Cell Motility and the Cytoskeleton 43:159–166 (1999) Poly(A) mRNA Is Attached to Insect Ovarian Microtubules In Vivo in a Nucleotide-Sensitive Manner Susan Stephen, Nicholas J. Talbot, and Howard Stebbings* School of Biological Sciences, Washington Singer Laboratories, University of Exeter, United Kingdom 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. Cell Motil. Cytoskeleton 43:159–166, 1999. r 1999 Wiley-Liss, Inc. Key words: insect oogenesis; mRNA transport; microtubule motor proteins INTRODUCTION The localization of mRNA to discrete domains has been reported in a number of polarised somatic cells such as neurones, fibroblasts, and epithelial cells, and particularly in the oocytes and eggs of different species [Wilhelm and Vale, 1993]. The distribution of mRNA allows the spatial control of gene expression and in oocytes asymmetric localization of specific mRNAs has been shown to underlie the establishment of morphogen protein gradients, which in turn determine cell fate during early development [for reviews see St Johnston, 1995; Bashirullah et al., 1998]. In Drosophila oogenesis, the example that has been most extensively studied, a large number of transcripts, including those encoding polarity determinants [St Johnston and Nusslein-Volhard, 1992] and cell-cycle regulatory proteins [Dalby and Glover, 1992], have been shown to possess localization signals in their 38 untranslated regions (UTRs) [see Spradling, 1993] and to pass selectively in a multistep process from a group of nurse cells, via intercellular bridges called ring canals, to precise locations within the adjacent oocyte [Theurkauf and Hazelrigg, 1998]. r 1999 Wiley-Liss, Inc. In Drosophila egg chambers, microtubules traverse the short ring canals between fifteen precisely arranged nurse cells and the oocyte, and are therefore appropriately placed to act as substrates for mRNA transport. The microtubules derive from a centrosome within the oocyte [Theurkauf et al., 1992], so that transport between the nurse cells and an oocyte is in the retrograde direction. The involvement of the microtubules in the passage of maternal mRNA from nurse cells to oocytes has been demonstrated, because if flies are fed with the antimicrotubule agent colchicine, the microtubules are disrupted and the mRNAs are incorrectly localized [Pokrywka and Stephenson, 1991; Theurkauf et al., 1993]. Mislocalization of transcripts also occurs in Drosophila Contract grant sponsor: The Wellcome Trust; Contract grant number: 047511; Contract grant sponsor: University of Exeter Research Fund. *Correspondence to: Howard Stebbings, School of Biological Sciences, Washington Singer Laboratories, Perry Road, Exeter EX4 4QG, UK. E-mail: H. Stebbings@exeter.ac.uk. Received 15 January 1999; accepted 22 March 1999. 160 Stephen et al. mutants showing abnormal arrangements of microtubules in their egg chambers [Lane and Kalderon, 1994]. Particles believed to contain maternal meassages have been tracked in Drosophila egg chambers and shown to move in a microtubule-dependent fashion [Theurkauf and Hazelrigg, 1998], and the co-localization of microtubule motor proteins with different maternal-effect gene products within the system [Clark et al., 1994; Li et al., 1994] has suggested that they could propel the mRNA along the microtubules. However, while it is apparent that microtubules are necessary for the localization of mRNAs, no direct evidence for their role in mRNA transport has been obtained, and a participating motor protein has not been identified. In this study, we have focused on oogenesis in the insect Notonecta glauca. By contrast with Drosophila, in Notonecta and hemipterans generally the nurse cells are retained at the anterior while the oocytes are displaced as a chain down an ovariole (see Fig. 1). As a consequence, the intercellular bridges between the nurse cell region and each of the oocytes become greatly extended into nutritive tubes. In Notonecta, the nutritive tubes may reach several millimeters in length. Each is packed with some thirty thousand aligned microtubules interspersed with ribosomes, and bounded by sparsely arranged