ANNOUNCEMENTS

Summary

The oogenesis of the calcareoous sponge Sycon ciliatum has been studied by electron microscopy. In this species, oogonia probably derive from choanocytes through loss of collar and flagellum and formation of phagosome-like inclusions. Oogonia can be occasionally found within flagellated chambers and show a prominent rough endoplasmic reticulum, several mitochondria and polygonal dense granules. The latter are also visible in choanocytes. Oocytes lie in the mesohyl beneath the choanoderm. They contain a nucleolated nucleus, vesiculated granules and phagosome-like inclusions involved in the formation of fibrillar yolk material. Mature eggs are large, irregularly shaped, and filled with fibrillar yolk inclusions. A second ultrastructural confirmation of the carrier-cell mediated fertilization of calcareous sponges is also given.     

Piwi expression in archeocytes and choanocytes in demosponges: insights into the stem cell system in demosponges

SUMMARY Little is known about the stem cells of organisms early in metazoan evolution. To characterize the stem cell system in demosponges, we identified Piwi homologs of a freshwater sponge, Ephydatia fluviatilis, as candidate stem cell (archeocyte) markers. EfPiwiA mRNA was expressed in cells with archeocyte cell morphological features. We demonstrated that these EfPiwiA‐expressing cells were indeed stem cells by showing their ability to proliferate, as indicated by BrdU‐incorporation, and to differentiate, as indicated by the coexpression of EfPiwiA with cell‐lineage‐specific genes in presumptive committed archeocytes. EfPiwiA mRNA expression was maintained in mature choanocytes forming chambers, in contrast to the transition of gene expression from EfPiwiA to cell‐lineage‐specific markers during archeocyte differentiation into other cell types. Choanocytes are food‐entrapping cells with morphological features similar to those of choanoflagellates (microvillus collar and a flagellum). Their known abilities to transform into archeocytes under specific circumstances and to give rise to gametes (mostly sperm) indicate that even when they are fully differentiated, choanocytes maintain pluripotent stem cell‐like potential. Based on the specific expression of EfPiwiA in archeocytes and choanocytes, combined with previous studies, we propose that both archeocytes and choanocytes are components of the demosponge stem cell system. We discuss the possibility that choanocytes might represent the ancestral stem cells, whereas archeocytes might represent stem cells that further evolved in ancestral multicellular organisms.     

The synthesis of α-amylase by rough and in vitro reconstituted rough endoplasmic reticulum derived from rat parotid gland

Summary The isolation of rough and smooth endoplasmic reticulum from rat parotid salivary gland is described. The rough membrane was stripped of its bound ribosomes using the KCl-puromycin method. Rough endoplasmic reticulum was reconstituted from stripped-rough membrane and polyribosomes. The reconstituted rough membrane resembled the native rough membrane in the following aspects: RNA/protein ratio, buoyant density in a continuous sucrose gradient and amino acid incorporation capacity. The in vitro synthesis of -amylase by both rough and in vitro reconstituted rough membrane was demonstrated using SDS polyacrylamide gel electrophoresis. The reconstituted rough membrane could be restripped by KCl-puromycin. The in vitro synthesized -amylase remained associated with the rough or the in vitro reconstituted rough membrane, even after these membranes were stripped of their bound ribosomes.Abbreviations Fp Free polyribosomes - Bp Membrane-bound polyribosomes released by DOC - RM Rough membrane - SM Smooth membrane - RMst Rough membrane stripped - RMrec In vitro reconstituted rough membrane - DOC Sodium deoxycholate     

Variable domain I of nematode CLEs directs post-translational targeting of CLE peptides to the extracellular space

