Abnormal spore cleavage: Abnormal spores of Allomyces macrogynus

In the aquatic phycomycete Allomyces macrogynus abnormal spore cleavage takes place in the presence of colchicine or benomyl resulting in multinucleate–multiflagellate spores due to failure in the formation of cytoplasmic microtubules after the induction of zoosporogenesis. The 27 cytoplasmic microtubules which normally surround the nucleus and nuclear cap of the mature spore are not formed in the presence of colchicine or benomyl. At high concentrations of colchicine (4–8 mg/ml) the spores do not have a flagellum. Colchicine or benomyl inhibit microtubule formation during zoosporogenesis and also appear to perturb the mobilization of the gamma bodies which are believed to be the source of the vesicles which form the axonemal membrane and cleavage furrows. These observations are discussed in relation to the hypothesis of Heath that cytoplasmic microtubules formed during zoosporogenesis determine cytoplasmic domains which will delimit the spore initials at cleavage. The observations presented here appear to confirm this hypothesis.     

Cell wall synthesis in Allomyces macrogynus

Scanning electron microscopy and freeze-etching/cleaving have been employed to examine events in the synchronized development of gametophytic germlings of the aquatic Phycomycete Allomyces macrogynus. Motile spores were induced to start synchronized development and the sequence of surface changes associated with the encystment process was studied. Time course studies show that small vesicles (apparently blebbed off from the gamma-particles) start to accumulate on the surface of the plasma membrane after 6 min of synchronized growth at the same time as the first cell wall material can be detected. The vesicles increase in number during encystment. After 15 min of synchronized growth the number of vesicles decrease and after 20 min of growth no vesicle can be observed on the cell surface. During this period the cell surface appears increasingly smooth, probably due to cell wall formation. In freeze-etching/cleaving electron micrographs from this period, both intact and what appear to be ruptured vesicles outside the cell surface, can be observed. The intact vesicle has a characteristic surface pattern presumably of membrane particles. This surface view of the encystment processes supports the hypothesis that the gamma-particles through gamma vesicle formation participate in the cell wall synthesis during encystment in Allomyces.     

Osmotic regulation of the uniflagellate phycomycete spore

In spores of the Blastocladiales there is a strict temporal correlation between the breakdown of the matrix of the gamma body and the swelling of the gamma body with the duration of the spore's motility. The swelling of the gamma body upon decay is interpreted as being due to water uptake. The swollen gamma bodies fuse with the plasmalemma and expel their content. We suggest that the swelling of the gamma body and its subsequent fusion with the plasmalemma of the spore are the means by which the spores of the Blastocladiales maintain osmotic balance with the medium in which they swim. The decay of the gamma body during the motile period of the spore is a separate and distinct process, not related to the mobilization of the remaining gamma bodies during encystment and cell wall formation.     

Allomyces neo-moniliformis gametogenesis

Summary InAllomyces neo-moniliformis meiosis takes place during resting sporangium germination. The meiospores are characteristically binucleate and biflagellate as described byEmerson (1938) andTeter (1944). A variation in the number of nuclei and flagella per meiospore from two is correlated with germination of the resting sporangia under reduced oxygen tension. The meiospores are extremely poor swimmers and are typically amoeboid. At encystment the gamma bodies of the cell are mobilized and appear involved in cyst wall synthesis. A single mitotic division of each nucleus gives rise to four nuclei. Gamete cleavage is as described for spore cleavage inBlastocladiella (Lessie andLovett 1968). The assembly of the nuclear cap and side body complex of the spore are extremely late processes in gametogenesis. The gametes are released when the single papilla dissolves. The gametes fuse in pairs and after zygote formation the cell is uninucleate with two flagella. The biflagellate zygote is an active swimming cell. The presence of homothallism or hetero-thallism inA. neo-moniliformis is discussed.     

