Isolation of microtubule motors from an insect ovarian system:

characterization using a novel motility substratum

ANGELA ANASTASI, CHERRYL HUNT and HOWARD STEBBINGS*

Department of Biological Sciences, Washington Singer Laboratories, University of Exeter, Perry Road, Exeter EX4 4QG, UK

* Author for correspondence

Summary

The ovaries of hemipteran insects contain massivemicrotubule-based translocation channels known asnutritive tubes, linking nurse cells to the developingoocytes. Translocation, which is in a retrograde di-rection along the nutritive tube microtubules, haspreviously been reactivated in vitro. Here, ATP-sensitive microtubule-associated proteins (MAPs)have been isolated from the insect ovaries, and beadscoated with such proteins applied to salt-treated,detergent-extracted nutritive tube microtubulesmicrodissected from the insect ovaries. These mo-tility substrata are composed of many thousands ofparallel microtubules, all with a common knownpolarity, so that not only are they easily observed,but the direction of any translocation along theirlength can be readily interpreted.

ATP extracts of insect ovarian MAPs, containingboth kinesin and dynein, were seen to promote bi-directional movements of beads. Movements in the

two directions differed in both rate and form. Onfractionation of the ATP extract, those fractionscontaining kinesin brought about bead movement inan anterograde direction. Fractions containingdynein failed to promote movement of beads, and nosingle fraction promoted movement of beads in aretrograde direction.

Kinesin, while clearly present in the insect ovary, isabsent from the nutritive tube translocation chan-nels. The nutritive tubes, however, contain a poly-peptide that co-electrophoreses with insect ovariandynein, making dynein a possible candidate for themotor that drives the retrograde translocation alongnutritive tubes.

Key words: microtubule-associated proteins, translocation,kinesin, cytoplasmic dynein.

Introduction

During oogenesis in hemipteran insects, components syn-thesized by nutritive cells making up an anterior trophicregion of the ovariole pass back to the chain of developingoocytes along a system of discrete and extensive cytoplas-mic channels - the nutritive tubes (see Stebbings, 1986).Nutritive tubes are typically some 20 /an in diameter,several millimetres in length and contain many thousandsof parallel microtubules. In common, therefore, with ver-tebrate and invertebrate nerve axons, fish pigment cellprocesses and amoeboid cell networks, but, perhaps to aneven greater extent, nutritive tubes exhibit an exagger-ated form of microtubule-associated intracellular translo-cation - a phenomenon that is believed to occur to someextent in all eukaryotic cells.

Apart from being greatly emphasized, translocationalong insect ovarian nutritive tubes would appear to be atits simplest, since, in contrast to other systems that havereceived attention, translocation is unidirectional, it oc-curs along microtubules of common polarity (Stebbingsand Hunt, 1983), and microtubules are the only cytoskel-etal elements present (Hyams and Stebbings, 1977).

As has been possible with permeabilized cell models offish pigment cells (Stearns and Ochs, 1982; Clark andRosenbaum, 1982; Rozdzial and Haimo, 1986), amoebanetworks (Koonce and Schliwa, 1986), isolated neuronalJournal of Cell Science 96, 63-69 (1990)Printed in Great Britain © The Company of Biologists Limited 1990

axoplasm (Allen et al. 1985; Schnapp et al. 1985), andextracts from cells grown in culture (Dabora and Sheetz,1988), movements of organelles along nutritive tubemicrotubules have been reactivated in vitro, thus demon-strating an active involvement of the microtubules intranslocation along nutritive tubes (Stebbings and Hunt,1987).

Motors that drive microtubule translocation have beenstudied mainly in neuronal material in which bidirec-tional movement occurs along the axons. With the devel-opment of in vitro assay systems based on the movement ofbeads along microtubules and the gliding of microtubuleson glass substrata (Vale et al. 19856) came the discovery ofthe anterograde motor, kinesin, which was found to bringabout movement towards the plus ends of microtubules(Vale et al. 1985a) and the retrograde motor, MAP-1C orcytoplasmic dynein, which drives movement towards theminus ends (Paschal et al. 1987). Both motors have beencharacterized to some extent and structural studies onkinesin have yielded an insight into the mechanism ofmotility and ire vivo function of this motor (Hirokawa et al.1989; Scholey et al. 1989; Yang et al. 1989).

