1. Volume 15, Issue 4, Pages 603-616 (October 2008)
An Afg2/Spaf-Related Cdc48-like AAA ATPase Regulates the Stability and Activity of the C. elegans Aurora B Kinase AIR-2 
Todd R. Heallen, Henry P. Adams, Tokiko Furuta, Koen J. Verbrugghe, Jill M. Schumacher 
Developmental Cell 
Volume 15, Issue 4, Pages (October 2008)
DOI: /j.devcel
Copyright © 2008 Elsevier Inc. Terms and Conditions

2. Figure 1
Loss of C. elegans CDC-48.3 Suppresses air-2(or207ts) Lethality and Mitotic Defects
(A) Viability of control and cdc-48.3(RNAi) treated air-2(or207ts) embryos reared at 15, 18, 20, 22, and 25°C.
(B) Embryos dissected from control-treated air-2(or207ts) and cdc-48.3(RNAi);air-2(or207ts) adult hermaphrodites reared at 22°C were fixed and stained with DAPI (blue), and α-tubulin (green) and AIR-2 (red) antibodies. One-cell embryos at different stages of mitosis are shown. Scale bar = 10 μm.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

3. Figure 2
AIR-2 Levels Are Increased at Mitotic Exit in cdc-48.3(RNAi) Embryos
(A) Protein extracts from wt, cdc-48.3(RNAi), air-2(RNAi), air-2(or207ts), and cdc-48.3(RNAi);air-2(or207ts) embryos reared at 22°C were subjected to western analysis with AIR-2 and α-tubulin-specific antibodies. Band intensities were measured and normalized to α-tubulin protein levels. Error bars represent standard deviation from three independent experiments.
(B) Embryos dissected from control and cdc-48.3(RNAi) treated adult hermaphrodites were fixed and stained with DAPI (blue), and α-tubulin (green) and AIR-2 (red) antibodies. One-cell embryos at each mitotic stage are shown. Arrow points to marked accumulation of AIR-2 at the midbody of a cdc-48.3(RNAi) embryo. Scale bar = 10 μm.
(C and D) The mean fluorescence intensity of chromosomal passenger-localized AIR-2 (prophase and metaphase chromosomes, anaphase central spindle, telophase midzone and midbody) was measured and plotted against spindle length in wt (C) and air-2(or207ts) (D) embryos reared at 22°C and treated with control and cdc-48.3(RNAi). Spindle length was measured from the center of one centrosome to the other. Each data point represents one embryo, and colored points mark the start of each mitotic phase as follows: prophase (black), prometaphase (orange), metaphase (purple), anaphase (green), telophase (red), and late telophase/G1 entry (blue). Number of embryos: wt;control(RNAi), n = 98; wt;cdc-48.3(RNAi), n = 87; air-2(or207ts);control(RNAi), n = 91; air-2(or207ts);cdc-48.3(RNAi), n = 94.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

4. Figure 3
CDC-48.3 Directly Interacts with and Inhibits AIR-2 Kinase Activity in an ATPase-Dependent Manner
(A) AIR-2 was immunoprecipitated from protein extracts of control, cdc-48.3, and air-2(RNAi) treated GFP-CDC-48.3 transgenic animals, and subjected to western blotting with AIR-2 (bottom) and GFP-specific (top) antibodies. L, 10% of protein extract loaded in each IP; IP, immunoprecipitated pellet.
(B) Recombinant AIR-2 was mixed with glutathione beads incubated with buffer, GST-CDC-48.3, or GST-CDC The beads were pelleted, washed, and subjected to western analysis with AIR-2 and GST antibodies. S, 25% of supernatant left after pelleting beads; p, washed glutathione bead pellet.
(C) Schematic illustrating CDC-48.3 protein fragments used in binding and kinase assays (Figures 3C, 3F, and 3G). Recombinant AIR-2 was mixed with full-length GST-CDC-48.3 or the depicted GST-tagged fragments bound to glutathione beads, and treated as described in (A).
(D) GST-AIR-2 or GST-AIR-1 were mixed with either GST-CDC-48.3 (lanes 2 and 5) or GST-CDC-48.1 (lanes 3 and 6) in kinase buffer supplemented with [γ32P]-ATP and MYBP as an exogenous substrate (lanes 1-6). [γ32P]-ATP incorporation was visualized by phosphoimaging, MYBP loading by Ponceau S staining, and AIR-2, AIR-1, GST-CDC-48.1, and GST-CDC-48.3 loading by western analysis with AIR-2, AIR-1, and GST antibodies, respectively.
(E) Quantitation of 32P incorporation into MYBP. Error bars represent standard deviation from three independent experiments. MYBP phosphorylation by AIR-2 did not change in the presence of GST-CDC-48.1 (p = 0.43). 32P incorporation into MYBP in the presence of AIR-2 was normalized to 1.
(F) GST-AIR-2 was mixed with full-length GST-CDC-48.3 or the GST-tagged fragments depicted in (C) in kinase buffer with MYBP as an exogenous substrate. Proteins were visualized as in (D).
(G) GST-CDC-48.3 (N+D1) mutants proteins were assayed for effects on AIR-2 kinase activity.
(H) Recombinant AIR-2 was mixed with GST-CDC-48.3 (N + D1) mutant proteins bound to glutathione beads, and treated as described in (A).
(I) GST-CDC-48.1 and wt or mutant GST-CDC-48.3 proteins were assayed for ATPase activity. Error bars represent standard deviation from three independent experiments.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

