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.     

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.     

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.     

The distribution and function of microtubules in nutritive tubes

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.     

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.     

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.     

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.     

Perturbed turnover of microtubule-based nutritive tubes in ovarioles of virgin and precocene-treated Dysdercus fasciatus

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.     

Microtubule-dependent microtubule nucleation based on recruitment of gamma-tubulin in higher plants

Despite the absence of a conspicuous microtubule-organizing centre, microtubules in plant cells at interphase are present in the cell cortex as a well oriented array. A recent report suggests that microtubule nucleation sites for the array are capable of associating with and dissociating from the cortex. Here, we show that nucleation requires extant cortical microtubules, onto which cytosolic gamma-tubulin is recruited. In both living cells and the cell-free system, microtubules are nucleated as branches on the extant cortical microtubules. The branch points contain gamma-tubulin, which is abundant in the cytoplasm, and microtubule nucleation in the cell-free system is prevented by inhibiting gamma-tubulin function with a specific antibody. When isolated plasma membrane with microtubules is exposed to purified neuro-tubulin, no microtubules are nucleated. However, when the membrane is exposed to a cytosolic extract, gamma-tubulin binds microtubules on the membrane, and after a subsequent incubation in neuro-tubulin, microtubules are nucleated on the pre-existing microtubules. We propose that a cytoplasmic gamma-tubulin complex shuttles between the cytoplasm and the side of a cortical microtubule, and has nucleation activity only when bound to the microtubule.     

Cytoplasmic dynein mediates adenovirus binding to microtubules

During infection, adenovirus (Ad) capsids undergo microtubule-dependent retrograde transport as part of a program of vectorial transport of the viral genome to the nucleus. The microtubule-associated molecular motor, cytoplasmic dynein, has been implicated in the retrograde movement of Ad. We hypothesized that cytoplasmic dynein constituted the primary mode of association of Ad with microtubules. To evaluate this hypothesis, an Ad-microtubule binding assay was established in which microtubules were polymerized with taxol, combined with Ad in the presence or absence of microtubule-associated proteins (MAPs), and centrifuged through a glycerol cushion. The addition of purified bovine brain MAPs increased the fraction of Ad in the microtubule pellet from 17.3% +/- 3.5% to 80.7% +/- 3.8% (P < 0.01). In the absence of tubulin polymerization or in the presence of high salt, no Ad was found in the pellet. Ad binding to microtubules was not enhanced by bovine brain MAPs enriched for tau protein or by the addition of bovine serum albumin. Enhanced Ad-microtubule binding was also observed by using a fraction of MAPs purified from lung A549 epithelial cell lysate which contained cytoplasmic dynein. Ad-microtubule interaction was sensitive to the addition of ATP, a hallmark of cytoplasmic dynein-dependent microtubule interactions. Immunodepletion of cytoplasmic dynein from the A549 cell lysate abolished the MAP-enhanced Ad-microtubule binding. The interaction of Ad with both dynein and dynactin complexes was demonstrated by coimmunoprecipitation. Partially uncoated capsids isolated from cells 40 min after infection also exhibited microtubule binding. In summary, the primary mode of Ad attachment to microtubules occurs though cytoplasmic dynein-mediated binding.     

Diverse effects of beta-tubulin mutations on microtubule formation and function

We have used in vitro mutagenesis and gene replacement to construct five new cold-sensitive mutations in TUB2, the sole gene encoding beta-tubulin in the yeast Saccharomyces cerevisiae. These and one previously isolated tub2 mutant display diverse phenotypes that have allowed us to define the functions of yeast microtubules in vivo. At the restrictive temperature, all of the tub2 mutations inhibit chromosome segregation and block the mitotic cell cycle. However, different microtubule arrays are present in these arrested cells depending on the tub2 allele. One mutant (tub2-401) contains no detectable microtubules, two (tub2-403 and tub2-405) contain greatly diminished levels of both nuclear and cytoplasmic microtubules, one (tub2-104) contains predominantly nuclear microtubules, one (tub2-402) contains predominantly cytoplasmic microtubules, and one (tub2-404) contains prominent nuclear and cytoplasmic microtubule arrays. Using these mutants we demonstrate here that cytoplasmic microtubules are necessary for nuclear migration during the mitotic cell cycle and for nuclear migration and fusion during conjugation; only those mutants that possess cytoplasmic microtubules are able to perform these functions. We also show that microtubules are not required for secretory vesicle transport in yeast; bud growth and invertase secretion occur in cells which contain no microtubules.     