mitochondria [Macgregor and Stebbings, 1970]. The extensive microtubule aggregates in nutritive tubes of Notonecta are long-lived and stable, and are not depolymerised by anti-microtubule agents or cold [Macgregor and Stebbings, 1970; Lane and Stebbings, 1994]. Importantly, microtubules are the only cytoskeletal elements present in nutritive tubes. They have a common polarity, with their minus ends towards the oocyte [Stebbings and Hunt, 1983], so that nurse cell/oocyte transport is in the retrograde direction. Large quantities of RNA pass from the nurse cells along the nutritive tubes to the oocytes, and autoradiographic studies with Notonecta have shown that the bulk of this travels at a slow rate (⬃30 µm/h), probably passively and as monomeric ribosomes [Macgregor and Stebbings, 1970]. Similar studies reported a comparable rate of RNA movement along the nutritive tubes of Pyrrhocoris, a different hemipteran, but also resolved a smaller faster component travelling at ⬃200 µm/h, believed to be actively transported mRNA [Mays, 1972]. The presence of mRNA in nutritive tubes was subsequently demonstrated in a further species, Oncopeltus, by in situ hybridization to sections of ovarioles [Capco and Jeffery, 1979]. Because of their size and stability, microtubule bundles can be isolated and detergent-extracted from isolated nutritive tubes of Notonecta [Hyams and Stebbings, 1979; Stebbings and Hunt, 1983] and this presents a unique opportunity to investigate directly the relation- Fig. 1. Diagrammatic representation of the disposition of the nurse cells and a developing oocyte within an ovariole of Notonecta. In Notonecta, each oocyte is connected to a large number of nurse cells by a greatly extended nutritive tube (NT). Large numbers of microtubules (MTS) pack the nutritive tube (plus and minus signs indicate their polarity). In Drosophila, by comparison, an oocyte is closely connected to fifteen nurse cells via short ring canals. ship between microtubules and components that translocate along them. Using hybridization techniques we confirm that poly(A) mRNA is present in the nutritive tubes of Notonecta. We provide direct evidence that the poly(A) mRNA is attached to microtubules extracted from isolated nutritive tubes. We also show that this attachment is nucleotide-sensitive, a property consistent with a motor protein-mediated interaction between poly(A) mRNA and the microtubules. The pattern of nucleotide sensitivity predicts kinesin-driven transport of mRNA, and this is supported by our identification of a kinesin within the nutritive tubes and, more specifically, mRNA/ microtubule bundles isolated from them. MATERIALS AND METHODS Animals and Dissection Notonecta glauca females were dissected in Locke’s modified insect Ringer [Hyams and Stebbings, 1979] and Attachment of mRNA to Microtubules their ovaries placed immediately into RNAse-free microtubule support buffer (PEM); 0.1 M PIPES pH 6.9, monosodium salt (Calbiochem, La Jolla, CA), 1 mM EGTA, 2.5 mM MgSO4 (BDH-Merck, Lutterworth, UK). All subsequent manipulations were performed in RNasefree PEM. All chemicals were from Sigma Chemical Co. (St Louis, MO) unless stated otherwise. Isolation and Detergent Extraction of Nutritive Tubes Nutritive tubes were isolated from ovarioles in polarised light, using tungsten needles [Hyams and Stebbings, 1979]. They were left attached to the nurse cell trophic regions for ease of handling. Extraction was performed with 0.5% Triton X-100 (Boehringer Mannheim UK Ltd., Lewes, UK) for 30–60 sec [Stebbings and Hunt, 1983]. Oligonucleotide Hybridization and Detection Isolated and isolated, extracted nutritive tubes were rinsed briefly in PEM and fixed in 3% paraformaldehyde in phosphate buffer for 15 min, and rinsed in PBS. Digoxygenin-labelled 24-mer oligo (dT) probe (R & D Systems Inc., Minneapolis, MN) was applied to the tubes at a concentration of 1.5 µg ml⫺1 in hybridization solution [Alison et al., 1994] in a humidified chamber at 37°C for at least 4 h [Sambrook et al., 1989]. After hybridization, tissues were washed to high stringency in 1 ⫻ SSC, 37°C for 30 min [Sambrook et al., 1989] and