1
Laboratorio di Biologia Cellulare, Istituto Superiore di
Sanità, Rome, Italy
2
Dipartimento di Biotechnologie Cellulari ed Ematologia,
Università La Sapienza, Rome, Italy
*
Author for correspondence (e-mail:
Gigliani{at}bce.med.uniroma1.it
)
Accepted May 1, 2001
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SUMMARY |
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Key words: Tat-tubulin interaction, Microtubule polymerization, Tat, Cell polarization
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INTRODUCTION |
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A wealth of emerging evidence points to the involvement of host cell
cytoskeleton in HIV infection (Cenacchi et al.,
1996; Delezay et al.,
1997
; Malorni et al.,
1997
; Bukrinskaya et al.,
1998
). HIV-encoded proteins
such as gp120 and Rev appear to affect cytoskeleton organization either by
inducing cellular ultrastructural changes and massive disruption of
microtubules (Cenacchi et al.,
1996
; Delezay et al.,
1997
; Malorni et al.,
1997
) or by depolymerizing
microtubules via a specific Revtubulin interaction (Watts et al.,
2000
). Furthermore, it has
been suggested that the degenerative neuronal changes described in
HIV-infected people are caused by neuronal cytoskeletal changes (Jacobson et
al., 1997
). The HIV
transactivator factor Tat, which can also be released by infected cells and
which plays a number of extracellular roles (Rubartelli et al.,
1998
), affects several
cellular functions by inducing angiogenesis (Mitola et al.,
2000
; Benelli et al.,
2000
), cell proliferation and
apoptosis (Chang et al., 1995
)
and by affecting the immune response of the host (Goldstein,
1996
). Also, Tat appears to be
involved in AIDS-associated neurodegenerative diseases (Cupp et al.,
1993
; Conant et al.,
1998
) and oncogenesis (Delli
Bovi et al., 1986
; Ensoli et
al., 1999
).
The Drosophila model allows us to use a novel approach to study
the action of viral gene products by analyzing their effects within a
territory and not just in the single cell; this is similar to the study of
gene expression restricted to a well defined territory during developmental
process (Garcia-Bellido et al.,
1973; Lawrence,
1973
). Any expansion or
restriction of the territory in which the gene is expressed results in mutated
phenotypes. This novel concept offers unique advantages, allowing the analysis
of the gene product interactions and the effects of the ectopic gene
expression in the developmental context. Thus, to examine the effects of Tat,
we considered this protein, expressed in Drosophila, as a gene
expressed in a foreign territory (i.e. as a gene ectopically expressed).
To test whether Tat is involved in the cytoskeleton organization, we produced Tat transgenic fly lines and analyzed the effect of Tat (under the control of the hsp70 promoter) by expressing it during Drosophila oogenesis. The oocyte of Drosophila is, in fact, a highly polarized cell and genetic, molecular and cytological studies have shed light on the specific functions of the cytoskeleton during oogenesis.
In this paper we show that: (1) Tat expressed during Drosophila oogenesis results in embryos with only one dorsal appendage, indicating that Tat affects oocyte polarization; and (2) this oocyte depolarization appears to be a consequence of a delay in the microtubule polymerization process caused by the specific interaction of Tat with the MAP binding domain of tubulin. These results indicate that Tat can interact with tubulin to alter the MT polymerization rate in HIV-infected cells and further our understanding of the molecular mechanism underlying Tat-mediated pathogenesis.
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MATERIALS AND METHODS |
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Flies homozygous for both hsp:tat and khc:lacZ constructs were obtained by
crossing the WG hsp:Tat line (yw; tatw+; TM3/Gl), which carries the hsp:tat
construct on the second chomosome, with the KZ503 strain (Clarck et al.,
1994).
Recombinant plasmids
The pCasPeR:Tat plasmid was constructed as follows: full-length Tat cDNA
(amplified by PCR) from pCMV-Tat plasmid, was cloned into the P-element vector
pCaSpeR-hs EcoRI-XbaI sites. The pCI-Tat plasmid was
constructed as described (Longo et al.,
1995). The two
pcI*-tub plasmids were constructed from pC169 plasmid (Longo et
al., 1995
) by replacing the
rop gene (excised as a HindIII-BamHI fragment) with
the PCR products of the I and the II+III domains of
-tubulin,
respectively.