5. Figure 4
pAIR-2 Levels Are Upregulated from Metaphase through Telophase in cdc-48.3(RNAi);air-2(or207ts) Embryos
(A) Embryos dissected from control and cdc-48.3(RNAi) treated air-2(or207ts) hermaphrodites reared at 22°C were fixed and stained with DAPI (blue), and α-tubulin (green) and pAurora (pAur) (red) antibodies. One-cell embryos at different mitotic stages are shown. Centrosome pAur staining reflects phosphorylated AIR-1. Scale bar = 10 μm.
(B) Mitotic chromosomal passenger (CPC) localized pAur immunostaining was measured in control (n = 89) and cdc-48.3(RNAi) (n = 92) treated one-cell air-2(or207ts) embryos (22°C) and plotted against spindle length.
(C) pAur immunostaining throughout the entire embryo was measured in control (n = 89) and cdc-48.3(RNAi) (n = 92) treated one-cell air-2(or207ts) embryos (22°C) and plotted against spindle length.
(D) CPC localized pAur immunostaining was measured in control (n = 267) (gray bars) and cdc-48.3(RNAi) (n = 269) (black bars) treated one-cell air-2(or207ts) embryos reared at the indicated temperatures. CPC pAur immunostaining measured in wt embryos reared at 25°C is included as a control (white bars). Spindle length and morphology were used to delineate the stages of mitosis. Error bars represent standard deviation.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

6. Figure 5 cdc-48.3(RNAi) Leads to Mitotic Delays and Spindle Defects
(A) Embryos from OD57 (mCherry::Histone H2B; GFP::α-tubulin) L4 hermaphrodites fed control and cdc-48.3(RNAi) were subjected to live imaging. Time 0 (c and b′) corresponds to metaphase chromosome alignment. (a and a′) Pronuclear meeting (−390 and −450 s respectively from metaphase), (b) prophase, (b′) metaphase chromosome alignment, (c) metaphase chromosome alignment and completion of spindle rotation, (c′) completion of spindle rotation, (d and d′) anaphase, (e and e′) telophase, (f and f′) metaphase of AB cell division. Scale bar = 10 μm.
(B) Graph of mitotic progression in control (n = 4) and cdc-48.3(RNAi) (n = 4) treated OD57 embryos undergoing the first mitotic division. AB Metaphase, metaphase of the AB cell (two-cell embryo). Error bars represent standard deviation.
(C) Wt and air-2(or207ts) L4 hermaphrodites were subjected to mock or cdc-48.3 dsRNA microinjection, and reared at 15°C or 22°C. Embryonic viability was scored 48 hr postinjection. Wt control 15°C, n = 992, 22°C, n = 1002; wt cdc-48.3(RNAi) 15°C, n = 1123, 22°C, n = 1099; air-2(or207ts) control 15°C, n = 1055, 22°C, n = 1048; air-2(or207ts);cdc-48.3(RNAi) 15°C, n = 1068, 22°C, n = Error bars represent standard deviation.
(D) Live imaging of embryos from OD57 hermaphrodites subjected to mock or cdc-48.3 dsRNA microinjection was performed (control, n = 3 embryos; cdc-48.3(RNAi), n = 7 embryos). Time 0 (c and c′): metaphase chromosome alignment. (a and a′) Pronuclear meeting (−330 and −630 s respectively from metaphase), (b) prophase, (b′) metaphase chromosome alignment, (c) metaphase chromosome alignment and completion of spindle rotation, (c′) completion of spindle rotation, (d and d′) anaphase, (e and e′) telophase, (f and f′) metaphase of AB cell division. Scale bar = 10 μm.
(E) Graph of mitotic progression in one-cell embryos from control (n = 3) and cdc-48.3 dsRNA (n = 5) injected OD57 hermaphrodites. AB Metaphase, metaphase of the AB cell (two-cell embryo). Error bars represent standard deviation.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions