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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.

^* Author for correspondence (e-mail: cdoe{at}uoneuro.uoregon.edu)

Accepted 13 June 2008

	SUMMARY

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The atypical protein kinase C (aPKC) is required for cell polarization of many cell types, and is upregulated in several human tumors. Despite its importance in cell polarity and growth control, relatively little is known about how aPKC activity is regulated. Here, we use a biochemical approach to identify Dynamin-associated protein 160 (Dap160; related to mammalian intersectin) as an aPKC-interacting protein in Drosophila. We show that Dap160 directly interacts with aPKC, stimulates aPKC activity in vitro and colocalizes with aPKC at the apical cortex of embryonic neuroblasts. In dap160 mutants, aPKC is delocalized from the neuroblast apical cortex and has reduced activity, based on its inability to displace known target proteins from the basal cortex. Both dap160 and aPKC mutants have fewer proliferating neuroblasts and a prolonged neuroblast cell cycle. We conclude that Dap160 positively regulates aPKC activity and localization to promote neuroblast cell polarity and cell cycle progression.

Key words: PKC, Cell cycle, Intersectin, Neuroblast, Polarity, Quiescence, Drosophila

	INTRODUCTION

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Asymmetric cell division has been proposed as a mechanism for maintaining stem cell numbers while allowing the generation of differentiated cell types (Morrison and Kimble, 2006). It is also been proposed that asymmetric cell division of a small number of tumor stem cells may be the origin of several tumor types (Morrison and Kimble, 2006). Asymmetric cell division typically involves establishment of a cell polarity axis, followed by alignment of the mitotic spindle with the polarity axis to produce daughter cells with different molecular composition. One evolutionarily conserved protein involved in both cell polarity and asymmetric cell division is atypical protein kinase C (aPKC; called PKC-3 in C. elegans and aPKC, / in mammals). aPKC is required for C. elegans blastomere cell polarity (Tabuse et al., 1998), Drosophila oocyte polarity (Tian and Deng, 2008); C. elegans, Drosophila and mammalian epithelial polarity (Aranda et al., 2006; Gopalakrishnan et al., 2007; Harris and Peifer, 2007; Helfrich et al., 2007; Hutterer et al., 2004; Nagai-Tamai et al., 2002; Rolls et al., 2003; Suzuki et al., 2004; Suzuki et al., 2002; Takahama et al., 2008; Yamanaka et al., 2006; Yamanaka et al., 2003; Yamanaka et al., 2001); and Drosophila planar cell polarity (Djiane et al., 2005); and has been implicated as an oncogene in several human tumors (Fields et al., 2007; Regala et al., 2005; Yi et al., 2008). aPKC is called `atypical' because unlike classical PKCs or novel PKCs, it is not activated by Ca²⁺ or diacylglycerol, but rather by a relatively poorly understood mechanism of protein-protein interactions.

Drosophila larval brain neural progenitors (neuroblasts) are a powerful system with which to study the role of asymmetric cell division and stem cell self-renewal and proliferation. aPKC is required for both cell polarity and stem cell self-renewal in Drosophila neuroblasts (Lee et al., 2006; Rolls et al., 2003). aPKC colocalizes with the polarity proteins Par-6, Cdc42 and Bazooka (Baz; Par3 in mammals) at the apical cortex of mitotic neuroblasts, and is segregated into the self-renewing neuroblast at each cell division. The apical cortical crescent of aPKC ensures exclusion of the differentiation factors Miranda, Prospero, Brain tumor (Brat) and Numb from the apical cortex, resulting in their restriction to the basal cortex and ultimate partitioning into the smaller ganglion mother cell (Doe, 2008; Knoblich, 2008), which typically divides once to form two postmitotic neurons. In aPKC mutants neuroblasts, cell polarity is disturbed such that Miranda and Numb basal proteins become distributed uniformly on the neuroblast cell cortex, both at metaphase and at telophase, resulting in a molecularly symmetric cell division and a decrease in neuroblast numbers (Lee et al., 2006; Rolls et al., 2003). aPKC has been proposed to regulate neuroblast cortical polarity by phosphorylating and inhibiting cortical localization of the Lethal giant larvae (Lgl) protein (Betschinger et al., 2003), which is required for basal targeting of Miranda and Numb proteins (Ohshiro et al., 2000; Peng et al., 2000). Overexpression of a membrane-targeted aPKC, but not a kinase dead version, leads to reduced basal protein localization and the formation of supernumerary neuroblasts (Lee et al., 2006), revealing the importance of aPKC kinase activity for promoting neuroblast cell polarity and self-renewal. Despite the central role of aPKC kinase activity in establishing neuroblast cell polarity, relatively little is known about how aPKC activity is regulated. Recently, we showed that the small G-protein Cdc42 is colocalized with aPKC and can stimulate aPKC activity by relieving Par-6 inhibition of aPKC (Atwood et al., 2007). However, the stimulation of activity is modest and the polarity phenotypes of cdc42 mutant are not fully penetrant, which suggests that additional aPKC activators may exist.