Expression of HIV-Tat protein by heat shock treatment
In all experiments the expression of Tat (under the control of hsp70
promoter) was induced by subjecting adults or embryos to heat shock treatment
for 1 hour at 37°C.
Immunoprecipitation and western blotting
To detect the expression of Tat in Tat-transgenic Drosophila,
18-hour-old embryos were subjected to heat shock, and protein extracts were
fractionated by 15% SDS-polyacrylamide slab gel (PAGE) electrophoresis and
transferred to nitrocellulose sheets. Membranes were incubated with 5% nonfat
dry milk in NET buffer (150 mM NaCl, 50 mM Tris, 5 mM EDTA, 0.05% Triton
X-100, pH 7.5) for 1 hour at room temperature. After incubation, the sheets
were washed three times with NET and then incubated with anti-Tat anti-serum
(kindly supplied by G. Imerio) at a dilution 1:500 for 2 hours at room
temperature. After three washes, blots were incubated with a secondary
antibody (goat anti-rabbit) conjugated to horseradish-peroxidase (Biorad,
1:15,000) and, after the final washing, the reaction was visualized by
incubation with ECL chemiluminescence reagent (Amersham). For the
immunoprecipitation experiments, protein extracts were immunoprecipitated with
anti--tubulin antibody, using protein A/G plus-agarose (Santa Cruz) as
recommended by the manufacturer. Precipitated proteins were resolved by 15%
SDS-PAGE and immunoblotted with both anti-
-tubulin and anti-Tat
antibodies. The secondary antibodies used to detected Tat and
-tubulin
were goat anti-rabbit (Biorad, 1:15,000) and goat anti-mouse (Biorad,
1:5,000), respectively.
Immunofluorescence microscopy
Testes from adult males were dissected in PBS plus 5% glycerol.
Spermatocytes were fixed in methanol for 10 minutes at -20°C and then in
acetone for 5 minutes at -20°C, washed in PBS and observed under a
phase-contrast microscope. Cytological preparations fixed on glass slides were
extensively washed in PBS plus 3% BSA, incubated for 40 minutes at room
temperature with anti-goat antibody, and then washed for 1 hour. PBS plus 3%
BSA was also used both for the antibody incubations and for washing. The
detection of Gurken in ovaries was as described (Neuman-Silberger and
Schüpbach,
1996). All preparations were
incubated overnight at 4°C in appropriate primary antibody dilutions.
After washing in PBS plus 3% BSA, samples were incubated for 1 hour with
secondary fluorescein- or rhodamine-conjugated antibodies and then extensively
washed in PBS plus 3% BSA. Primary antibody dilutions were: monoclonal mouse
anti-Tat, 1:200; monoclonal mouse anti-
-tubulin (Amersham), 1:150;
anti-Gurken antibody, 1:3000. Secondary antibody dilutions were:
fluorescein-conjugated goat F(ab')2 fragment to mouse IGG
(Cappel), 1:100; rhodamine-conjugated goat F(ab')2 fragment
to mouse IGG (Cappel), 1:300; fluorescein-conjugated goat
F(ab')2 fragment to rat IGG (Cappel) 1:100. All preparations
were examined with a Nikon optiphot fluorescence microscope equipped with the
Biorad MRC1024ES laser scanning confocal attachment.
In vitro tubulin polymerization assay
To polymerize microtubules, a solution containing 2 mg/ml of tubulin
(Sigma), 10-4 M GTP, 10-2 M sodium phosphate,
10-3 M EGTA, 1.6x10-2 M MgCl2 and 3.4 M
glycerol at pH 7 was incubated at 37°C for 30 minutes. The absorbance was
continuously monitored at 350 nm. Tat was added to a final concentration of
0.10 mM. Aprotinin (Sigma) control protein was added to a final concentration
of 0.10 mM.
Phage immunity test
Bacterial cells transformed with plasmids expressing different
repressor fusion proteins were tested for sensitivity to
phages.
Phages of different virulent phenotypes were assayed by spot tests, at
concentrations varying from 10 to 106 phages per spot, on lawns of
transformed bacteria. The
phages used are as described (Longo et al.,
1995
).