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     

Microtubules in the microspikes and cortical cytoplasm of isolated cells

In thin sections through microspikes extending from the surface of isolated cells, a core has been seen which may contain microtubular elements. The differences between these and microtubules seen elsewhere in the cytoplasm are attributed to their rapid growth and exposed location which make them especially vulnerable to injury by preparative treatment. In support of this view it is shown that cytoplasmic microtubules may be altered or even destroyed by exposing the cells to changes in osmotic pressure. Associated with these straight microtubules in the cytoplasm were also found solid microfilaments. The form of these components and their location and alignment in portions of cells which are under tension or in motion suggest that they function in the structural support of the cell and its microspikes and in the transmission of tension in the cytoplasm. A second type of microtubule, smaller in diameter and tortuous in form, was also seen in certain cells and is presumed, from its shape, to have little to do with cytoplasmic support.     

Developmental reorientation of transverse cortical microtubules to longitudinal directions: a role for actomyosin-based streaming and partial microtubule-membrane detachment

Transversely oriented cortical microtubules in elongating cells typically reorient themselves towards longitudinal directions at the end of cell elongation. We have investigated the reorientation mechanism along the outer epidermal wall in maturing leek (Allium porrum L.) leaves using a GFP-MBD microtubule reporter gene and fluorescence microscopy. Incubating leaf segments for 14-18 h with the anti-actin or anti-actomyosin agents, 20 microm cytochalasin D or 20 mM 2,3-butanedione monoxime, inhibited the normal developmental reorientation of microtubules to the longitudinal direction. Observation of living cells revealed a small subpopulation of microtubules with their free ends swinging into oblique or longitudinal directions, before continuing to assemble in the new direction. Electron microscopy confirmed that longitudinal microtubules are partly detached from the plasma membrane. Incubating leaf segments with 0.2% 1 degree-butanol, an activator of phospholipase D, which has been implicated in plasma membrane-microtubule anchoring, promoted the reorientation, presumably by promoting microtubule detachment from the membrane. Stabilizing microtubules with 10 microm taxol also promoted longitudinal orientation, even in the absence of cytoplasmic streaming. These results were consistent with confocal microscopy of live cells before and after drug treatments, which also revealed that the slow (days) global microtubule reorientation is superimposed over short-term (hours) regional cycling in a clockwise and an anti-clockwise direction. We propose that partial detachment of transverse microtubules from the plasma membrane in maturing cells exposes them to hydrodynamic forces of actomyosin-driven cytoplasmic streaming, which bends or shifts pivoting microtubules into longitudinal directions, and thus provides an impetus to push microtubule dynamics in the new direction.     

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.     

Interaction of an Overexpressed gamma-Tubulin with Microtubules In Vivo and In Vitro

gamma-Tubulin is an ubiquitous MTOC (microtubule-organizing center) component essential for the regulation of microtubule functions. A 1.8 kb cDNA coding for gamma-tubulin was isolated from CHO cells. Analysis of nucleotide sequence predicts a protein of 451 amino acids, which is over 97% identical to human and Xenopus gamma-tubulin. When CHO cells were transiently transfected with the gamma-tubulin clone, epitope-tagged full-length, as well as truncated polypeptides (amino acids 1-398 and 1-340), resulted in the formation of cytoplasmic foci of various sizes. Although one of the foci was identified as the centrosome, the rest of the dots were not associated with any other centrosomal components tested so far. The pattern of microtubule organization was not affected by induction of such gamma-tubulin-containing dots in transfected cells. In addition, the cytoplasmic foci were unable to serve as the site for microtubule regrowth in nocodazole-treated cells upon removal of the drug, suggesting that gamma-tubulin-containing foci were not involved in the activity for microtubule formation and organization. Using the monomeric form of Chlamydomonas gamma-tubulin purified from insect Sf9 cells (), interaction between gamma-tubulin and microtubules was further investigated by immunoelectron microscopy. Microtubules incubated with gamma-tubulin monomers in vitro were associated with more gold particles conjugated with gamma-tubulin than in controls where no exogenous gamma-tubulin was added. However, binding of gamma-tubulin to microtubules was not extensive and was easily lost during sample preparation. Although gamma-tubulin was detected at the minus end of microtubules several times more frequently than the plus end, the majority of gold particles were seen along the microtubule length. These results contradict the previous reports (; ), which might be ascribed to the difference in the level of protein expression in transfected cells.     

Unidirectional microtubule assembly in cell-free extracts of Spisula solidissima oocytes is regulated by subtle changes in pH

Most, if not all, microtubules in vivo grow unidirectionally from a nucleation site such as the centrosome. This organized growth of microtubules can generate and maintain the radially symmetrical array of interphase microtubules as well as the bipolar mitotic apparatus. To investigate the regulation of polarized microtubule growth, we have prepared a cell-free extract from surf clam oocytes that exhibits unidirectional microtubule assembly. Immunofluorescence microscopy was used to visualize the net assembly of microtubules onto the fast (plus)- and slow (minus)- growing ends of isolated ciliary axonemes. All detectable microtubule growth in these cytoplasmic extracts occurred at the plus (+) ends and the extent of (+) end growth was regulated by subtle changes in pH. Microtubule assembly in these crude extracts was highly favored at pH 7.3, the pH of the post-fertilization cytoplasm. In contrast, when tubulin was purified from these oocyte extracts, integral components were lost, and microtubule growth became predominantly bidirectional and was favored at acidic pH. These results indicate that cytoplasmic factors may inhibit bidirectional growth in vivo and that temporal or local changes in cytoplasmic pH may influence microtubule assembly during the cell cycle.     

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.     

Identification with cellular microtubules of one of the co-assemlbing microtubule-associated proteins.

In this paper we describe a procedure for detecting proteins associated with cytoplasmic microtubules in vivo. Detergent-extracted cytoskeletons of NIL8 hamster cells are prepared under conditions which preserve the microtubules. The cytoskeletons are then extracted in the presence of calcium, which depolymerizes the microtubules and quantitatively extracted cytoskeletons are prepared from cells that have been incubated with colchicine. The cytoskeletons from these cells contain no microtubules or tubulin. Electrophoretic analysis of the calcium extracts of the colchicine-treated and untreated cells reveals several radioactively labeled polypeptides. There is, however, no apparent quantitative or qualitative difference between the two extracts other than the tubulin polypeptides. Each of the extracts is mixed with an excess of unlabeled calf brain microtubule protein and carried through cycles of temperature-dependent microtubule assembly. Distinct species from each extract co-assemble at a constant ratio, but only one polypeptide is uniquely derived from cells containing intact microtubules. The molecular weight of this polypeptide is similar to that proposed for the tau species detected in brain microtubule preparations.     

Dynein, dynactin, and kinesin II's interaction with microtubules is regulated during bidirectional organelle transport

The microtubule motors, cytoplasmic dynein and kinesin II, drive pigmented organelles in opposite directions in Xenopus melanophores, but the mechanism by which these or other motors are regulated to control the direction of organelle transport has not been previously elucidated. We find that cytoplasmic dynein, dynactin, and kinesin II remain on pigment granules during aggregation and dispersion in melanophores, indicating that control of direction is not mediated by a cyclic association of motors with these organelles. However, the ability of dynein, dynactin, and kinesin II to bind to microtubules varies as a function of the state of aggregation or dispersion of the pigment in the cells from which these molecules are isolated. Dynein and dynactin bind to microtubules when obtained from cells with aggregated pigment, whereas kinesin II binds to microtubules when obtained from cells with dispersed pigment. Moreover, the microtubule binding activity of these motors/dynactin can be reversed in vitro by the kinases and phosphatase that regulate the direction of pigment granule transport in vivo. These findings suggest that phosphorylation controls the direction of pigment granule transport by altering the ability of dynein, dynactin, and kinesin II to interact with microtubules.