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. 2002 Jul;7(3):297–308. doi: 10.1379/1466-1268(2002)007<0297:hatpmd>2.0.co;2

Hsp70 and thermal pretreatment mitigate developmental damage caused by mitotic poisons in Drosophila

Olga A Isaenko 1,*, Timothy L Karr 1, Martin E Feder 1,2,1
PMCID: PMC514829  PMID: 12482205

Abstract

To assess the ability of the heat-inducible molecular chaperone heat-shock protein 70 (Hsp70) to mitigate a specific developmental lesion, we administered the antimicrotubule drugs vinblastine (VB) and colchicine (COL) to larvae of Drosophila engineered to express differing levels of Hsp70 after heat pretreatment (HP). VB and COL decreased survival during metamorphosis, disrupted development of the adult eye and other structures as well as their precursor imaginal disks, and induced chromosome nondisjunction in the wing imaginal disk as indicated by the somatic mutation and recombination test (SMART) assay. Hsp70-inducing HP reduced many of these effects. For the traits viability, adult eye morphology, eye imaginal disk morphology, cell death in the eye imaginal disks, and single and total mutant clone formation in the SMART assay, HP reduced the impact of VB to a greater extent in Drosophila with 6 hsp70 transgenes than in a sister strain from which the transgenes had been excised. Because the extra-copy strain has higher levels of Hsp70 than does the excision strain but is otherwise almost identical in genetic background to the excision strain, these outcomes are attributable to Hsp70. The hsp70 copy number had a variable interaction with HP and COL administration.

INTRODUCTION

Molecular chaperones are a class of proteins that participate in the maturation and quality control of other proteins and minimize the tendency of other proteins to aggregate when in nonnative conformations (Feder and Hofmann 1999). The latter function is particularly important with respect to protein-damaging stresses such as heat and numerous toxic substances, which themselves induce many but not all molecular chaperones and other proteins. To date, most experimental work has examined the protective function of molecular chaperones in vitro, in isolated cells, or at a particular developmental stage (eg, embryo, adult); how chaperones protect complex processes such as development itself against stress is less well known. Clearly, many chaperones are essential for proper development; indeed, numerous chaperone mutants fail to develop or develop abnormally (Heikkila 1993a, 1993b; Angelier et al 1996; Krone et al 1997; Morange et al 1998; Rutherford and Lindquist 1998; Luft and Dix 1999; Vega-Nunez et al 1999). These studies, however, seldom determine the precise molecular lesion against which chaperones protect. Accordingly, we examined the developmental damage of the microtubule poisons vinblastine (VB) and colchicine (COL), whose mechanisms of toxicity are known in detail (Hamel 1996; Nogales 2001). We used both heat-inducible protein synthesis and genetic manipulation of a specific chaperone, the Drosophila DnaK–heat shock protein (Hsp) family member Hsp70, to ascertain whether Hsps in general and Hsp70 in particular protect the developmental process against the effects of these mitotic poisons.

Appropriate microtubule function is critical throughout development, particularly but not exclusively with respect to its role in normal chromosome segregation. Even in unstressed cells, molecular chaperones interact with the microtubule machinery, participating in its assembly, normal function, and repair (Gupta 1990; Lavoie et al 1993; Marchesi and Ngo 1993). We challenged these functions by administering VB and COL, which bind to tubulin, causing it to aggregate or changing its enzymatic properties (Galloway and Ivett 1986; Hamel 1996; Nogales 2001). COL inhibits polymerization of tubulin by site-specific binding to β-monomers of tubulin within dimers (Margolis and Wilson 1977; Uppuluri et al 1993); VB induces depolymerization and aggregation of tubulin polymers (Bensch and Malawista 1969). These effects disrupt microtubules and microtubule-dependent processes (Liang and Satya-Prakash 1985) and efficiently induce developmental abnormalities in the adult eye (Clayton and Francoeur 1971; Wolsky 1983; Drozdovskaya and Rapoport 1988; Isaenko et al 1994; Isaenko and Shvartsman 1999) and sperm (Wilkinson et al 1975), sex chromosome nondisjunction (Shvartsman and Isaenko 1999a, 1999b), embryonic lethality (Shvartsman and Isaenko 1999a, 1999b), and mutagenesis (Graf et al 1984; Isaenko et al 1994) in Drososphila. To test whether chaperones protect against such damage, we examined viability, specific markers of normal development (morphology of adult eye and eye imaginal disk, cell death and nuclear position in developing neurons of the eye imaginal disk), and mutagenesis in larvae treated with mitotic poisons in which we manipulated molecular chaperone levels. We quantified mutagenic activity with the somatic mutation and recombination test (SMART) assay, which infers frequencies of distinct genetic events (eg, mitotic recombination, point mutation, deletion, and chromosome nondisjunction) from the size and type of mutant clones expressing recessive marker alleles of mwh (multiple wing hairs) and flr3 (flare; Graf et al 1984).