Effector proteins expressed in the esophageal gland cells of cyst nematodes are delivered into plant cells through a hollow, protrusible stylet. Although evidence indicates that effector proteins function in the cytoplasm of the syncytium,^1–3 technical constraints have made it difficult to directly determine where nematode effector proteins are initially delivered: cytoplasm, extracellular space, or both. Recently, we demonstrated that soybean cyst nematode CLE (HgCLE) propeptides are delivered to the cytoplasm of syncytial cells. Genetic and biochemical analyses indicate that the variable domain (VD) sequence is then required for targeting cytoplasmically delivered nematode CLEs to the apoplast where they function as ligand mimics of endogenous plant CLE peptides.⁴ The fact that nematode CLEs are targeted through the gland cell secretory pathway and delivered as mature propeptides into plant cells makes it impossible for these proteins to be subsequently delivered to the extracellular space via co-translational translocation through the endoplasmic reticulum (ER) secretory pathway of the host cell. However, when expressed in transgenic plants, if the mature nematode CLE propeptide harbored a functional cryptic signal peptide, it could possibly traffic to the apoplast through the ER secretory pathway by co-translational translocation. Here, we present evidence that VDI, the N-terminal sequence of the VD of HgCLE2,⁴ is sufficient for trafficking CLE peptides to the apoplast and that trafficking is indeed through an alternative pathway other than co-translational translocation.Key words: cyst nematode, effector, CLE, variable domain, trafficking, endoplasmic reticulum, co-translational translocation, post-translational     

Untersuchungen zur Ultrastruktur der Choanocyte von Ephydatia fluviatilis L.

Zusammenfassung Aufgrund elektronenmikroskopischer Befunde wird die Morphologie der Choanocyten des Süßwasserschwammes Ephydatia fluviatilis beschrieben. Die Choanocyte besteht aus Zelleib, Geißel und Kragen. Der Zelleib ist gekennzeichnet durch einzelne Zisternen des endoplasmatischen Reticulums, die der basalen und zum Teil der lateralen Zellmembran parallel anliegen. Die kontraktilen Vakuolen der Choanocyten entleeren ihren Inhalt in das Lumen der Geißelkammer. In einigen Choanocyten kann senkrecht zum Basalkörper ein Procentriol nachgewiesen werden. Die Geißel zeichnet sich durch Plasmaleisten und Fahnen aus. Die den Kragen aufbauenden etwa 35 Fibrillen werden als Mikrovilli gedeutet. Vereinzelt tritt an der Basis des Kragens ein Faltenmuster auf.

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Ultrastructure of choanocytes in Ephydatia fluviatilis L.

Summary The morphology of the choanocytes of the freshwater sponge, Ephydatia fluviatilis, is described on the basis of electron microscope studies. The cell body of the choanocytes bears a cilium and a collar. In the cell body characteristic single cisternae of the endoplasmic reticulum are found in juxtaposition with the basal and lateral plasmalemmata. The contractile vacuoles extrude their contents into the lumen surrounded by the collar chamber. In some choanocytes a procentriole is found in addition to the typical basal body. The cilium of the choanocytes is characterized by cytoplasmic crests and thread-like extensions. The collar is formed by approximately 35 microvilli which show a peculiar arrangement. Occasionally, the basis of the collar displays cytoplasmic folds.

Die Arbeit wurde teilweise mit Unterstützung durch die Deutsche Forschungsgemeinschaft durchgeführt.     

Structural organization of the ommatidium in the ventral compound eye of the dragonflySympetrum

Summary In the dragonflySympetrum, the circumferential sequence of retinular cells *R5*R4, R3*R2, R1*R8, R7, R6 (in which R5 & 8, R2 & 3, R1 & 4 comprise three receptor pairs, R7 and R6 an unmatched pair with long visual fibres, and asterisks denote the positions of cone cell processes) is homologized to the general pattern of odonate retinulae. This sequence runs in an anticlockwise direction for ommatidia of the right ventral retina viewed from outside inwards, that in the left retina runs clockwise. The proximo-distal sequence of contributions of these cells to the retinula (presence of nucleus, contribution to the tiered rhabdom, Fig. 1) has R1 & 4 in the basal third (Fig. 10) beneath R5 & 8, and R2 & 3 (Fig. 6); R7 has a large distal rhabdomere beneath which R6 contributes a few microvilli for most of the rhabdom's length. There is no twist to the rhabdom, and neighbouring ommatidia have consistent orientations. R1 is dorsal and R2 & 3 anterior. Rhabdom diameters are shown in Table 1; individual rhabdomere volumes are as follows: R7, 320 m³; R5 & 8, 650 m³ each; R2 & 3, 430 m³ each; R1 & 4, 230 m³ each.Abbreviations LA light-adapted - DA dark-adapted - ER endoplasmic reticulum - BM basement membrane     

