Observations on cytoplasmic transport along ovarian nutritive tubes of polyphagous coleopterans

Summary The ovarioles of Coccinella and Tenebrio are shown to be telotrophic — a characteristic normally associated with hemipterans rather than coleopterans. They possess an anterior region of trophic cells and a chain of oocytes. The trophic cells are connected with the latter by a series of nutritive tubes, and autoradiography has shown that RNA is transported along the tubes to the oocytes. However, the system in these beetles differs markedly from that of hemipterans in that the nutritive tubes do not contain an extensive complement of aligned microtubules. The significance of this to both the mechanism and the selectivity of transport is discussed.     

The mechanism of microtubule associated cytoplasmic transport

Summary The nutritive tubes of telotrophic insect ovaries are cytoplasmic channels along which ribosomes are transported over distances of several mm from trophic cells to the developing oocytes. The presence within the nutritive tubes of a massive number of orientated microtubules renders them strongly birefringent in polarised light, a property which, together with their size, rendered them amenable to isolation by microdissection. Ultrastructurally the isolated tubes were indistinguishable from undissected controls. Polyacrylamide gels revealed a consistent pattern of some 30 bands of which tubulin was the most prominent. The tubes also contained a band which comigrated with the major high molecular weight micro tubule associated protein (MAP) from mouse brain but no detectable actin, myosin or dynein. Microtubules in the isolated tubes were not depolymerised by treatments (cold, calcium and colchicine) which typically disrupt cytoplasmic microtubules. Following extraction of the membrane enclosing the tubes and the cytoplasmic matrix the microtubule cytoskeleton persisted, retaining its cylindrical organisation although no bridges between the microtubules were detected in the electron microscope. The possibility that the stability and spatial deployment of the nutritive tube microtubules is conferred by specific microtubule accessory proteins is discussed.     

Direct evidence for the nature of the binding of mitochondria to microtubules in ovarian nutritive tubes of an hemipteran insect

Interactions between mitochondria and microtubules have been investigated in the nutritive tubes that link the nurse cells to the oocytes in ovarioles of the hemipteran insect, Notonecta. The nutritive tubes comprise large numbers of microtubules, which can be dissected manually from ovarioles. This approach, which retains the intrinsic components of the system, has allowed the in vivo interactions between the microtubules and mitochondria, which are also present in the nutritive tubes, to be studied directly. Static binding occurred between mitochondria and microtubules, and investigations of its nucleotide and salt-sensitivities have indicated its microtubule-associated protein (MAP)-dependency. Received: 30 September 1996 / Accepted: 1 February 1997     

Organisation of the endosperm and endosperm–placenta syncytia in bladderworts (Utricularia, Lentibulariaceae) with emphasis on the microtubule arrangement

Multinucleate cells play an important role in higher plants, especially during reproduction; however, the configurations of their cytoskeletons, which are formed as a result of mitosis without cytokinesis, have mainly been studied in coenocytes. Previous authors have proposed that in spite of their developmental origin (cell fusion or mitosis without cytokinesis), in multinucleate plant cells, radiating microtubules determine the regular spacing of individual nuclei. However, with the exception of specific syncytia induced by parasitic nematodes, there is no information about the microtubular cytoskeleton in plant heterokaryotic syncytia, i.e. when the nuclei of fused cells come from different cell pools. In this paper, we describe the arrangement of microtubules in the endosperm and special endosperm–placenta syncytia in two Utricularia species. These syncytia arise from different progenitor cells, i.e. cells of the maternal sporophytic nutritive tissue and the micropylar endosperm haustorium (both maternal and paternal genetic material). The development of the endosperm in the two species studied was very similar. We describe microtubule configurations in the three functional endosperm domains: the micropylar syncytium, the endosperm proper and the chalazal haustorium. In contrast to plant syncytia that are induced by parasitic nematodes, the syncytia of Utricularia had an extensive microtubular network. Within each syncytium, two giant nuclei, coming from endosperm cells, were surrounded by a three-dimensional cage of microtubules, which formed a huge cytoplasmic domain. At the periphery of the syncytium, where new protoplasts of the nutritive cells join the syncytium, the microtubules formed a network which surrounded small nuclei from nutritive tissue cells and were also distributed through the cytoplasm. Thus, in the Utricularia syncytium, there were different sized cytoplasmic domains, whose architecture depended on the source and size of the nuclei. The endosperm proper was isolated from maternal (ovule) tissues by a cuticle layer, so the syncytium and chalazal haustorium were the only way for nutrients to be transported from the maternal tissue towards the developing embryo.     

