Drosophila Strains and Constructs
UAS constructs were expressed in SOP cells by using neur
> Gal4 and in neuroblasts by using insc
> Gal4, except for experiments shown in Figure S1A
, in which act5C
> Gal4 was used. GAL80ts
was used to suppress the expression of aPKCΔN
prior to pupariation. All fly stocks and constructs are described in detail in the Supplemental Experimental Procedures.
Lgl-GFP is functional because it complements lgl1/4
mutants in the larval brain (Figure S8L
). The Par-6-GFP genomic rescue construct is functional because it rescues par-6Δ226
mutants to viability (data not shown).
Live imaging of SOP cells was performed essentially as described (Bellaiche et al., 2001a
). Images were acquired on a Zeiss LSM510 Meta confocal microscope with a Plan-Apochromat 633/1.40 oil immersion objective at 53 zoom (single cells) or 13 zoom (whole notum). For two-dimensional (2D) recordings, images were acquired at intervals of 6–12 s. For 3D recordings, a z-stack of 10 or 20 slices at 1 μm distance was acquired at intervals of 42 s or 84 s, respectively. Images were processed in LSM software (Zeiss), ImageJ (NIH), and Imaris (Bitplane).
For FRAP analysis, a Plan-Apochromat 633/1.40 oil immersion objective was used at 53 zoom with a pinhole of 2 Airy units. The bleach pulse consisted of 10 passes of a 488 nm laser at 100% power. Quantitative experiments were performed in bidirectional mode at 8 fps, and 8 prebleach frames and 480 postbleach frames were acquired. Integrated densities of the bleached area, a whole-cell, and a background region were determined for each time point by using LSM software (Zeiss). Values were corrected for background in Microsoft Excel. In addition, the double normalization method was used to correct for loss of total fluorescence due to bleach pulse and acquisition bleaching (Phair et al., 2004
). Each experiment was normalized to the average prebleach fluorescence, which allowed averages and standard deviations to be calculated for the individual time points.
Antibodies and Immunohistochemistry
Phosphospecific antibodies against Ser34 of Par-6 were raised in rabbits against the peptide EFRRWpSFKRNEAE and were affinity purified. Baz antibodies were raised in rabbits against the N-terminal 318 amino acids of Baz fused to MBP.
Antibodies used in western blotting, immunoprecipitation, and/or immunohistochemistry were rabbit anti-p-Ser34-Par-6 and anti-Baz (1:200; this study); rabbit anti-Par-6 (1:200; Petronczki and Knoblich, 2001
); rabbit anti-Lgl (1:100) and rabbit anti-p-Ser660-Lgl (1:200; Betschinger et al., 2003
); mouse anti-cmyc (sc-40) and rabbit anti-PKCζ (1:1,000; sc-216, Santa Cruz); rabbit anti-p-Thr555-PKCζ (1:100; ab5813) and rabbit anti-GFP (1:1,000; ab6556, Abcam); mouse anti-GFP (1:100; Roche); rabbit anti-p-Ser7-Numb (1:200;Nishimura and Kaibuchi, 2007
); rabbit anti-Miranda (1:200; Betschinger et al., 2006
); rabbit anti-p-H3 (1:1000; Millipore); mouse anti-Prospero (1:20; MR1A, DSHB); rabbit anti-Numb (1:100; Schober et al., 1999
); and rabbit anti-α-adaptin (1:200; Dornan et al., 1997
). Rabbit anti-GFP (A-6455, Molecular Probes) was used for immunoprecipitation of Baz-GFP.
For immunohistochemistry, third-instar wandering larvae were dissected in PBS, and brains with attached ventral nerve cords were fixed for 10 min in 3.7% formaldehyde in PBS containing 0.2% Triton X-100 and processed as described (Betschinger et al., 2006
). Images were acquired on a Zeiss LSM510 confocal microscope and were processed in Photoshop (Adobe Systems).
For in vitro kinase assays, bacterially produced proteins were incubated with either 30 ng recombinant human AurA (Millipore) for 30 min at 30°C or with 50 ng recombinant PKCζ (Calbiochem) for 10 min at 25°C in kinase buffer (25 mM Tris-HCl [pH 7.5], 25 mM NaCl, 5 mM MgCl2,10 mM β-glycerophosphate, 1 mM EGTA, 1 mM DTT, 100 mM ATP) containing 1.5 mCi [γ-32P]ATP. For IP kinase assays, the immunoprecipitates were incubated for 30 min at 30°C in kinase buffer (20 mM Tris-HCl [pH 7.5], 5 mM MgCl2, 0.1 μM Calyculin A, 1 mM EGTA, 1 mM DTT, 0.1% Triton X-100, 100 mM ATP). A total of 100 ng recombinant AurA was used in the assay illustrated in . A total of 40 μg/ml of the peptide SIYRRGARRWRKL was used as a pseudosubstrate inhibitor against aPKC in the experiment illustrated in . Reactions were terminated by the addition of SDS sample buffer.
Immunoprecipitations and Binding Assays
For immunoprecipitations, embryos or larval brains were extracted with lysis buffer (20 mM Tris-HCl [pH 7.5], 1 mM EDTA, 100 mM NaCl, 1% Triton X-100, 10 μg/ml PMSF, Complete Protease Inhibitor Cocktail [Roche]), cleared by centrifugation at 16,000 3 g for 15 min, and incubated with antibodies for 1–2 hr at 4°C. The immunocomplexes were then precipitated by using Protein A-Sepharose 4B beads (Amersham), washed three times with lysis buffer, and eluted by boiling in SDS sample buffer. For in vitro binding assays, GST fusion proteins were immobilized onto glutathione Sepharose 4B beads (Amersham) and incubated with embryonic lysate or larval brain lysate in lysis buffer for 1–2 hr at 4°C. The beads were washed three times with lysis buffer and were eluted by boiling in SDS sample buffer.
Embryonic myc-Lgl and myc-Numb immunoprecipitates were washed three times with high-salt buffer (20 mM Tris-HCl [pH 7.5], 1 mM EDTA, 500 mM NaCl, 1% Triton X-100, 10 mg/ml PMSF) and twice with kinase buffer (25 mM Tris-HCl [pH 7.5], 100 mM NaCl, 5 mM MgCl2,10mM β-glycerophosphate, 1 mM DTT, 1 mM EGTA, 0.1% Trition X-100) at 4°C and were incubated with 100 ng recombinant PKCζ (Calbiochem) for 30 min at 30°C in kinase buffer containing 100 μM ATP. Subsequently, the samples were washed three times with high-salt buffer and twice with lysis buffer.
Phosphorylated samples were incubated with larval brain extract in phosphatase buffer (50 mM Tris-HCl [pH 7.5], 100 mM NaCl, 0.1 mM EGTA, 2 mM DTT, 1 mM MnCl2, 0.5% Trition X-100, 10 mg/ml PMSF, EDTA-free Complete Protease Inhibitor Cocktail [Roche]) with or without phosphatase inhibitors (0.1 μM Calyculin A, 20 mM β-glycerophosphate, 5 mM NaF, 1 mM sodium pyrophosphate) for the indicated durations at 20°C, terminated by the addition of chilled lysis buffer with phosphatase inhibitors, centrifuged, and eluted by boiling in SDS sample buffer.