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First published online 9 July 2008
doi: 10.1242/dev.024059

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Dap160/intersectin binds and activates aPKC to regulate cell polarity and cell cycle progression

Institute of Neuroscience, Institute of Molecular Biology, Howard Hughes Medical Institute, University of Oregon, Eugene, OR 97403, USA.

View larger version (29K): [in this window] [in a new window]   Fig. 1. Dap160 interacts with aPKC. (A) Drosophila Dap160 and vertebrate intersectin protein domains. (B) Dap160 antibody detects two bands in wild-type lysate (top arrow, 160 kDa; bottom arrow, 120 kDa) that may correspond to the two predicted isoforms shown in A; and these bands are absent in lysate from dap160 mutants, illustrating the specificity of the antibody. An independently generated antibody gives the same result (see Fig. 2N). (C) Immunoprecipitation from larval lysate using an aPKC antibody and a control antibody (Bgal) and blotted with a Dap160 antibody shows aPKC co-immunoprecipitates Dap160 protein (arrowhead). (D,E) In vitro protein interaction experiments. (D) In vitro generated Dap160 protein coupled to glutathione S-transferase (GST) beads can bind in vitro produced aPKC protein (arrowhead). Beads alone do not bind aPKC; input lane shown at left. (E) In vitro generated Dap160 protein coupled to glutathione S-transferase (GST) beads can bind in vitro produced Par-6 protein (arrowhead). Beads alone do not bind Par-6; input lane shown at left. (F,G) Dap160 directly stimulates aPKC activity and this effect can be partially blocked by Par6. Top: presence (+) or absence (-) of each protein; middle: phosphorylation of the aPKC substrate peptide. Histogram shows quantification of the phosphorylation (bars) over a schematic depiction of protein levels (see Materials and methods for protein concentrations). Note that aPKC alone can have high activity immediately after its synthesis (F) or much lower activity after storage (G), but can still be stimulated by Dap160.

View larger version (62K): [in this window] [in a new window]   Fig. 2. Dap160 co-localizes with aPKC in neuroblasts. (A-L) Neuroblasts co-stained for Dap160 (A-F) and aPKC (G-L); merged images below. Genotypes, developmental stages and cell cycle stages labeled at top. (B,H) Dap160 colocalizes with aPKC at the apical cortex of mitotic embryonic neuroblasts. (E,K) Endogenous Dap160 is undetectable at the site of aPKC apical cortical localization in larval brain metaphase neuroblasts. (F,L) Overexpression of Dap160 reveals colocalization with aPKC at the apical cortex in larval brain metaphase neuroblasts (90%, n=12). Scale bar: 5 µm. (M) aPKC can immunoprecipitate Dap160 from embryonic lysates. Right lane: aPKC antibody can immunoprecipitate Dap160 (top arrow) and aPKC (bottom arrow). Left lane: the GFP control antibody does not immunoprecipitate either Dap160 or aPKC. (N) Two independently generated Dap160 antibodies detect apical crescents of Dap160 in wild-type neuroblasts or Dap160 overexpression neuroblasts (arrowheads) but not in dap160 mutant neuroblasts (brackets). Roos/Marie antibody from Roos and Kelly (Roos and Kelly, 1998). Scale bar: 5 µm. (O) Dap160 overexpression results in the formation of Dap160/Sec15 double-positive puncta in neuroblasts (arrowheads; left pair of panels) and Dap160/aPKC double-positive puncta (arrowheads; right pair of panels). Scale bar: 5 µm.

View larger version (24K): [in this window] [in a new window]   Fig. 3. Dap160 regulates embryonic neuroblast cortical polarity. (A-E) Wild type metaphase embryonic stage 15 neuroblasts stained for the indicated proteins (top labels) have apical crescents of aPKC, Bazooka, Par-6 (100%, n=23; 100%, n=34; and 96%, n=23, respectively) and basal crescents of Miranda (93%, n=43) and Numb (100%, n=21) (white arrowheads). (F-J) dap160 metaphase embryonic stage 15 neuroblasts. (F) aPKC is mostly ectopic cortical aPKC (71%, n=41, red arrow) or cytoplasmic (not shown). (G) Par-6 is ectopic cortical (50%, n=12, red arrow) or normal (not shown). (H) Baz is cytoplasmic (21.8%, n=96; H), ectopic cortical or normal (not shown). (I) Miranda is ectopic cortical (red arrow, 36%, n=55) or normal (not shown). (J) Numb is ectopic cortical (red arrow, 38%, n=18) or normal (not shown). Proteins are delocalized and may also be at lower levels than in wild-type neuroblasts. (K-M) dynamin mutant mitotic larval neuroblasts (shibirets/shibirets at restrictive temperature; see Materials and methods) have aPKC apical crescents (K) and basal crescents of Miranda (L) and Numb (M). (N) Quantification of the neuroblast cortical polarity phenotypes. Number of neuroblasts scored is shown as a number at the bottom of each bar. Scale bar: 5 µm.

View larger version (75K): [in this window] [in a new window]   Fig. 4. Dap160 positively regulates neuroblast pool size. (A) Neuroblast numbers scored wild-type (green bars), dap160 mutants (red bars) and Dap160 misexpression larvae at second instar (L2) or wandering third instar (L3). Numbers inside bars indicate numbers of brains analyzed. See Materials and methods for genotypes and growth temperatures. (B-E) Wild-type (B,C), dap160 (D) and aPKC (E) mutant clones (n50 and n=35 respectively) always contain a single Deadpan-positive neuroblast (arrowhead). (F-L) Neuroblast cortical polarity in larval neuroblasts. (F,G) Wild-type neuroblasts have aPKC apical crescents (arrowhead) and Miranda (Mira) basal crescents. (H,I) dap160 mutants have weak ectopic cortical aPKC (arrows) and normal Mira basal crescents (15%, n=13). (J,K) Dap160 overexpression in neuroblasts leads to weak ectopic cortical aPKC (arrow), increased cytoplasmic and reduced cortical Mira (19%, n=21 as shown; remainder normal basal crescents); Dap160 is detected in cortical patches and cytoplasmic puncta (L). (M,N) Live imaging of wild-type neuroblasts with GFP::Mira and Cherry::Jupiter. (M) Wild-type neuroblasts show basal GFP::Mira at metaphase (brackets) and partition GFP::Mira to the GMC at telophase (arrowhead; 100%, n=37). (N) Dap160 misexpressing neuroblasts show cytoplasmic GFP::Mira at metaphase (brackets) and occasionally do not segregate GFP::Mira to the GMC at telophase (arrow; 8%, n=40). Scale bars: 5 µm.

View larger version (55K): [in this window] [in a new window]   Fig. 5. Dap160 is required for neuroblast cell cycle progression. Wild-type, dap160 mutant and aPKC mutant neuroblasts imaged with Jupiter::GFP from nuclear envelope breakdown (NEBD) to anaphase onset (AO). (A) Wild-type neuroblasts have a NEBD-AO interval of 7.76±2.04 minutes; n=15. (B) dap160 mutant neuroblasts have a NEBD-AO interval of 13.37±4.4 minutes; n=10. (C) aPKC mutant neuroblasts have a NEBD-AO interval of 17.84±4.52 minutes; n=11. Scale bar: 5 µm.

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