The effect of colchicine on the sleeve element of microtubules

The microtubules in the nutritive tubes of telotrophic insect ovaries, like those in many other situations, are surrounded by an electron clear zone or ‘sleeve’ after conventional preparative procedures for electron microscopy. Ribosomes, which also pack the nutritive tubes, do not encroach into this region, and although microtubules are often closely opposed, they are rarely seen to touch. The composition of the microtubule sleeve zone is unknown. This study shows that colchicine not only destroys the microtubules in the nutritive tubes, but also the sleeve zone which surrounds them, suggesting that the integrity of the microtubules is essential for the existence of the sleeves.     

Independent regulation of microtubule spacing and microtubule stability following redundancy of nutritive tubes in telotrophic ovaries in hemiptera (insecta)

The nutritive tubes that act as conduits between the nutritive cells and the developing oocytes within the ovaries of hemipteran insects, contain vast aggregates of aligned microtubules. During the previtellogenic stages of oogenesis, components synthesised in the nutritive cells pass within the nutritive tubes and accumulate in the oocytes. Using polarised light and electron microscopy, we have monitored the changes in both the spacing and stability of the microtubules which occur when, at the onset of vitellogenesis, translocation within the nutritive tube ceases and the tube becomes redundant. Having investigated nutritive tube redundancy in the ovaries of 4 species of hemipterans, we have discovered the outcome to be similar in each case, with the microtubules losing their characteristic spacing and becoming closely packed prior to their depolymerisation. The feature that differs is the timing of these changes because, in certain species, microtubule depolymerisation closely follows the microtubule rearrangement, while in other species, depolymerisation of the microtubules occurs some considerable time after their change in pattern. This evidence demonstrates that microtubule spacing and stability are regulated independently following redundancy of nutritive tubes, and we speculate upon how this regulation might be achieved within the insect ovaries.     

Phosphorylation of microtubule-associated proteins from the ovaries of hemipteran insects by MPF and MAP kinase: Possible roles in the regulation of microtubules during oogenesis

Nutritive tubes that link the developing oocytes to the nurse cells in ovarioles of hemipteran insects contain extensive arrays of microtubules. These are established, then later depolymerised, by developmentally regulated processes. Breakdown of the microtubules corresponds with the activation of M-phase promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase), late in oogenesis, as the oocytes proceed to arrest at the first meiotic metaphase [Lane and Stebbings, Roux's Arch Dev Biol 205:150–159 (1995)]. The mechanisms that lead to the breakdown of nutritive tube microtubules are unknown. Here, we have investigated the possibility that the insect ovarian microtubules are regulated by MPF- or MAP kinase-dependent phosphorylation, focusing upon the prominent high molecular weight microtubule-associated protein (HMW MAP) enriched in this system, which is a potential target for protein kinase activity in vivo. We have purified the prominent HMW MAPs from the ovaries of two species of hemipterans, and have shown them to be substrates in vitro for the activities of MPF and MAP kinase. However, although the catalytic component of MPF (p34^cdc2) is present within microtubule-rich portions of hemipteran ovarioles, we have found that neither this protein nor its regulatory partner (cyclin B) co-purify with microtubules during taxol-mediated microtubule isolation. Arch. Insect Biochem. Physiol. 39:81–90, 1998.© 1998 Wiley-Liss, Inc.     

Binding of axonemal dynein to microtubules comprising the cytoplasmic transport system in insect ovarioles

Dynein isolated from ciliary axonemes of Tetrahymena is shown to bind in a characteristic fashion as arms to microtubules dissected from the nutritive tubes of insect ovarioles. The microtubules in nutritive tubes are associated with the transport of cytoplasmic components along their length, and the significance of their ability to bind axonemal dynein, to the possibility that microtubule/dynein interactions are involved in microtubule-associated movements, generally, is discussed.     

Quinacrine-induced dilation of the rat cecum and degeneration of large granular vesicle-containing neurons in the myenteric plexus

Summary An electrophoretic analysis of the microtubule-containing transport channels, known as nutritive tubes, which link the nutritive cells with the chain of developing oocytes in the telotrophic ovarioles of the hemipteran Notonecta glauca, has been carried out. The major polypeptide components resolved have been identified tentatively as -and -tubulin subunits by their comparable electrophoretic mobility to tubulin subunits from purified mammalian brain microtubule protein. Co-migration of some of the minor components with proteins resolved from insect ribosomes (which are the only other components of the nutritive tubes as seen in ultrastructural studies) indicates that these may be ribosomal proteins. Also characterized by electrophoresis were the nutritive cells, which are the source of synthesis of the components transported via the nutritive tubes, and the oocytes, the sites of their accumulation.     