Despite the importance of aPKC in cell polarity and growth control, little is known about how aPKC activity is regulated. Here, we have sought to identify aPKC-interacting proteins using a biochemical approach, and then assay their function in Drosophila neuroblasts. We performed immunoprecipitation experiments coupled to mass spectrometry analysis (IP/MS) using aPKC as the bait protein and identified Dap160 (dynamin associated protein-160), a member of the intersectin protein family (Adams et al., 2000). Dap160 was originally identified by its ability to interact with the endocytic protein Dynamin in Drosophila head extracts (Roos and Kelly, 1998). dap160 mutants have nerve terminals with reduced levels of endocytic proteins (Koh et al., 2004; Marie et al., 2004), consistent with a role in endocytosis. However, dap160 mutants were also isolated in a genetic screen for modifiers of the Sevenless receptor tyrosine kinase (Roos and Kelly, 1998), showing that Dap160 also regulates signal transduction, similar to mammalian intersectin (Malacombe et al., 2006; Martin et al., 2006; Tong et al., 2000a; Tong et al., 2000b). Here, we show that Dap160 binds aPKC, increases its kinase activity, and that both Dap160 and aPKC are required for neuroblast cell polarity and cell cycle progression.

	MATERIALS AND METHODS

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Fly stocks and MARCM clones
The wild-type fly stock was yellow white (y w). The dap160^Q24, Df(2)10523^#35 and UAS-Dap160 stocks were gifts from Graeme Davis (UCSF). dap160¹ was a gift from Hugo Bellen (Koh et al., 2004). aPKC^k0643 flies lacking the early embryonic lethal background mutation have been described previously (Rolls et al., 2003). Analysis of cell polarity was carried out in stage 15-16 dap160^Q24/Df(2)10523 trans-heterozygotes embryos as previously described (Marie et al., 2004). The hypomorphic dap160^Q24/dap160¹ hetero-allelic combination was used to assay larval brain neuroblast numbers. The shi^ts2 chromosome was a gift from Mani Ramaswami (Arizona). To generate positively marked MARCM clones, we recombined FRT40 onto the dap160^Q24 chromosome using standard techniques, and used the previously described FRTG13 aPKC^k06403 chromosome (Rolls et al., 2003). dap160 neuroblast mutant clones were generated by mating dap160^Q24 FRT40/CyO, actGFP to y w hsFLP; FRT40A tubP-GAL80/CyO ActGFP; tubP-GAL4 UAS-mCD8::GFP/TM6 Tb Hu (Bello et al., 2008). aPKC^k06403 neuroblast mutant clones were generated by mating FRTG13 aPKC^k06403/CyO to y w hsFLP; FRTG13 tubP-GAL80/CyO actGFP; tubP-GAL4 UAS-mCD8::GFP/TM6 Tb Hu (C. Cabernard and C.Q.D., unpublished). Clones were induced at 24-28 hours after larval hatching for 1 hour at 37°C and aged for 4 days at 25°C.

