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First published online 11 March 2009
doi: 10.1242/dev.026963

Development 136, 1283-1293 (2009)
Published by The Company of Biologists 2009
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A novel role for an APC2-Diaphanous complex in regulating actin organization in Drosophila

Rebecca L. Webb, Meng-Ning Zhou and Brooke M. McCartney*

Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.


Figure 1
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  		Fig. 1. Schematics of APC proteins and cytoskeletal rearrangements in the Drosophila syncytial embryo. (A) Multiple domains of mammalian APC interact with the cytoskeleton, either directly or indirectly. (B) Drosophila APC2 with relevant alleles indicated. Blue arrows, stop codons. A and B are modified with permission from McCartney et al. (McCartney et al., 2006Go). (C) At interphase, cortical actin is organized into apical caps above each nucleus. Centrosomes are located between each nucleus and its actin cap and nucleate microtubules. (D) Actin caps in a wild-type (WT) embryo expressing MoesinGFP, an actin marker. (E) At metaphase, actin has reorganized into rings (see surface view) that extend into actin pseudocleavage furrows (see cross-section). Centrosomes and microtubules have reorganized into the spindle. (F) Actin rings in the same embryo as in D, 6 minutes 30 seconds later. Scale bar: 10 µm.

 

Figure 2
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  		Fig. 2. Apc2 mutants exhibit defects in actin rings and furrow extension; dia5 mutants exhibit actin defects similar to those of Apc2 mutants. (A) Apical views of cycle 12 metaphase Drosophila embryos. Apc2d40 and Apc2g10 embryos display incomplete actin rings (arrows) that are occasionally associated with spindle collisions (arrowhead). A surface (0 µm) view is shown. (B) Cross-sectional views of metaphase actin show that Apc2{Delta}S furrows appear largely WT, whereas furrow extension in Apc2d40 andApc2g10 mutants is grossly defective (yellow arrows). (C) Quantification of furrow extension. The percentage of incomplete actin rings at four depths for five embryos of each genotype is shown. The increase in incomplete actin rings in Apc2g10 embryos as compared with WT is significant at all depths. (D-G'') Apical (D-G), subapical (D'-G') and cross-sectional (D''-G'') views of actin in cycle 12 metaphase WT (D-D''), Apc2g10 (E-E'') and dia5 (F-G'') embryos. Defects in dia5 embryos range from actin primarily in caps during metaphase (F,F') to embryos with incomplete actin rings (G,G', arrowheads) that are similar to, but more severe than, those in Apc2g10 (E,E', arrowheads). Arrows in F'',G'' indicate effective furrow extension Scale bars: 10 µm.

 

Figure 3
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  		Fig. 3. Syncytial embryos do not contain acetylated microtubules and Apc2 mutants have no visible microtubule defects. (A-B'') Staining for tubulin (A,B, red in merge) versus acetylated tubulin (A',B', green in merge) in gastrulated (A-A'') and syncytial (B-B'') Drosophila embryos. Unlike the acetylated microtubules in gastrulated embryos (A'), anaphase microtubules in syncytial embryos are not acetylated (B'). Overexposure (insets) reveals weak staining for acetylated microtubules at spindle poles (arrow) in syncytial embryos. (C) Pole-pole distance measurements in live cycle-12 embryos; n=30 for each. (D-E') Anaphase microtubules in WT (D,D') and Apc2g10 (E,E') cycle-12 embryos have similar densities and organization. (F,F') Schematics indicating the plane of section in D-E'. (G-H') Three-dimensional volume-rendered views of deconvolved cycle 9 anaphase asters reveals qualitatively similar astral microtubule organization in WT (G,G') and Apc2g10 (H,H') embryos. Arrows in G and H indicate the asters that are shown at high magnification in G' and H'. Scale bars: 10 µm.

 

Figure 4
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  		Fig. 4. APC2 and DIA proteins colocalize together and with actin in WT embryos. (Aa-Fe) APC2 (Ab-Fb) and DIA (Ac-Fc) colocalize with actin (Aa-Fa) throughout the cell cycle in WT cycle-12 Drosophila embryos. Colocalization of APC2 and DIA (yellow in Ad-Fd and white in Ae-Fe) is not uniform throughout the cell cycle, but peaks during prometaphase (Cd,Ce) and metaphase (Dd,De). (G) Metaphase; in cross-section APC2 (green) and DIA (red) colocalize with actin (blue) as the furrows extend. DIA is enriched at the furrow tip (arrow). Scale bars: 10 µm.