Heat pretreatment (HP), which induces expression of many molecular chaperones, mitigates many developmental and mutagenic events caused by VB and COL (Isaenko and Shvartsman 1999; Shvartsman and Isaenko 1999a, 1999b). Studies such as those cited here do not, however, allow assignment of these effects to a specific heat-inducible chaperone. Thus, in the present study, we manipulated Hsp expression in 2 different ways: with HP and by comparing strains of Drosophila differing in hsp70 copy number. Specifically, we exploited an allelic series of hsp70 created by means of germ-line transformation and unequal homologous recombination (Welte et al 1993). These techniques create sister strains, an “extra-copy” strain with 10 wild-type and 12 transgenic hsp70 copies in the diploid and an “excision” strain with only the 10 wild-type copies but derived from the same transformation event. Thus, the excision strain controls for insertional mutagenesis; the extra-copy strain, however, has higher cellular Hsp70 concentrations than does the excision strain under many conditions of comparable heat shock (Welte et al 1993; Feder et al 1996; Krebs and Feder 1997, 1998). Comparative data for these 2 strains indicate that both HP and Hsp70 protect against the damage caused by mitotic poisons.

MATERIALS AND METHODS

Fly strains and culture

Flies were cultured in instant medium (Carolina Biological Supply) at 22.5°C. Strains included were Oregon-R (wild-type, 10 diploid hsp70 copies) and the hsp70 extra-copy and excision strains described in the Introduction (Welte et al 1993); Hsp70 expression in these strains has been characterized previously (Lindquist 1980; Welte et al 1993; Feder et al 1996; Krebs and Feder 1998). We constructed 4 other strains for use in the SMART assay. These strains contained mwh and flr3 wing markers, with genotypes as follows:

  1. w1118; P{FRT w+hs 3Xhsp70+}; mwh

  2. w1118; P{FRT w+hs 3Xhsp70+}; flr3/Ser

  3. w1118; P{FRT w+hs}; mwh

  4. w1118; P{FRT w+hs}; flr3/Ser

Treatment

VB (Sigma, catalog no. V1377) or COL (Sigma, catalog no. C9754) was added to the medium to yield final concentrations of 0.4, 1, 2.5, and 5 μg/mL (VB) and 1, 3, 5, 8, and 10 μg/mL (COL). Synchronous larvae (±1 hour) were counted and transferred to this medium or control medium at indicated developmental stages. Survivors were counted and analyzed for morphological defects either as wandering third-instar larvae or adults enclosing from puparia as described subsequently. Preliminary studies of viability (Fig 1) and eye abnormality in surviving adults (Fig 2) identified 2.5 μg/mL VB and 8 μg/mL COL as equally effective doses; subsequent studies used these concentrations. In some cases larvae underwent heat treatment before (HP) or after (heat posttreatment [HT]) transfer by immersing the vials for 30 minutes in a circulating water bath thermostatted at 36°C.

Fig 1.

Fig 1.

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 Effect of heat pretreatment (HP) or vinblastine (VB) (or both) ingestion on heat shock protein 70 levels in larvae of the extra-copy and excision strains. Lane 1: controls; lane 2: 1 hour after 30 minutes HP at 36°C; lane 3: 6 hours after 30 minutes HP at 36°C; lane 4: 24 hours after 30 minutes HP at 36°C; lane 5: 30 minutes HP at 36°C followed by 2.5 μg/mL VB present in medium for 6 hours; lane 6: 30 minutes HP at 36°C followed by 2.5 μg/mL VB present in medium for 24 hours; lanes 7 and 8: 2.5 μg/mL VB present in medium for 6 and 24 hours, respectively. Upper row: extra-copy strain; lower row: excision strain

Fig 2.

Fig 2.

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 Colchicine (COL) and vinblastine (VB) affect viability of larvae and do so differently in strains of Drosophila differing in hsp70 copy number. (A, B) Effect of drug concentration in medium throughout larval development on pupal death in wild-type Drosophila. (C, D) Effect of 2.5 μg/mL VB or 8 μg/mL COL in third-instar larval medium on pupal death in an extra hsp70 copy strain and a control “excision” strain. As indicated, some larvae underwent Hsp-inducing heat pretreatment (36°C, 30 minutes)

Analyses

Hsp70 immunoblots

Third-instar larvae (3–4) were frozen in liquid nitrogen and then stored at −80°C until further analysis. For isolation of protein, samples were thawed, transferred to 1 mL phosphate-buffered saline (PBS) containing protease inhibitor cocktail (Boehringer-Mannheim, catalog no. 1697498), ground with a disposable plastic pestle while ice-cold, and centrifuged at 4°C 14 000 revolutions/min for 30 minutes. The protein concentration of the supernatant was determined in triplicate by the BCA assay (Pierce, catalog no. 23227). Equivalent amounts of proteins (10 μg soluble protein per gel lane) were separated electrophoretically on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels and electroblotted to Immobilon-P membrane (Millipore, catalog no. IPVH07850). After extensive blocking, membranes were incubated with the Drosophila Hsp70-specific antibody 7FB (Velazquez et al 1980; Velazquez and Lindquist 1984) (1:1000), and bound antibody was detected with 1:1000 peroxidase-conjugated goat anti-rat IgG antibody (Jackson ImmunoResearch Lab, catalog no. 112-035-003), Pierce SuperSignal chemoluminescent reagents, and Kodak X-ray film.

Survival and morphology

We estimated the level of pupal death as the ratio of nonemerged pupae to the total number of pupae and scored surviving adults under the dissecting microscope for any abnormalities, including abnormal eye morphology and size, reduced number of ommatidia, and wing or thorax abnormalities.

Gross morphology of eye imaginal disks

Disks were dissected from wandering third-instar larvae, fixed in 3.7% paraformaldehyde in PBS for 20 minutes, washed 3 times in PBS + 0.1% Triton X-100 (PBST), transferred to PBS with ribonuclease A (2 mg/mL) for 2 hours, washed again 3 times in PBST, and stained with Sytox Green (Molecular Probes Inc, catalog no. S-7020) according to the manufacturer's protocol. After 3 washes with PBST, eye disks were mounted in mounting medium (0.25% n-propylgallate, 70% glycerol in PBS, pH 8.6) and examined under the fluorescent microscope.

Scanning electron microscopy

Flies were dehydrated through 25%, 50%, 75%, and 100% ethanol for at least 24 hours per session. After dehydration, samples were transferred to a shallow dish and allowed to dry. They were then mounted onto metal stubs and gold-coated for viewing.

Cell death

Eye imaginal disks were dissected from wandering third-instar larvae in 1-μM acridine orange (Sigma, catalog no. A6014). The samples were then rinsed in PBS, mounted on slides, and examined immediately with a Zeiss LSM 510 confocal microscope.

Number of developed neurons and nuclear position

Eye imaginal disks were dissected from wandering third-instar larvae, fixed in 3.7% formaldehyde, washed in PBS, and then blocked with 5% normal donkey serum in PBST. After blocking, the disks were incubated in 1:1000 anti-tubulin monoclonal antibody (Dm1a; Amersham) and 1:20 rat anti-elav antibody (Mab, catalog no. 7E8A10; Patel et al 1994) in PBST with 5% normal donkey serum at room temperature for 1 hour. These sera recognize cytoskeleton and neuronal nuclei, respectively. Disks were washed 3 times in PBST and then incubated with 1:400 anti-mouse (CY5 Dams IgG 715-175-150, Jackson ImmunoResearch Lab Inc) and 1:400 anti-rat (LRSC-Darat IgG 712-085-150, Jackson ImmunoResearch Lab Inc) antibodies at room temperature for 30 minutes. After 3 washes in PBST, disks were mounted and examined with a Zeiss LSM 510 confocal microscope.

SMART assay

The transheterozygous mwh/flr progeny from crosses between strains 1 and 2 or 3 and 4 were treated as described subsequently, and wings of surviving adult flies were dissected, prepared, and analyzed as in Graf et al (1984). Statistical conclusions were based on SELBY-OLSON analysis, and all calculations used the SMART statistical program on the basis of the formulae of Frei and Wurgler (1988). Clone formation frequencies were calculated as in Szabad et al (1983).

RESULTS

Heat shock protein 70

Hsp70 was not detectable by immunoblot assay in larvae not undergoing HP and in larvae ingesting VB without HP (Fig 1). HP increased levels of Hsp70, and VB ingestion either did not affect or slightly augmented this increase. When Hsp70 was detectable, its level was greater in the extra-copy strain than in the excision strain.

In each of the following sections, we first report the effect of ingestion of mitotic poisons by larvae, then present the impact of Hsp-inducing HP on this effect, and finally compare results for an allelic series of Drosophila differing in hsp70 copy number.

Viability

In wild-type flies, larval ingestion of either VB or COL caused death during the pupal stage in dose-dependent fashion (Fig 2 A,B). In the hsp70 allelic series (Fig 2 C,D), feeding of VB increased pupal death from 5–6% to 80–85%; the extra-copy and excision strains did not differ significantly. By contrast, HP decreased the death of extra-copy pupae to 54%, whereas it decreased that of excision pupae to only 72% (P < 0.05). COL alone and HP before COL administration had similar effects, but the extra-copy and excision strains did not differ in these effects (P > 0.05).

Eye development

In wild-type flies, VB and COL concentrations resulting in incomplete mortality disrupted development of the eyes of surviving adults (Fig 3 A,B). Disruptions included reduction in ommatidia number, change in facet structure (deviation from the regular hexagonal shape), and reduction or doubling of interommatidial trichomes (Fig 4). Interestingly, the eye imaginal disks display anomalies corresponding to those in adult eyes (Fig 5). The disks are smaller and have fewer developed clusters, fewer regularly oriented photoreceptors, and less regular cell and tissue polarity than do disks of controls. Proportions of VB-treated larvae with normal disks and normal eyes as adults (21% and 27%, respectively), proportions of those with abnormal disks and abnormal eyes (21% and 24%), and proportions of those with disks absent and dying while pupae (58% and 49%) did not differ significantly (Fisher's ϕ angular transformation with Yates' correction for continuity, P > 0.01). Eye developmental disruption increased with the developmental stage at which larvae ingested VB or COL (Fig 3 C,D). Wing and thorax anomalies were also evident but rare (data not shown).

Fig 3.

Fig 3.

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 Frequencies of adult eye abnormalities induced by vinblastine (VB) and colchicine (COL) depend on dose, stage of larvae development, hsp70 copy number, and heat treatment. As indicated, some larvae underwent heat shock protein–inducing heat treatment (36°C, 30 minutes) before (HP) or after (HT) drug exposure. (A, B) Effect of drug concentration in medium throughout larval development on eye malformation in adult wild-type Drosophila. (C, D) Effect of 2.5 μg/mL VB or 8 μg/mL COL at different stages of larvae development on adult eye abnormalities in wild-type Drosophila. (E, F) Frequencies of eye abnormalities in the hsp70 extra-copy and excision strains treated at the third instar

Fig 4.

Fig 4.

