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Specific domains drive VM32E protein distribution and integration in Drosophila eggshell layers

Davide Andrenacci¹, Filippo M. Cernilogar¹, Carlo Taddei¹, Deborah Rotoli^2,*, Valeria Cavaliere¹, Franco Graziani² and Giuseppe Gargiulo^1,

¹ Dipartimento di Biologia Evoluzionistica Sperimentale, Via Selmi 3, 40126 Bologna, Italy
² Istituto Internazionale di Genetica e Biofisica; Via Marconi 10, 80125 Napoli, Italy
^* Present address: Centro di Oncologia Sperimentale, CNR, Via Pansini 2 Napoli, Italy
Author for correspondence (e-mail: gargiulo_g{at}biblio.cib.unibo.it )

View larger version (7K): [in a new window]   Fig. 1. Structure of the primer used to construct chimeric VM32E-MYC genes, showing the MYC epitope and the SnaBI restriction site.

View larger version (39K): [in a new window]   Fig. 4. Western blot analysis of VM32E and VM26A.2 vitelline membrane proteins. Staged egg chambers (stages 9-14) and ovaries (Ov) were processed as described in Materials and Methods. The pellet phase (P) contains the proteins integrated into the eggshell whereas the supernatant phase (S) contains those not yet incorporated into the eggshell. The VM32E protein was detected using the anti-CVM32E antibody, whereas the VM26A.2 protein was detected by anti-VMP antibody. (A) By the end of oogenesis (stages 13-14), a significant reduction of the VM32E signal is detected compared with the earlier stages (10-12). (B) At stage 10, the VM26A.2 protein is detected mainly in the pellet phase. At stage 14, this protein is still fully releasable from the pellet phase. (C) Stage 14 egg chambers and ovaries were extracted in presence of 2% SDS.

View larger version (23K): [in a new window]   Fig. 9. Western blot analysis of various truncated VM32E-MYC chimeric proteins. (A) Staged egg chambers treated as described in Materials and Methods. The VM32E-MYC and 1 constructs show the same protein extraction pattern as the wild-type VM32E protein. The 2 protein shows a strong signal in the S phase of stage 10B, indicating that a significant proportion of its molecules are not yet integrated into the membrane. A remarkable feature of 3 protein is the high amount extracted from the pellet of stage 14. (B) Stage 14 egg chambers processed in presence of 2% SDS. The 2 protein is fully solubilized by SDS, whereas the 3 protein appears cross-linked by disulfide bridges. (C) Western blot analysis of 2 and 3 proteins in laid eggs. As with the wild-type VM32E protein, the 3 protein appears tightly integrated in the vitelline membrane. The released amount of 2 protein is the same from whole (+Ch.) or dechorionated (-Ch.) eggs, suggesting tight cross-linking only in the chorion layer. (D) Structure of the three VM32E deletions analyzed here.

View larger version (30K): [in a new window]   Fig. 5. Analysis of the VM32E protein in fs(2)QJ42 egg chambers. (A) Stage 10B egg chamber stained with anti-CVM32E antibody and examined laterally by confocal microscopy. The sagittal section shows the antibody staining in the anterior (to the left) and posterior domains. (B) Western blot analysis of VM32E protein in wild-type and fs(2)QJ42 ovaries extracted in the absence of SDS (the first two lanes from the left) and with increasing amounts of this detergent. The pellet (P) and supernatant (S) phases were separated and analyzed by western blot using the anti-CVM32E antibody. In the fs(2)QJ42 mutant, the VM32E protein appears to be quickly solubilized by SDS at low concentration.

View larger version (41K): [in a new window]   Fig. 2. VM32E gene expression and VM32E protein distribution. (A,B) Whole mount in situ hybridization showing the spatial distribution of the VM32E transcript. (A) At stage 10A, the expression is evident in a group of ventral columnar follicle cells. (B) At stage 10B, all main body columnar follicle cells express the gene; most anterior (arrows) and posterior (arrowhead) follicle cells are silent. (C-H) Whole-mount egg chambers stained with anti-CVM32E antibody and examined laterally by confocal microscopy. The white arrows indicate the posterior polar domain. (C) Surface view of a stage 10B egg chamber in which the VM32E protein appears within the main body follicle cells, except the most posterior ones. (D) Section of the same egg chamber showing the VM32E protein in the extracellular space between the follicle cells and the oocyte, and the absence of this protein from the posterior domain. (E) Sagittal section of a stage 10B egg chamber at a lower magnification. (F) Sagittal section of a stage 11 egg chamber showing, at a lower magnification, the presence of the VM32E protein in the polar domains. (G) The posterior polar domain of a stage 10B egg chamber at a higher magnification. (H) The posterior polar domain of a stage 11 egg chamber at a higher magnification. (I,L) Whole-mount egg chambers stained with anti-VMP antibody and examined laterally by confocal microscopy. (I) Surface view of stage 10B egg chamber in which the VM26A.2 protein appears within all the main body follicle cells. (L) Sagittal section of the same egg chamber showing the VM26A.2 protein in the extracellular space between the follicle cells and the oocyte.

