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During early oogenesis in Dysdercus fasciatus, anteriorly positioned nurse cells supply each oocyte with mRNA, ribosomes, and proteins via a microtubule-rich nutritive tube that lengthens as the oocyte is displaced backwards down an ovariole. Nurse cell-dependent development of an oocyte continues until the latter reaches a particular stage of oogenesis after which the nutritive tube supplying it becomes redundant and breaks down. The signal for nutritive tube breakdown is believed to derive from the oocyte, and to be developmental stage-specific. To explore this, nutritive tube turnover has been investigated following the experimental inhibition of oocyte maturation both by the prevention of mating, and also the topical application of precocene II. In each case, the nutritive tubes with their component microtubules continued to extend and failed to show normal tube redundancy, typified by microtubule rearrangement and then depolymerisation. This provided an in vivo demonstration that the dynamics of a large microtubule aggregate are influenced by the developmental state of the cytoplasm., (Copyright 2002 Wiley-Liss, Inc.)

Messenger RNAs are localized in both somatic and germ cells as a means of focusing the translation of proteins at specific cellular sites. The signals for this lie within the mRNA, and these are recognized by proteins in the cell. The latter appear to be attached via linker proteins to the transport machinery for localization. In some instances it is a myosin motor which translocates along actin microfilaments, and in others kinesin or dynein motors appear to be responsible for driving the movement of mRNA along microtubule substrates. The way that cytoskeleton-based mRNA translocation is regulated is speculated upon.

In Drosophila melanogaster the staufen gene encodes an RNA-binding protein that is essential for the correct localization of certain nurse cell-derived transcripts in oocytes. Although the mechanism underlying mRNA localization is unknown, mRNA-staufen complexes have been shown to move in a microtubule-dependent manner, and it has been suggested that staufen associates with a motor protein which generates the movement. We have investigated this possibility using Notonecta glauca in which nurse cells also supply the oocytes with mRNA, but via greatly extended nutritive tubes comprised of large aggregates of parallel microtubules. Using a staufen peptide antibody and RNA probes we have identified a staufen-like protein, which specifically binds double-stranded RNA, in the nutritive tubes of Notonecta. We show that while the staufen-like protein does not co-purify with microtubules from ovaries using standard procedures it does so under conditions of motor-entrapment, specifically in the presence of AMP-PNP. We also show that the staufen-like protein is subsequently removed by ATP and GTP, but not ADP. Nucleotide-dependent binding to microtubules is typical of a motor-mediated interaction and the pattern of attachment and detachment of the staufen-like protein correlates with that of a kinesin protein within the ovaries. Our findings indicate that the staufen-like RNA-binding protein attaches to, and is transported along, Notonecta ovarian microtubules by a kinesin motor.

In ovarioles of hemipteran insects, RNA passes from anteriorly positioned nurse cells to the chain of developing oocytes via extended nutritive tubes. These intercellular connections may reach several millimeters in length. Each nutritive tube is comprised of many thousands of parallel microtubules. We have extracted microtubule bundles from isolated nutritive tubes of Notonecta glauca and, using hybridization techniques, provide evidence of poly(A) mRNA attachment to microtubules in vivo. We also show this attachment to be nucleotide-sensitive, which is typical of a motor protein-mediated interaction. The pattern of nucleotide sensistivity is indicative of a kinesin motor mechanism. We provide evidence that a kinesin is present in the nutritive tube translocation channels and is a component of the mRNA/microtubule bundles isolated and extracted from them. Our findings are consistent with kinesin-driven transport of mRNA along the nutritive tube microtubules.

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), later 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 (p34cdc2) 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.

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.

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.

The nurse cells in insect ovarioles supply the developing oocytes with various cellular components, including mRNAs, which pass from one cell to the other through intercellular bridges traversed by microtubules. Best studied of these mRNAs are those that encode the axis-determining factors in Drosophila embryos. These mRNAs are further translocated and localized within the oocyte to sites where the products of their translation will ultimately function. This article explores the evidence supportive of a role for microtubules and motor proteins in these processes.

Bundles of native microtubules isolated from the ovarioles of hemipteran insects are seen to shimmer when observed using dark-field microscopy. This novel form of microtubule motility becomes even more obvious when the isolated bundles are detergent-extracted and reactivated. We have studied the nucleotide-specificity and the drug-sensitivity of microtubule shimmering in order to obtain information regarding the nature of the motor protein responsible, and to compare its properties with those of previously characterised microtubule motors. The involvement of structural MAPs in the shimmering and in maintenance of microtubule bundles in this system has also been investigated.