rinsed 3 ⫻ 3 min in Tris buffered detergent (TBD); 50 mM Tris, 150 mM sodium chloride, 0.1% Triton X-100, pH 7.6. Tissues were blocked in TBDB (TBD with 5% non-fat dried milk) for 10 min prior to incubation with 1:200 dilution alkaline phosphatase-conjugated sheep anti-digoxygenin fab fragments (Boehringer Mannheim) in TBDB for 30 min at 37°C. Tissues were rinsed for 2 ⫻ 3 min in TBD and 2 ⫻ 3 min in deionised water. Detection of hybridization was carried out in 100 µl Western Blue Stabilized Substrate (Promega, Madison, WI) for 30 min in the dark at room temperature (RT), and the reaction stopped by washing 3 ⫻ 5 min in deionised water. In some control preparations, extracted nutritive tubes were treated for 30 min at 37°C with 0.1 Uµl⫺1 RNase One (Promega UK Ltd., Southampton, UK) prior to hybridization. In others, either the oligo (dT)digoxygenin probe or the antidigoxygenin-alkaline phosphatase detection conjugate were omitted. In still further preparations, 24-mer oligo (dA) replaced the oligo (dT). Following the hybridization procedures, isolated nutritive tubes or isolated, extracted tubes were located by differential interference contrast (DIC) microscopy using a Zeiss (Oberkochen, Germany) Axiophot photomicroscope with ⫻20 and ⫻40 objectives, and then the presence or absence of hybridization label in such 161 preparations was observed using bright field (BF) microscopy. Application of Nucleotides Isolated extracted nutritive tubes were incubated in 10 mM ADP, 2–10 mM ATP, or 2–10 mM GTP for 2 min, and then rinsed briefly 3 times in PEM prior to fixation and hybridization with oligo (dT), as previously described. Other tubes were pre-incubated in 0.5 mM AMP-PNP or 2 Uml⫺1 apyrase for 30 min. Electrophoresis and Western Blot Analysis Notonecta ovarian clarified supernatant was prepared as per Anastasi and coworkers . Ovaries from 25 Notonecta were dissected and placed in 0.1 M PIPES. Following homogenization on ice in the presence of 1.5 mM CaCl2 to depolymerise the microtubules, 2 mM EGTA was added to chelate the calcium and the suspension was spun at 33,000g for 30 min at 4°C. The supernatant was removed and ultracentrifuged at 132,000g for a further hour at 4°C. The clarified supernatant was mixed with Laemmli buffer [Laemmli, 1970] and separated by one-dimensional SDS-PAGE on 5–10% polyacrylamide gels under non-reducing conditions. Proteins were either detected using 5% Coomassie Blue R-250 or transferred to Immobilon-P membrane (Millipore, Bedford, MA) using a semi-dry blotting system (Schleider and Schuell Ltd., Dassel, Germany) for Western blot analysis. Membranes were washed in PBS and incubated in 5% Marvel (Nestle, Basel, Switzerland) in PBS for 1 h at RT. After rinsing in PBS, blots were treated with 1:100 dilution rabbit anti-kinesin (a gift from Dr. P.J. Hollenbeck, Purdue University, W. Lafayette, IN) for 1 h at RT in the presence of 0.1% Nonidet P-40 (NP-40). Blots were washed three times for 5 min with PBS/0.1% NP-40 and then incubated for 1 h at RT with 1:300 dilution horse radish peroxidase-conjugated goat anti-rabbit IgG (DAKO Laboratories, High Wycombe, UK) followed by further washings in PBS/NP-40 buffer. Antibody binding was detected using L-chloronaphthanol/H2O2 reaction [LaRochelle, 1996]. Immunohistochemistry Ovarioles were processed for immunofluorescence according to the methods of Anastasi and co-workers . Ovarioles were fixed overnight in 4% paraformaldehyde in PBS buffer, washed three times in PBS, transferred to 10% sucrose in PBS for 1 h and then mounted and frozen in Oct compound (Gurr, BDH, Poole, UK). Transverse sections of ovarioles (10 µm) were cut using a Reichart-Jung (Vienna, Austria) Cryocut E, and collected on glass slides coated with 0.01% poly-l-lysine. Before labelling, sections were permeabilized with 0.1% Triton X-100 in PBS buffer, pH 7.4, for 10 min, washed 162 Stephen et al. Fig. 2. a–f: Isolated nutritive tubes after hybridization with digoxygeninconjugated oligo (dT), detected with alkaline phosphatase. Top: Bright field (BF) images. Bottom: Equivalent differential interference contrast (DIC) images for positional