View larger version (78K): [in a new window]   Fig. 3. Multilayered distribution of VM32E protein detected by immunoelectron microscopy using anti-CVM32E antibody. (A) At stage 10A of egg chamber development, immunogold particles are seen in the vitelline bodies and in secretory vesicles of the follicle cells (arrow). (B) At the same stage, in the posterior domain, a very low density of gold particles is visible inside the vitelline body. (C) At stage 10B, immunogold particles are clearly visible in the vitelline membrane. (D) At stage 12, the gold particles are localized in the vitelline membrane and in the forming endochorion pillars (arrow). (E) Stage 14 egg chamber showing immunogold particles on the endochorion layer and on the vitelline membrane. Abbreviations: en, endochorion; fc, follicle cell; oc, oocyte; vb, vitelline body; vm, vitelline membrane.

View larger version (23K): [in a new window]   Fig. 6. (A) Sequence of the VM32E protein deletions analyzed. (B) Alignment of the protein sequences of the conserved regions in D. melanogaster and A. aegypti vitelline membrane proteins. Residues that are conserved between the two species are indicated by bars. Highly conserved residues are indicated with asterisks. Small gaps that improve the alignment are shown as dots. References: VM32E (Gigliotti et al., 1989; Adams et al., 2000), VM34C (Mindrinos et al., 1985), VM26A.2 (Popodi et al., 1988; Adams et al., 2000), VM26A.1 (Burke et al., 1987), 15a-1 and 15a-2 (Lin et al., 1993), 15a-3 (Edwards et al., 1988).

View larger version (144K): [in a new window]   Fig. 7. Immunolocalization of the VM32E-MYC protein. (A,B) Stage 10B egg chamber stained with anti-MYC monoclonal antibody and examined by confocal microscopy. The white arrows indicate the posterior polar domain. (A) Surface view in which the VM32E-MYC protein appears within the main body follicle cells except the most posterior ones. (C) Immunoelectron microscopy of VM32E and VM32E-MYC proteins by double immunogold staining in stage 10B VM32E-MYC egg chamber. The 10-nm gold particles label the VM32E-MYC protein, whereas the 20 nm particles label both the wild-type and the VM32E-MYC proteins. The two proteins appear uniformly distributed in the vitelline membrane. The scale bar in the insert is 0.1 µm. Abbreviations: fc, follicle cell; oc, oocyte; vm, vitelline membrane. (D) In a stage 14 egg chamber, the VM32E-MYC protein is localized in both the vitelline membrane and endochorion. (E) At the same stage, the gold particles are also detected in vesicles localized in the subcortical region of the oocyte (arrows).

View larger version (81K): [in a new window]   Fig. 8. Distribution of 2 and 3 proteins. (A) Stage 10A egg chamber from a female expressing the 2 protein stained with anti-MYC monoclonal antibody and examined laterally by confocal microscopy. The sagittal section of the egg chamber clearly shows the presence of the 2 protein in the anterior and posterior domains. (B) Higher magnification of the posterior pole of the same egg chamber. (C) Electron microscopy of stage 14 egg chamber showing the presence of the 2 protein in both the vitelline membrane and endochorion layer. (D) Stage 10B egg chamber from a female expressing the 3 protein stained with anti-MYC monoclonal antibody and examined laterally by confocal microscopy. The sagittal section of the egg chamber clearly shows the proper localization of the 3 protein. (E) Electron microscopy analysis of stage 14 egg chamber showing the absence of 3 protein from the endochorion layer. Strong positive labeling is visible in the vitelline membrane. The white arrows indicate the posterior polar domain. Abbreviations: en, endochorion; oc, oocyte; vm, vitelline membrane. (F) Structures of the VM32E deletions analyzed here.