Results presented here indicate that the high molecular weight microtubule-associated proteins isolated from the ovaries of the hemipterans, Oncopeltus fasciatus and Notonecta glauca, while absent from nervous tissue, are present in the testes of the respective species. Here they are seen to be located to extensive microtubule aggregates within the cells surrounding the sperm, but not obviously to the sperm themselves.

MAPs and microtubule motor proteins from the massive microtubule translocation complexes within the ovaries of hemipteran insects have been identified and characterized. Both classes of proteins have been compared with those of other systems, and the function of both in the insect ovaries is speculated upon.

The enormous numbers of microtubules within the nutritive tubes of hemipteran ovarioles are amenable to the hook-decoration technique for determining microtubule structural polarity, as they can be microdissected from ovarioles intact. This has allowed the correlation between the polarity of this continuously elongating complex of microtubules within a nutritive tube and the direction of transport along its length; and has shown that the plus or fast growing ends of the microtubules are all situated at the anterior end of a nutritive tube proximal to the trophic region from which synthesised materials are passed back towards the developing oocytes.

The movement of radioactively labelled polypeptides into the microtubule-associated transport channels in the ovaries of a hemipteran insect has been analysed using SDS-polyacrylamide slab gel electrophoresis and fluorography. The patterns of label suggest that the microtubules which pack the transport channels form a relatively static cytoskeleton while other components move independently from them along the channels. As well as illustrating the functional organisation of microtubule-associated transport in this system our studies of labelled proteins have also provided clues as to the mechanism of transport itself.

Microtubule-based translocation channels within the ovaries of the hemipteran insect Notonecta have been isolated by microdissection, and then detergent-extracted to leave a bundle of some 30,000 aligned microtubules. On addition of ATP and other hydrolysable nucleotides the microtubule bundle contorts into a helical configuration, a property we have called 'corkscrewing', before straightening again. This we believe to be indicative of force generation within the bundle. Electrophoretic analysis of the bundle of native microtubules reveals many polypeptides apart from the tubulins, and a number of these comigrate with microtubule-associated proteins (MAPs), which copolymerize with tubulins in reassembled microtubules from the same system. Corkscrewing does not occur if the microtubule bundle is pretreated with salt, a procedure that removes MAPs from microtubules, suggesting that the force is generated by a MAP or MAPs. In addition, certain minor polypeptides comprising the native microtubules are ATP-sensitive, a property expected of a microtubule motor.

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.

In this study we have applied microtubule-associated proteins (MAPs) from mammalian brain to both native and reassembled insect ovarian microtubules. Such microtubules, which are normally smooth walled, become decorated with projections similar to those observed when mammalian brain MAPs are added back to assembling or assembled mammalian brain microtubules. The mammalian MAPs were also detected as components of insect microtubules when analyzed by polyacrylamide gel electrophoresis. Our observations suggest that mammalian brain MAPs have common binding sites on microtubules from two widely different sources and indicate the degree of evolutionary conservation of such sites.

1. The trophozoites of Selenidium fallax propagate bending waves at rates of up to 35 microns s-1, of a similar character to those manifested by eukaryotic cilia and flagella. A beat frequency of 0.12-0.15 Hz appears average, though rates outside this range have been recorded. Translatory locomotion at up to 6 microns s-1 has been observed. The protozoan demonstrates the presence of an active bending mechanism, probably along its entire length, and a means of coordinating adjacent bends. 2. The Reynolds number for the motion in 10(-5)-10(-4), suggesting that the hydrodynamic aspects of the trophozoite movement are amenable to analysis by similar means to those already employed for cilia and flagella. 3. It is possible that the protozoans exhibit a sliding microtubule mechanism, which could be very usefully compared with that occurring in the ciliary axoneme.