In vitro binding assays and immunoprecipitations
GST-tagged Dap160 was engineered by polymerase chain reaction from a P-spaceneedle-Dap160-dsred construct (a gift from Graeme Davis) followed by subcloning into pGEX-4T1 vector (Pharmacia). The construct was verified by DNA sequencing. GST-Dap160 was expressed in BL21 cells overnight at 25°C, adsorbed onto glutathione agarose (Sigma), washed three times with binding buffer [10 mM HEPES (pH 7.5), 100 mM NaCl, 1 mM DTT, 0.1% Tween-20]. We co-incubated GST-Dap160 with Par-6 or aPKC proteins at room temperature for 15 minutes followed by five washes in binding buffer. Interactions were tested by eluting proteins in SDS sample buffer, SDS-PAGE and western blotting. Par-6 and aPKC proteins were a gift from Scott Atwood (Prehoda laboratory, OR). For immunoprecipitation experiments, a 12-hour collection of y w embryos was homogenized in lysis buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 0.1% Tween-20, 1 mM EDTA, 2.5 mM EGTA, 10% glycerol, supplemented with protease inhibitor tablets; Roche] to produce 1 ml of lysate. Lysates were pre-cleared with protein agarose-A beads for 1 hour at 4°C and subsequently divided equally (500 µl each) in two Eppendhorf tubes and incubated with 2 µl of either anti-βgal or anti-aPKC antibodies for 4 hours at 4°C. Lysates were then incubated with protein agarose A beads for 1 hour at room temperature. For pulldowns, beads were precipitated and washed three times in modified lysis buffer containing 1 mM NaCl. Samples were sent to the Fred Hutchinson Cancer Research Center proteomic facility (Seattle, WA) for mass-spectrometry analysis.

Kinase activity assays
aPKC activity assays were carried out as described previously (Atwood et al., 2007). Briefly 0.16 µM aPKC was co-incubated with 50 µM peptide substrate, no Par-6 or 1.14 µM Par-6, no Dap160 or increasing molar concentration of Dap160 protein (0.34, 0.68, 1.02 µM). Phosphorylated peptides were resolved by gel electrophoresis, quantified using ImageJ and normalized to total peptide input.

Antibodies, immunostaining and imaging
An affinity-purified anti-Dap160 antibody was raised against the C-terminal peptide sequence GPF VTS GKP AKA NGT TKK (Alpha Diagnostic). Guinea pig or chicken anti-Dap160 was used at 1:100 (this study); guinea pig or rat anti-Miranda at 1:500 (Doe laboratory); rabbit anti-aPKC at 1:1000 (Sigma), guinea pig anti-Numb at 1:1000 (a gift from Jim Skeath, Missouri); rabbit anti-Scrib at 1:2500 (Doe laboratory); rat monoclonal anti-Dpn (Doe laboratory) at 1:1; mouse anti-Pros monoclonal (purified MR1A at 1:1000; Doe laboratory); rabbit anti-phosphohistone H3at 1:1000 (Sigma, St Louis, MO); rabbit anti-GFP at 1:1000 (Sigma, St Louis, MO); rat anti-Par-6 at 1:250 (Atwood et al., 2007); mouse anti-β galactosidase at 1:500 (Promega); mouse anti- tubulin (at 1:1500, Sigma); guinea pig anti Sec15 at 1:1500 (Hugo Bellen); rabbit anti-Rab11 at 1-1000 (Donald Ready); and rabbit anti-Dap160 at 1:500 (Marie et al., 2004; Roos and Kelly, 1998). Secondary antibodies were obtained from Molecular Probes (Eugene, OR). Embryos were fixed and stained as described previously (Siegrist and Doe, 2005), except that fixing was carried out in 9% paraformaldehyde for 15 minutes. shi^ts2 embryos were shifted to restrictive temperature (37°C) for 30 minutes and subsequently fixed and stained. Larval brains were dissected, fixed and stained as described previously (Siller et al., 2005), and analyzed with a BioRad Radiance 2000 or Leica TCS SP laser scanning confocal microscope using a 60x1.4 NA oil immersion objective. Images were processed with Illustrator software (Adobe).