Kinesin has also been identified and characterized inincreasing numbers of non-neuronal sources that showmicrotubule-based motility, including sea-urchin eggs(Scholey et al. 1985; Porter et al. 1987), Drosophila em-bryos (Saxton et al. 1988) and bovine adrenal medulla

63

(Murofushi et al. 1988). Cytoplasmic dynein has similarlybeen identified in non-neuronal material, such as chickembryo fibroblasts (Schroer et al. 1989), HeLa cells and thenematode Caenorhabditis elegans (Lye et al. 1989). How-ever, it still remains to be determined whether kinesin anddynein are universal motors and evidence is emerging toshow that further microtubule motors exist (Shpetner andVallee, 1989). Also, the one motor-one direction conceptmay be an oversimplification, since in the giant amoeba,Reticulomyxa, a dynein-like motor has been found to bringabout bidirectional movement (Euteneuer et al. 1988).

Prompted by the extensive microtubule-associatedtranslocation channels they contain, we set out to identifymicrotubule motors in the ovaries of an hemipteran insectOncopeltus fasciatus - the milkweed bug. In doing so wehave developed a new motility substratum for studyingmicrotubule translocation and translocation motors thathas considerable advantages over those used in existingassays. It employs beads and massive bundles of nativemicrotubules from the nutritive tubes, and is analogous tothe actin filament model from internodal cells of Nitella,which has proved so valuable in studying actin-myosininteractions in cytoplasmic streaming (Sheetz andSpudich, 1983).

Materials and methods

Dissection of ovariesA culture of Oncopeltus fasciatus was maintained in the labora-tory. Dissection and subsequent processing of ovaries was carriedout in 0 . 1 M Pipes buffer, pH6.9, lmM EGTA, 2.5 mil MgSO4(PEM), containing lmM dithiothreitol 0 . 1 M phenylmethylsul-phonyl fluoride (PMSF) 10//gml~l soybean trypsin inhibitor,100 ng ml"1 N-tosyl-L-phenylalanine chloromethyl ketone,lO/zgrnl"1 iV-a^benzoyl-L-arginine methyl ester, 10/igml"1 N-a-p-tosyl-L-arginine methyl ester, l/zgrnl"1 leupeptin, 1/igml"1

aprotinin, lpgml"1 pepstatin A. All chemicals used were ofAnalar grade. After excision of the ovaries, the vitellogenicoocytes were discarded to avoid contamination of the extract withyolk. The remaining tissue, comprising the trophic regions andprevitellogenic oocytes, together with the nutritive tubes, waspooled in buffer and kept on ice. The complete sample was made1.5 mM in Ca2+ to depolymerize the microtubules and homogen-ized in a hand-held homogenizer. At this stage 2 mM EGTA wasadded to chelate the Ca2+. Homogenates were usually then frozenat -70°C.

Isolation of motorsFor the isolation of motors, ovaries from about 400 O. fasciatuswere used. The method employed was essentially that ofEuteneuer and co-workers (1988), except that AMP-PNP wasadded to facilitate the isolation of kinesin. The frozen homogen-ates were pooled after thawing, O.lmgml"1 hexokinase and10 mM glucose were added and the homogenate was spun at33 000g for 30 min at 4°C. The supernatant was removed and re-centrifuged at 132000 g for l h at 4°C. The clarified supernatantwas removed and incubated for 20 min at room temperature with5mM AMP-PNP and an equal volume of a Smgml"1 solution ofpig brain tubulin, which had been purified using DEAE-Sepha-dex chromatography and polymerized with 10 fat taxol. This wasthen layered over a 25 % sucrose cushion prepared in PEM bufferplus 5 mM AMP-PNP and 10 /*M taxol, and centrifuged at 33 000 gfor 1 h at 15 °C. The microtubule pellet was washed by resuspend-ing in PEM buffer plus 10 /.IM taxol and 50 mM NaCl, and spun at33 000 g for 20 min at 15 °C. The pellet was resuspended in 10 mMMgATP, left incubating for 30 min at room temperature andcentrifuged at 33 000g for 20min at 15°C. The ATP eluate wascarefully removed and loaded in the cold on a 5 % to 30 % sucrose-density gradient made in PEM and sedimented at 900004*(32 000 revs min"1) in a Beckman SW 50.1 rotor for 16h at 4°C.

For size calibration, identical sucrose gradients were loaded withlmg of each of the following: thyroglobulin (19 S), catalase(11.3 S) and bovine albumin (3S), and centrifuged together withgradients loaded with sample. Fractions (300-350 u\) were col-lected by upward displacement using Maxidens (Nyegaard andCo. Diagnostics, Oslo, Norway). The fractions were concentratedfivefold using Amicon Centricon 30 filters (Stonehouse, Glos).

Biochemical methodsVanadate cleavage. Ultraviolet (u.v.) irradiation was per-

formed in the presence of 100 UM vanadate and 10 mM MgATP,according to the method of Gibbons and co-workers (1987). For themotility assay, 5mM norepinephrine was added after u.v. ir-radiation to reduce the vanadate (Gibbons et al. 1978).

Preparation of mouse brain kinesin. Mouse brain kinesin wasprepared according to the method of Vale and co-workers (1985a).

Nutritive tube preparations for immunoblotting. Nutritivetubes were microdissected and prepared for electrophoresis asdescribed previously (Sharma and Stebbings, 1986).

Gel electrophoresis and Western blotting. Analytical gel electro-phoresis was performed in SDS-polyacrylamide slab gels withlinear 5% to 10% acrylamide gradients according to Laemmli(1970). The gels were stained for protein using either CoomassieBlue (Fairbanks et al. 1971) or silver (Merril et al. 1981). All gelscontained the following molecular mass (xlO~3) markers (SigmaChemical Co., Poole, Dorset): rabbit muscle myosin (205),/3-galactosidase (116), phosphorylase b (97.4), bovine serum albu-min (66), ovalbumin (45) and carbonic anhydrase (29). Dyneinheavy-chain concentrations were determined by densitometry ofthe Coomassie-stained gel.

For immunoblotting 5% to 10% gradient gels were run andelectrophoretically transferred to nitrocellulose sheets (Towbin etal. 1979) for 4h using the Bio-Rad Transblot ElectrophoreticTransfer System (Bio-Rad Labs, Watford, Herts). Polyclonalantibodies to chicken brain kinesin were used for probing. Theseantibodies were kindly donated to us by Dr P. Hollenbeck,Harvard Medical School. Horseradish peroxidase-conjugated goatanti-rabbit IgG (Bio-Rad) was used as the secondary antibody andthe peroxidase activity was localized by using 4-chloro-l-naphthol.

Motility assayInsect ovaries provided the microtubule substrata for the motilityassays. Bundles of native parallel microtubules were obtainedfrom isolated and extracted nutritive tubes. For this, nutritivetubes of another hemipteran, Notonecta glauca, were preferred asthey are more numerous in the ovariole, longer, contain moremicrotubules and are more readily isolated than those of Oncopel-tus.

Notonecta were collected from ponds in the vicinity of Exeterand their ovaries dissected in insect Ringer. Individual ovarioleswere teased apart, desheathed and microdissected in PEM, 0.1 %Triton X-100 to reveal demembranated, extracted nutritive tubes.To remove endogenous microtubule-associated proteins (MAPs),0.6 M NaCl was added to the extraction buffer. In nutritive tubesit has been shown previously that the microtubules have acommon polarity, with their plus ends directed towards thetrophic region (Stebbings and Hunt, 1983). Using tungstenneedles, nutritive tubes were scissored away from the trophicregion, leaving a small amount of trophic tissue attached to act asa polarity reference. Polybead carboxylated microspheres (Poly-sciences, Ltd, Northampton) of 0.23 ̂ m diameter were added topotential cytoplasmic motors and left on ice for varying times(15 min to 2h). The protein-coated latex spheres were thenapplied to the microtubule substrata and made 10 mM in MgATP.A coverslip was added and sealed with VALAP (1:1:1, by wt,vaseline, lanolin, paraffin wax). Microtubule-substrata bundleswere located by virtue of their being birefringent, using a Zeissphotomicroscope fitted with polarization optics and then beadmovement was viewed with a X100 objective and a dark-fieldsystem. Video recordings were made by fitting the microscopewith a Hamamatsu C2400-01 Chalnicon video camera, possessinga contrast-enhancement circuit, connected to a Sony Unimaticvideo recorder. The characteristics and the rates of bead translo-

64 A. Anastasi et al.

cations (15 estimations in each case) were assessed from videorecordings.

During the development of our nutritive tube motility assay,insect ovarian motors and chicken brain motors (provided by DrP. Hollenbeck) were used to confirm its general applicability.

Results

Protein purificationWhen insect ovary homogenates were depleted of endogen-ous ATP by the addition of hexokinase/glucose and mixedwith purified MAP-free taxol-stabilized porcine brainmicrotubules, a high molecular weight protein, similar insize to microtubule-associated protein 1 (MAP-1) boundspecifically to the microtubules (Fig. 1). This protein wasreleased from the microtubules with MgATP. When AMP-PNP was added, a 116K (K=103Mr) polypeptide wasbound to the microtubules. This was also extractable withMgATP. In a typical preparation, hexokinase/glucose andAMP-PNP were added together to isolate both proteinspecies (Fig. 1).