reference of the unlabelled tubes. a: Isolated nutritive tube showing hybridization signal throughout its length. b: After detergent extraction, hybridization signal labelled the extracted microtubule bundle. c: No signal was seen when oligo (dT) was substituted by oligo (dA). d: Pretreatment of the extracted microtubule bundle with RNase resulted in no hybridization signal. e: No signal was seen when oligo (dT) was omitted, or (f) when alkaline phosphatase-conjugated antibody was absent from the detection procedure. Bar ⫽ 50 µm. three times with PBS and then incubated with 5% BSA/5% normal goat serum in PBS buffer for 1 hour at RT to block non-specific binding, before rinsing with PBS containing 0.1% BSA. Sections were incubated with 1:100 dilution rabbit anti-kinesin polyclonal antibody, or mouse anti-kinesin heavy chain monoclonal antibody (Sigma Chemical Co.) for 1 h at RT. Sections were then washed three times in PBS/BSA buffer and incubated with 1:100 dilution of either rhodamine-conjugated goat anti-rabbit IgG (DAKO) or fluorescein isothiocyanateconjugated goat anti-mouse IgG (DAKO) as appropriate, for 1 h at RT. After rinsing three times in PBS/BSA, preparations were mounted in Vectashield (Vector Labs. Inc., Burlingame, CA) and viewed using a Zeiss Axiophot fluorescence microscope. Nutritive tubes were isolated and their microtubule bundles extracted and fixed, before being processed as described above for sections. RESULTS Electron Microscopy Nutritive tubes were isolated and extracted as above and prepared for electron microscopy [Stebbings and Hunt, 1987]. In ovarioles of N. glauca, nurse cells are retained anteriorly and supply a chain of developing oocytes via extended intercellular connections called nutritive tubes. Nutritive tubes are approximately 30 µm in diameter, reach several millimeters in length, and are strongly birefringent. Stripping away of the oocytes freed the nutritive tubes from the ovarioles, and isolated tubes remained birefringent for many hours. Electron micrographs of nutritive tubes revealed many thousands of aligned microtubules interspersed with ribosomes, with a few mitochondria at the tube peripheries. As seen in previous studies [Stebbings and Hunt, 1983], isolated nutritive tubes remained birefringent on extraction with non-ionic detergents, indicating that the microtubules persist as stable bundles following this procedure. This was confirmed in electron micrographs that showed that on removal of the membrane surrounding the tube, the ribosomes had been solubilised, and the microtubules, which packed more closely together, were the only structures remaining. After the incubation of isolated nutritive tubes with oligo (dT) followed by alkaline phosphatase detection of Attachment of mRNA to Microtubules 163 Fig. 3. a–f: Microtubule bundles from isolated, extracted nutritive tubes exposed to different nucleotide regimes prior to hybridization with oligo (dT). Top: Bright field. Bottom: Differential interference contrast. a: Nutritive tube extracted in the presence of apyrase, which appeared to have little effect on the hybridization signal. b: In the presence of AMP-PNP, the signal was consistently more intense. c: Pretreatment with 10 mM ADP had no effect on the hybridization signal. d: 5 mM ATP resulted in greatly reduced, although detectable, signal, while (e) no signal was seen after 10 mM ATP or (f) 5 mM GTP. Bar ⫽ 50 µm. the annealed probe, hybridization precipitation signal was seen throughout lengths of all the tubes examined (Fig. 2a). No signal was seen after RNase One pretreatment or with oligo (dA), suggesting specific hybridization of the oligo (dT) probe to polyadenylated RNA species within the nutritive tubes. To investigate the relationship between poly(A) mRNA and the microtubules within nutritive tubes, further hybridizations were carried out using microtubule bundles extracted from isolated tubes. These microtubule bundles consistently hybridized with oligo (dT) (Fig. 2b), demonstrating that poly(A) mRNA is attached to the microtubules. No hybridization occurred with oligo (dA) (Fig. 2c). In an additional series of