Redundant nutritive tubes in insect ovarioles: the fate of an extensive microtubule transport system

The developing oocytes in the ovarioles of hemipteran insects receive materials from nutritive cells by way of channels known as nutritive tubes. The tubes contain an extensive system of microtubules which are thought to be involved in the transport between the two cell types. At the onset of vitellogenesis the connection is discontinued. Redundant nutritive tubes have been identified, compared with functional tubes, and their fate discussed.     

Reorganisation of microtubule arrays in the telotrophic ovaries of hemipteran insects: Correlation with meiotic reinitiation

We have shown that, in the ovaries of hemipteran insects, microtubule reorganisation and depolymerisation in the oocytes, and in the nutritive tubes supplying them, correlate with the activation of M-phase promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase) as the oocytes proceed to arrest at the first meiotic metaphase. The application, however, of Xenopus egg extracts with high MPF activities to isolated nutritive tube microtubules failed to result in their depolymerisation, suggesting that a novel factor may be required for the breakdown of these highly stable microtubule arrays in vivo.     

Free and centrosome-attached microtubules: Quantitative analysis and modeling of two-component system

In cultivated in vitro interphase animal cells, microtubules form a network whose density is highest in the central cell area, in the region of centrosome, and decreases towards the cell periphery. Since identification of individual microtubules in the central cell area is significantly difficult and more often is impossible, there are several approaches to studying microtubules in the internal cell cytoplasm. These approaches are based on a decrease of microtubule density—both real, due to their partial depolymerization (by the action of cold temperatures or cytostatics), or apparent, due to a decrease of cell thickness (by photobleaching of preexisting microtubules and analysis of newly formed ones). In the present work, we propose a method based on the determination of optical density which allows evaluation of the state of the cytoplasmic microtubule system as a whole. The method consists of a comparison of the dependences describing changes of the microtubule optical density from the cell center to the periphery in controls and in experiments. Analysis of living cells by the proposed method has shown that the character of curves describing the decrease of optical density from the cell center to its periphery is different for various cell types; the dependence can be described both as an exponential regression (the CHO cell line) and as a linear regression (the NIH-3T3 and REF cell lines). Our previous studies have allowed the suggestion that the character of the dependence is determined by the ratio of free and centrosome-attached microtubules and by the position of their ends in the cell cytoplasm. To test this hypothesis, we considered model systems with all microtubules assumed to be in a straight orientation and divergent radially from the centrosome, but with different arrangements of plus-and minus-ends. In the model system, in which all the microtubule minus-ends are attached to the centrosome while the plus-ends are at different distances from it, the microtubule density is described by the exponential (f(x) = ae ^?bx ). Introduction of free microtubules into the system leads to a change of the character of this dependence, and the system in which the concentration of free microtubules with minus ends located at different distances from the cytoplasm is 5 times higher than that of the centrosome-attached microtubules is described by the linear regression equation (f(x) = k ^* x + b), which corresponds to the experimentally obtained dependences for 3T3 and REF cells. Thus, we believe that even in cells with a radial microtubule system, free microtubules may constitute the majority.     

THE EFFECTS OF ISOPROPYL N-PHENYL CARBAMATE ON THE GREEN ALGA OEDOGONIUM CARDIACUM : I. Cell Division

Cell division in vegetative filaments of the green alga Oedogonium cardiacum is presented as an experimental system. We report on how we have used this system to study the effects of isopropyl N-phenylcarbamate (IPC) on the mitotic apparatus and on the phycoplast, a planar array of cytokinetic microtubules. Polymerization of microtubules was prevented when filaments, synchronized by a light/dark regime and chilled (2°C) while in metaphase or just before phycoplast formation, were exposed to 5.5 x 10^-4 M IPC and then returned to room temperature. Spindles reformed or phycoplasts formed when these filaments were transferred to growth medium free of IPC. However, the orientation of both microtubular systems was disturbed: the mitotic apparatus often contained three poles, frequently forming three daughter nuclei upon karyokinesis; the phycoplast was often stellate rather than planar, and it sometimes was displaced to the side of both daughter nuclei, resulting in a binucleate and an anucleate cell upon cytokinesis. Our results suggest that IPC (a) prevents the assembly of microtubules, (b) increases the number of functional polar bodies, and (c) affects the orientation of microtubules in O. cardiacum. High voltage (1,000 kV) electron microscopy of 0.5-µm thick sections allowed us to visualize the polar structures, which were not discernible in thin sections.     

