August Ruthmann

The ventral epithelium of Trichoplax adhaerens (Placozoa): Cytoskeletal structures, cell contacts and endocytosis

SummaryAll cilia emerge from ciliary pits supported along their circumference by 22–24 dense rodlets that are connected by filaments to a surrounding sheath of endoplasmic reticulum. The proximal part of the basal body is provided with two short lateral rootlets and one long terminal rootlet, all associated with microtubules. The lateral rootlets are in turn connected by fine fibrous material to the dense supporting rodlets which follow the contour of the ciliary pit and extend along the ciliary membrane beyond the level of the basal plate where the central pair of microtubules originates. The proximal part of the basal body has fine fibrous connections to the endoplasmic reticulum while its distal portion is surrounded by nine curved sheets. The terminal cell contactions are by belt desmosomes that are accompanied by a bundle of microfilaments which encircle the apical region of the cell and insert at the cell membrane. Tight junctions are lacking. Endocytosis was demonstrated by the uptake of cationized ferritin. The structures associated with the ciliary pits are probably associated with the firm anchorage of the ciliary base since Trichoplax adheres to the substrate as it moves propelled by its ventral cilia. The marginal bundle of microfilaments may be involved in folding of the organism during feeding.

Mitosis-like macronuclear division in a ciliate

The macronucleus of a small marine ciliate of the genus Protocrucia consists of a cluster of ten vesicles which give rise to 20 distinct chromosomal elements in the course of prophase-like condensation stages. Size differences of vesicles and chromosomes are cytological indications of their genetic individuality. In an anaphase-like stage, the chromosomal elements are separated in two daughter groups which re-form 10 vesicles each. The micronucleus divides simultaneously. The existence of a precisely functioning mode of chromosome distribution is also indicated by DNA measurements. Since the macronucleus contains much more DNA than the micronucleus, the macronuclear chromosomes are thought to be oligotenic. This hypothesis is supported by the much larger size of the macronuclear chromosomes. In contrast to other modes of macronuclear division known so far, this ciliate has retained some essential features of mitosis.

Spindel und kinetochoren in der Mitose und Meiose der Baumwollwanze Dysdercus intermedius (Heteroptera)

In spermatogonial mitoses of Dysdercus intermedius, chromosomal spindle microtubules insert in discrete kinetochores. The latter consist of two electron dense plates of about 0,3 μm diameter located at opposite sites at the poleward surfaces of the daughter chromatids. The evidence presented permits the interpretation that a localized kinetochore owes its layered substructure to successive binding sites for kinetochore material at the ends of bundled microtubules. A working hypothesis, based upon different distributions of genetic loci for kinetochore material in species with localized and diffuse kinetochores is proposed. In Dysdercus, holokinetic chromosome behavior in mitotic anaphase movement is not due to truly diffuse kinetochores but to chromosomal interconnections which are established at prometaphase. Such interconnections are absent in the first meiotic division when each bivalent is enclosed by a sheath of endoplasmic reticulum. Spindle microtubules penetrate through terminal openings in this sheath and end singly in dense spots of kinetochore material.

Holokinetic Composite Chromosomes with "Diffuse" Kinetochores in the Micronuclear Mitosis of a Heterotrichous Ciliate

During micronuclear mitosis of the heterotrichous ciliate Nyctotherus ovalis Leidy rod-shaped composite chromosomes are formed by lateral association of telokinetic chromosomes. The formation of these composite chromosomes seems to be a highly ordered process since only nuclei with either 18 or 24 such chromosomes can be observed, and nuclei with the same chromosome number show a similar length distribution of their chromosomes. Further, these data indicate that we examined two otherwise indistinguishable races. During metaphase the composite chromosomes become arranged in the spindle equator in a holokinetic fashion, their entire poleward surfaces being covered by kinetochore material. These “diffuse” kinetochores have a trilaminar appearance comparable to those of monokinetic chromosomes. Their electron density after employing Bernhards procedure revealed the same ribonucleoprotein distribution as reported for the localized kinetochores formed during the extranuclear mitosis in other cells. During early anaphase the outer kinetochore layer remains continuous while the individual chromosomes in the composite group show a tendency to separate leaving chromatin-free spaces of about 40 nm diameter. Kinetochore microtubules which are still anchored in the outer kinetochore layer seem to elongate and to extend into the interpolar spindle region predominantly through these “holes” in the chromatin. These observations suggest a like polarity of kinetochore and interpolar microtubules in the polar spindle region while microtubules in the interpolar space seem to interdigitate in an antiparallel fashion. The activity of the kinetochore to act as a microtubule-organizing center (MTOC) seems to be modulated by the chromatin underlying the outer kinetochore layer which may prevent further outgrowth of kinetochore microtubules.