For live imaging we used the GFP protein-trap line G147 (GFP::Jupiter) that expresses a microtubule-associated GFP fusion protein (Morin et al., 2001), worniu-GAL4, UAS-GFP::Miranda, UAS-CHERRY::Jupiter (C. Cabernard and C.Q.D., unpublished) and worniu-GAL4; UAS-Dap160/TM6B Tb. Live imaging was performed as previously described (Siller et al., 2005), except that temporal resolution was either 15 seconds for 2-hour imaging intervals or 3 minutes for overnight imaging sessions. The 4D data sets were processed in ImageJ (NIH) and Imaris (Bitplane, Switzerland) software.

Statistical analysis
To test the significance in neuroblast number variation between wild-type, dap160 mutant and Dap160-overexpressing brains we used a t-test (two-tailed distribution and two samples, equal variance). To test the significance in NEBD to AO length variation between wild type, dap160 and aPKC mutants we used a t-test (two-tailed distribution and two samples, unequal variance). To test the significance of the differences in polarity protein localization between wild type and dap160 mutant neuroblasts, we performed a z-test using the percent normal crescent for each polarity protein comparing wild type and dap160 mutants; the difference in percent normal between wild type and dap160 mutants was significant with a one-tail confidence level greater than 99.6 % for all polarity proteins analyzed.

	RESULTS

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Dap160 and aPKC are part of the same protein complex
To identify novel components in aPKC pathways of neuroblast cell polarity and neuroblast self-renewal, we sought to identify aPKC/Par-6 interacting proteins by IP/MS using aPKC as our bait protein. IP eluates containing complex mixtures of proteins from a non-specific pull-down (anti-green fluorescent protein; GFP) or anti-aPKC pull down were analyzed by Multiple Dimensions Protein Identification Technology (MUDPIT). These experiments returned Dap160 as a potential aPKC-interacting protein with the highest score of tryptic products hits. Dap160 contains two Eps15 homology domains (EH), a coiled-coil domain (CC) and four Src homology 3 domains (SH3) (Fig. 1A). These domains are conserved in the vertebrate Dap160 homologue intersectin (Fig. 1A), although Dap160 lacks the DH, PH, C2 domains present in the long isoform of intersectin and consequently is not predicted to have GTPase activity (Fig. 1A). Dap160 has two alternative splice variants: a long isoform, which migrates at 160 kDa and a shorter isoform migrating at 120 kDa (Fig. 1A,B).

Next, we sought to verify that Dap160 and aPKC are part of the same protein complex in vivo. An affinity-purified peptide antibody raised against Dap160 C terminus, which recognizes a 160 kDa band in wild-type larval lysate but not in dap160 mutant lysate (Fig. 1B), was used to perform aPKC/Dap160 pull-down experiments. We found that Dap160 and aPKC reproducibly co-immunoprecipitate (Fig. 1C), thus indicating that Dap160 and aPKC are present in the same protein complex. We obtained similar results with a second, independently generated Dap160 antibody (Marie et al., 2004; Roos and Kelly, 1998) (Fig. 2M).

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.

We next tested whether Dap160 increased aPKC activity (similar to the Par-6 binding protein Cdc42) or repressed aPKC activity (similar to the aPKC binding protein Par-6) (Atwood et al., 2007). We co-incubated aPKC protein and a proven fluorescent peptide substrate (Atwood et al., 2007) with increasing amounts of Dap160 protein in the in the presence of Par-6. By measuring the amount of phosphorylated substrate relative to total peptide input, we found that Dap160 increased aPKC activity in a dose-dependent manner (Fig. 1F). To test whether Dap160 stimulation of aPKC is dependent on Par-6, we compared the ability of Dap160 to stimulate aPKC in the presence or absence of Par-6. We found that Dap160 directly stimulates aPKC and that Par-6 could almost completely suppress the ability of Dap160 to activate aPKC (Fig. 1G). Note that aPKC basal activity is variable between experiments (compare Fig. 1F with Fig. 1G) but we found that Dap160 was always able to increase aPKC basal activity. We conclude that the Dap160/aPKC or Dap160/Par-6/aPKC complex is more active than the aPKC/Par-6 complex.