Differential requirement for phospholipase D/Spo14 and its novel interactor Sma1 for regulation of exocytotic vesicle fusion in yeast meiosis

During sporulation and meiosis of budding yeast a developmental program determines the formation of the new plasma membranes of the spores. This process of prospore membrane (PSM) formation leads to the formation of meiotic daughter cells, the spores, within the lumen of the mother cell. It is initiated at the spindle pole bodies during meiosis II. Spore formation, but not meiotic cell cycle progression, requires the function of phospholipase D (PLD/Spo14). Here we show that PLD/Spo14 forms a complex with Sma1, a meiotically expressed protein essential for spore formation. Detailed analysis revealed that both proteins are required for early steps of prospore membrane assembly but with distinct defects in the respective mutants. In the Deltaspo14 mutant the initiation of PSM formation is blocked and aggregated vesicles of homogenous size are detected at the spindle pole bodies. In contrast, initiation of PSM formation does occur in the Deltasma1 mutant, but the enlargement of the membrane is impaired. During PSM growth both Spo14 and Sma1 localize to the membrane, and localization of Spo14 is independent of Sma1. Biochemical analysis revealed that Sma1 is not necessary for PLD activity per se and that PLD present in a complex with Sma1 is highly active. Together, our results suggest that yeast PLD is involved in two distinct but essential steps during the regulated vesicle fusion necessary for the assembly of the membranous encapsulations of the spores.     

The ultrastructure of cell wall formation and of gamma particles during encystment of Allomyces macrogynus zoospores

Structural changes during cell wall formation by populations of semisynchronously germinating zoospores were studied in the water mold Allomyces macrogynus. Fluorescence microscopy using Calcofluor white ST (which binds to -1,4-linked glycans) demonstrated that Calcofluor-specific material was deposited around most cells between 2–10 min after the induction of encystment (beginning when a wall-less zoospore retracts its flagellum and rounds up). During the first 15 min of encystment there was a progressive increase in fluorescence intensity. Ultrastructural analysis of encysting cells showed that within 2–10 min after the induction of encystment small vesicles 35–70 nm diameter were present near the spore surface, and some were in the process of fusing with the plasma membrane. The fusion of vesicles with the zoospore membrane was concomitant with the appearance of electron-opaque fibrillar material outside the plasma membrane. Vesicles similar to those near the spore surface were found within the gamma () particles of encysting cells. These particles had a crystalline inclusion within the electron-opaque matrix. During the period of initial cyst cell wall formation numerous vesicles appeared to arise at the crystal-matrix interface. Approximately 15–20 min was required for the cell wall to be formed. We suggest that the initial response of the zoospore to induction of encystment is the formation of a cell wall mediated by the fusion of cytoplasmic vesicles with the plasma membrane.Non-Standard Abbreviations GlcNac N-Acetylglucosamine - DS sterile dilute salts solution - PYG peptone-yeast extract-glucose broth     

Liberation and Development of Allomyces arbuscula Mitospores Viewed by Scanning Electron Microscopy

Scanning electron microscopy has been employed to examine events in the release and development of mitospores of the aquatic fungus, Allomyces arbuscula. Among the salient features of spore release from the mitosporangium is the digestion of the inner matrix of the exit papillum. Hydrolysis appears to begin at the outer layer of the papillum plug matrix and probably results from activation of localized hydrolytic enzymes. The plug clearly consists of at least two different component layers. Elaboration of mitospores from the mitosporangium is depicted in several micrographs. Motile spores were induced to begin development, and the sequence of surface changes associated with the encystment process was studied. Time course studies show the retraction of the flagellum, the change from elipsoidal to spherical shape, and the deposition of the cell wall. Early in encystment, small vesicles accumulate on the surface of the plasma membrane. These enlarge and fuse to form the mature cyst wall. This surface view of cell wall deposition appears to support the possible role of gamma particles in cell wall synthesis during encystment.     

End3p-mediated endocytosis is required for spore wall formation in Saccharomyces cerevisiae

During sporulation in Saccharomyces cerevisiae, vesicles transported to the vicinity of spindle pole bodies are fused to each other to generate bilayered prospore membranes (PSMs). PSMs encapsulate the haploid nuclei that arise from the meiotic divisions and serve as platforms for spore wall deposition. Membrane trafficking plays an important role in supplying vesicles for these processes. The endocytosis-deficient mutant, end3Delta, sporulated poorly and the spores produced lost resistance to ether vapor, suggesting that END3-mediated endocytosis is important for sporulation. End3p-GFP localized to cell and spore peripheries in vegetative and sporulating cells and colocalized with actin structures. Correspondingly, the actin cytoskeleton appeared aberrant during sporulation in end3Delta. Analysis of meiosis in end3Delta mutants revealed that the meiotic divisions occurred with wild-type kinetics. Furthermore, PSMs were assembled normally. However, the levels of proteins required for spore wall synthesis and components of the spore wall layers at spores were reduced, indicating that end3Delta mutants are defective in spore wall synthesis. Thus, END3-mediated endocytosis is important for spore wall formation. Additionally, cytological analyses suggest that trafficking between the plasma membrane and PSMs is important earlier during sporulation.     

