A novel F-box protein is required for caspase activation during cellular remodeling in Drosophila

Abstract

Terminal differentiation of male germ cells in Drosophila and mammals requires extensive cytoarchitectural remodeling, the elimination of many organelles, and a large reduction in cell volume. The associated process, termed spermatid individualization, is facilitated by the apoptotic machinery, including caspases, but does not result in cell death. From a screen for genes defective in caspase activation in this system, we isolated a novel F-box protein, which we termed Nutcracker, that is strictly required for caspase activation and sperm differentiation. Nutcracker interacts through its F-box domain with members of a Cullin-1-based ubiquitin ligase complex (SCF): Cullin-1 and SkpA. This ubiquitin ligase does not regulate the stability of the caspase inhibitors DIAP1 and DIAP2, but physically binds Bruce, a BIR-containing giant protein involved in apoptosis regulation. Furthermore, nutcracker mutants disrupt proteasome activity without affecting their distribution. These findings define a new SCF complex required for caspase activation during sperm differentiation and highlight the role of regulated proteolysis during this process.

Fig. 1.

ms771 is a male-sterile mutant defective in caspase activity during sperm differentiation. (A) Diagram (left) and immunostaining (right) of spermatid individualization in Drosophila. At the final stage of differentiation, an actin-based individualization complex (IC) is formed around the elongated nuclei of 64 spermatids that are connected by cytoplasmic bridges. As the IC moves down the length of the spermatids, it expels the excess cytoplasm and unneeded organelles, leaving each spermatid engulfed in its own membrane. During this movement, the excess material accumulates around the IC to create the cystic bulge (CB). When the complex reaches the end of the tails, the CB turns into the waste bag (WB), which eventually degrades. In the right-hand panel, confocal images of several wild-type (yw) cysts illustrate the individualization (ind.) process. The cysts were stained with DAPI (nuclei, blue), phalloidin (IC, red) and for cleaved Caspase-3 (cytoplasm, green). (B) In contrast to cysts from yw testes, cysts from ms771 homozygotes do not stain for cleaved Caspase-3. The nuclei (DAPI stained) in the mutant elongate, but the IC does not form. Like wild-type cysts, ms771 cysts stain positively with AXO 49 antibody, a late differentiation marker, indicating that these deficiencies are not caused by a global differentiation defect. (C) Electron micrographs of cysts during individualization. Each spermatid within the cyst contains an axoneme (red arrow) and two mitochondrial derivatives (smaller and larger round structures, orange arrows). The ms771 mutant displays normal formation of these structures, but the space between the spermatids indicates that the overall cyst structure preceding individualization is defective (black arrows).

Fig. 2.

ms771 maps to nutcracker (CG10855), which encodes an uncharacterized F-box protein expressed in testes. (A) nutcracker genomic region. nutcracker is located at position 63F1, within the region removed by the deficiency Df(3L)Exel6097. Two piggyback insertions (inverted triangles) are annotated for nutcracker: PBac{WH}CG10855^f03797 is inserted upstream of the ATG start site, and PBac{WH}CG10855⁰⁷²⁵⁹ (nutcracker⁰⁷²⁵⁹) is inserted in the intronic region. An orange star indicates the location of the premature stop codon mutation in nutcracker^ms771 that results in the truncation of the F-box domain. (B) The ms771 mutation maps to nutcracker. The deficiency Df(3L)Exel6097 fails to complement ms771 sterility and staining, and both these defects are rescued by reintroducing nutcracker ORF. nutcracker⁰⁷²⁵⁹ also has individualization defects and is hypomorphic for Caspase-3 staining. The staining is slightly reduced, but not eliminated, in the transheterozygote nutcracker^ms771/07259. (C) The F-box protein Nutcracker shares sequence homology with the F-box-only protein FBXO7. Alignment of the Drosophila Nutcracker protein against human FBXO7 (accession number CAG30377). The proteins exhibit highest similarity in their F-box domains. Overall, they share 17 identical amino acids (5% of Nutcracker, 2% of FBXO7), and 36 similar amino acids (11% of Nutcracker, 7% of FBXO7). Not shown are the last ~100 amino acids of FBXO7, as it is a longer protein.

Fig. 3.

ms771 harbors a truncated Nutcracker protein. (A) Western blot of testes lysates using the Nutcracker antibody detects a band of the predicted size (~35 kDa). Analysis of testes lysates taken from the mutant flies indicate that whereas nutcracker^ms771 (ms771) harbors a truncated form of Nutcracker protein, the nutcracker⁰⁷²⁵⁹ (07259) mutant is a protein null. The asterisk indicates a non-specific band that serves as a loading control. (B) RT-PCR analysis of nutcracker mRNA transcripts collected from wild-type (yw) or nutcracker⁰⁷²⁵⁹ (ntc⁰⁷²⁵⁹) adult testes. Compared with the transcript of predicted size found in wild-type testis, the nutcracker⁰⁷²⁵⁹ transcript contains a ~600 nt insertion. Sequencing analysis of the corresponding band revealed that this insertion is located in the middle of the transcript, resulting in a frame shift in the ORF. (C) nutcracker is expressed in the germ line. Semi-quantitative RT-PCR of nutcracker mRNA using total mRNA taken from either wild-type or son-of-oskar males. More transcripts were detected in the wild type, indicating that it is preferentially expressed in testes.