Microtubule-dependent streaming
Bulk ooplasmic movements within living oocytes were assayed as follows:
adult females were transferred to a cover glass and covered with halocarbon
oil, and egg chambers were removed and dissected. The cover glass was then
transferred to the confocal microscope, and autofluorescent yolk granules were
directly imaged with a BHS filter set provided with the Bio-Rad MRC1024ES
laser scanning confocal microscope. Single frame images were collected at 10
second intervals with the use of a confocal microscope with fluorescent
filters. Temporal projections were created by summing 10 frames from a
time-lapse sequence with the project (maximum) utility of the COSMOS software
provided with the Bio-Rad confocal microscope. Each projection represents 100
seconds of total elapsed time.
Characterization of mutated phenotypes
To analyze chorion and embryonic cuticular phenotypes, embryos from both
w67c23 and w67c23 strains that are
transgenic for Tat were processed as described (Wieschaus and
Nusslein-Volhard, 1986).
Drosophila virgin females (5-6-days old) were subjected to heat shock
for 1 hour at 37°C and then mated with males (5-6-days old) on apple
juice-agar plates at 25°C for 39-40 hours (Wieschaus and Nusslein-Volhard,
1986
). Embryos were then
collected and observed under a microscope.
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RESULTS |
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Expression of Tat during Drosophila oogenesis causes
abnormality in embryo dorsal appendage formation
Homozygous Tat transgenic lines of D. melanogaster were used to
test whether HIV-Tat protein, expressed at different developmental stages by
heat shock promoter, is capable of inducing particular phenotypes. To this
end, Tat was expressed during Drosophila oogenesis (stages 8-10) by
subjecting Drosophila females to heat shock treatment. 5000
fertilized eggs from different experiments were collected and 10% to 13%
(depending on experiments) of eggs showed only one fused dorsal appendage
(Fig. 2A) instead of the two
normally present (Fig. 2B). In
the control sample (non-transgenic line subjected to the same heat shock
treatment) <1% of eggs exhibited this phenotype. This phenotype resembled
that observed in mutations that alter the dorso-ventral patterning of the egg
shell (Nilson and Schüpbach,
1999) and may be caused by
either mislocalization of determinants for oocyte axis specification
(Theurkauf et al., 1993
) or
inhibition of the microtubule polymerization process (Koch and Spitzer,
1983
).
|
To shed light on the molecular mechanism by which Tat interferes with two apparently different mechanisms to produce this particular phenotype, we first tested whether Tat interacts with microtubules. We used Drosophila spermatocytes for these experiments since they are relatively large and the organization of the microtubules differs during the cell cycle stages.
Tat was induced by heat shock in adult males, testes were dissected and
fixed, and spermatocytes at interphase and anaphase were immunostained with
both anti--tubulin and anti-Tat monoclonal antibodies. Immunochemical
fluorescent confocal microscopy showed that Tat colocalizes to microtubules
both in interphase (Fig. 3A-C) and in division spermatocytes (Fig.
3D-F).
|
Delaying effect of Tat on tubulin polymerization process
Since Tat appears not to affect the microtubule structural organization of
spermatocytes at interphase or anaphase
(Fig. 3), we wished to
determine the effect of the Tat-tubulin interaction by testing the reaction
rate of tubulin polymerization in the presence or absence of Tat. Tubulin (the
main component of microtubules) was polymerized in vitro in the presence of
GTP (Mitchison and Kirschener,
1984) and Tat, and the
polymerization reaction rate was monitored on the spectrophotometer. The
results show that Tat causes a delay in the tubulin polymerization process by
negatively affecting the cooperative effect of
and ß tubulin
monomers in the polymerization reaction
(Fig. 4). Tat appears to affect
the sigmoid trait of the curve, but not the lag phase or the final
concentration of polymerized tubulin. The protein aprotinin (a protease
inhibitor that, in common with Tat, has low molecular weight and cysteine
residues) used as control does not affect the polymerization rate of tubulin
(Fig. 4). This result
demonstrates that Tat binds to tubulin and that this binding acts on
microtubule assembly by delaying the tubulin polymerization process.
|
Tat and tubulin co-immunoprecipitation
To confirm that Tat associates with tubulin in vivo, we carried
out a co-immunoprecipitation assay (Fig.