Involvement of bristle coat structures in surface membrane formations and membrane interactions during coenocytotomic cleavage in caps of Acetabularia mediterranea

In the multinucleate cap rays of the green alga Acetabularia mediterranea the cell surface increases dramatically within a short time period during the final stages of coenocytotomic cleavage. In early stages of cyst formation the cytoplast is traversed by numerous large and prolate cleavage vesicles which are characterized by typical columellar or spinous coat structures. The cleavage vesicles are closely associated with the surface of plastids and, to a lesser degree, of mitochondria. This intimate association seems to be mediated by regularly spaced, densely stained intermembranous cross-bridge structures and is maintained throughout cleavage. These cleavage vesicles contain a finely fibrillar material structurally similar to the hyaline layer of mucilage that fills the space between the plasma membrane and cell wall. They line up with invaginations of the plasmalemma and vacuole membranes and, together with smaller vesicles interspersed, constitute preformed "perforation lines" for the final separation of the coenoblast portions. Equidistantly spaced plaques of attachment of such vesicles with surface membrane are described. We hypothesize (a) that the cleavage vesicle membrane is the immediate precursor to the new postcoenocytotomic surface membrane, (b) that the cleavage vesicle coat structures are integrated into the subsurface coat of the plasma membrane, (c) that growth of the laterally attached cleavage vesicles by intussusception of small fuzzy-coated vesicles is confined to their "free ends," (d) that the intermembranous cross- bridge elements are related to bristle coat structures and play a role in the establishment of the cleavage lines, and (e) that the coenocytotomic cleavage process is organized so that adjacent plastids are separated in a way that guarantees the inclusion of several plastids in each cyst.     

SSP2 and OSW1, two sporulation-specific genes involved in spore morphogenesis in Saccharomyces cerevisiae

Spore formation in Saccharomyces cerevisiae requires the synthesis of prospore membranes (PSMs) followed by the assembly of spore walls (SWs). We have characterized extensively the phenotypes of mutants in the sporulation-specific genes, SSP2 and OSW1, which are required for spore formation. A striking feature of the osw1 phenotype is asynchrony of spore development, with some spores displaying defects in PSM formation and others spores in the same ascus blocked at various stages in SW development. The Osw1 protein localizes to spindle pole bodies (SPBs) during meiotic nuclear division and subsequently to PSMs/SWs. We propose that Osw1 performs a regulatory function required to coordinate the different stages of spore morphogenesis. In the ssp2 mutant, nuclei are surrounded by PSMs and SWs; however, PSMs and SWs often also encapsulate anucleate bodies both inside and outside of spores. In addition, the SW is not as thick as in wild type. The ssp2 mutant defect is partially suppressed by overproduction of either Spo14 or Sso1, both of which promote the fusion of vesicles at the outer plaque of the SPB early in PSM formation. We propose that Ssp2 plays a role in vesicle fusion during PSM formation.     

