APC16 depletion prevents exit from mitosis. (A) Immunoblots of cells transfected with the indicated siRNA oligonucleotides. UTR (untranslated region) and ORF (open reading frame) refer to location of the siRNA targeting sequence within endogenous APC16 mRNA. (B) Selected frames of a live cell imaging series of HeLa-H2B^EYFP cells either mock transfected or transfected with APC16 siRNA (#11, ORF). The arrows and arrowheads each point to a cell with a metaphases of ~8 hours (arrow) and at least 10 hours (arrowhead). (C) Graph of the cumulative duration of mitotic phases in cells transfected and analyzed as in B. Each bar represent one cell. Bars indicate time from nuclear envelope breakdown (NEB) to metaphase (white), from the first frame of metaphase to either anaphase, death (black, usually preceded by loss of chromosome alignment for 1-2 hours), or the end of the time-lapse (TL) experiment (gray). Red indicates postmetaphase stages. (D) Whisker diagram of the duration of mitosis (NEB to anaphase) in HeLa cells transfected with the indicated siRNA oligonucleotides and imaged with DIC microscopy. Cells never entering anaphase were included provided they had entered mitosis at least 6 hours before the experiment was terminated. The number of cell analyses (n) is indicated. Data are of at least three independent experiments. (E) The percentage of cells (transfected and imaged as in D) in which mitosis took longer than 4 hours. Values are means ± s.d. from at least three independent experiments. (F) The percentage of HeLa cells, transfected with plasmids and siRNA oligonucleotides as indicated, in which mitosis took longer than 4 hours. Values are means ± s.d. of transfected (EYFP-positive) and untransfected (EYFP-negative) cells in the same well; data are from two independent experiments.

APC16 is required for phosphorylation of APC3/Cdc27 but not for assembly of the APC/C holocomplex. (A) Immunoblots of whole cell lysates of the experiments shown in B and C. (B,C) Immunoblots of cellular fraction of (B) mock siRNA- or (C) APC16 siRNA-transfected HeLa cells after gel filtration. Migration of markers is indicated on top and the fractions containing APC/C are indicated at the bottom. Lines in the APC16 immunoblots indicate that the samples on either side were run on different gels. (D) Diagram of APC/C structure, according to Herzog et al. (Herzog et al., 2009). Approximate location of APC4 and APC3/Cdc27 are indicated. IP, immunoprecipitation. IB, immunoblot. (E) Immunoblots (APC3/Cdc27 and APC4) of anti-APC3/Cdc27 immunoprecipitates or whole cell lysates (wcl) from mitotic lysates of HeLa cells transfected with the indicated siRNA oligonucleotides. Mock siRNA-transfected cells were collected after 12 hours of nocodazole treatment, whereas APC16 siRNA-transfected cells were collected by mitotic shake-off. (F) Immunoblots (APC3/Cdc27) of whole cell lysates from mitotic HeLa cells transfected with the indicated siRNA oligonucleotides and left untreated (shake-off) or treated with nocodazole for 12 hours (noco) or MG132 for 90 minutes (MG132 + shake-off). Mock siRNA-transfected cells were treated with MG132 to enrich for mitotic cells in metaphase. Two chemiluminescence exposures (long and short exp.) are shown.

zgc:110659 is a functional D. rerio orthologue of APC16. (A-D) Larvae were stained in whole-mount for phospho-histone H3 (pH3, red) at the 18-somite stage. Lateral views of embryos show that many cells are pH3-positive in the head region of embryos in which APC11 (B) or APC16 (C) has been knocked down compared with (A) control larvae. Inset in C shows high power view of mitotically arrested cell in APC16 knocked down embryo. (D) APC16 knockdown is rescued by mRNA of human APC16 fused to EYFP. Scale bar: 200 μm. (E,F) A significant increase in (E) pH3-positive (normalized to DAPI-positive cells) and (F) apoptotic cells [per high power field (HPF)] was found in both APC11 and APC16 knocked down embryos and was rescued with human APC16^EYFP mRNA.

Tandem-affinity purifications of MCC-APC/C identifies the APC/C interactor C10orf104 (APC16). (A,B) Expression levels of TAP-tagged Mad2 (A) and LAP-tagged BubR1 (B) relative to the endogenous proteins. (C,D) SDS-PAGE silver staining of tandem-affinity purifications of Mad2 (C) and BubR1 (D) from mitotic HeLa cells. Molecular mass markers as well as some of the interactors are indicated. (E) Primary amino acid sequence of APC16. Blue indicates sequences covered in the various mass spectrometry analyses. Red asterisks indicates phosphorylated residues. (F) Immunoblots (APC4, APC16, BubR1 or APC3/Cdc27) of the various immunoprecipitates (IP) and of whole-cell lysates (wcl) from mitotic (nocodazole, N) or interphase (thymidine, T) HeLa cells. (G) Anti-GFP immunoprecipitates from APC16^EYFP-expressing HeLa cells incubated with a recombinant securin substrate resulted in a HA-ubiquitin banding pattern consistent with ubiquitylation. Anti-APC3 immunoprecipitates also contained ubiquitylation activity whereas control IgG precipitates did not. The immunoprecipitates with ubiquitylation activity also contained Cdc20 and APC6/Cdc16.

Stabilization of APC/C substrates following depletion of APC16. (A,B) Immunoblots [securin and tubulin (A) or cyclin A and tubulin (B)] of lysates from HeLa cells transfected with the indicated siRNA oligonucleotides. In A, all cells were collected after 12 hours of nocodazole treatment or after an additional 2 hours following nocodazole washout. In B, mitotic APC/C siRNA cells were collected by shake-off prior to nocodazole addition, then treated with nocodazole for 12 hours followed by either nocodazole washout or treatment with the cdk1-inhibitor RO-3306 for an additional 2 hours. (C,D) Graph of relative fluorescence intensity of mCherry-D-Box (cyclin B^1-90) in individual HeLa-mCherry-cyclinB^1-90 cells transfected with the indicated siRNA oligonucleotides. In APC/C siRNA samples, only cells that were delayed in mitosis for at least 4 hours were analyzed for stability of mCherry-D-Box. Plots are representative of at least two independent experiments, at least five cells per experiment. Immunoblot in D shows APC8 and tubulin protein levels in the imaged cells.

K10D2.4 is a functional C. elegans orthologue of APC16. (A) Diagram of APC16-like protein sequences of the indicated organisms and the relative location of AH4 domain. (B) Sequence alignment of a part of the AH4 domain of the candidate APC16-like proteins. The C. elegans sequence is that of K10D2.4. GenBank entries for the corresponding nucleotide designated sequences are: NM_173473.2 (H. sapiens); BC025117.1 (M. musculus); NM_001017878.1 (D. rerio); NM_065697.4 (C. elegans); NM_139559.2 (D. melanogaster); NM_117569.2 (A. thaliana). (C) K10D2.4 knockdown results in a metaphase-arrest phenotype. C. elegans gonad showing embryos arrested at the one-cell stage (outlined) filling the uterus after K10D2.4 and mat-2 RNAi. Arrows point to recently fertilized eggs with a metaphase spindle. Asterisks indicate spindles that have been in a prolonged metaphase arrest. Scale bars: 10 μm. Green, GFP–α-tubulin; red, mCherry–H2B. (D) Frames from time-lapse movies of meiotic spindles of a wild-type and K10D2.4 embryo. Exit from the spermatheca was taken as t=0. The cortex is to the left, background fluorescence was removed with the ImageJ ‘subtract background’ command. Scale bars: 2 μm. (E) Quantification of anaphase onset in wild-type and K10D2.4 RNAi embryos.