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 Electron micrographs of eye malformations induced by mitotic poisons. (A) Normal eye. (B–F) The spectrum of eye defects caused by vinblastine (VB) (2.5 μg/mL). The most common cases were reduced (B) or totally missing eye (F). In some cases the shape of the eye was also modified, phenocopying eyeless or bar mutations (C, D). The eyes of the 2 sides are usually affected quite differently (E). In extreme cases the eye may be completely absent and its place covered with bristles, often unusually large (F). (G) Normal ommatidia and interommatidial trichomes. (H) Disruption of the shape of ommatidia and doubling of trichomes caused by VB (arrows indicate defective ommatidia and trichomes). Scale bar = 10 μm (A–F) and 100 μm (G, H)

Fig 5.

Fig 5.

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 Both heat pretreatment (HP) and hsp70 copy number affect the impact of vinblastine (VB) on the gross morphology of Sytox Green–stained eye imaginal disks. (A, C, E) Excision strain. (B, D, F) Extra-copy strain. (A, B) Control disks with normal morphology and regular cell number. (C, D) HP (as shown above) reduced eye disk abnormality, including number and size of ommatidia in VB-treated larvae as compared with unpretreated controls (E, F). Anterior is to the left. Magnification 250×

In third-instar larvae fed VB and COL, Hsp-inducing HP significantly reduced the proportion of adults with aberrant eyes (P < 0.05 for wild-types; Fig 3 C,D), more so in the extra-copy strain than in the excision strain (Fig 3 E,F): without HP, VB-induced eye anomalies were significantly (P < 0.05) less prevalent in the extra-copy strain than in the excision strain (26% vs 46%, respectively). HP reduced these percentages to 7% and 29% respectively, which differ significantly (P < 0.05). Similarly, COL feeding alone resulted in significantly less frequent eye abnormalities in extra-copy larvae than in excision larvae (9% vs 17%). In contrast to the result for VB treatment, HP reduced abnormality frequency in the excision strain (to 4%; P < 0.05) but had no effect in the extra-copy strain.

Eye disk development

Both HP and hsp70 copy number affected the impact of VB on the gross morphology of eye imaginal disks (Fig 5). Morphology and cell number were normal in control disks of the extra-copy and excision strains (Fig 5 A,B). VB-treated disks (Fig 5 E,F) were of abnormal shape, size, and cell number. In such disks, developed ommatidia were about 25% as numerous as in controls. These effects were more severe in the excision strain than in the extra-copy strain. HP protected overall disk morphology but not the size and number of developed ommatidia against the effects of VB exposure (Fig 5 C,D).

Cell death

The differing cell numbers of eye disks from control and VB- and COL-treated larvae suggest that both mitotic poisons may increase cell death during eye development. In extra-copy and excision disks, cell death (as indicated by acridine orange staining) was about 4 times more prevalent in disks from VB-treated (Fig 6 C,F) and COL-treated (data not shown) third-instar larvae than in disks from controls (Fig 6 A,D). HP decreased cell death (P < 0.01) induced by both agents (Fig 6 B,E)—more efficiently in extra-copy disks.

Fig 6.

Fig 6.

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 hsp70 copy number affects cell death in eye disks from third-instar larvae fed vinblastine (VB). Third-instar eye imaginal disks were stained with acridine orange and examined under the confocal microscope. (A–C) Extra-copy strain. (D–F) Excision strain. (A, D) In untreated disks, scattered cell death is anterior to the morphogenetic furrow, with little or none posterior to the furrow. (C, F) In VB-treated disks, cell death increases. (B, E) Heat pretreatment before VB-feeding reduces cell death. Anterior is to the left. Magnification 630×. Images were analyzed and numbers of pixels were calculated to evaluate statistical differences. Mean pixel number was calculated from 3 different replicates in all samples and was (A) 3413.13 (n = 29); (B) 2188.08 (n = 184); (C) 14 991.04 (n = 314); (D) 3263.25 (n = 32) (E) 8589.56 (n = 237); (F) 13 452.34 (n = 265)

Nuclear position

As photoreceptor progenitor cells commit to a specific fate and begin to differentiate, their nuclei migrate apically (Tomlinson 1985; Fan and Ready 1997). To examine the microtubule-dependent migration of photoreceptor nuclei, the impact of VB and COL (which disrupt microtubules) on this migration, and the putative protective effects of HP and Hsp70, we immunostained neuronal nuclei and cytoskeleton of the third-instar eye disks. In control disks, photoreceptor nuclei were typically in clusters near the apical surface (Fig 7, top row). In VB- and COL-treated disks, photoreceptor nuclei were distributed randomly throughout the eye disk; few occupied the normal apical region and many had “fallen” into the optic stalk, having migrated along their axons (Fig 7, middle). In disks pretreated before VB or COL administration (Fig 7, bottom row), the nuclear position resembled that in control disks. Thus, HP mitigated the effect of VB or COL on nuclear position.

