. In the distal part of spermatids, a zone of differentiation develops with a row of microtubules beneath the plasmalemma. Adjacent to these microtubules, an intercentriolar body is flanked by two basal bodies that give rise to two axonemes (each with a 9+“1” microtubular pattern) that face in opposite directions. The Golgi complex appears in the central portion of the spermatid and produces numerous small and large electron-dense bodies. The small bodies surround the nucleus, whereas the large bodies cluster along with the mitochondria in the central part of the spermatid. Development of the spermatid leads to cell elongation and formation of a filiform, biflagellate mature spermatozoon with cortical microtubules all along the sperm shaft. The male canal system consists of paired vasa deferentia that separately enter a single seminal vesicle. A single prostatic canal connects the seminal vesicle to the prostatic vesicle. Ultrastructurally, the seminal vesicle and prostatic canal are very similar, and along with the prostatic vesicle and stylet pocket, are lined by a ciliated epithelium. The ultrastructure of the prostatic vesicle indicates that it probably produces a large volume of seminal fluid that, along with spermatozoa, is transferred to the mating partner through a stylet. Some of the findings, particularly on sperm ultrastructure, may provide characters useful for phylogenetic analysis.     

FINE STRUCTURE OF ANNELLOPHORES. I. SCOPULARIOPSIS BREVICAULIS AND S. KONINGII

Fine-structure observations of annelloconidium production in filamentous Hyphomycetes are reported for the first time. The difference in conidium morphology between Scopulariopsis brevicaulis and S. koningii was quite distinct. In S. brevicaulis, verrucosities appeared early in conidium ontogeny and formed an integral part of the primary wall layer of mature conidia. In S. koningii, verrucosities were absent. In S. brevicaulis, annellations did not invariably result on conidiophore necks with the production of each conidium in the basipetal sequence, but alternatively could be left on subapical regions of subsequently formed conidia. In S. koningii, annellations were more distinct, and the position of a conidium-delimiting septum was variable. If a septum were formed at a position proximal to previous septa, a portion of the annellophore neck remained attached to the base f the seceding conidium. In both species, a spherical electron-dense body, perhaps analogous to septal pore plugs in vegetative hyphae, plugged the pore between conidia and conidiophores and remained embedded in the base of seceded conidia.     

Developmental features of protophloem sieve elements in roots of wheat (Triticum aestivum L.)

Summary Protophloem sieve elements (PSEs) in roots of wheat (Triticum aestivum L.) are arranged in single vertical files. The number of PSEs within the files increases by symmetrical divisions, which take place after the completion of asymmetrical (formative) divisions and before the initiation of differentiation. The divisions are preceded by well defined pre-prophase bands (PPB) of microtubules, which surround the nucleus in an equatorial position. In the cytoplasmic region between the nuclear surface and the PPB, perinuclear and endoplasmic microtubules were observed. The perinuclear microtubules are considered as part of the developing spindle, while the endoplasmic ones interlink the perinuclear microtubules with the PPB. Dividing cells do not show any signs of incipient differentiation. The first and most reliable indication of a commencing differentiation is provided by the sieve-element plastids that begin to accumulate dense crystalloid inclusions in the very young PSEs. In mature PSEs plastids contain two kinds of crystalloid inclusions, dense and thin, in a translucent stroma. Depending on the plastid-inclusions criterion it was shown that: (a) the PSEs of a given root do not initiate differentiation at exactly the same stage, (b) the developmental sequence extends to a span of 7–9 actively differentiating PSEs arranged in a single vertical file, and (c) each PSE needs about 16–21 h to pass through the whole developmental sequence. In the last two differentiating PSEs of a file, mitochondria were found to be enveloped by single cisternae of ER. The association is temporary as it is lost in the first PSEs with an autolysed lumen. During differentiation, Golgi bodies were abundant and active in producing vesicles involved in cell wall development. Golgi vesicles were also found among the microtubules of the PPB, but no local thickening was observed. Golgi bodies disorganize in the last stages of autolysis and disappear in mature sieve elements.Abbreviations ER endoplasmic reticulum - MSE metaphloem sieve element - PPB pre-prophase band - PSE protophloem sieve element Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Ultrastructural studies on oogenesis in the shrew (Crocidura russula): I. The preantral follicle