5). We incubated protein extracts from Drosophila embryos
with anti--tubulin monoclonal antibody for immunoprecipitation,
followed by immunoblotting with anti-Tat polyclonal antibody. As shown in
Fig. 5 (lane 1), Tat is
detected in immunoprecipitate obtained from Tat-induced transgenic embryos,
whereas, both in non-Tat-induced embryos and in non-transgenic control embryos
(lanes 2 and 3, respectively), no immunoreactive band is observed.
|
Tat and tubulin heterodimerization assay
To identify any domain(s) of tubulin involved in the linking between Tat
and tubulin, we performed heterodimerization assays based on cI phage
repressor properties (Longo et al.,
1995
). The cI repressor
functions as a dimer; thus, the fusion of the cI DNA-binding domain to a
heterologous protein region capable of forming dimers, is expected to produce
a functional
repressor, and render bacterial cells expressing it
immune to
phage infection. If no dimerization occurs the cells are
phage sensitive. The
and ß tubulin amino acid sequences are
highly conserved among species (Theurkauf et al.,
1986
) and each monomer can be
divided into three functional domains: the N-terminal domain I containing the
nucleotide binding region, the intermediate domain II containing the taxol
binding site and the C-terminal domain III, which appears to constitute the
binding surface for motor proteins and for microtubule-associated proteins
(MAPs) (Nogales et al., 1998
).
Recently, Chau et al. reported that the tubulin amino acid sequence found
between domains II and III, contains the binding site for the MAP-Tau (Chau et
al., 1998
). On the basis of
these data, we performed experiments to test whether domains of
Drosophila
-tubulin interact with Tat and, if so, which. For
this purpose,
4-tubulin DNA coding for domain I (amino acids 1-215) and
for domains II and III (amino acids 216-462) was amplified by PCR and each
-tubulin fragment was cloned into the pC169 vector (Longo et al.,
1995
) that contained a
sequence coding for the
cI DNA-binding domain (cI*)
carrying a mutation that prevents its binding to the operator. The recombinant
plasmids were used to transform E. coli cells containing the cI-Tat
fusion cloned in the pC168 low copy number compatible plasmid (Longo et al.,
1995
). The transformants were
tested for
immunity. If the interaction between the two proteins
occurs, the functional chimeric repressor (cI-Tat) should be titrated out by
the heterodimerizing cI*-Tub fusion protein and should become
inactive, making the transformed cells sensitive to
infection. The
results show that E. coli cells transformed with Tat and plasmid
carrying the
-tubulin domain I, are immune to
phage, whereas
E. coli cells co-transformed with Tat and plasmid carrying the
-tubulin domain II+III are sensitive to
infection. Thus, we
can conclude that Tat specifically interacts with the tubulin domains II+III
but is unable to form dimers with tubulin domain I alone.
On the whole, these results demonstrate that: (1) Tat and tubulin interact with each other; and (2) the interaction specifically involves the MAPs-binding domain of tubulin and strongly suggests that the Tat-induced delay in tubulin polymerization depends on competition between Tat and MAPs in binding to tubulin.
Microtubule-dependent cytoplasmic streaming is prematurely blocked by
Tat
To ascertain whether the delaying effect of Tat on microtubule
polymerization occurs in vivo and to test the eventual consequences, we used
Drosophila oocytes as the experimental model. During
Drosophila gametogenesis, female gametes develop as syncitia
connected by large cytoplasmic bridges called ring canals, which allow the
flow of nutrients between cells in a syncitium (Robinson and Cooley,
1996). This transport is
essential for the development of normal oocytes. The cytoskeleton plays an
integral role in cytoplasm transport as shown by the fact that disruption of
the cytoskeleton by mutation or by drugs, such as colchicine, causes defective
transport (Theurkauf et al.,
1993
; Koch and Spitzer,
1983
). During stages 10b-13 of
oogenesis the molecules are distributed in the ooplasm by cytoplasmic
streaming generated by microtubules to avoid the formation of the
anterior-posterior particle-gradient and allow the binding of particles to
localized specific anchors (Theurkauf,
1994a
; Theurkauf,
1994b
; Clarck et al.,
1997
; Glotzer et al.,
1997
).