Fig. 4.

Nutcracker and Cullin-1 form an SCF complex and colocalize with actin at the individualization complex. (A) The F-box protein Nutcracker co-immunoprecipitates (co-IPs) with members of the SCF complex. Protein A (PrA)-tagged Nutcracker (PrA-ntc) was expressed in testes and its endogenous interacting partners were identified by western blot. Co-IP followed by western blot analysis using either Cullin-1 or SkpA antibodies demonstrates that Nutcracker binds both Cullin-1 and SkpA, which are both part of the SCF ubiquitin ligase (the middle lane of the co-IP). The interaction is hindered by the removal of the F-box domain (PrA-ntcΔF), suggesting that Nutcracker is part of this complex (right-hand lane of the co-IP). The input panel indicates that equal amounts of either SkpA or Cullin-1 were present in the lysate at the beginning of the experiment. (B) Nutcracker antibody staining (green). As the IC forms around the elongated nuclei, Nutcracker protein accumulates around it (upper left). After the IC begin to move, Nutcracker localizes around the bulge, intertwined within the complex, as can be seen in the two planes shown (lower left). The staining completely disappears in nutcracker⁰⁷²⁵⁹, which occasionally displays formed (yet defective) ICs (right-hand panels). The ICs are stained with phalloidin (red), and the nuclei are stained with DAPI (blue). (C) Cullin-1 staining (green) of yw testes showing similar staining pattern to Nutcracker (arrows point to the staining around the nuclei in the left panel, and to the staining around the IC in the right panel). (D) Nutcracker misexpression affects IC integrity. DJ-PrA-ntc was expressed in the nutcracker^ms771 background. The DJ promoter drives high levels of expression at later stages of differentiation, so Nutcracker is overexpressed and mistimed. This misexpression does not rescue sterility, but does restore some cleaved Caspase-3 staining (not shown). The IC, which does not form in nutcracker^ms771, does form in this background (phalloidin, red), but its formation is altered and the actin cones are scattered (white arrows).

Fig. 5.

Nutcracker physically interacts with Bruce, a giant IAP-like protein. (A) Western blots of total protein lysates from yw or nutcracker^ms771 testes using either DIAP1 or DIAP2 antibodies indicate that Nutcracker does not affect the levels of these direct caspase inhibitors. (B) The F-box protein Nutcracker physically binds Bruce. PrA, PrA-ntc or PrA-ntcΔF were expressed in S2 cells together with the Bruce ‘mini-gene’ construct. Western blot analysis of this co-IP experiment revealed that Bruce binds both full-length and truncated Nutcracker, suggesting that Bruce is an interacting protein but that its interaction with Nutcracker is independent of the SCF complex. (C) Genetic interaction between Bruce and nutcracker. Caspase staining (green) of the hypomorphic allele nutcracker⁰⁷²⁵⁹ is unchanged in double mutants with Bruce^8-1e or Bruce^10-1e.

Fig. 6.

Nutcracker affects proteasome activity but not distribution or numbers. (A) Proteasome distribution during individualization. GFP-tagged Alpha6T is a marker for proteasome localization (Zhong and Belote, 2007). Alpha6T is detected in elongated nuclei before the IC forms (top left), and after IC formation moves to the base of it (bottom left). Testes of nutcracker^ms771 display no Alpha6T outside the nuclei (top right). By contrast, in the nutcracker⁰⁷²⁵⁹ hypomorphic allele, in which occasional ICs are formed, Alpha6T is detected at its normal location ahead of the IC (bottom right). (B) Testes from nutcracker^ms771 display decreased proteasome activity. Total protein was extracted from the indicated genotype and proteasome activity detected by measuring fluorogenic peptide hydrolysis after 10 minutes. Measurements are plotted as relative luminescence units (RLU). Each genotype sample was split and a proteasome inhibitor (MG132) was added to half the sample to confirm the specificity of the read-out. Wild type, yw; nutcracker^ms771, ms771; nutcracker^ms771;Hsp83-nutcracker, ms771-rescue. (C) The levels of the proteasome subunit Alpha7 are unchanged in the nutcracker^ms771 mutant. Total protein was extracted from either wild-type (yw) or nutcracker^ms771 (ms771) testes and the amounts of the indicated proteins were determined by western blot analysis. Amounts of other SCF members (Cullin-1 and SkpA) were also assayed and used as loading controls.

Fig. 7.

A model for caspase activation by the Drosophila Nutcracker SCF complex. Our model suggests that Nutcracker forms a complex with members of an SCF ubiquitin ligase complex. This complex can then regulate caspase activation, possibly by modifying proteins that activate the proteasome; these could include, for example, components of the proteasome regulatory particle or other associated proteins. Caspase activation could then be facilitated by ubiquitin-mediated regulation.