Development of labellar taste hairs in the blowfly,Calliphora vicina (Insecta,Diptera)

Summary The early development of taste sensilla has been studied with special emphasis on cilia, dendrite, and pore formation.In the 39-h stage (the first stage investigated) differentiation of sensilla is already under way. The mechanisms of differentiation of dendrites (39–48 h) deviate from the mechanisms described for differentiation of true cilia. In taste hairs the centrioles meet in the tip of the narrowed apical region of the sensory neuron. Together they sink deeper into this region and line up coaxially, thus forming the basal body complex. Thereafter, lateral contacts between this complex and the plasma membrane of the neuron are established. Formation of open connections between the trichogen lumen of the hair and the environment, or the dendrite lumen, was not observed.Electrophysiological data indicate that the sensilla become functional from 3 days before emergence onwards.List of Abbreviations B Basal body complex - bc Basal compartment - bs Basal sheath - bl Basement lamina - D Dendrite(s) - EPC Non-sensillar epithelial cells - ES Ecdysial sheath - GERL Granular endoplasmatic reticulum-lysosomes - HS Hemolymph space - P Protrusions - PRN Prospective receptor neuron - RER Rough endoplasmatic reticulum - RN Receptor neuron - n Neck - THC Thecogen cell - TOC Tormogen cell - TRC Trichogen cell - TRS Trichogen sprout     

The pituitary of Aphanius dispar (Rüppell) from hypersaline marshes and freshwater

Summary An ultrastructural study of the rostral pars distalis of the pituitary of Aphanius dispar specimens taken from freshwater or hypersaline marshes revealed significant structural differences which indicate higher activity of the prolactin cells in the hypotonic medium. Prolactin cells from freshwater specimens had larger secretory granules, a higher amount of endoplasmic reticulum, and expanded intercellular spaces with many secretory lakes. These cells contained an unusual cytoplasmic structure, consisting of twisted canals with vesicular lumina, connected to the endoplasmic reticulum of the cell. This structure is about 1–2 m in diameter.Stellate cells are characterized by extracellular spacing junctions which are particularly noticeable at the confluence of the interstellate cell canaliculi and the pericapillary space.Abbreviations FW freshwater - HS hypersaline - NS neurosecretory - PCB paracrystalline body - PNH proximal neurohypophysis - RPD rostral pars distalis - SG secretory granule - SW seawater This paper is dedicated with affectionate respect to Professor Berta Scharrer on the occasion of her 70th birthdayThe assistance of Cynthia Bellon in editing this paper is gratefully acknowledged     

Drosophila embryo syncytial blastoderm cellular architecture and morphogen gradient dynamics: Is there a correlation?

During embryo development in many metazoan animals, the first differentiated cell type to form is an epithelial cell. This epithelial layer is modified by developmental cues of body axes formation to give rise to various tissues. The cells that arise are mesenchymal in nature and are a source of other tissue types. This epithelial to mesenchymal transition is used for tissue type formation and also seen in diseases such as cancer. Here we discuss recent findings on the cellular architecture formation in the Drosophila embryo and how it affects the developmental program of body axes formation. In particular these studies suggest the presence of compartments around each nucleus in a common syncytium. Despite the absence of plasma membrane boundaries, each nucleus not only has its own endoplasmic reticulum and Golgi complex but also its own compartmentalized plasma membrane domain above it. This architecture is potentially essential for morphogen gradient restriction in the syncytial Drosophila embryo. We discuss various properties of the dorso-ventral and the antero-posterior morphogen gradients in the Drosophila syncytium, which are likely to depend on the syncytial architecture of the embryo.     