To test whether Tat affects microtubule-mediated transport, we examined
bulk cytoplasmic movements inside living egg chambers after heat-shock both in
oocytes expressing Tat and in control oocytes. In these experiments,
autofluorescent yolk granules within the ooplasm were followed with time-lapse
laser scanning confocal microscopy (Theurkauf,
1994b). In all oocytes that
expressed Tat (57 oocytes examined from different experiments), the
cytoplasmic streaming gradually decreased to terminate immediately, or at most
1 hour, after Tat expression (Fig.
6A,B), whereas the cytoplasmic flow observed in control oocytes
was not affected by heat shock and normally terminated in 2 hours and 30
minutes.
|
Therefore, we can conclude that Tat interacts with microtubules in vivo and that the consequence of this interaction produces a premature stop of the microtubule-dependent cytoplasmic streaming.
Tat depolarizes Drosophila oocytes
Egg polarization depends on the correct localization of the determinants of
the antero-posterior and dorso-ventral axes which, in turn, depend on the
microtubule cytoskeleton organization (Nilson and
Schüpbach,
1999). During stages 8 through
10, microtubules associate preferentially with the anterior cortex of the
oocyte, so that a broad anterior to posterior cortical gradient is formed at
stage 9 (Theurkauf,
1994b
).
To verify whether Tat can affect cytoskeletal functions that mediate axis
specification, we tested the position, after the expression of Tat in oocytes,
of the TGF--like protein Gurken, which is the basic determinant of the
dorsal-ventral axis (Neuman-Silberger and
Schüpbach,
1996
) and that of the
plus-end-directed microtubule motor protein Kinesin, which mirrors
antero-posterior polarity of the Drosophila oocyte (Clarck et al.,
1994
; Clarck at al.,
1997
).
In Drosophila wild-type stage 9-10a egg chambers, the Gurken
protein is spatially localized on the dorsal-anterior corner of oocytes
(Neumann-Silberger and Schupbach,
1996). After Tat induction by
heat shock treatment, the Gurken protein (Grk), detected by specific anti-Grk
antibody (Neuman-Silberger and Schupbach,
1996
), was mislocalized in
oocytes (Fig. 7). It appears,
in fact, to be distributed either along the anterior border of the oocyte, or
along the anterior and dorsal border (Fig.
7B,C), whereas the heat shock treatment in non-transgenic lines
did not affect the localization of the Gurken protein
(Fig. 7A).
|
Kinesin is normally localized in the posterior of the oocytes during stages 8 and 9 of oogenesis. We examined the localization of Kinesin in egg chambers from Drosophila females transgenic both for kinesin:ßgal fusion and for hsp:Tat construct following heat shock treatment. As shown in Fig. 8 by ß-galactosidase staining, the expression of Tat resulted in the mislocalization of Kinesin to the middle of the oocyte (Fig. 8B). By contrast, in the oocyte in which Tat was not expressed, kinesin was localized normally (Fig. 8A).
|
Therefore, the interaction of Tat with microtubules determines the mislocalization of axis determinants. Thus, these results account for the mutated phenotype occurring in embryos after Tat induction.
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DISCUSSION |
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The role of Tat in the microtubule polymerization process
Here we have shown that when Tat is expressed in Drosophila
oocytes at stage 10b-13, oocyte cytoplasmic streaming is prematurely blocked.
A similar effect has been observed (Theurkauf,
1994b) by treating oocytes
with colchicine, a drug that inhibits microtubule polymerization. Thus, Tat
exhibits the same effect as colchicine but via a different mechanism.
Colchicine, in fact, inhibits the polymerization of microtubules by binding to
tubulin monomers during the nucleation process and is ineffective on
polymerized tubulin. By contrast, Tat binds to already polymerized tubulin as
shown by the in vivo experiments (Figs
3,
4). This binding occurs through
the tubulin domain needed for tubulin-MAPs binding. MAPs contribute to
microtubule stabilization by inhibiting tubulin dissociation at the
microtubule ends (Drewes et al.,
1998
); therefore, we suggest
that the polymerization delaying effect caused by Tat depends on the Tat-MAPs
competition at the tubulin-MAPs binding site. Interestingly, the tubulin:Tat
relative concentration in the in vitro experiment to measure the tubulin
polymerization reaction rate, is 100:1. This condition is very similar to that
present in HIV-infected cells, where the concentration of Tat is certainly
lower compared with the concentration of tubulin. It is known that many human
neurodegenerative conditions involve a reorganization of the neuronal
cytoskeleton, which seems due to the loss of MAP-tubulin binding (Chau et al.,
1998
; Drewes et al.,
1998
). Therefore, besides the
mechanisms already described, the Tat-tubulin association makes it possible
for Tat to be involved in the pathogenesis of the AIDS-associated neurologic
disorders, destabilizing the MTs through competition with MAPs.