AMPHIESMAL ULTRASTRUCTURE OF DINOFLAGELLATES: A REEVALUATION OF PELLICLE FORMATION1

The ultrastructure of the amphiesma during pellicle formation was investigated in two species of Dinophyceae, Amphidinium rhynchocephalum Anissimowa and Heterocapsa niei (Loeblich) Morrill & Loeblich using thin sections. In both species the amphiesma consists of an outermost membrane (i.e. the plasma membrane) underlain by amphiesmal vesicles. In A. rhynchocephalum the latter appear empty whereas each amphiesmal vesicle in H. niei contains a thecal plate and a thin, amorphous layer (dark-staining layer) located between, the thecal plate and the inner amphiesmal vesicle membrane. When cells of both taxa are carefully fixed, amphiesmal vesicles are always separate entities (i.e. the sutures are undisrupted). During ecdysis the following amphiesmal components are shed: the plasma membrane, the outer amphiesmal vesicle membrane, and in H. niei the thecal plates. The inner membranes of the amphiesmal vesicles then fuse with each other and form a continuous membrane (termed pellicle membrane) that remains tightly oppressed to an underlying amorphous layer (pellicular layer). In A. rhynchocephalum the pellicular layer is already present in vegetative non-ecdysed cells, whereas in H. niei it forms during ecdysis beneath the pellicle membrane. During ecdysis in H. niei, material from the dark-staining layer precipitates on the outer surface of the pellicle membrane, where it forms a characteristic honeycomb pattern. The new observations are incorporated into a revised model of pellicle formation in dinoflagellates and contrasted with earlier proposals.     

Functional necessity of the cytoskeleton during cleavage membrane development and zoosporogenesis in Allomyces macrogynus

Cleavage membrane development and cytokinesis were examined in zoosporangia of Allomyces macrogynus treated with cytoskeletal inhibitors and compared to zoosporogenesis under control conditions. Developing membranes were visualized in living zoosporangia with laser-scanning confocal microscopy using the lipophilic membrane dye FM4-64. Under control conditions, cleavage membranes developed in four discrete stages, ultimately interconnecting to delimit the cytoplasm into polygonal uninucleate domains of near uniform size. Disruption of microtubules did not impede the normal four-stage development of cleavage membranes, and cytokinesis occurred with only minor detectable anomalies, although zoospores lacked flagella. Disruption of actin microfilaments did not inhibit membrane formation but blocked nuclear migration and significantly disrupted membrane alignment and cytoplasmic delimitation. This resulted in masses of membrane that remained primarily in cortical regions of the zoosporangia, as did nuclei, throughout zoosporogenesis. Zoospores formed in the absence of microtubules had only a slightly larger mean diameter than control zoospores, although nearly 50% of spores contained two or more nuclei. Microfilament inhibitor treatments produced spores with substantially larger mean diameters and correspondingly larger numbers of nuclei per spore, with greater than 85% containing three or more nuclei. These results showed that a functional actin microfilament cytoskeleton was required for proper alignment of cleavage elements and cytokinesis in Allomyces zoosporangia while microtubules played a less significant role.     

THE DEVELOPMENT OF BASAL BODIES AND FLAGELLA IN ALLOMYCES ARBUSCULUS

The development of basal bodies and flagella in the water mold Allomyces arbusculus has been studied with the electron microscope. A small pre-existing centriole, about 160 mµ in length, was found in an inpocketing of the nuclear membrane in the vegetative hypha. Thus, formation of a basal body does not occur de novo. When the hyphal tip started to differentiate into gametangia, the centrioles were found to exist in pairs. One of the members of the pair then grew distally to more than three times its original length, whereas the other remained the same size. The larger centriole would correspond to the basal body of a future gamete. Gametogenesis was usually induced by transferring a "ripe" culture to distilled water. Shortly after this was done, a few vesicles were pinched off from the cell membrane of the gametangium and came in contact with the basal body. Apparently, they fused and formed a large primary vesicle. The flagellum then started to grow by invaginating into it. Flagellar fibers were evident from the very beginning. As the flagellum grew so did the vesicle by fusion with secondary vesicles, thus coming to form the flagellar sheath. The different stages of flagellar morphogenesis are described and the possible interrelationships with other processes are discussed.     

Exocytosis of catecholamine (CA)-containing and CA-free granules in chromaffin cells

Recent evidence suggests that endocytosis in neuroendocrine cells and neurons can be tightly coupled to exocytosis, allowing rapid retrieval from the plasma membrane of fused vesicles for future use. This can be a much faster mechanism for membrane recycling than classical clathrin-mediated endocytosis. During a fast exo-endocytotic cycle, the vesicle membrane does not fully collapse into the plasma membrane; nevertheless, it releases the vesicular contents through the fusion pore. Once the vesicle is depleted of transmitter, its membrane is recovered without renouncing its identity. In this report, we show that chromaffin cells contain catecholamine-free granules that retain their ability to fuse with the plasma membrane. These catecholamine-free granules represent 7% of the total population of fused vesicles, but they contributed to 47% of the fusion events when the cells were treated with reserpine for several hours. We propose that rat chromaffin granules that transiently fuse with the plasma membrane preserve their exocytotic machinery, allowing another round of exocytosis.     