A three dimensional model of the photosynthetic membranes of Ectothiorhodospira halochloris

The three dimensional organization of the complete photosynthetic apparatus of the extremely halophilic, bacteriochlorophyll b containing Ectothiorhodospira halochloris has been elaborated by several techniques of electron microscopy. Essentially all thylakoidal sacs are disc shaped and connected to the cytoplasmic membrane by small membraneous bridges. In sum, the lumina of all thylakoids (intrathylakoidal space) form one common periplasmic space. Thin sections confirm a paracrystalline arrangement of the photosynthetic complexes in situ. The ontogenic development of the photosynthetic apparatus is discussed based on a structural model derived from serial thin sections.Abbreviations E. Ectothiorhodospira - bchl bacteriochlorophyll - R. Rhodopseudomonas - RC reaction center     

Worming Our Way In and Out of the Caenorhabditis elegans Germline and Developing Embryo

The germline and embryo of the nematode Caenorhabditis elegans have emerged as powerful model systems to study membrane dynamics in an intact, developing animal. In large part, this is due to the architecture of the reproductive system, which necessitates de novo membrane and organelle biogenesis within the stem cell niche to drive compartmentalization throughout the gonad syncytium. Additionally, membrane reorganization events during oocyte maturation and fertilization have been demonstrated to be highly stereotypic, facilitating the development of quantitative assays to measure the impact of perturbations on protein transport. This review will focus on regulatory mechanisms that govern protein trafficking, which have been elucidated using a combination of C. elegans genetics, biochemistry and high‐resolution microscopy. Collectively, studies using the simple worm highlight an important niche that the organism holds to define new pathways that regulate vesicle transport, many of which appear to be absent in unicellular systems but remain highly conserved in mammals .     

An ultrastructural examination of developing and mature paraspermatozoa in Pyrazus ebeninus (Mollusca,Gastropoda, Potamididae)

Summary The mesogastropod Pyrazus ebeninus, produces true spermatozoa (here termed euspermatozoa) and multi-flagellate, mobile cells (here termed paraspermatozoa). The mature paraspermatozoon consists of an elongateconical head (6.5–8.5 m in length), constructed of an electron-dense mosaic sheath surrounding a similarly dense, rod-shaped nuclear core (which runs almost the full length of the head). An acrosome-like structure forms the apex of the head. Five to eight axonemes are fixed to the posterior extremity of the nuclear core, each by means of an attachment complex (dense attachment rod, centriolar cap and centriole). A short (3–4 m) midpiece zone follows the head and consists of the multiple axonemes interspersed with very elongate mitochondria. A tuft of short (20 m) tails (termed minor tails) emerges from the midpiece in addition to one very long tail (termed the major tail) ensheathed in dense granules which resemble glycogen granules. A single membrane surrounds head, midpiece and tails whilst the nuclear core retains the original double nuclear membrane.Developmentally, the multiple axonemes arise from one of a pair of wheel-shaped arrangements of centrioles and attach to posterior indentations in the nucleus prior to its transformation into the nuclear core. Dense vesicles, derived apparently from the endoplasmic reticulum, accumulate along and around the developing nuclear core and (in the presence of microtubules) condense into the mosaic head sheath. Cytoplasmic mitochondria elongate and collect at the posterior axis of the cell, where, together with the axonemes, they form the midpiece.Features not previously reported in other ultrastructural studies of paraspermatozoa include the acrosome-like structure of the head, the structure of the midpiece zone, the glycogen sheath of the major tail, the dense annular structure at the junction of the midpiece and major tail and the presence of microtubules in the final phase of head and midpiece maturation. Some features of the euspermatozoon are also described and the comparative ultrastructure of mature and developing paraspermatozoa and their possible functions in the Gastropoda, are reviewed.Abbreviations ac euspermatozoon acrosomal cone - ar euspermatozoon axial rod - ax axoneme - b dense block of mosaic sheath - c centriole - cc centriolar cap - co cone of acrosome-like structure - dr dense attachment rod - dv dense vesicle - g glycogen granules - G Golgi complex - GER granular endoplasmic reticulum - H head of paraspermatozoon - m mitochondrion - M midpiece (euspermatozoon, paraspermatozoon) - maj major tail - min minor tails - mt microtubules - n nucleus - nc nuclear core - p dense plug of acrosome-like structure - pm plasma membrane - sGv small Golgi vesicles - Z transition of centriole to centriolar cap of attachment complex     