Fig 7.

Fig 7.

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 Heat pretreatment mitigates nuclear mispositioning induced by vinblastine (VB) in third-instar eye disks. Eye disks were double-labeled with anti-elav (red) and anti-microtubule (blue) and examined by confocal microscopy. A series of optical planes separated by 2.5 μm were recorded from apical to basal, in normal (top, magnification 630×), VB-treated (middle, magnification 1000×), and heat-pretreated disks (bottom, magnification 1000×). In normal disks, photoreceptor nuclei are regularly ordered at the apical surface. In VB-treated disks, nuclei are disordered and distributed throughout the depth of the retinal epithelium. In disks of larvae heat pretreated before VB administration, nuclear position resembled that in control disks. Anterior is to the right

Mutagenesis

As the wing-spot test of SMART reveals, both VB and COL induced mosaic mutant spots in extra-copy and excision Drosophila strains (Table 1). The strains were similar in several ways: both drugs induced only small single spots and both increased clone formation; VB had a greater mutagenic activity than did COL. HP decreased the frequency of VB-induced spots. The strains differed in other respects: HP decreased the frequency of COL-induced small single spots only in extra-copy flies, and had a much greater effect on VB-induced single and total spots in the extracopy strain than in the excision strain (F1,1 = 3.9); both agents did not alter the cell cycle divisions in the extra-copy strain. The frequency of clone formation was greater in the extra-copy strain than in the excision strain, probably consistent with a higher mutability.

Table 1.

 VB and COL-induced spots in wings of Drosophila differing in hsp70 copy number

graphic file with name i1466-1268-7-3-297-t01.jpg

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DISCUSSION

Spatial and temporal regulation of cell division is fundamental for normal development, and that mitotic poisons such as VB and COL disrupt development is neither novel (Gelei and Csik 1940) nor surprising. In the present study, VB and COL affected survival during metamorphosis, development of the adult eye and other structures as well as their precursor imaginal disks, and chromosome disjunction in the wing imaginal disk. These findings, moreover, are consistent with long-held expectations that developmental processes or stages with high mitotic activity should be most sensitive to microtubule-disruptive drugs such as VB and COL. Likewise, both the Hsp70-DnaK family and other families of molecular chaperones have long been known to play diverse and essential roles in normal development in Drosophila (Michaud et al 1997; Elefant and Palter 1999) and in other multicellular eukaryotes (Heikkila 1993a, 1993b; Angelier et al 1996; Krone et al 1997; Morange et al 1998; Luft and Dix 1999; Vega-Nunez et al 1999).

These established aspects of development intersect in a novel fashion in the present study, which demonstrates that HP in a Drosophila line engineered for heat-inducible overexpression of Hsp70 reduces both the frequency and severity of developmental defects caused by VB and COL. HP alone, which induces a diverse suite of Hsps in Drosophila, is sufficient for partial abatement of VB and COL effects, as has also been shown previously (Isaenko and Shvartsman 1999; Shvartsman and Isaenko 1999a, 1999b). Significantly, in the extra-copy strain, with 5 natural and 6 transgenic hsp70 copies in the haploid genome, this abatement is many times greater than in its sister strain from which the transgenic hsp70 copies have been excised. Both our own (Fig 1) and numerous prior determinations (see Introduction) establish that these strains differ in Hsp70 levels in relation to hsp70 copy number. As the genomes of these strains are otherwise almost identical, this outcome strongly suggests Hsp70 as the cause of the mitigation of developmental defects. Indeed, Roberts and Feder (1999) reported a similar result for protection against heat-induced developmental defects.