AN ULTRASTRUCTURAL BASIS FOR HYPHAL TIP GROWTH IN PYTHIUM ULTIMUM

Hyphae of the fungus Pythium ultimum extend by tip growth. The use of surface markers demonstrates that cell expansion is limited to the curved portion of the hyphal apex. Growing and non-growing regions are reflected in internal organization as detected by light and electron microscopy. The young hypha consists of three regions: an apical zone, a subapical zone and a zone of vacuolation. The apical zone is characterized by an accumulation of cytoplasmic vesicles, often to the exclusion of other organelles and ribosomes. Vesicle membranes are occasionally continuous with plasma membrane. The subapical zone is non-vacuolate and rich in a variety of protoplasmic components. Dictyosomes are positioned adjacent to endoplasmic reticulum or nuclear envelope, and vesicles occur at the peripheries of dictyosomes. A pattern of secretory vesicle formation by dictyosomes is described which accounts for the formation of hyphal tip vesicles. Farther from the hyphal apex the subapical zone merges into the zone of vacuolation. As hyphae age vacuolation increases, lipid accumulations appear, and the proportional volume of cytoplasm is reduced accordingly. The findings are integrated into a general hypothesis to explain the genesis and participation of cell components involved directly in hyphal tip growth: Membrane material from the endoplasmic reticulum is transferred to dictyosome cisternae by blebbing; cisternal membranes are transformed from ER-like to plasma membrane-like during cisternal maturation; secretory vesicles released from dictyosomes migrate to the hyphal apex, fuse with the plasma membrane, and liberate their contents into the wall region. This allows a plasma membrane increase at the hyphal apex equal to the membrane surface of the incorporated vesicles as well as a contribution of the vesicle contents to surface expansion.     

Exocyst Subcomplex Functions in Autophagosome Biogenesis by Regulating Atg9 Trafficking

During autophagy, double-membrane vesicles called autophagosomes capture and degrade the intracellular cargo. The de novo formation of autophagosomes requires several vesicle transport and membrane fusion events which are not completely understood. We studied the involvement of exocyst, an octameric tethering complex, which has a primary function in tethering post-Golgi secretory vesicles to plasma membrane, in autophagy. Our findings indicate that not all subunits of exocyst are involved in selective and general autophagy. We show that in the absence of autophagy specific subunits, autophagy arrest is accompanied by accumulation of incomplete autophagosome-like structures. In these mutants, impaired Atg9 trafficking leads to decreased delivery of membrane to the site of autophagosome biogenesis thereby impeding the elongation and completion of the autophagosomes. The subunits of exocyst, which are dispensable for autophagic function, do not associate with the autophagy specific subcomplex of exocyst.     

Vesicle production and fusion during lobe formation inMicrasterias visualized by high-pressure freeze fixation

Summary Two different types of Golgi vesicles involved in wall formation can be visualized during lobe growth inMicrasterias when using high-pressure freeze fixation followed by freeze substitution. One type that corresponds to the dark vesicles (DV) described in literature seems to arise by a developmental process occurring at the Golgi bodies with the single vesicles being forwarded from one cisterna to the next. The other vesicle type appears to be produced at thetrans Golgi network without any visible precursors at the Golgi cisternae. A third type of vesicle, produced by median andtrans cisternae, contains slime; these are considerably larger than those previously mentioned and they do not participate in wall formation. The distribution of the two types of cell wall vesicles at the cell periphery and their fusion with the plasma membrane are shown for the first time, since chemical fixation is too slow to preserve a sufficient number of vesicles in the cortical cytoplasm. The results indicate that fusions of both types of vesicles with the plasma membrane are possible all over the entire surface of the growing half cell. However, the DVs are much more concentrated at the growing lobes, where they form queues several vesicles deep behind zones on the plasma membrane thought to be specific fusion sites. The structural observations reveal that the regions of enhanced vesicle fusion correspond in general to the sites of calcium accumulation determined in earlier studies. By virtue of the absence of the DVs in the region of cell wall indentations the second type of wall forming vesicle appears prominent; they too fuse with the plasma membrane and discharge their contents to the wall.     