Three‐dimensional fate mapping of larval tissues through metamorphosis in the glass sponge Oopsacas minuta

The tissue of glass sponges (Class Hexactinellida) is unique among metazoans in being largely syncytial, a state that arises during early embryogenesis when blastomeres fuse. In addition, hexactinellids are one of only two poriferan groups that already have clearly formed flagellated chambers as larvae. The fate of the larval chambers and of other tissues during metamorphosis is unknown. One species of hexactinellid, Oopsacas minuta, is found in submarine caves in the Mediterranean and is reproductive year round, which facilitates developmental studies; however, describing metamorphosis has been a challenge because the syncytial nature of the tissue makes it difficult to trace the fates using conventional cell tracking markers. We used three‐dimensional models to map the fate of larval tissues of O. minuta through metamorphosis and provide the first detailed account of larval tissue reorganization at metamorphosis of a glass sponge larva. Larvae settle on their anterior swimming pole or on one side. The multiciliated cells that formed a belt around the larva are discarded during the first stage of metamorphosis. We found that larval flagellated chambers are retained throughout metamorphosis and become the kernels of the first pumping chambers of the juvenile sponge. As larvae of O. minuta settle, larval chambers are enlarged by syncytial tissues containing yolk inclusions. Lipid inclusions at the basal attachment site gradually became smaller during the six weeks of our study. In O. minuta, the flagellated chambers that differentiate in the larva become the post‐metamorphic flagellated chambers, which corroborate the view that internalization of these chambers during embryogenesis is a process that resembles gastrulation processes in other animals.     

Membrane enzyme reactor with simultaneous separation using electrophoresis

A membrane enzyme reactor with simultaneous separation was investigated. Enzymes, urease and aspartase, were immobilized by a porous polytetrafluoroethylene membrane. Electrical field was applied in the medium while the reaction was carried out. Products with electrical charge could be separated through the membrane from the reaction medium as they were formed. Reaction behavior was analyzed by a simple model considering both pore-migration and reaction in the skelton of the membrane. According to the analysis the inherent reaction rate of the immobilized enzymes decreases significantly. This is probably caused by the structural variation of enzymes. For the case of urease, the change of pH inside the membrane may also cause the decrease of the reaction rate. The model analysis showed that the enzyme content in the membrane and the residence time of the substrate in the membrane governed overall extent of reaction.List of Symbols e g (dm³)^–1 enzyme concentration in the membrane - L cm membrane thickness - K _m mM Michaelis constant - Rate mmol · min^–1 · g^–1 rate of product formation per unit weight of enzyme - S mM substrate concentration - S _in mM inlet substrate concentration - S _out mM outlet substrate concentration - u cm · min^–1 migration rate - V V voltage between the electrodes - V _m mmol · min^–1 · g^–1 maximum reaction rate - X conversion - z cm distance from the surface inside the membrane - void fraction of the porous membrane - tortuosity of the membrane - min space time     

Ontogeny of microbodies (glyoxysomes) in cotyledons of dark-grown watermelon (Citrullus vulgaris Schrad.) seedlings