In addition, we have shown that Tat colocalizes with tubulin throughout the
cell cycle, including cells at phase S and anaphase
(Fig. 3). Thus, the association
of Tat with microtubules appears to be cell-cycle independent. This
association seems to keep Tat far from receptors to which it may associate
when secreted, and far from nuclear DNA to which Tat can associate via
transcriptional complex of cellular genes and thus affect normal cell
functions. Microtubules, by capturing Tat, may control both the translocation
of Tat into the nucleus and the secretion of Tat from the cells (Battaglia et
al., 1997). However, this
Tat-microtubule interaction affects cell polarization and may result in
further damage to the cell.
The role of Tat in oocyte polarization
The expression of Tat during Drosophila oogenesis results in
embryos that present only one dorsal appendage. This mutated phenotype in
Drosophila arises from dislocation of the dorso-ventral axis and is
caused by mutations affecting the spindle genes, which are involved
in patterning, and in DNA repair in mitosis and meiosis. One of the
spindle mutations affects the gurken gene expression by
drastically reducing gurken mRNA translation but seems not to
influence the microtubule polymerization process (Gonzalez-Reyes et al.,
1997; Ghabrial, et al.,
1998
). On the contrary, we
observe that, after Tat expression, Gurken is still produced in the egg, but
it is abnormally localized. Thus, the mechanism by which Tat causes
dorso-ventral axis mislocalization differs from that of spindle
mutation and appears to depend on microtubule delayed polymerization. This
result raises the possibility that the interaction of Tat with microtubules
induces defects in mitotic and/or meiotic spindle formation that may result in
chromosome aneuploidies.
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ACKNOWLEDGMENTS |
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REFERENCES |
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---|
Battaglia, P. A., Regoli, E. and Gigliani F. (1997). Measurement of the range of HIV-LTR transactivating activity of HIV-TAT in vitro. Int. J. Oncology. 11,1007 -1011.
Benelli, R., Barbero, A., Ferrini, S., Scapini, P., Cassatella, M., Bussolino, F., Tacchetti, C., Noonan, D. M. and Albini, A. (2000). Human immunodeficiency virus transactivator protein (Tat) stimulates chemotaxis, calcium mobilization, and activation of human polymorphonuclear leukocytes: implications for Tat-mediated pathogenesis. J. Infect. Dis. 182,1643 -1651.[Medline]
Bukrinskaya, A., Brichacek, B., Mann, A. and Stevenson, M.
(1998). Establishment of a functional human immunodeficiency
virus type 1 (HIV-1) reverse transcription complex involves the cytoskeleton.
J. Exp. Med. 188,2113
-2125.
Cenacchi, G., Guiducci, G., Pasquinelli, G., Re, M. C., Ramazzotti, E., Furlini, G., Malorni, W., DeLuca, M. and Martinelli, G. N. (1996). Early ultrastructural changes of human keratinocytes after HIV-1 contact: an in vitro study. Eur. J. Dermatol. 6,213 -218.
Chang, H. K., Gallo, R. C. and Ensoli, B. (1995). Regulation of cellular gene expression and function by tha human immunodeficiency virus type-1 Tat protein. J. Biomed. Sci. 2,189 -202.[Medline]
Chau, M. F., Radeke, M. J., de
Inés, C., Barasoain, I., Kohlstaedt, L. A. and
Feinstein, S. C. (1998). The microtubule-associated protein
tau cross-links to two distinct sites on each and ß tubulin
monomer via separate domains. Biochemistry
37,17692
-17703.[Medline]
Clarck I., Giniger, E., Ruhola-Baker, H., Jan, L. Y. and Jan Y. N. (1994). Transient posterior localization of a kinesin fusion protein reflects anteroposterior polarity of the Drosophila oocyte. Curr. Biol. 4,289 -300.[Medline]
Clarck, I. E., Jan, L. Y. and Jan, Y. N.
(1997). Reciprocal localization of Noad and kinesin fusion
proteins indicates microtubule polarity in the Drosophila oocyte,
epithelium, neuron and muscle. Development
124,461
-470.