control experiments, the application of RNase One to the microtubules prevented the hybridization with oligo (dT) (Fig. 2d). No hybridization signal was seen when the digoxygenin-conjugated oligo (dT) probe was omitted (Fig. 2e), thereby removing the antigen and showing that the antibody did not bind non-specifically to the microtubules. Similarly, no signal was observed when the alkaline phosphatase-conjugated antibody was absent from the detection procedure (Fig. 2f), showing that the signal was not due to endogenous alkaline phosphatase activity. To investigate the nature of mRNA attachment to the microtubules, the mRNA-microtubule complexes extracted from isolated nutritive tubes were subjected to different nucleotide regimes prior to the application of oligo (dT) probe. Extraction under conditions of ATP depletion using apyrase appeared to have little or no effect and resulted in the same levels of oligo (dT) hybridization to the microtubule bundles (Fig. 3a). Microtubule bundles extracted in the presence of AMP-PNP were strongly labelled with oligo (dT) hybridization signal (Fig. 3b). Signal was also present after the application of ADP (Fig. 3c). ATP (5 mM) resulted in greatly reduced hybridization signal (Fig. 3d), and no signal was detected in preparations treated with 10 mM ATP (Fig. 3e). The results were similar with GTP (Fig. 3f). Nucleotide sensitivity provides strong evidence that the attachment of poly(A) mRNA to nutritive tube microtubules is mediated by a microtubule motor-protein, and the pattern of sensitivity indicates involvement of a kinesin. Anti-kinesin antibodies identified a single protein band of ⬃120 kDa in Western blots of Notonecta ovarian proteins (Fig. 4a,b) and immunofluorescence studies of transverse sections of ovarioles of Notonecta, using two different anti-kinesin antibodies, showed strong labelling in the nutritive tubes (Fig. 4c). Similar microtubule bundles to those extracted from isolated nutritive tubes 164 Stephen et al. Fig. 4. a: Notonecta ovarian proteins stained with Coomassie Blue and (b) probed with anti-kinesin antibody in a corresponding Western blot, which detected a protein of ⬃120 kDa. c: Transverse section of an ovariole of Notonecta probed with anti-kinesin antibody showing bright fluorescent labelling in the nutritive tubes (arrowheads) Bar ⫽ 30 µm. d: Microtubule bundles extracted from isolated nutritive tubes also label strongly with anti-kinesin antibodies, while (e) no fluorescence is seen in controls when the primary antibody is omitted. Bar ⫽ 30 µm. for hybridizations studies also labelled strongly with the anti-kinesin antibodies (Fig. 4d,e). DISCUSSION A number of mechanisms could underlie the localization of mRNAs during oogenesis in Drosophila [St Johnston, 1995; Glotzer and Ephrussi, 1996], but transport by microtubule-dependent motors is most frequently proposed to play a part in the passage of mRNAs from the nurse cells to the oocytes. However, direct evidence for microtubule-mediated mRNA transport in this and other systems has been difficult to obtain, and the motor molecules involved have not been identified. Although mRNA and polysomes have been shown to associate with microtubules reassembled in vitro [Hamill et al., 1994; Han et al., 1995] a specific cellular attachment between mRNA and microtubules has not previously been demonstrated. In situ hybridization at the light and ultrastructural levels has, however, revealed the colocalization of mRNA with microtubules, as well as with microfilaments and intermediate filaments, in cultured neurones and fibroblasts [Taneja et al., 1992; Bassell et al., 1994a,b]. Also, following the biochemical detergent-extraction of many cell types, including Drosophila oocytes [Pokrywka and Stephenson, 1994], mRNA and polysomes have been found to be associated with the insoluble cytoskeletal fraction, with monomeric ribosomes in the soluble fraction [Lenk et al., 1977; Jeffery, 1982; Hesketh and Pryme, 1991]. Attempts have then been made to distinguish the relative involvement of microtubules and microfilaments by perturbing these cytoskeletal organelles with either