Evidence for three "classes" of microtubules in the interpolar space of the mitotic micronucleus of a ciliate and the participation of the nuclear envelope in conferring stability to microtubules.

Exposure of Nyctotherus ovalis to low temperatures or vinblastine caused similar reactions of “classes” of microtubules (mt) present in the mitotic micronucleus of this ciliate towards both treatments. However, differences of sensitivity between certain “classes” of mt at individual mitotic stages exist. Unlike the kinetochore mt (kmt) of most other eukaryotic cells, kmt in Nyctotherus completely disassemble after incubation at 6–8 ° C (60 min) and most disappear after prolonged exposure to vinblastine (10−5 M, 16 h). The depolymerization of kmt causes the collapse of the spindle and a dislocation of chromosomes at metaphase, yet the reduced number of kmt after vinblastine-treatment still allows an alignment of composite complexes at the spindle equator. The data suggest that three individual sets of mt exist in the interpolar spindle region during ana- and telophase: 1) interpolar mt (int mt), which are assembled during anaphase, are cold- and vinblastine sensitive; 2) manchette mt (ma mt), which are first observed underneath the nuclear envelope during mid-anaphase, are cold-stable and insensitive to vinblastine treatment (10−5 M); after prolonged treatment (16 h) they form spiral structures; 3) stembody mt (st mt), comprising the interpolar region of the nucleus during telophase, are cold- and vinblastine insensitive. Paracrystalline structures resembling a stembody are formed in telophase-like division stages after prolonged vinblastine exposure (16 h, 10−5 M). Since kmt and int mt possess the same sensitivity under depolymerizing conditions, they probably have a similar composition. Thus the idea that the int mt in this organism arise by elongation of kmt is supported. However, st mt apparently do not originate from an extension of preexistent int mt, but appear to represent a new set of stable mt. This is emphasized not only by their greater stability compared to the int mt but also by the distribution of cold-stable mt in late anaphase micronuclei. The ma mt may be an intermediary step in formation of st mt since their stability resembles that of the st mt. A comparison of the substructure of vinblastine-induced paracrystals in Nyctotherus with those observed in in vitro systems with known composition suggests that a turnover of MAPs may be responsible for the different stability of mt and thus could specify and regulate mt sensitivity and function. Another organelle, possibly involved in conferring stability to mt, is the nuclear membrane. The assumption that the nuclear envelope possesses an intrinsic property to nucleate mt and thus aid in the alignment of mt is supported.

Alternative modes of asexual reproduction inTrichoplax adhaerens (Placozoa)

SummaryHollow swarmers are budded off at the dorsal surface ofTrichoplax and are covered by dorsal epithelium. Their inner cavity is lined with the flagellated cells of the ventral epithelium. There is no indication that the fiber cells included between the epithelia take any part either in morphologenesis or the separation of the bud from the mother animal. The early primordium forms in the interspace. A single layer of cells derived from both epithelia surrounds a cavity filled with granular matter that stains like proteins. The latter is used up during the floating phase of the swarmers that may last for a week. After settling at the bottom, the hollow sphere opens at one point. The concave ventral epithelium gradually flattens as more cells become incorporated in it. The latter form new flagella and flagellar pits. More frequently found than swarmers are small spherical forms that are unable to float and possess a distinct polarity. Their upper half is covered by dorsal epithelium and their lower half by ventral epithelium. Large fiber cells are in the center. Their site and mode of formation is unknown. Rarely observed are dorsal stolons whose bulbous end flattens upon touching the substrate. Since they are totally covered by the flat cells of the dorsal epithelium, they may have to undergo a transformation, like the hollow swarmers, to bring the ventral epithelium into contact with the substrate.