These results are consistent with several nonexclusive mechanisms of developmental protection. First, Hsp70 may act directly on microtubules to repair or prevent the damage caused by VB and COL. In the normal cell, all cytoplasmic chaperones (Csermely 2001), including some of mitochondrial origin (Soltys and Gupta 1999), associate with microtubules and bind tubulin. These include Hsp70 family members, which are implicated in the assembly of microtubules (Gupta 1990; Marchesi and Ngo 1993; Eggers et al 1997; Liang and MacRae 1997). In the cell, VB binds to a specific domain of β-tubulin (Rai and Wolff 1996), resulting in the disruption of normal tubulin polymers (Bensch and Malawista 1969; Roobol et al 1976, 1977; Galloway and Ivett 1986; Hamel 1996). We hypothesize that Hsp70 (1) through prior binding to tubulin, impedes VB access to its binding domain; (2) helps minimize the formation of tubulin aggregates or helps disaggregate them (Diamant et al 2000); or (3) promotes the de novo (re)assembly of tubulin polymers (Liang and Satya-Prakash 1985; Gupta 1990; Marchesi and Ngo 1993; Eggers et al 1997). In support of the first proposed mechanism, we note that HP was relatively effective in reducing the effects of VB, whereas similar treatment after exposure to COL was relatively ineffective. With respect to the second, genetic evidence indicates that Hsp70 and its cochaperones are sufficient to suppress several diseases of protein aggregation in Drosophila (Warrick et al 1999; Chan et al 2000; Fernandez-Funez et al 2000; Kazemi-Esfarjani and Benzer 2000). In contrast to VB, COL disrupts de novo assembly of microtubules (Roobol et al 1976; Margolis and Wilson 1977; Uppuluri et al 1993). The extra-copy and excision hsp70 lines differed dramatically in their susceptibility to VB but were similar in their susceptibility to COL, suggesting that Hsp70 does not respond to the damage caused by COL. Indeed, COL has numerous effects besides those on tubulin (Fulton 1984), and Hsp70 may not affect tolerance of these pleiotropic effects.

The failure of VB and COL to affect Hsp70 levels in the present study is surprising, given that unfolded, damaged, or aggregated proteins are key inducers of molecular chaperone expression (Cotto and Morimoto 1999). This failure, however, is consistent with several prior reports (Clark and Brown 1987; Ait-Aissa et al 2000; O.A. Issaenko, data not shown). By contrast, COL induces expression of hsr-ω, a normally heat-inducible noncoding gene that putatively functions in transcriptional regulation during heat shock (Lakhotia and Sharma 1996).

Hsp70 may also affect cell proliferation and development through means unrelated to its direct interaction with tubulin. Hsp70 is a component of the centrosome, which it protects from heat damage, and participates in the regulation of both apoptosis and the cell cycle (Helmbrecht et al 2000; Garrido et al 2001). Moreover, Hsp70 is by no means the only molecular chaperone interacting with tubulin (Liang and MacRae 1997). TCP-1 or CCT, the eukaryotic cytosolic chaperonin, is essential for the de novo folding of tubulin (Yaffe et al 1992; Gao et al 1993). Hsp90 is a component of microtubules and cilia and may regulate tubulin dimer formation (Liang and MacRae 1997; Garnier et al 1998; Stephens and Lemieux 1999). Indeed, some of the developmental abnormalities resulting from the genetic or pharmacological inhibition of Hsp90 (Rutherford and Lindquist 1998) are reminiscent of those in the present study.

As stated in the Introduction, the precise means by which molecular chaperones protect the development process against damage caused by stress has been enigmatic. We have used well-characterized drugs to produce a specific developmental lesion and then shown that genetic and acute manipulation of Hsp70 can mitigate this lesion. This paradigm may be applicable to other chaperones, stresses, and aspects of development.

Acknowledgments

We thank N. Patel (University of Chicago) for antibody, A. Mahowald and K. Williams for comments on the manuscript, and the University of Chicago Confocal Microscopy Image Facility for advice and assistance. This work was supported by NSF grant 99-86158 to M.E.F. and NSF-NATO Postdoctoral Fellowship DGE 98-04537 on behalf of O.A.I.

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