Dap160 and aPKC co-localize in neuroblasts
To determine whether Dap160 has the potential to activate aPKC in neuroblasts, we examined Dap160 localization during neuroblast asymmetric cell division. In wild-type neuroblasts, Dap160 protein is cytoplasmic at interphase, but becomes enriched at the apical cortex during mitosis (Fig. 2A-D); this is identical to aPKC localization (Fig. 2G-K) (Rolls et al., 2003). Two different antibodies produced similar staining in wild-type neuroblasts, and showed no apical Dap160 enrichment in dap160 mutants (Fig. 2B,N), confirming specificity of the antibodies. In larval neuroblasts, Dap160 was undetectable at the cortex at mitosis (Fig. 2E), possibly owing to low protein levels and/or reduced antibody detection sensitivity. Therefore, we used a UAS-dap160 transgene to overexpress Dap160 in larval neuroblasts, and observed enrichment of Dap160 at the apical cortex (90%, n=12; Fig. 2F,L) although weaker ectopic cortical patches could also be observed (66%, n=12; Fig. 4K). This provides further support for the specificity of our antibodies for Dap160 protein. We conclude that Dap160 and aPKC are cytoplasmic in interphase neuroblasts, and can be colocalized at the apical cortex of mitotic neuroblasts.

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.

We further investigated Miranda localization by time-lapse analysis of larval neuroblasts expressing GFP::Miranda and the microtubule-binding protein Cherry::Jupiter (C. Cabernard and C.Q.D., unpublished). In wild-type neuroblasts, GFP::Miranda forms basal crescents during mitosis (100%, n=37; Fig. 4M). In neuroblasts with overexpression of Dap160, we could observe divisions where Miranda was cytoplasmic (8%, n=40 divisions from 19 neuroblasts; Fig. 4N), which we never observed in wild type. We conclude that Dap160 positively regulates aPKC localization and activity, and is required for aPKC-mediated neuroblast cortical polarity.

Dap160 regulates the number of proliferating larval neuroblasts
An important function of aPKC is to maintain larval neuroblast pool size: aPKC mutants have fewer proliferating larval brain neuroblasts and overexpression of a membrane-tethered aPKC increases the number of larval brain neuroblasts (Lee et al., 2006). Here, we test whether decreasing or increasing Dap160 levels has a similar effect on brain neuroblast numbers. We find that wild-type third larval instar brain lobes contain 96±5 (n=5) Deadpan-positive (Dpn⁺) neuroblasts, whereas similarly staged dap160 mutants contain only 72.6±6 (n=5); P=0.0002 Dpn⁺ neuroblasts (Fig. 4A). We conclude that both Dap160 and aPKC are required to maintain the normal number of proliferating neuroblasts in the larval brain. The loss of neuroblasts in the dap160 mutant brain could be due to neuroblast death, differentiation or the failure of neuroblasts to exit from quiescence during larval stages (see Discussion).

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.

Dap160 and aPKC are required for neuroblast cell cycle progression
We next tested whether Dap160 and aPKC are required for neuroblast cell cycle progression. We performed time lapse imaging of neuroblast cell cycle progression in both dap160 and aPKC mutant larval neuroblasts expressing the spindle marker GFP::Jupiter. Wild-type neuroblasts take 7.76±2.04 (n=15) minutes to transit from nuclear envelope breakdown (NEBD) to anaphase onset (AO; Fig. 5A), consistent with previous reports (Siller et al., 2006; Siller and Doe, 2008; Siller et al., 2005). By contrast, progression through mitosis (NEBD-AO) was delayed in both dap160 mutants and aPKC mutants: 13.37±4.4 minutes, n=10; P=0.006 in dap160 mutant neuroblasts (Fig. 5B) and 17.84±4.52 minutes, n=11; P=7.6x10^-6 in aPKC mutant neuroblasts (Fig. 5C). In addition, dap160 mutant neuroblasts had a longer interphase length (often over 12 hours; data not shown) compared with an average of 2 hours for wild-type neuroblasts (Siller and Doe, 2008) (C. Cabernard and C.Q.D., unpublished). We conclude that Dap160 and aPKC promote cell cycle progression in larval neuroblasts.