Juveniles stick to adults: recruitment of the tube-dwelling polychaete Lanice conchilega (Pallas, 1766)

Juveniles of the tube-dwelling polychaete L. conchilega were found attached to adult tubes at an exposed beach in South Wales (U.K.). They attached to the upper part of tubes protruding above the sediment. The aim of this study was to determine the timing of juvenile settlement on to adult tubes, their abundance and size distribution, and the time of detachment and densities in the sediment after re-settlement. Furthermore, to establish whether the sediment composition of juvenile tubes differed from those of adults and the surrounding sediment. In 1998, 1999 and 2000, tubes of adult L. conchilegawere sampled and numbers of attached juveniles were quantified and their sizes measured. Densities of solitary L. conchilega in the sediment were estimated, as well as the size distribution of solitary tubes. On average, 5–13 juveniles were attached to single adult tubes in spring, but numbers varied greatly with up to 72 juveniles on a single tube in April 1998. Availability of a hard substratum and chemical cues are discussed as potential factors triggering the attachment of juveniles to adult tubes. Juveniles started to detach and re-settle in the sediment 1 month after attachment. Once settled in the sediment, juvenile numbers decreased steeply, but the size distribution of solitary L. conchilega suggested that some juveniles survived in the population. However, the extent to which numbers of juvenile recruits contribute to the density of adults remained unclear. Juveniles used a smaller mean particle size for tube building than adults, but both actively selected particles.     

The nature of the clear zone around microtubules

Summary The clear zones seen around microtubules in transverse sections of nutritive tubes vary in size depending on whether a microtubule is bordered by ribosomes or by another microtubule. We consider that such a finding is not consistent with the current view, that the clear zone is maintained by microtubule-associated material. It can, however, be accounted for by an electrostatic repulsion between the surfaces of negatively charged microtubules and between microtubules and ribosomes which are also negatively charged. The experiments presented here, involving on the one hand the addition of cationic substances to microtubules and on the other the alteration in charge of the microtubules, support this hypothesis.     

Distribution of callose synthase, cellulose synthase, and sucrose synthase in tobacco pollen tube is controlled in dissimilar ways by actin filaments and microtubules

Callose and cellulose are fundamental components of the cell wall of pollen tubes and are probably synthesized by distinct enzymes, callose synthase and cellulose synthase, respectively. We examined the distribution of callose synthase and cellulose synthase in tobacco (Nicotiana tabacum) pollen tubes in relation to the dynamics of actin filaments, microtubules, and the endomembrane system using specific antibodies to highly conserved peptide sequences. The role of the cytoskeleton and membrane flow was investigated using specific inhibitors (latrunculin B, 2,3-butanedione monoxime, taxol, oryzalin, and brefeldin A). Both enzymes are associated with the plasma membrane, but cellulose synthase is present along the entire length of pollen tubes (with a higher concentration at the apex) while callose synthase is located in the apex and in distal regions. In longer pollen tubes, callose synthase accumulates consistently around callose plugs, indicating its involvement in plug synthesis. Actin filaments and endomembrane dynamics are critical for the distribution of callose synthase and cellulose synthase, showing that enzymes are transported through Golgi bodies and/or vesicles moving along actin filaments. Conversely, microtubules appear to be critical in the positioning of callose synthase in distal regions and around callose plugs. In contrast, cellulose synthases are only partially coaligned with cortical microtubules and unrelated to callose plugs. Callose synthase also comigrates with tubulin by Blue Native-polyacrylamide gel electrophoresis. Membrane sucrose synthase, which expectedly provides UDP-glucose to callose synthase and cellulose synthase, binds to actin filaments depending on sucrose concentration; its distribution is dependent on the actin cytoskeleton and the endomembrane system but not on microtubules.     