The development of glyoxysomal marker enzyme activities and concomitant ultrastructural evidence for the ontogeny of glyoxysomes has been studied in cotyledons of dark-grown watermelon seedlings (Citrullus vulgaris Schrad., var. Florida Giant). Catalase (CAT, EC 1.11.1.6) was stained in glyoxysomal structures with the 3,3-diaminobenzidine procedure. Serial sections and high-voltage electron microscopy were used to analyze the three-dimensional structure of the glyoxysomal population. With early germination CAT was localized in three distinct cell structures: spherical microbodies already present in freshly imbibed cotyledons; in appendices on lipid bodies; and in small membrane vesicles between the lipid bodies. Due to their ribosome-binding capacity, both appendices and small vesicles were identified as derivatives of the endoplasmic reticulum (ER). In the following period, glyoxysome formation and lipid body degradation were found to be inseparable processes. The small CAT-containing vesicles attach to a lipid body on a restricted area. Both lipid body appendices and attached cisternae enlarge around and between tightly packed lipid bodies and eventually become pleomorphic glyoxysomes with lipid bodies entrapped into cavities. The close contact between lipid body and glyoxysomes is maintained until the lipid body is digested and the glyoxysomal cavity becomes filled with cytoplasm. During the entire period of increase in glyoxysomal enzyme activities, no evidence was obtained for destruction of glyoxysomes, but small CAT-containing vesicles were observed from day 2 through day 6 after imbibition, indicating a continuous de novo formation of glyoxysomes. This study does not substantiate the hypothesis that glyoxysomes bud directly from the ER. Rather, ER-derivatives, e.g., lipid body appendices or cisternae attached to lipid bodies are interpreted as being glyoxysomal precursors that grow in close contact with lipid bodies both in volume and surface membrane area.Abbreviations CAT catalase - DAB 3,3 diaminobenzidine tetrahydrochloride - ER endoplasmic reticulum - GOX glycolate oxidase - HPR hydroxypyruvate reductase - HVEM high-voltage electron microscopy - ICL isocitrate lyase - MS malate synthase - RER rough endoplasmic reticulum In the figures bars represent 0.1 m (if not stated otherwise)     

The polypeptide structure and assembly of Ly-2/3 heterodimers

Mild reduction of mature, thymic Ly-2/3 heterodimers of M _r 67 000 resulted in dissociation into three individual polypeptide chains, , , and , of respective M _r values 38000, 35000, and 30000. The and chains were both immunoprecipitated by a monoclonal antibody directed to the Ly-2.1 epitope whereas the Ly-3.1 antibody bound only the chain. The possibility that the and chains of each heterodimer established their interchain links within a labile precursor protein in which a and segments were fused was considered but discounted by the finding that in mice heterozygous for both Ly-2 and Ly-3 loci, the Ly-2 product of one chromosome was not exclusively joined to Ly-3 structures coded by the same chromosome. By utilizing ionic detergents which selectively alter the charge of intrinsic membrane proteins, both Ly-2 and Ly-3 polypeptides were shown to have membrane insertion sites. It is suggested that as a consequence of their likely synthesis on membrane-bound polysomes, newly synthesized Ly-2 and Ly-3 structures accumulate within the same subcellular compartment — the membranes of the rough endoplasmic reticulum. Their elevated concentration within this space may facilitate a low affinity binding interaction between Ly-2 and Ly-3 which is later stabilized by interchain disulfide bond formation.Abbreviations used in this paper BSA bovine serum albumin - DOC sodium deoxycholate - DTT dithiothreitol - HA hemagglutinin - HTAB hexadecyltrimethylammonium bromide - RER rough endoplasmic reticulum - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TX100 Triton X-100     