colchicine and or cytochalasin. Unlike most cells, the nutritive tube transport channels linking the nurse cells to the oocytes in Notonecta have an uncomplicated cytoskeleton comprised solely of large bundles of stable microtubules. Moreover, the microtubule bundles are sufficiently large to be dissected manually from ovarioles, while viewed in polarised light. They have, therefore, proved to be an ideal system for investigating the relationship between microtubules and transported RNA. In this study, we have found that oligo (dT) probes hybridized evenly throughout the lengths of isolated nutritive tubes, confirming the presence of mRNA. As found previously [Stebbings and Hunt, 1983], detergent-extraction of isolated nutritive tubes resulted in the release of the massive microtubule bundles, and the solubilisation of the ribosomes. The latter are, therefore, free within the nutritive tubes and probably translocate passively along them, as previously proposed [Macgregor and Stebbings, 1970]. Hybridization of oligo (dT) to the extracted microtubule bundles showed that, by contrast, poly(A) mRNA remained attached to the microtubules, suggesting that its transport is microtubule-based. These findings provide evidence that mRNA is attached to microtubules isolated directly from cells, and therefore in vivo. We set out to investigate the nature of the mRNA/ microtubule attachment and found it to be disrupted by the addition of ATP and also GTP, but unaffected by ADP and possibly even enhanced by the nonhydrolyzable ATP analogue, AMP-PNP. The attachment was also apparently unaffected by apyrase, which results in ATP depletion. Nucleotide-sensitive binding to microtubules is a property of microtubule-based motor molecules [Vallee and Shpetner, 1990], and our results indicate that mRNA may Attachment of mRNA to Microtubules be attached to the nutritive tube microtubules via a motor protein. Kinesin and dynein motors have both been identified in hemipteran ovaries [Anastasi et al., 1990] and, while suggesting that motor proteins drive mRNA transport along the microtubules in the ovaries, our results do not resolve the precise identity of the motor involved. However, the ‘‘nucleotide fingerprint’’ of the mRNA attachment to microtubules is more consistent with the involvement of a kinesin motor, since the latter is removed from microtubules by both ATP and GTP, while dynein is only removed by ATP [Paschal et al., 1987]. This is supported by our immunolocalization of a kinesin within the nutritive tubes and more specifically as a component of mRNA/microtubule complexes isolated and extracted from them. The polarity of microtubules in nutritive tubes of Notonecta [see Stebbings and Hunt, 1983] means that a minus end-directed kinesin would be required for mRNA transport to the oocytes. It would appear, therefore, to be similar to Drosophila, where dynein is not involved in morphogen localization [McGrail and Hays, 1997] and a minus end-directed kinesin has been shown to co-position with localized mRNAs [Clark et al., 1997]. Genetic and molecular biological screens have resulted in the discovery of large numbers of kinesin-like motor proteins, with more than thirty genes encoding members of the kinesin superfamily in Drosophila alone [see Hirokawa, 1998]. Each kinesin, with some redundancy, is believed to have its own cargo with many being involved in cell division and others in transporting specific membranous organelles, and also DNA. Kinesins for transporting mRNA have been predicted [Moore and Endow, 1996]. The molecular mechanism for mRNA/motor protein associations also remains unclear. It has been shown that the non-motor domains of plus and putative minus end-directed chromokinesins bind directly to DNA [Afshar et al., 1995; Wang and Adler, 1995]. It appears, however, that additional components are involved in mRNA transport and localization, since a number of RNA-binding proteins have been found—the most studied being Drosophila staufen [Ferrandon et al., 1994]— which bind to localization signals in the 38 UTRs of mRNAs and mediate interactions with the cytoskeleton [Ainger et al., 1993]. When the 38 UTR of bicoid mRNA was injected into Drosophila embryos, it was found to associate with staufen and form large and easily visible particles that moved in a microtubule-dependent manner [Ferrandon et al., 1994]. However, these macromolecular particles may be artefactual because they were not seen with endogenous bicoid mRNA or injected full-length message, and we did not observe such particles in our preparations. 