The Nutrimentary Egg Development of the Mite,Varroa jacobsoni (Acari,Arachnida), an Ectoparasite of Honey Bees

The fine structure of the female gonad of Varroa jacobsoni is described. There are two components: the ovary proper and the so-called lyrate organ. The ovary is the place where oocytes mature, embedded in a supporting tissue composed of two cell types: somacells 1 and somacells 2. The lyrate organ has a nutrimentary function and is comprised of two components: supporting cells and nutritive tissue. The supporting cells are similar to the somacells 2 in that they contain abundant microtubules. The nutritive tissue is an extensive syncytium. It is connected with the oocytes via intercellular bridges, the nutritive cords. Oocytes and nutritive tissue are thought to have derived from common stem cells. From fine structural evidence it is concluded that ribosomes are one of the most important components to be transported via the nutritive cords into the oocytes. However, an increase in number of mitochondria in the middle-stage oocytes may also be a consequence of transport of these organelles from the nutritive tissue into the oocytes. Further characteristics make plausible that the interdependences of oocytes and nutritive tissue are comparable to those found in meroistic ovarioles of insects. The somatic components do not seem to be as important as the follicle cells of insects, however. It is assumed that the evolution of a nutrimentary oogenesis speeds up embryogenesis. Thus, the differentiation of the female gonad of Varroa jacobsoni may have facilitated the species' adaptation to a development completed in a short and limited time within the shelter of the covered brood cell of the bee.     

Corn Mitochondrial Swelling and Contraction-an Alternate Interpretation

Mitochondria isolated from 3-day-old etiolated corn shoots (Zea mays L.) can be categorized into three separate groups, each group characteristic of the cell type from which the mitochondria were isolated. Phloem sieve tubes and some adjacent parenchyma cells contain mitochondria that have few cristae and little amorphous matrix. Mitochondria from meristematic and undifferentiated cells have more cristae and matrix. Vaculate and differentiated cells have mitochondria with well-developed cristae and abundant matrix. Each mitochondrial type exhibits typical in vitro spontaneous swelling and substrate-induced contraction responses. characterized by change or lack of change in cristae size and in density of amorphous material. For the second and third types of mitochondria, swelling and contraction are characterized by a change in degree of cristae size and in matrix density. The first type undergoes few changes upon swelling or contraction. Radical changes of the inner membrane, withdrawal and infolding, are associated with cell differentiation and not with swelling and contraction of isolated corn shoot mitochondria.     

One-Dimensional Brownian Motion of Charged Nanoparticles along Microtubules: A Model System for Weak Binding Interactions

Various proteins are known to exhibit one-dimensional Brownian motion along charged rodlike polymers, such as microtubules (MTs), actin, and DNA. The electrostatic interaction between the proteins and the rodlike polymers appears to be crucial for one-dimensional Brownian motion, although the underlying mechanism has not been fully clarified. We examined the interactions of positively-charged nanoparticles composed of polyacrylamide gels with MTs. These hydrophilic nanoparticles bound to MTs and displayed one-dimensional Brownian motion in a charge-dependent manner, which indicates that nonspecific electrostatic interaction is sufficient for one-dimensional Brownian motion. The diffusion coefficient decreased exponentially with an increasing particle charge (with the exponent being 0.10 k_BT per charge), whereas the duration of the interaction increased exponentially (exponent of 0.22 k_BT per charge). These results can be explained semiquantitatively if one assumes that a particle repeats a cycle of binding to and movement along an MT until it finally dissociates from the MT. During the movement, a particle is still electrostatically constrained in the potential valley surrounding the MT. This entire process can be described by a three-state model analogous to the Michaelis-Menten scheme, in which the two parameters of the equilibrium constant between binding and movement, and the rate of dissociation from the MT, are derived as a function of the particle charge density. This study highlights the possibility that the weak binding interactions between proteins and rodlike polymers, e.g., MTs, are mediated by a similar, nonspecific charge-dependent mechanism.     

Interaction of human serum high density lipoprotein apoprotein with phospholipids

The interaction of the apoprotein of human serum high density lipoprotein-3 (apo HDL₃)¹¹ with aqueous dispersion of natural and synthetic phospholipids (PL) was investigated at a temperature above the transitions of the PL hydrocarbon chains and also above their critical micellar concentration. This protein is known to contain two major polypeptides: apo A-I and apo A-II. The protein:PL mixtures (weight ratio, 2, 1 or 0.5) were subjected to sonic irradiation and then fractionated by either CsCl or D₂O-sucrose density gradient ultracentrifugation. Usually three bands were obtained, the relative mass distribution of which depended upon the nature of the PL and the ratio of the interacting components: one band contained the PL-poor protein (d 1.28 g/ml), another, the uncombined PL (d ? 1.08 g/ml), and the third band, both protein and PL. This band, which was considered to represent the actual complex, had a hydrated density intermediate between those of either apo HDL₃ or PL alone, a particle weight of 80,000 to 170,000 (i.e., less than that of intact HDL₃), a morphology by electron microscopy which depended on the materials and experimental conditions employed and circular dichroic spectra