The material released from the microtubules withMgATP was sedimented in a 5 % to 30 % sucrose densitygradient, and the resulting fractions were analysed forpolypeptide composition by gel electrophoresis (Fig. 2).The high molecular weight protein sedimented at around20 S. It co-electrophoresed with MAP-1C isolated from pigbrain microtubules (data not shown). Like dynein heavychains, this protein showed susceptibility to cleavage at asingle site when irradiated with u.v. in the presence ofvanadate and ATP, yielding two polypeptides of molecularweights 190K and 230K, as shown in Fig. 3. About 70 % ofthe protein was cleaved within 60min and no furthercleavage occurred with longer irradiation (data not

- D Y N

205K-f I--.JI-.JI5 •]

. I

- K I N

irTUB

45K

29K-

2 3 4 5 6 7 8 9 10

Fraction number

Fig. 2. Fractions from a 5 % to 30 % sucrose density gradientloaded with an ATP-released supernatant (as in Fig. 1, lane C)and centrifuged, were analysed by gel electrophoresis and silverstained. Fractions are numbered from the top to the bottom ofthe gradient. (Kinesin is separated on the gradient from dyneinand tubulin. A doublet at approx. 64-66K co-sedimented withthe kinesin.) Sedimentation standards, thyroglobulin, catalaseand bovine albumin, sedimented at fractions 9, 6 and 3,respectively. Motile activity is indicated by a + at the bottom ofthe lane. DYN, dynein; KIN, kinesin; TUB, tubulin.

205K-

116K-97K-

66K-

45K-

29K-

a b

Fig. 1. Binding ofpolypeptides from insectovary homogenates tomicrotubules in the absenceof ATP and their subsequentrelease from microtubulesby MgATP. Lane A, clarifiedovary homogenate to whichhexokinase/glucose hadbeen added. Lane B,polypeptides that bind totaxol-stabilized, purifiedbrain microtubules in theabsence of ATP. Lane C,polypeptides released by10 nun MgATP frommicrotubule pellets asprepared in lane B.

shown). The 116K species sedimented at around 9S and adoublet of around 64-66K co-sedimented with it. The116K polypeptide showed immunoreactivity with anti-chicken brain kinesin (Fig. 4).

Motility assayThe insect ovary extract, the material released from thepig brain microtubules with MgATP and the sucrosegradient fractions of this ATP eluate were incubated withlatex beads and then tested to see whether they producedbead movement along microtubule substratum bundles.The polarity of the latter was defined by their attachmentto a small amount of trophic tissue (Fig. 5). Duringdissection, nutritive tubes were detergent-extracted andsalt-treated to remove their endogenous MAPs, and soproduce large bundles of clean, parallel, native micro-tubules that are readily visible by dark-field microscopy(Fig. 6A) and stable over periods of several hours.

The clarified homogenate of insect ovaries broughtabout no movement of beads on the microtubule bundles.In the case of beads incubated with insect ovarian materialreleased from the brain microtubules with MgATP therewas considerable bead attachment to the microtubulebundles (cf. Fig. 6A and B). At any one time as many as

Microtubule motors in insect ovaries 65

205K-

116K-

97K-i

66K-

fcs

0 30 60min

Fig. 3. Ultraviolet-induced cleavage of the high molecularweight polypeptide (large arrowhead) isolated from insectovaries in the presence of vanadate and MgATP. The ATP-released supernatant was exposed to u.v. for 30 and 60 min.Smaller arrowheads show positions of the cleavage products.

half of the attached beads were seen to be moving alongthe microtubule bundle. Bead translocations towards thetrophic region ends - or in an anterograde direction -predominated. Anterograde movements were uninter-rupted, and at a rate of 0.28±0.05/xms~1 over distances oftens of micrometres. In such movements beads appeared tofollow each other along distinct pathways on the micro-tubule bundle with no obvious overtaking or sidewaysdisplacement. Bead movements in the opposite, retrogradedirection, towards the oocyte ends of the nutritive tubemicrotubules also occurred. These movements were quitedifferent, appearing jerky and intermittent, with beadstraversing 1 /an or so before stopping. Neither is themovement so linearly defined, and beads appear lessfirmly associated with the microtubule surfaces, in someinstances making lateral 'jumps' during translocation.Movement of beads in the retrograde direction occurred atrates of 0.67±0.2/mis~1. Preliminary experiments haveshown that the addition of 5-10 min AMP-PNP to theMgATP eluate preparations resulted in the cessation ofboth anterograde and retrograde bead movements. Theyhave also shown that u.v. irradiation of the same eluate inthe presence of vanadate, while not affecting anterograde,brought about some reduction but not a total inhibition ofretrograde movement. This is probably due to the fact thatonly partial cleavage (70 %) of the dynein was achieved.We are developing methods for analysing accurately smallvariations in bead movements on our new substrata.