Description of Hyalinocysta expilatoria n. sp., a microsporidian parasite of the blackfly Odagmia ornata

Hyalinocysta expilatoria n. sp. is described from a larva of Odagmia ornata collected in Sweden. Infection was restricted to the adipose tissue which was transformed into a syncytium. The earliest stage observed was diplokaryotic merozoites, which mature directly into diplokaryotic sporonts. Each sporont produces a sporophorous vesicle (pansporoblast), which persists, also enclosing mature spores. Usually nuclear divisions result in a plasmodium with 8 nuclei, which fragments into 8 sporoblasts, each of which develops into a spore without further division. Occasionally an aberrant number of spores (2, 4, 6) is formed. The spores are pyriform with a flattened area at the posterior pole. Spores in sporophorous vesicles with 8 spores are 4.0–6.0 μm long, in vesicles with 4 spores 4.0–5.0 μm, and in vesicles with 2 spores 7.0–8.0 μm. In some vesicles the spores develop asynchronously, and 2, 4, or 6 mature spores are found together with 6, 4, or 2 immature. There was also a small number of vesicles with supernumerary spores, less than 8 normally developed. The 325–350 nm thick spore wall is composed of three layers. The polar filament is anisofilar with 7 coils in a single layer. The anterior 5–6 coils are wide, the posterior 2-1 thin. The angle of tilt of the anterior filament coil is approximately 50°. The spore has a single nucleus. The sporophorous vesicle is delimited by a thin membrane, also visible in haematoxylin stained preparations. Vesicles with mature spores are void of metabolic inclusions.     

Acrosome differentiation in the ascidians Clavelina lepadiformis and Ciona intestinalis

The spermatozoa of both Clavelina lepadiformis and Ciona intestinalis have architectural features characteristic of ascidian spermatozoa that have been previously described. They have an elongated head (6 microm and 3 microm long, respectively) and a single mitochondrion that is closely applied laterally to the nucleus; they lack a midpiece. The acrosome of Clavelina lepadiformis spermatozoa is a moderately electron-dense, pear-shaped flattened vesicle, approx. 300 nm x 200 nm x 40 nm in length, width, and height, respectively. The acrosome of Ciona intestinalis spermatozoa is a moderately electron-dense, round flattened vesicle with an electron-dense plate in its central region. It is approx. 200 nm x 200 nm x 50 nm in length, width, and height, respectively. During spermiogenesis in both ascidians, several proacrosomal vesicles (50-70 nm in diameter) appear in a blister at the future apex of the spermatids. These vesicles appear to be associated with the inner surface of the plasma membrane enclosing the blister. They come into contact with each other along the inner surface of the plasma membrane and fuse to form a horseshoe-shaped acrosomal vesicle, which becomes a round, flattened vesicle during further differentiation. Some speculations about the mechanism of acrosome differentiation, the possible role of the acrosome during fertilization, and in the speciation of ascidians are presented.     

Serotonin release from the neuronal cell body and its long-lasting effects on the nervous system

Serotonin, a modulator of multiple functions in the nervous system, is released predominantly extrasynaptically from neuronal cell bodies, axons and dendrites. This paper describes how serotonin is released from cell bodies of Retzius neurons in the central nervous system (CNS) of the leech, and how it affects neighbouring glia and neurons. The large Retzius neurons contain serotonin packed in electrodense vesicles. Electrical stimulation with 10 impulses at 1 Hz fails to evoke exocytosis from the cell body, but the same number of impulses at 20 Hz promotes exocytosis via a multistep process. Calcium entry into the neuron triggers calcium-induced calcium release, which activates the transport of vesicle clusters to the plasma membrane. Exocytosis occurs there for several minutes. Serotonin that has been released activates autoreceptors that induce an inositol trisphosphate-dependent calcium increase, which produces further exocytosis. This positive feedback loop subsides when the last vesicles in the cluster fuse and calcium returns to basal levels. Serotonin released from the cell body is taken up by glia and released elsewhere in the CNS. Synchronous bursts of neuronal electrical activity appear minutes later and continue for hours. In this way, a brief train of impulses is translated into a long-term modulation in the nervous system.