 

Figure 5
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  		Fig. 5. DIA is required for normal APC2 localization, but APC2 is not required for DIA localization. (Aa-Jc) Stage-matched cycle-12 WT (A,C,E,G,I) and dia5 (B,D,F,H,J) Drosophila embryos showing the localization of actin (Aa-Ja), APC2 (Ab-Jb) and Anillin (Ac-Jc). APC2 (Fb,Hb, arrow) fails to localize to actin rings (Fa,Ha, arrow) during metaphase and anaphase in dia5 mutant embryos, whereas Anillin continues to colocalize with actin (Fc,Hc, arrow). During telophase, APC2 localizes to remnant actin rings and reforming caps in dia5 embryos (Ja,Jb, arrow). (K,L) In WT (K) and Apc2g10 (L) cycle-12 metaphase embryos, DIA localizes to actin rings. (K',L') In cross-section (at the yellow lines in K,L), DIA (green) is enriched at the furrow tips in Apc2g10 embryos (L', arrow) as in WT (K', arrow). Scale bars: 10 µm.

 

Figure 6
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  		Fig. 6. Apc2 and dia genetically interact, and APC2 and DIA bind directly. (A-D) Actin in cycle-12 metaphase Drosophila embryos at 0 µm. In comparison to WT (A), Apc2g10 (B) and diak/CyO (C) embryos, diak/CyO; Apc2g10 (D) embryos have increased cap-like actin (arrow). Scale bar: 10 µm. (E) Quantification of furrow extension at -0.8 µm reveals enhancement of the Apc2g10 phenotype with reduction of dia. diak is diak07135. (F) His-DIAC519 binds directly to APC2C and to Chickadee, EB1, human APC1Basic and Drosophila APC1Basic (positive controls) in RIPA buffer. (G) DIA co-immunoprecipitated with APC2, but not with Myc (negative control) from 0-2 hour embryo lysates. (H) Schematic summary of APC2 and DIA constructs and interactions. B, bead; S, supernatant.

 

Figure 7
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  		Fig. 7 . EB1 may function independently of APC2-DIA. (A-B'') Apical views of actin (A,A'',B,B'') and microtubules (A',A'',B',B'') in cycle-12 metaphase Eb1B13 embryos. Arrows in A and B indicate actin mats; yellow lines indicate plane of sections in C. (C) Partial furrow extension (arrows) in Eb1B13 embryos in cross-section. Eb1B13 mutants exhibit a wide array of spindle morphologies, including apparently normal spindles (A') and severely disrupted spindles (B'). (D-G) Actin in cycle-12 metaphase embryos of the indicated genotypes. (H) Quantification of furrow extension shows that there is no significant effect of reduction of Eb1 on the Apc2g10 phenotype. (I) Free MBP-EB1 binds directly to DIAC484 and human APCEB1bd (positive control), but to neither Drosophila APC2 fragment in HKT buffer. B, bead; S, supernatant.

 

Figure 8
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  		Fig. 8. Rho1 and RhoGEF2 mutants. (A-F) Cycle-12 metaphase Drosophila embryos. Rho1 (D) and RhoGEF2 (E) mutants display areas of decreased actin (D,E, arrows), as well as areas of excessive accumulation of actin associated with rings (D,E, insets, arrowheads), which are not observed in WT (A), Apc2g10 (B) or dia5 (C) embryos. (F) Cross-sections stained for actin show defective thickened furrows in Rho1 and RhoGEF2 embryos (arrows). (G-G'') APC2 (G') and DIA (G'') localize to cortical actin (G) in a RhoGEF204291 mutant cycle-12 anaphase embryo. Scale bars: 10 µm.

 

Figure 9
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  		Fig. 9. Model for Drosophila APC2-DIA function. (A) We propose that APC2 promotes the activity of DIA by facilitating the interaction between DIA and an activator. This interaction enhances the efficiency of actin nucleation in the pseudocleavage furrow. In the absence of APC2, DIA and its activator interact less efficiently, resulting in defective furrow extension. (B) Many proteins affect syncytial actin rearrangments (see Discussion).

 

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