Properties of single chloride selective channel from sarcoplasmic reticulum

The behavior of single chloride channels in sarcoplasmic reticulum of rabbit and trout skeletal muscle was examined by fusing isolated vesicle fractions into planar lipid bilayers. The channel exhibited a full open state with a unit conductance of 65 pS (in 100 mM Cl^-) and several subconductance states with reversal potentials which were dependent on the chloride gradient across the bilayer. Open probability was 0.6–0.95 for membrane potentials ranging from-60 to+60 mV. The kinetic behaviour could be described by assuming one time constant for the fully conducting channel, and at least two time constants for the non-conducting channel. In the presence of methane sulfonate, sulfate and phosphate anions, a decrease in the unit current amplitude but not open time argued in favor of a competition between these anions and Cl^- at the transport site of the channel. Chloride channel activity was not affected by variations of Ca²⁺ concentration in both chambers or by the presence of Mg²⁺. Similarly, neither millimolar ATP nor the presence of the drugs taurine (up to 10 mM), lidocaine (2–40 M) or the calmodulin antagonist W7 (5–150 M), modified channel behavior. Finally, pH variations between 6.8 to 8 were without effect.Abbreviations DIDS 4,4-Diisothiocyanostilbene-2,2-disulfonic acid - EGTA Ethylene glycol bis-(-aminoethyl ether) N,N,N',N'-tetraacetic acid - HEPES N-2-Hydroxyethyl-piperazine-N'-2-ethane sulfonic acid - SR Sarcoplasmic reticulum - TRIS Tris(hydroxymethyl)aminomethane     

Ultrastructure of spermatogenesis and sperm ofMulticotyle purvisi (Plathelminthes,Aspidogastrea)

Summary The ultrastructure of spermatogenesis is described for the first time in an aspidogastrean. The zone of differentiation which is usually formed during digenean spermiogenesis was not observed inMulticotyle purvisi. Instead, spermatid components are assembled within the common cytoplasmic mass before the outgrowth of spermatids. Microtubules, mitochondrion, nucleus and axonemes including their basal body regions, migrate from the cytoplasm into the spermatid which is pinched off at the level of the arching membrane. An unusual, complex structure of the basal body region is described. Intercentriolar bodies and striated rootlets are left behind and quickly disappear from the residual cytoplasm. Despite these atypical aspects, spermiogenesis results in the formation of mature sperm with the classical structure common to Digenea and Monogenea Polyopisthocotylea with the addition of some extra, non-cortical microtubules and a dense rod along part of the length of the sperm.Abbreviations used in the figures A cell type A, primary spermatogonium - AM arching membrane - AX axoneme - AZ attachment zone - B cell type B — spermatogonium - BB basal body region - C cell type C — spermatogonium - CEL central element - CI cisternae - CY cytophore - D cell type D — primary spermatocyte - DO doublet of microtubules - DR dark rod - E cell type E — multinucleate condensed cytophore - ER endoplasmic reticulum - G glycogen - GO Golgi body - I intercentriolar body - LB lamellate body - M mitochondrion - ME remnants of arching membranes - MT microtubules - N nucleus - R rootlet - S spermatid     

Rise in chlorotetracycline fluorescence accompanies tracheary element differentiation in suspension cultures ofZinnia

Summary Developing tracheary elements in suspension cultures ofZinnia elegans fluoresce intensely relative to non-differentiating cells when stained with chlorotetracycline (CTC), a fluorescent chelate probe for membrane associated calcium. This suggests that a change in calcium uptake or subcellular distribution accompanies the onset of tracheary element differentiation. A few cells in early differentiating cultures were brightly fluorescent, but did not have visible cell wall thickenings, suggesting that a rise in sequestered calcium may precede visible differentiation. Diffuse CTC fluorescence in early differentiation most likely results from sequestration of calcium in the endoplasmic reticulum. Late in differentiation, CTC fluorescence becomes punctate in appearance, probably due to loss of plasma membrane integrity occurring at the onset of autolysis.Zinnia suspension culture cells were found to be very sensitive to CTC and low concentrations (10 M) were used to assure accurate localization of membrane-associated calcium in healthy cells.Abbreviations CTC chlorotetracycline - DIC differential interference contrast - DiOC₆ 3,3-dihexyloxacarbocyanine iodide - ER endoplasmic reticulum - EGTA ethylene glycol bis-(amino-ethyl ether) N,N,N¹N¹-tetraacetic acid - NPN n-phenylnaphthylamine - OsFeCN osmium tetroxide and potassium ferricyanide - TE tracheary element - TEM transmission electron microscopy