165 The number and identity of the proteins involved in transporting mRNA along nutritive tubes of Notonecta is not presently known. Detailed characterization of the mRNA/microtubule complexes from nutritive tubes, which clearly possess the essential components for transporting mRNA, will resolve this question. ACKNOWLEDGMENTS N.J.T. is a Nuffield Science Research Fellow. S.S. is in receipt of a BBSRC studentship. REFERENCES Afshar K, Barton NR, Hawley RS, Goldstein SLB. 1995. DNA binding and meiotic chromosomal localization of the Drosophila Nod kinesin-like protein. Cell 81:129–138. Ainger K, Avossa D, Morgan F, Hill SJ, Barry C, Barbarese E, Carson JH. 1993. Transport and localization of exogenous myelin basic protein mRNA microinjected into oligodendrocytes. J Cell Biol 123:431–441. Alison M, Chaudry Z, Baker J, Lauder I, Pringle JH. 1994. Liver regeneration: a comparison of in situ hybridization for histone mRNA with bromodeoxyuridene labelling for the detection of S-phase cells. J Histochem Cytochem 42:1603–1608. Anastasi A, Hunt C, Stebbings H. 1990. Isolation of micotubule motors from an insect ovarian system: characterization using a novel motility substratum. J Cell Sci 96:63–69. Anastasi A, Hunt C, Stebbings H. 1991. Characterization of a nucleotide-sensitive high molecular weight microtubuleassociated protein in the ovary of a hemipteran insect. Cell Motil Cytoskeleton 19:37–48. Bashirullah A, Cooperstock RL, Lipshitz HD. 1998. RNA localization in development. Annu Rev Biochem 67:335–394. Bassell GJ, Powers CM, Taneja KL, Singer RH. 1994a. Single mRNAs visualized by ultrastructural in situ hybridization are principally localized at actin filament intersections in fibroblasts. J Cell Biol 126:863–876. Bassell GJ, Singer RH, Kosik KS. 1994b. Association of poly(A) mRNA with microtubules in cultured neurons. Neuron 12:571– 582. Capco DG, Jeffery WR. 1979. Origin and spatial distribution of maternal RNA during oogenesis of an insect, Oncopeltus fasciatus. J Cell Sci 39:63–76. Clark IE, Giniger E, Ruohola-Baker H, Jan LY, Jan YN. 1994. Transient posterior localization of a kinesin fusion protein reflects anteroposterior polarity of the Drosophila oocyte. Curr Biol 4:289–300. Clark IE, Jan LY, and Jan YN. 1997. Reciprocal localization of Nod and kinesin fusion proteins indicates microtubule polarity in the Drosophila oocyte, epithelium, neuron and muscle. Development 124:461–470. Dalby B, Glover DM. 1992. 38 non-translated sequences in Drosophila cyclin B transcripts direct posterior pole accumulation in oogenesis and peri-nuclear association in syncitial embryos. Development 115:989–997. Ferrandon D, Elphick L, Nusslein-Volhard C, and St Johnston D. 1994. Staufen protein associates with the 38UTR of bicoid mRNA to form particles that move in a microtubule-dependent manner. Cell 79:1221–1232. Glotzer JB, Ephrussi A. 1996. mRNA localization and the cytoskeleton. Semin Cell Dev Biol 7:357–365. 166 Stephen et al. Hamill D, Davis J, Drawbridge J, Suprenant KA. 1994. Polyribosome targeting to microtubules: enrichment of specific mRNAs in a reconstituted microtubule preparation from sea urchin embryos. J Cell Biol 127:973–984. Han JR, Yiu GK, Hecht NB. 1995. Testis/brain RNA-binding protein attaches translationally repressed and transported mRNAs to microtubules. Proc Natl Acad Sci USA 92:9550–9554. Hesketh JE, Pryme IF. 1991. Interaction between mRNA, ribosomes and the cytoskeleton. Biochem J 277:1–10. Hirokawa N. 1998. Kinesin and dynein superfamily proteins and the mechanism of organelle transport. Science 279:519–526. Hyams JS, Stebbings H. 1979. The mechanism of microtubule associated cytoplasmic transport: isolation and preliminary characterization of a microtubule transport system. Cell Tissue Res 196:103–116. Jeffery WR. 1982. Messenger RNA in the cytoskeletal framework: analysis by in situ hybridization. J Cell Biol 95:1–7. Laemmli UK. 1970. Cleavage of structural proteins during the asembly of the head of bacteriophage T4. Nature 227:680–685. Lane JD, Stebbings H. 1994. Independent regulation of microtubule spacing and microtubule stability following redundancy of nutritive tubes in telotrophic ovarioles of Hemiptera (Insecta). Int J Morphol Embryol 23:297–309. Lane ME, Kalderon D. 1994. RNA localization along the anterioposterior axis of the Drosophila oocyte requires PKA-mediated signal transduction to direct normal microtubule organization. Genes & Devel 8:2986–2995. LaRochelle WJ. 1996. Detection of proteins on blots using avidin- or streptoavidin-biotin. In: Walker JM, editor. The protein protocols handbook. Totowa: Humana Press Inc. p 323–327. Lenk R, Ransom L, Kaufmann Y, Penman S. 1977. A cytoskeletal structure with associated polyribosomes obtained from HeLa cells. Cell 10:67–78. Li M-G, McGrail M, Serr M, Hays TS. 1994. Drosophila cytoplasmic dynein, a microtubule motor that is asymmetrically localized in the oocyte. J Cell Biol 126:1475–1494. Macgregor HC, Stebbings H. 1970. A massive system of microtubules associated with cytoplasmic movement in telotrophic ovarioles. J Cell Sci 6:431–449. Mays U. 1972. Stofftransport in ovar von Pyrrhocoris apterus. Z Zellforsch Mikrosk Anat. 123:395–410. McGrail M, Hays TS. 1997. The microtubule motor cytoplasmic dynein is required for spindle orientation during germ line cell divisions and oocyte differentiation in Drosophila. Development 124:2409–2419. Moore JD, Endow SA. 1996. Kinesin proteins: a phylum of motors for microtubule-based motility. Bioessays 18:207–219. Paschal BM, Shpetner HS, Vallee RB. 1987. MAP 1C is a microtubuleactivated ATPase that translocates microtubules in vitro and has dynein-like properties. J Cell Biol 105:1273–1282. Pokrywka NJ, Stephenson EC. 1991. Microtubules mediate the localization of bicoid RNA during Drosophila oogenesis. Development 113:55–66. Pokrywka NJ, Stephenson EC. 1994. Localized RNAs are enriched in cytoskeletal extracts of Drosophila oocytes. Dev Biol 166:210– 219. Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbour Laboratory Press. Spradling AC. 1993. Developmental genetics of oogenesis. In: Bate M, Martinez-Arias A, editors. The development of Drosophila melanogaster. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. p 1–70. St Johnston D. 1995. The intracellular localization of messenger RNAs. Cell 81:161–170. St Johnston D, Nusslein-Volhard C. 1992. The origin of pattern and polarity in the Drosophila embryo. Cell 68:201–219. Stebbings H, Hunt C. 1983. Microtubule polarity in nutritive tubes of insect ovarioles. Cell Tissue Res 233:133–141. Stebbings H, Hunt C. 1987. The translocation of mitochondria along insect ovarian microtubules from isolated nutritive tubes: a simple reactivated model. Cell Tissue Res 233:133–141. Taneja KL, Lifshitz LM, Fay FS, Singer RH. 1992. Poly(A) RNA codistribution with microfilaments: evaluation by in situ hybridization and quantitative digital imaging microscopy. J Cell Biol 119:1245–1260. Theurkauf WE, Hazelrigg TI. 1998. In vivo analyses of cytoplasmic transport and cytoskeletal organization during Drosophila oogenesis: characterization of a multi-step anterior localization pathway. Development 125:3655–3666. Theurkauf WE, Smiley S, Wong ML, Alberts BM. 1992. Reorganization of the cytoskeleton during Drosophila oogenesis: implications for axis specification and intercellular transport. Development 115:923–936. Theurkauf WE, Alberts BM, Jan YN, Jongens TA. 1993. A central role for microtubules in the differentiation of Drosophila oocytes. Development 118:1169–1180. Vallee RB, Shpetner HS. 1990. Motor proteins of cytoplasmic microtubules. Annu Rev Biochem 59:909–932. Wang S-Z, Adler R. 1995. Chromokinesin: a DNA-binding, kinesinlike nuclear protein. J Cell Biol 128:761–768. Wilhelm JE, Vale RD. 1993. RNA on the move: the mRNA localization pathway. J Cell Biol 123:269–274.