Ultrastructural analysis of the ovarian follicle prior to antrum formation in the shrew, Crocidura russula, shows gradual differentiation of mitochondria, endoplasmic reticulum, Golgi complexes, vesicles, and multivesicular bodies correlated with the growth of the oocyte from primordial to tertiary follicle and the development of the follicular wall. The growth rate of the follicle in relation to that of the oocyte was found to be biphasic.     

Ultrastructure of freeze-substituted hyphae of the basidiomyceteLaetisaria arvalis

Summary The ultrastructure of freeze-substituted (FS) hyphae ofLaetisaria arvalis is described and compared to that of similar hyphae preserved by conventional chemical fixation (CF). The outline of membrane-bound organelles as well as the plasma membrane was smooth in FS cells. In contrast, hyphae preserved by CF exhibited membrane profiles that were extremely irregular. Centers of presumed Golgi activity were best preserved by FS. Microvesicles, 27–45 nm diameter and hexagonal in transverse section, were observed most readily in FS cells. Filasomes (= microvesicles within a filamentous matrix) were only observed in FS cells. Apical vesicles, 70–120 nm diameter, associated with the centers of Golgi activity and within the Spitzenkörper region exhibited finely granular matrices in FS hyphae, whereas in CF hyphae the contents were coarsely fibrous and less electron-dense. Microvesicles were present at hyphal apices and regions of septa formation. Filasomes were also found at regions of septa formation as well as along lateral hyphal tip cell walls. Microvesicles, but not filasomes, were observed in membrane-bound vesicles (= multivesicular bodies) and in larger vacuoles. Filaments, 5.2–5.4 nm wide, were juxtaposed with centripetally developing septa. Cytoplasmic inclusions, 20–40 m in length, composed of bundles of 6.7–8.0 nm wide filaments were observed in both FS and CF hyphae.     

Improved preservation of the form and contents of wall vesicles and the golgi apparatus in freeze substituted hyphae ofSaprolegnia

Summary Secretory vesicles involved in cell wall synthesis (wall vesicles) and the Golgi apparatus have been compared in conventionally fixed and freeze substituted hyphae of the oomycete fungusSaprolegnia ferax. Wall vesicles freeze substituted in various fluids range from spherical to tubular and contain an intensely staining, phosphorous rich matrix. In contrast diverse conventional fixations cause artefactual constrictions in most tubular vesicles and loss of their intensely staining contents. These data are interpreted to show the existence of an intravesicular skeletal system, with cellular regulation, to determine vesicle morphology and intravesicular synthesis of a hypothetical phosphorylated glycolipid cell wall precursor. Whilst freeze substitution gives superior preservation of wall vesicle morphology, it does not demonstrate any preferential association between wall vesicles and microtubules thus suggesting that microtubules are only indirectly involved in wall vesicle transport. Freeze substitution is superior to conventional fixation for analysis of the Golgi apparatus because it uniquely reveals both differentiation of a specific single cisterna in each Golgi body and greater differences in membrane thicknesses throughout the endomembrane system.     

Convergence of the endocytic and lysosomal pathways in soybean protoplasts

Ultrastructural analysis of endocytosis of cationized ferritin (CF) has been combined with ultrastructural localization of acid phosphatases (AcPase) in soybean (Glycine max (L.) Merr.) protoplasts. While CF is an electron-dense marker of organelles of the endocytic pathway, ultrastructural histochemistry of AcPase identifies the organelles involved in the synthesis, transport, and storage of lytic-compartment enzymes, i.e. the lysosomal pathway. Acid phosphatases have been localized using both lead- and cerium-precipitation techniques. Protoplasts have been exposed to CF for 5 min, 30 min, or 3 h and processed for AcPase localization. At 5 min, smooth vesicles contain both CF and AcPase. By 30 min, Golgi cisternae and multivesicular bodies contain both labels. By 3 h, vacuoles become labelled with both CF and AcPase. The large central vacuoles contain intraluminal membranes which are associated with both AcPase and CF. These observations extend the analogy between plant vacuoles and animal lysosomes and demonstrate the points at which the endocytic pathway of plants converges with the lysosomal pathway.Abbreviations AcPase acid phosphatase - CF cationized ferritin - ER endoplasmic reticulum - MVB multivesicular body - PCR partially coated reticulum - PM plasma membrane     

On the ultrastructure of differentiating secondary xylem in willow

Summary Studies of differentiating xylem inSalix fragilis L. show the immediate cambial derivatives to be ultrastructurally similar. The Golgi apparatus is important at all stages of wall synthesis, possibly producing (amongst other substances) hemicellulose material which is carried to the wall in vesicles or multivesicular bodies. The endoplasmic reticulum also contributes one or more components to the developing wall: at some stages during differentiation the endoplasmic reticulum produces electron opaque bodies which appear to be guided towards the wall by microtubules. Compact structures formed from concentric membranes (myelin-like bodies) have been found joined to rough endoplasmic reticulum, but their presence is not explained.Two types of plasmalemma elaboration occur: invagination of the plasmalemma itself to form vesicles which may contain cytoplasmic material; and vesicles between the plasmalemma and cell wall which are the result of single vesicles or multivesicular bodies traversing the plasmalemma. Both systems provide a means for transporting cytoplasmic material across the plasmalemma.Microtubules have been seen associated with all vesicles derived from the cytoplasm which appear to be moving towards the wall. The presence of microtubules may generally be explained in terms of orientation of vesicles, even if they also happen coincidentally to parallel the supposed orientation of microfibrils in the wall itself. It is possible to resolve connections between the microtubules and the plasmalemma.     

Organization of the actin cytoskeleton during pollen development inGasteria verrucosa (Mill.) H. Duval visualized with rhodamine-phalloidin

The three-dimensional organization of the microfilamental cytoskeleton of developingGasteria pollen was investigated by light microscopy using whole cells and fluorescently labelled phalloidin. Cells were not fixed chemically but their walls were permeabilized with dimethylsulphoxide and Nonidet P-40 at premicrospore stages or with dimethylsulphoxide, Nonidet P-40 and 4-methylmorpholinoxide-monohydrate at free-microspore and pollen stages to dissolve the intine.Four strikingly different microfilamentous configurations were distinguished. (i) Actin filaments were observed in the central cytoplasm throughout the successive stages of pollen development. The network was commonly composed of thin bundles ramifying throughout the cytoplasm at interphase stages but as thick bundles encaging the nucleus prior to the first and second meiotic division. (ii) In released microspores and pollen, F-actin filaments formed remarkably parallel arrays in the peripheral cytoplasm. (iii) In the first and second meiotic spindles there was an apparent localization of massive arrays of phalloidin-reactive material. Fluorescently labelled F-actin was present in kinetochore fibers and pole-to-pole fibers during metaphase and anaphase. (iv) At telophase, microfilaments radiated from the nuclear envelopes and after karyokinesis in the second meiotic division, F-actin was observed in phragmoplasts.We did not observe rhodamine-phalloidin-labelled filaments in the cytoplasm after cytochalasin-B treatment whereas F-actin persisted in the spindle. Incubation at 4° C did not influence the existence of cytoplasmic microfilaments whereas spindle filaments disappeared. This points to a close interdependence of spindle microfilaments and spindle tubules.Based on present data and earlier observations on the configuration of microtubules during pollen development in the same species (Van Lammeren et al., 1985, Planta165, 1-11) there appear to be apparent codistributions of F-actin and microtubules during various stages of male meiosis inGasteria verrucosa.Abbreviation DMSO dimethylsulfoxide