Hook decoration with pig brain tubulin was used to assess the polarity of microtubules which mainly have 15 protofilaments in the transcellular bundles of late pupal Drosophila wing epidermal cells. The microtubules make end-on contact with cell surfaces. Most microtubules in each bundle exhibited a uniform polarity. They were oriented with their minus ends associated with their hemidesmosomal anchorage points at the apical cuticle-secreting surfaces of the cells. Plus ends were directed towards, and were sometimes connected to, basal attachment desmosomes at the opposite ends of the cells. The orientation of microtubules at cell apices, with minus ends directed towards the cell surface, is opposite to the polarity anticipated for microtubules which have elongated centrifugally from centrosomes. It is consistent, however, with evidence that microtubule assembly is nucleated by plasma membrane-associated sites at the apical surfaces of the cells (Mogensen, M. M., and J. B. Tucker. 1987. J. Cell Sci. 88:95-107) after these cells have lost their centriole-containing, centrosomal, microtubule-organizing centers (Tucker, J. B., M. J. Milner, D. A. Currie, J. W. Muir, D. A. Forrest, and M.-J. Spencer. 1986. Eur. J. Cell Biol. 41:279-289). Our findings indicate that the plus ends of many of these apically nucleated microtubules are captured by the basal desmosomes. Hence, the situation may be analogous to the polar-nucleation/chromosomal-capture scheme for kinetochore microtubule assembly in mitotic and meiotic spindles. The cell surface-associated nucleation-elongation-capture mechanism proposed here may also apply during assembly of transcellular microtubule arrays in certain other animal tissue cell types.

Root tip mitosis in both Vinca rosea and the chosen control Lepidium sativum is susceptible to the effects of colchicine at and above 5X10(-4)M.--Root tip mitosis in L. sativum is susceptible to the effects of vinblastine at concentrations equal to and greater than 10(-4)M, while root tip mitosis in V. rosea, the plant which is the source of vinblastine, is unaffected by exogenously-applied VBL at concentrations up to and including 10(-2)M.--The possible mechanisms by which the resistance of V. rosea to vinblastine might be conferred are discussed.

The density and distribution of microtubules in the nutritive tubes of three hemipteran insects, Corixa punctata, Notonecta glauca and Dysdercus cingulatus, were analysed from electron micrographs by computer. Both parameters varied amongst all three species, the density of microtubules in Corixa being approximately three times that seen in Dysdercus. The density and distribution of microtubules within the nutritive tubes correlated directly with the size of particle transported by them, suggesting that the microtubules may act as a sieve to preclude certain cellular components from entering the tubes. If microtubules are further involved in the generation of motive force for cytoplasmic transport along the nutritive tubes, this function is not dependent upon the number of microtubules, or upon their arrangement with respect either to each other or to the particle being transported.

Effects of glycerol and freezing on microtubules in three different systems viz. the ovaries of Notonecta glauca, the sperm tails of Notonecta glauca and the hyphae of the fungus Phanaerochaete velutina have been studied by means of freeze-substitution. The protofilamentous substructure of microtubules is visible in positive image whether or not the tissues are treated with glycerol prior to freezing, suggesting that it is the freezing which allows this by the weakening of hydrophobic bonding between protofilaments. Glycerol, used as a cryoprotectant, causes a re-arrangement of the microtubules which is proportional to the concentration of, and the duration of exposure to, glycerol. This rearrangement of microtubules is greater in systems where the microtubules are not inter-linked than those where microtubules have obvious intertubule linkages. Rates of cooling on freezing affected the degree of rearrangement and was less with propane than with Freon 22. Possible explanations for these effects have been discussed.

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.

The ovarioles of Coccinella and Tenebrio are shown to be telotrophic--a characteristic normally associated with hemipterans rather than coleopterans. The 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 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.

The massive system of microtubules in the ovarioles of Notonecta glauca has been prepared for electron microscopy by means of freeze-substitution. With this technique the protofilaments comprising the microtubule walls can be observed in positive image, indicating that the procedure provides a valuable method for observing microtubule substructure. Surprisingly though, the cryoprotectants, which are an integral part of the method, caused a rearrangement of the microtubules from their usual pattern in the ovarioles. These observations, we believe, provide an insight into intra- and intermicrotubule interactions respectively.

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 microtubule 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.

Nutritive tubes, the microtubule-based translocation channels that link the trophic tissue to the developing oocytes in the ovaries of hemipteran insects, have been isolated and examined using video-enhanced differential interference contrast microscopy. When viewed in this way the nutritive tubes are seen to fray into linear strands, which, on the addition of exogenous ATP, support the translocation of particles along their lengths. The movement is also seen with GTP but not AMP-PNP. It is not affected by the addition of inhibitors of dynein or of energy metabolism. Electron microscopy shows the strands to consist of bundles of parallel microtubules of different sizes and the moving particles to be mitochondria. Comparisons are drawn between the movement of mitochondria along isolated insect ovarian microtubules and the reported translocation of vesicles along microtubules from squid axoplasm. The simplicity of the insect system is emphasized. The fact that it can be isolated easily and characterized biochemically makes it potentially valuable for investigating microtubule-based translocation.