Fine structure of the accessory glands of the female genital tract of the ichneumonid Pimpla turionellae (Insecta, Hymenoptera)

SummaryThe female accessory glands include the tubular poison gland, the paired, lemon-shaped uterus glands, and Dufours gland, an unbranched tubular organ. They consist essentially of a single layer of epithelium cells surrounded by a basement membrane. The lumen is lined by cuticle. The proteinaceous secretion of the poison gland is released into intracellular ducts provided with microvilli, each connected to a channel lined with cuticle which leads to the central lumen of the gland. The channel is formed by special canal cells. Nerve endings are interspersed among the gland cells. The uterus gland consists of four cell types derived from a single type of precursor cell found in newly hatched wasps. Type I cells are covered by type II cells and are thus without contact to the luminal surface of the gland. They contain stacks or whorls of mitochondria and smooth cisternae in an alternating arrangement. Vesicles with a secretory product are found in cells of types II and III. Deep anastomosing infoldings of the plasmalemma, stabilized by microtubules and dense material at the branchings, are characteristic for type II cells. Most secretory vesicles are found in type III cells, the prevalent cell type which is thought to be the source of the lipoprotein secretion. Coated vesicles are present at deep infoldings of the plasmalemma. The greatly enlarged apical surface area of type IV cells and the presence of mitochondria in slender outgrowths is suggestive of an osmoregulatory function. In Dufours gland, two cell types appear in succession, the first with a very dense cytoplasm, the second with dense inclusions and many seemingly empty vesicles of smooth endoplasmic reticulum. The secretion products, lecithin and a cholesterol ester, are thought to be formed by the second cell type. The dense inclusion might be lecithin, which reacts with osmium tetroxide. The cholesterol ester could have been washed out of the “empty” vesicles by the embedding procedure.

The cytoskeleton of the fiber cells of Trichoplax adhaerens (Placozoa)

SummaryThe cytoskeleton of Trichoplax adhaerens fiber cells was studied after chemical fixation, freeze-substitution, lysis of attached cells with nonionic detergents and by immunofluorescence. Cytoskeletal elements present in the cell bodies and reaching into the extensions include microtubules, intermediate filaments, 6–7 nm and 2–3 nm microfilaments. The latter seem to interconnect other cytoskeletal elements. Actin-like microfilaments are found both as networks and parallel strands. Immunofluorescence with antiactin shows the presence of actin in the cell body, underneath the plasmalemma and within the extensions. Both the results of immunofluorescence and the identification of 6–7 nm actin-like microfilaments support the concept of contractility of the fiber cells as the cause of the rapid shape changes of Trichoplax. Anti-tubulin fluorescence corresponds to the location of microtubules in the extensions as well as the cell bodies of the fiber cells. The extensions are withdrawn upon depolymerization of the microtubules by colchicine.

Trichoplax adhaerens F.E. Schulze (placozoa) in the scanning electron microscope

Summary1.The dorsal epithelium consists of flattened polygonal cells with various surface modifications including ruffles, microvilli-like projections, and small protruding vesicles. A double ridge arises where adjacent cells meet. The refractile lipid globules reach almost to the surface not by a pore, but by a slit between adjoining cells. There is some suggestion that the globules might be discharged through the slit.2.In both epithelia, the flagellum arises from an excentrically located ciliary pit. In the dorsal epithelium, the inner wall of the pit is connected to the basal portion of the flagellum by radiating filaments. In both epithelia, the flagella may bear ‘hoods’ as localized bulges in restricted areas. Their role is still unknown.3.Attached food organisms are found both on the ventral and the dorsal surface. The latter might function in food capture.4.Surface views of the fiber cells of the intermediate layer reveal many branched extensions and intercellular connections. The number of connections must far exceed the cell number of this layer and is thought to be instrumental in coordination.5.The bulging margin ofTrichoplax may contain a space of its own.

Spherical forms ofTrichoplax adhaerens (Placozoa)

SummaryTwo types of spherical forms of this normally flattened organism appear sporadically in our cultures. Hollow spheres have an outer wall of flagellated ventral epithelium. The large fiber cells protrude into the central cavity which can include a closed compartment of flagellated dorsal epithelium. Cells of the outer wall that withdraw their flagellum and leave the epithelium are phagocytozed by fiber cells. Solid spheres consist of an outer layer of dorsal epithelium and densely packed fiber cells in the interior that may also include a closed compartment of ventral epithelium cells. Closely apposed fiber cells may form special cell contacts or pores connecting the cells.