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A Drosophila model of HIV-Tat-related pathogenicity

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 )

View larger version (55K): [in a new window]   Fig. 1. Immunoblot of anti-Tat anti-serum with proteins separated by SDS-PAGE. Lane 1, protein extracts from Tat-transgenic embryos following heat-shock treatment. Lane 2, protein extracts from Tat-transgenic embryos without heat-shock treatment. Lane 3, protein extracts from non-transgenic embryos. Molecular weights of marker are indicated by arrows.

View larger version (43K): [in a new window]   Fig. 2. Drosophila embryos showing only one fused dorsal appendage following Tat expression (A); wild-type Drosophila embryo (B).

View larger version (61K): [in a new window]   Fig. 3. In vivo interaction between Tat and microtubules.(A-C) Drosophila spermatocytes at late S phase double-labelled with anti-Tat (A) and with anti--tubulin (B); merged image (C) reveals that Tat and -tubulin colocalize. (D-F) Drosophila spermatocytes at anaphase double-labelled with anti-Tat (D) and with anti--tubulin (E); merged image (F) reveals the colocalization of Tat with both -tubulin and centrosome (arrowhead).

View larger version (20K): [in a new window]   Fig. 4. In vitro effect of Tat on tubulin polymerization process. Upper curves, tubulin polymerization rate in presence of GTP at 37°C (red curve) and in presence of aprotinin and GTP at 37°C (green curve). Lower curve, tubulin polymerization rate in presence of Tat and GTP at 37°C.

View larger version (34K): [in a new window]   Fig. 5. Tat co-immunoprecipitation with -tubulin. Protein extracts immunoprecipitated with anti--tubulin and immunoblotted with anti--tubulin (top) and with anti-Tat (bottom) antibodies. Lane 1, protein extracts from Tat-transgenic embryos following heat-shock treatment. Lane 2, protein extracts from Tat-transgenic embryos without heat-shock treatment. Lane 3, protein extracts from non-transgenic embryos.

View larger version (54K): [in a new window]   Fig. 6. Confocal images of microtubule-dependent streaming in Drosophila oocyte before (A) and after (B) Tat expression.

View larger version (26K): [in a new window]   Fig. 7. Distribution pattern of Grk protein in egg chambers from Tat transgenic females. Confocal images of indirect immunofluorescent staining of egg chambers with anti-Grk antibody using a Cy3-conjugated anti-rat secondary antibody. The staining of the Grk protein is in green. F-actin is visualized with rhodamine-conjugated phalloidin in red; regions where labels overlap are yellow. All egg chambers are at stage 10 of oogenesis. (A) Egg chamber from a female not expressing Tat. The Grk protein is normally localized to the anterior-dorsal cortex of oocyte. (B,C) Egg chambers from females after Tat induction. Grk is localized around the entire circumference (B) or at the anterior margin (C).

View larger version (21K): [in a new window]   Fig. 8. Kinesin:ß-gal localization in oocytes after heat shock treatment from both wild-type and Tat transgenic females. Both panels show X-gal staining of stage 10 egg chambers. (A) Egg chamber from a wild-type female not expressing Tat subjected to heat-shock. Kin:ß-gal is normally localized at the posterior. (B) Egg chamber from a female after Tat induction. Kin:ß-gal is abnormally localized in the middle of the oocyte.