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First published online September 18, 2007
doi: 10.1242/10.1242/jcs.004762

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Structurally related TPR subunits contribute differently to the function of the anaphase-promoting complex in Drosophila melanogaster

Institute of Biochemistry, Biological Research Center, H-6726 Szeged, Hungary

View larger version (36K): [in this window] [in a new window]   Fig. 1. Distribution and sequence comparisons of TPR motifs in putative Apc7 orthologues. (A) Distribution of TPRs in the human Apc7 subunit (Hs), the Drosophila CG14444 gene product (Dm), and predicted proteins from Arabidopsis thaliana (At; accession no. NP 850309) and Apis mellifera (Am; accession no. XP 396165). The asymmetric rectangular symbols represent single TPR motifs. (B) Sequence alignment of seven TPR motifs of the Drosophila Apc7 protein. The TPR consensus sequence is shown below the alignment. Residues matching the consensus are in bold type and highlighted in grey (also in C). Residues are conserved only at the consensus positions but, even there, there is no invariant position. (C) Sequence comparison of the TPR10 motif from the proteins shown at the top of this figure. Conserved residues outside the consensus are highlighted in yellow. Residue conservation is more extensive both within and outside of the consensus.

View larger version (12K): [in this window] [in a new window]   Fig. 2. Gene silencing by RNA interference. (A) Scheme for transgenic RNA interference used in this work to knock down the expression of the TPR subunits of Drosophila APC/C. Inverted exon repeats of these genes (white bars) were cloned into pWIZ transformation vector under the control of the UAS promoter (white circles) and transgenic lines were made. These were crossed to the Act5C-GAL4 driver lines that constitutively expressed the GAL4 transcription activator in all cells throughout development. Progeny that contained both Act5C-GAL4 and UAS transgenic constructs expressed double-stranded hairpin RNAs that triggered gene silencing. (B) Monitoring TPR transcript levels in RNAi induced larvae (I) relative to uninduced controls (C). The rpL17A transcript was used as a calibration control. TPR gene-specific transcript levels appeared significantly lower in all RNAi induced samples.

View larger version (137K): [in this window] [in a new window]   Fig. 3. Silencing of the Apc6/Cdc16, Apc7 and Apc8/Cdc23 genes by RNAi results in mitotic abnormalities. Wild-type mitotic cells in prometaphase (A), metaphase (B) and anaphase (C). Neuroblast cells from both Apc6/Cdc16 (E-G) and Apc8/Cdc23 (I-L) knocked down larvae show metaphase-like arrest with overcondensed chromosomes. The chromosomes in most of the arrested cells appear scattered (E,G,I,K), in about 10% of mitotic cells the chromosomes are locked at the metaphase plate (F,J). Some degree of chromosome overcondensation could also be observed in anaphase figures (L) together with irregular chromosome segregation. Some cells appear polyploid (G,K), and they invariantly show chromosome overcondensation. Overcondensed chromosomes sometimes appear to be in the process of decondensation (G). Induction of Apc7-specific RNAi causes a mild mitotic phenotype. Neuroblast cells from Apc7RNAi larvae show signs of uneven chromosome condensation and telomeric fusions (D) and occasionally chromosome bridges in anaphase (H).

View larger version (105K): [in this window] [in a new window]   Fig. 4. Cyclin A is degraded in RNAi silenced Apc6/Cdc16 and Apc8/Cdc23 cells. Columns of images show tubulin (green and second column), DNA (blue and third column) and cyclin A (red and fourth column) localisations in mitotic cells. Cyclin A staining is visible in wild-type prophase or prometaphase (A-D) but it is undetectable in most of the wild-type metaphase (E-H) cells (n=12). Similarly to this, in the majority (80%) of both Apc6/Cdc16 (I-L) and Apc8/Cdc23 (M-P) RNAi induced cells (n=19 and 26, respectively), there is no detectable cyclin A at the onset of metaphase, indicating that cyclin A degradation is not affected in these lines.

View larger version (113K): [in this window] [in a new window]   Fig. 5. Cyclin B is not degraded in RNAi silenced Apc6/Cdc16 and Apc8/Cdc23 cells. Colours are the same as on Fig. 4. In wild-type cells (n=10) the cyclin B level is high in prophase and prometaphase (A-D) and it starts to diminish at or after the onset of metaphase (E-H). Cyclin B staining is quite pronounced in more than 80% of RNAi silenced Apc6/Cdc16 (I-L) and Apc8/Cdc23 (M-P) cells (n=20 and 17, respectively) arrested in metaphase indicating that cyclin B is not degraded.

View larger version (51K): [in this window] [in a new window]   Fig. 6. FLAG affinity chromatography of FLAG-tagged Apc7 and Apc8 complexes. (A) Western blots of total protein extracts from S2 cells transfected with HA-tagged Apc8 (H8, lane 1) or FLAG-tagged Apc7 (F7, lane 2) plasmids. Strip 1 was reacted with anti-HA (H, lane 1), and strip 2 with anti-FLAG (F, lane 2) monoclonal antibodies. (B) Western blots of proteins affinity-purified on anti-FLAG-M2 antibody beads from S2 cells co-transfected with FLAG-Apc7 and HA-Apc8 (F7-H8, lanes 3 and 4), FLAG-Apc8 and HA-Apc7 (F8-H7, lanes 5 and 6) or FLAG-Apc7 and HA-Apc3 (F7-H3, lanes 7 and 8) plasmids. Blots were reacted with anti-FLAG (lanes 3, 5 and 7) or anti-HA (lanes 4, 6 and 8) monoclonal antibodies. The immunoreactive bands labelled with an asterisk are proteolytic degradation products of Apc8. (C) Blots of anti-FLAG-M2 column-bound (lane 10) and unbound (flow-through, lane 9) proteins from S2 cells transfected with HA-Apc8 (H8) plasmid and incubated with anti-HA monoclonal antibody. Even after heavily overloading the anti-FLAG column, no nonspecific binding of HA-Apc8 could be detected. Similarly, HA-Apc3 did not bind to the anti-FLAG-M2 column (data not shown) E, eluate; FT, flow-through.

View larger version (57K): [in this window] [in a new window]   Fig. 7. Loss of both Apc6/Cdc16 and Apc8/Cdc23 functions induces apoptosis. Orcein stained preparations from brains of Apc6/Cdc16 (A) and Apc8/Cdc23 (B) RNAi L3 larvae show small, rounded cells usually in pairs with intense but uneven nucleolar staining resembling apoptotic cells. Acridine Orange staining further indicates the high incidence of dying cells in brains of RNAi larvae (D, only an Apc8/Cdc23 RNAi sample is shown) relative to wild-type larvae (C). (E) Bar graph showing the apoptotic index in neuroblast preparations of the respective genotypes. The apoptotic index is given as the mean number of apoptotic cells in a microscope field.