Conant, K., Garzino-Demo, A., Nath, A., McArthur, J. C.,
Halliday, W., Power, C., Gallo, R. C. and Major, E. O.
(1998). Induction of monocyte chemoattractant protein-1 in HIV-1
Tat-stimulated astrocytes and elevation in AIDS dementia. Proc.
Natl. Acad. Sci. USA 95,3117
-3121.
Cupp, C., Taylor, J. P., Khalili, K. and Amini, S. (1993) Evidence for stimulation of the transforming growth factor beta 1 promoter by HIV-1 Tat in cells derived from CNS. Oncogene 8,2231 -2236.[Medline]
Delezay, O., Yahi. N., Tamalet, C., Baghdiguian, S., Boudier, J. A. and Fantini, J. (1997). Direct effect of type 1 human immunodeficiency virus (HIV-1) on intestinal epithelial cell differentiation: relashionship to HIV-1 enteropathy. Virology 238,231 -242.[Medline]
Delli Bovi, P., Donti, E., Knowles, D. M., Fruedman-Kien, A., Luciw, P. A., Dina, D., Della-Favera, R. and Basilico, C. (1986). Presence of chromosomal abnormalities and lack of AIDS retrovirus DNA sequences in AIDS-associated Kaposi's sarcoma. Cancer Res. 46,6333 -6338.[Abstract]
Drewes, G., Ebneth, A. and Mandelkow, E. (1998). MAPs, MARKs and microtubules dynamics. Trends Biochem. Sci. 23,307 -311.[Medline]
Ensoli, B., Monini, B. and Sgadari, C. (1999). Pathogenesis and cell biology in Kaposi's sarcoma. In HIV and the New Viruses. 2nd edn (ed. A. Dalgleish and R. Weiss), pp.385 -413. Academic Press, San Diego, CA.
Feany, M. B. and Bender, W. W. (2000). A Drosophila model of Parkinson's disease. Nature 404,394 -398.[Medline]
Fortini, M. E. and Bononi, N. M. (2000). Modeling human neurodegenerative diseases in Drosophila. Trends Genet. 16,161 -167.[Medline]
Garcia-Bellido, A., Morata, G. and Ripoll, P. (1973). Developmental compartimentalization of the wing disk of Drosophila. Nature New Biol. 245,251 -253.[Medline]
Ghabrial, A., Ray, R. P. and
Schüpbach, T. (1998).
okra and spindle-B encode components of the RAD52 DNA repair
pathway and affect meiosis and patterning in Drosophila oogenesis.
Genes Dev. 12,2711
-2723.
Glotzer, J. B., Saffrich, R., Glozer, M. and Ephrusssi, A. (1997). Cytoplasmic flows localize injected oskar RNA in Drosophila oocytes. Curr. Biol. 7, 326-337.[Medline]
Goldstein, G. (1996). HIV-1 Tat protein has a potential AIDS vaccine. Nat. Med. 2, 960-964[Medline]
Gonzalez-Reyes, A., Elliot, H. and St Johnston, D.
(1997). Oocyte determination and origin of polarity in
Drosophila: the role of the spindle genes. Development
124,4927
-4937.
Jacobson, S., Henriksen, S. J., Prospero-Garcia, O. Phillips, T. R., Elder, J. H., Young, W. G., Bloom, F. E. and Fox, H. S. (1997). Cortical neuronal cytoskeletal changes associated with FIV infection. J. Neurovirol. 3, 283-289.[Medline]
Karn, J. (1999). Tackling Tat. J. Mol. Biol. 293,235 -254.[Medline]
Koch, E. A. and Spitzer, R. H. (1983). Multiple effects of colchicine on oogenesis in Drosophila: induced sterility and switch of potential oocyte to nurse-cell developmental pathway. Cell Tissue Res. 228,21 -32.[Medline]
Lawrence, P. A. (1973). A clonal analysis of segment development in Oncopeltus (Hemiptera). J. Embryol. Exp. Morph. 30,681 -699.[Medline]
Longo, F., Marchetti, M. A., Castagnoli, L., Battaglia, P. A. and Gigliani, F. (1995). A novel approach to protein-protein interaction: complex formation between the p53 tumor suppressor and the HIV Tat proteins. Biochem. Biophys. Res. Comm. 206,326 -334.[Medline]
Malorni, W., Guiducci, G., Pasquinelli, G., Rivabene, R., Re, M. C., Ramazzotti, E., DeLuca, M, LaPlaca, M. and Cenacchi, G. (1997). HIV-type 1 induces specific cytoskeleton alterations in human epithelial cells in culture. Eur. J. Dermatol. 7, 263-269.
