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First published online 10 January 2007
doi: 10.1242/dev.02766

Development 134, 703-712 (2007)
Published by The Company of Biologists 2007
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The Ovhts polyprotein is cleaved to produce fusome and ring canal proteins required for Drosophila oogenesis

Lisa N. Petrella1, Tracy Smith-Leiker1 and Lynn Cooley1,2,3,*

1 Departments of Genetics, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA.
2 Departments of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA.
3 Department of Molecular, Cellular and Developmental Biology, Yale University, 260 Prospect Avenue, New Haven, CT 06511, USA.


Figure 1
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  		Fig. 1. Wild-type and mutant Hts protein domains and expression. (A,B) For each wild-type and mutant protein, the Flybase designation, Whittaker mRNA designation (Whittaker et al., 1999Go), predicted size, RNA expression and protein localization are given. (A) All wild-type proteins contain the Head (H) and Neck (N) domains of Adducin (homology shown in red). ShAdd contains a truncated Tail (T) domain and then 23 novel amino acids at its C-terminus (purple). Ovhts, Add1 and Add2 share a common Tail. Alternative splicing at the codon for amino acid 658 (circled) results in three transcripts with different C-terminal domains. Ovhts has a novel domain designated the RC domain (blue). Add1 and Add2 maintain homology to mammalian Add, with Add2 splicing into an exon encoding 23 amino acids and both encoding the C-terminal MARCKS domain. The regions used as antigens for antibody production are underlined. (B) Two truncation mutants lack the wild-type C-terminal domains. hts{Delta}G has a single base pair deletion of G2346 resulting in a frameshift, six novel amino acids and a premature stop codon. In htsW532X Trp532 of both Ovhts and Add1/2 is mutated to a stop codon in the Tail domain.

 

Figure 2
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  		Fig. 2. hts antibodies and tagged proteins show differential localization of Hts proteins. (A-D) Germaria and (E-H) stage 9 egg chambers labeled with the designated antibody. (A) Regions of the germaria are underlined (King, 1970Go). Region 1 (R1), all dividing cells; Region 2, 16-cell cysts not surrounded by follicle cells (R2a, cysts are spherical; R2b cysts are lens shaped and span the width of the germarium); Region 3 (R3), cysts surrounded by follicle cells. (A,B) htsF and 1B1 antibodies label fusomes in the germline (arrow) and follicle cell membranes (arrowhead). (C) htsM antibody labels only follicle cell membranes. (D) htsRC antibody labels puncta and then RCs starting in Region 2a. (E-G) htsF, 1B1 and htsM antibodies label follicle cell membranes exclusively; there is no localization in the germline. (H) htsRC antibody labels only RCs. (I-K) Transgene expression in germaria co-stained with 1B1 (red). (I) ShAdd::Ven localizes to the fusome until Region 3 and then is present at a very low level in the cytoplasm. (J) Cer::Ovhts localizes to the fusome in the germaria based on staining with anti-GFP antibodies. (K) Ovhts::GFP starts as puncta in Region 2 and then localizes to RCs throughout oogenesis. (L,L') Higher magnification of a germaria expressing Ovhts::GFP. Puncta appear in Region 2a (arrowhead) near the fusome and start to become RCs (arrow) in Region 2b. Scale bars: A-D,I-L,10 µm; E-H, 20 µm.

 

Figure 3
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  		Fig. 3. Western immunoblot analysis of Hts proteins in ovary and S2 cell extracts. (A,B) Ovary extracts from virgin w1118 flies blotted with htsF and htsRC. (A) Both the Add1/2 doublet band at 90 kDa and the 80 kDa Ovhts-Fus band can be seen. (B) Only the Ovhts-RC doublet is detected. (C) Ovary extract from flies expressing tagged hts transgenes blotted with anti-GFP antibody. A 75 kDa nonspecific band is present in all lanes. The ShAdd::Ven band is of the predicted size. The bands in Cer::Ovhts-Fus (~110 kDa, *) and Ovhts-RC::GFP (~90 kDa, **) are of the sizes predicted for the respective cleavage products fused to the fluorescent proteins. (D,E) Protein extracts from S2 cells expressing Ovhts blotted with 1B1 and htsRC. (D) S2 cells express endogenous Add1/2 proteins that can be seen at 90 kDa. Bands representing both full-length Ovhts protein and Ovhts-Fus are indicated. (E) Bands representing both full-length Ovhts protein and Ovhts-RC are indicated. All Hts proteins consistently ran 10-20 kDa larger than predicted.

 

Figure 4
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  		Fig. 4. Expression of transgenes can rescue RCs but not the fusome. (A-F) Germaria of hts{Delta}G mutants expressing the designated hts transgenes. (A,B) Expression of Cer::Ovhts results in no fusome rescue by either anti-GFP antibody (A, green) or htsF (B, red) staining. However, there is rescue of Ovhts-RC localization to the RC shown by staining with htsRC antibodies (B, green). (C) Expression of Ovhts::GFP results in no fusome rescue (1B1 staining, red) but does rescue Ovhts-RC::GFP to RCs (green). (D) Expression of ShAdd::Ven results in no fusome rescue (1B1 staining, red). ShAdd::Ven (green) is cytoplasmic. (E,F) Expression of ShAdd::Ven (green) with either Cer::Ovhts (E) or Ovhts::GFP (F, green) does not rescue the fusome (E and F, green) or fusome staining with 1B1 (F, red), but does rescue RCs as seen with either transgenic GFP (F, green) or staining with htsRC antibody (E, red). Scale bars: 10 µm.

 

Figure 5
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  		Fig. 5. Localization of Ovhts::GFP to the RC is delayed in hts mutants. (A-C) Expression of Ovhts::GFP in wild-type, htsW532X and hts{Delta}G ovaries. (A) In the control background, Ovhts::GFP localizes to the RCs in Region 2a. (B,C) In htsW532X and hts{Delta}G, Ovhts::GFP localization is delayed until Region 3. (D) Quantitation of the localization timing of Ovhts::GFP at either Region 2a (black bars) or at a later stage (gray bars). There is a 30% and 91% delay in htsW532X and hts{Delta}G, respectively. *Significant {chi}2, P<0.001. Scale bars: 10 µm.

 

Figure 6
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  		Fig. 6. Expression of an uncleavable form of Ovhts disrupts the transition from fusome to RC. (A) Schematic of the location of the four 20-amino acid deletions. (B) Western blot of deletion constructs {Delta}1 through {Delta}4 expressed in S2 cells, probed with htsRC antibody. Ovhts-{Delta}3 protein is not cleaved. (C) Ovary extracts from flies expressing either Ovhts::GFP or Ovhts-{Delta}3::GFP blotted with htsRC antibody. Whereas only a cleavage product is present in Ovhts::GFP extract (open arrow), Ovhts-{Delta}3::GFP is only present in a full-length band (arrowhead). (D-D") Germaria from flies expressing Ovhts::GFP show a GFP signal on RCs but not on fusome (arrow D'), and 1B1 localization only to fusome and not to RCs (arrow D"). (E-E") Flies expressing Ovhts-{Delta}3::GFP show localization of GFP (E') and 1B1 (E") to both the fusome and RCs (arrows). (F,G) Stage 2 egg chambers expressing either Ovhts::GFP or Ovhts-{Delta}3::GFP stained with 1B1 antibody (red). (F) RCs show localization of Ovhts::GFP in tight clear rings that do not have 1B1 staining. (G) RCs show a colocalization of 1B1 staining and Ovhts-{Delta}3::GFP fluorescence on RCs (yellow). Additionally, RCs are malformed. (G',G") Magnification of boxed region in G shows that rings are not round, appear thicker and less organized, and can be fully occluded with fusome material that does (arrow) or does not (arrowhead) include Ovhts-{Delta}3::GFP. Scale bars: C,D, 20 µm; F,G, 10 µm; G',G", 5 µm.

 

Figure 7
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  		Fig. 7. Continuous Ovhts expression is necessary for Ovhts-RC and actin localization to RCs. (A) Expression of P{UASH-Ovhts::GFP} with MTD-Gal4 results in expression of Ovhts::GFP during all stages of oogenesis and continuous localization to RCs. (B) Expression of P{UASH-Ovhts-GFP} with the P{bam-GAL4} driver in wild-type ovaries shows expression and localization of Ovhts::GFP to RCs in the germaria and faintly in stage 2 (arrowhead). There is no Ovhts::GFP protein on RCs at later stages that are actinpositive (arrow). (C-D) Expression of P{UASH-Ovhts::GFP} in hts{Delta}G with P{nos-Gal4}. The P{nos-Gal4} driver produces Ovhts::GFP expression (green) in early and late stages, but no expression in intermediate stages. Staining with Rhodamine-conjugated phalloidin shows that F-actin localization to RCs mirrors Ovhts::GFP expression. (D) Higher magnification of an intermediate stage egg chamber that is not expressing Ovhts::GFP. F-actin is in clumps that may be degenerating RCs. Scale bars: A,B, 20 µm; C,D, 10 µm.

 

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© The Company of Biologists Ltd 2007