The sucrose density fractions of the MgATP eluate thatcontained kinesin (see Fig. 2) caused translocation of

r

Fig. 4. Immunoblots (above) with corresponding area ofCoomassie-stained gels (below) showing reactions to anti-chicken brain kinesin. Arrowhead shows position of kinesin.Lane A, clarified ovary homogenate. Lane B, ATP-releasedsupernatant. Lane C, nutritive tube proteins. Lane D, mousekinesin used as control.

bound beads in the anterograde direction only and at asimilar rate to the unfractionated MgATP eluate. With thedynein fractions, on the other hand, very few beads boundand no movement was observed.

To investigate whether the insect microtubule substratawould support translocation produced by non-insectmotors, beads incubated in preparations containing bothchicken brain kinesin and dynein were applied. Thesewere seen to bind and show bidirectional bead movementwith similar characteristics to the insect ovarian motors.

Location of motor proteins in insect ovariesSince our results have indicated that both kinesin andcytoplasmic dynein are present in the insect ovaries, wewished to determine whether they are present specificallyin the nutritive tube translocation channels. Immunoblot-ting of nutritive tube extract against anti-chicken brainkinesin showed no reactivity (Fig. 4).

A suitable dynein antibody has not been available to usas yet, but a polypeptide component of nutritive tubes(shown previously to be an ATP-sensitive MAP; Stebbingsand Sharma, 1989) comigrates on gels with the dyneinband identified in whole ovaries.

Discussion

Using a technique that embodies both the addition ofAMP-PNP and the depletion of ATP, methods that havebeen used by others in the isolation of microtubule motors,we have obtained MAPs and in particular the motor MAPsfrom the ovaries of hemipteran insects. These organs are

•if A. Anastasi et al.

Fig. 5. Isolated nutritive tubes of N. glauca with some trophic tissue left attached as a polarity marker, viewed using dark-fieldoptics. Bar, 40 /an.Fig. 6. A. Detergent-extracted nutritive tube viewed using dark-field optics. B. Nutritive tube as above, to which have been addedlatex beads coated with ATP-sensitive MAPs from insect ovaries. Bar, 20 /on.

known to contain extensive microtubule-based translo-cation systems (Stebbings, 1986).

We have studied the ability of an ATP extract of insectovarian MAPs to promote translocation of latex beads onmassive bundles of native microtubules from hemipteraninsect ovarian nutritive tubes. The bundles are composedof some 30000 parallel microtubules and are approxi-mately 20 fan in diameter and many millimeters in length.They are readily obtained from insect ovaries, and easilyviewed and handled using polarizing optics. Bead move-ment on the microtubule bundles was best observed usingdark-field optics, and with such optics and because of thesize of the microtubule bundles no image processing wasnecessary. In addition, the microtubules are all of acommon known polarity (Stebbings and Hunt, 1983), sothat microtubule substrata with a 'built-in' polaritymarker can be routinely achieved. Beads of different sizesand with different properties, as well as various organ-elles, can be applied separately or collectively to themicrotubule bundles so that large numbers of putativemotor samples, both purified and otherwise, can be as-sayed directly and rapidly.

Using this system, we observed that an ATP extract ofMAPs from the insect ovary homogenate caused latexbeads to bind to, and move bidirectionally along, nutritivetube microtubule substrata. Purification of the proteins inthe ATP extract by sucrose density gradient centrifugationshowed that kinesin was responsible for movement of

beads in the anterograde direction. Hemipteran insectovary kinesin showed characteristics similar to kinesinsstudied from a variety of sources (Hollenbeck, 1988)including Drosophila (Saxton et al. 1988), in terms ofmolecular weight, sedimentation coefficient and the direc-tion and nature of the movement it produced. It alsoshowed immunoreactivity with chicken brain kinesin.

Retrograde movement could not be definitely ascribed toa particular protein or proteins, but a polypeptide in theATP extract, comparable in size to dynein, which likeaxonemal dynein (Gibbons et al. 1987) is susceptible tocleavage by u.v. light, is a likely candidate. The dynein inthe insect ovary is therefore similar to the cytoplasmicdyneins recently purified from mammals through to proto-zoans, with examples that include bovine brain (Paschal etal. 1987), chicken brain and fibroblasts (Schroer et al.1989), squid optic lobes (Schnapp and Reese, 1989), thenematode Caenorhabditis (Lye et al. 1989) and the freshwater amoeba, Reticulomyxa (Euteneuer et al. 1988).

Although cytoplasmic dynein has been shown to supportmicrotubule gliding (Paschal et al. 1987), purified fractionscontaining insect ovarian dynein did not generate beadtranslocation. A similar result has been reported withpurified dynein from squid optic lobes, which also failed topromote bead movements (Schnapp and Reese, 1989). Asignificant finding of our study was that while beadsincubated in kinesin fractions bound to the microtubulemotility substrata, those incubated in dynein did not. This

Microtubule motors in insect ovaries 67

observation together with the lack of motility usingpurified dynein in our system could be due to there beingsub-threshold levels of insect dynein in the sucrose densityfractions, as has been found to be critical for axonemaldynein (Sale and Fox, 1988) and kinesin (Cohn et al. 1989;Howard et al. 1989); and with the insect ovaries it did notprove possible to concentrate the dynein farther. It ispossible that insect dynein may be comparable to bovinebrain kinesin, which has been shown to lose its capacityfor microtubule motility substrata at low protein concen-trations (Howard et al. 1989), possibly as a result ofdenaturation.

The retrograde movement of beads that we observedwith insect ovarian samples shares a number of featureswith the movement produced by axonemal as well as othercytoplasmic dyneins. Translocation has been generallyfound to occur at a faster rate than for kinesin (see Valleeet al. 1989), to be more intermittent (Paschal et al. 1987;Schnapp and Reese, 1989), and to be less linearly directedthan kinesin-produced movement, which is thought tooccur along a single microtubule protofilament (Gelles etal. 1988).

The small amounts of ovarian sample that it is feasibleto obtain, and the fact that only anterograde movementwas produced after fractionation of the ATP extracts, havemeant that only a limited number of inhibitor studies havebeen possible, and these were done with the unfrac-tionated ATP extracts. AMP-PNP, which has been shownto inhibit kinesin-based motility, brought about the cess-ation of both anterograde and retrograde translocation, ashas been shown in other systems (Dabora and Sheetz,1988). This is not surprising in view of the likely mixedand multiple binding of translocators to the beads, alsopointed out by others (Schnapp and Reese, 1989).

The demonstration of anterograde and retrograde trans-locators in insect ovary homogenates clearly leads to thequestion of their location and role in these organs. Thereason that such ovaries were chosen for investigation wasbecause the nutritive tubes, which are so conspicuouswithin them, represent extensive microtubule-associatedtranslocation channels. Translocation along the nutritivetubes at rates of up to S.Sjanmin"1 (Mays, 1972) isunidirectional from the nutritive cells to the oocytes, inwhat has been demonstrated to be a retrograde directionin relation to the component microtubules (Stebbings andHunt, 1983). Moreover, the translocation of mitochondriaalong isolated nutritive tube microtubules has been reacti-vated in vitro, again in the retrograde direction and at therate of 7.0/an s"1 (Stebbings and Hunt, 1987), therebydemonstrating the existence of an endogenous retrogrademotor.

Our immunological studies have shown that, althoughpresent within the ovaries, kinesin does not occur innutritive tubes. While not therefore involved in translo-cation along these channels, kinesin must play a roleelsewhere in the ovary — possibly being associated with thenumerous dividing cells of the trophic region. A dynein-like protein, on the other hand, has been detected innutritive tubes (see also Stebbings and Sharma, 1989), afinding that correlates with the retrograde translocationalong their length, and which points to the possibility ofdynein being responsible for retrograde organelle translo-cation within the system in vivo.

This research was supported by grants from the Science andEngineering Research Council, UK (GR/E 65500) and The Well-come Trust. We thank Dr Matthew Suflhess of the Natural

Products Branch of the National Cancer Institute USA for the giftof taxol. We are indebted to K. Sharma and A. Rijnenberg for theirpatient dissection of many thousands of insect ovaries.

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(Received 24 November 1989 - Accepted 5 February 1990)

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