Mitchison, T. and Kirschner, M. W. (1984). Microtubule assembly nucleated by isolate centrosome. Nature 312,232 -237.[Medline]
Mitola, S., Soldi, R., Zanon, I., Barra, L., Gutierrez, M. I.,
Berkhout, B., Giacca, M., Bussolino, F. (2000).
Identification of specific molecular structures of human immunodeficiency
virus type 1 Tat relevant for its biological effects on vascular endothelial
cells. J. Virol. 74,344
-353.
Moore, M. S., DeZazzo, J., Luk, A. Y., Tully, T., Singh, C. M. and Heberlain, U. (1998). Ethanol intoxication in Drosophila and pharmacological evidence for regulation by the cAMP signaling pathway. Cell 93,997 -1007.[Medline]
Neuman-Silberger, F. S. and
Schüpbach, T. (1996). The
Drosophila TGF- like protein Gurken: expression and cellular
localization during Drosophila oogenesis. Mech.
Dev. 59,105
-113.[Medline]
Nilson L. A. and Schüpbach T. (1999). EGF receptor signalling in Drosophila oogenesis. Curr. Top. Dev. Biol. 44,203 -243.[Medline]
Nogales, E., Wolf, S. G. and Downing, K. H.
(1998). Structure of the ß tubulin dimer by electron
crystallography. Nature
391,199
-203.[Medline]
Potter, C. J., Turenchalk, G. S. and Xu, T. (2000). Drosophila in cancer research. Trends Genet. 16,33 -39.[Medline]
Robinson, D. N. and Cooley, L. (1996). Stable intercellular bridges in development: the cytoskeleton lining the tunnel. Trends Cell Biol. 6,474 -479
Rubartelli, A., Poggi, A., Sitia, R. and Zocchi, M. R. (1998). HIV-1 Tat: a polypeptide for all season. Immunol. Today 19,543 -545.[Medline]
Spradling, A. C. (1986). P element-mediated transformation. In Drosophila: A Practical Approach (ed. D. B. Roberts), pp. 175-197. IRL press, Oxford, UK.
Theurkauf, W. E. (1994a). Immunofluorescence analysis of the cytoskeleton during oogenesis and early embryogenesis. In Methods in Cell Biology (ed. S. B. Goldstein and E. A. Fryberg), pp. 489-505. Academic Press.
Theurkauf, W. E. (1994b). Premature microtubule-dependent cytoplasmic streaming in cappucino and spire mutant oocytes. Science 265,2093 -2096.[Medline]
Theurkauf, W. E., Baum, H., Bo, J. and Wensink, P. C.
(1986). Tissue-specific and constitutive -tubulin genes of
Drosophila melanogaster code for structurally distinct proteins.
Proc. Natl. Acad. Sci. USA
83,8477
-8481.[Abstract]
Theurkauf, W. E., Alberts, B. M., Jan, Y. N. and Jongens, T.
A. (1993). A central role for microtubules in the
differentiation of Drosophila oocytes.
Development 118,1169
-1180.
Warrick, J. M., Paulson, H. L., Gray-Board, G. L., Bui, Q. T., Fischbeck, K. H., Pittman, R. N. and Bonini, N. M. (1998). Expanded polyglutamine protein forms nuclear inclusions and causes neural degeneration in Drosophila. Cell 93,939 -949.[Medline]
Watts, N. R., Sackett, D. L., Ward, R. D., Miller, M. W.,
Wingfield, P. T., Stahl, S. S. and Steven, A. C. (2000).
HIV-1 Rev depolymerizes microtubules to form stable bilayered rings.
J. Cell. Biol. 150,349
-360.
Wieschaus, E. and Nüsslein-Volhard, C. (1986). Looking at embryos. In Drosophila: A Pratical Approach (ed. D. B. Roberts), pp. 199-227, IRL press, Oxford, UK.