	DISCUSSION

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
aPKC plays an important role in regulating cell polarity, neural stem cell identity and neural stem cell proliferation in vertebrates and invertebrates (Costa et al., 2008; Grifoni et al., 2007; Lee et al., 2006; Rolls et al., 2003). Here, we show that Dap160 positively regulates aPKC activity and localization in neuroblasts, and is required for effective aPKC function in establishing neuroblast cell polarity and cell cycle progression.

Dap160 binds aPKC and increases kinase activity
Par6 directly interacts with aPKC to suppress aPKC activity, while Cdc42 binds Par-6 and modestly upregulates aPKC activity (Atwood et al., 2007; Etienne-Manneville and Hall, 2001; Henrique and Schweisguth, 2003; Hirano et al., 2005). Our study shows that Dap160 directly interacts with both aPKC and Par-6, and can stimulate aPKC activity independent of Par-6. However, Par-6 reduces the ability of Dap160 to stimulate aPKC, suggesting that the Dap160/aPKC complex is more active than Dap160/aPKC/Par6 complex, which in turn is more active than the aPKC/Par6 complex. The exact binding sites for the Dap160/aPKC interaction are unknown; good candidates would be the Dap160 SH3 domains and the two SH3-binding motifs (P-X-X-P) in aPKC.

Dap160 regulates aPKC localization
How does Dap160 promote aPKC localization and activity? Because Dap160 is known to bind Dynamin to regulate endocytosis, it is possible that Dap160 may promote apical aPKC localization by clearing aPKC from the basal cortex. Arguing against this model is our finding that a dynamin mutant (shi^ts2) does not affect the apical localization of aPKC or basal localization of Miranda and Numb (Fig. 3K-M; compare with dap160 phenotype shown in Fig. 3F,I,J). We favor a model in which Dap160 regulates aPKC localization via an endocytosis-independent mechanism. In support of endocytosis-independent functions for Dap160, its vertebrate homolog, intersectin, has both endocytosis and signaling functions. The signaling function requires protein domains shared between Dap160 and intersectin, and includes binding and recruiting mammalian son-of-sevenless (Sos) to the plasma membrane where it regulates Ras signaling (Tong et al., 2000a; Tong et al., 2000b).

Dap160 may promote aPKC localization indirectly, by increasing aPKC kinase activity. aPKC is required to stabilize the Par complex (Baz/Par-3, Par-6, aPKC) in many cell types, including neuroblasts (Atwood et al., 2007), and it is likely that lowered aPKC activity would destabilize anchoring proteins such as Bazooka from the neuroblast apical cortex. In support of this model, we observe a weakening of the Bazooka crescent in dap160 mutant embryonic neuroblasts.

Another possibility is that Dap160 regulates aPKC cortical polarity via vesicle transport. Consistent with this model, Dap160 controls synaptic vesicle transport in Drosophila nerve terminals (Marie et al., 2004), and aPKC (PKC-3), Par-6 and Cdc42 regulate vesicle transport in C. elegans embryos and mammalian cells (Balklava et al., 2007). In fact, Dap160 overexpression in neuroblasts results in enlarged vesicles that are positive for aPKC and the exocyst marker Sec15 (Fig. 2O and data not shown), suggesting that Dap160 may direct aPKC-positive vesicles to the apical cortex. One appealing model that awaits testing is that polarized vesicle transport localizes Par proteins to the neuroblast apical cortex, and in turn Par proteins restrict differentiation factors such as Miranda and Numb to the basal cortex.

dap160 mutant embryonic neuroblasts have defects in Baz localization, but dap160 mutant larval neuroblasts show normal Baz localization (data not shown). This may reflect a difference in the mechanism of Baz localization between embryonic and larval neuroblasts, because aPKC mutants also have normal Baz localization in larval neuroblasts (Rolls et al., 2003).

Dap160 and aPKC promote neuroblast cell cycle progression
We provide the first evidence that Dap160 and aPKC promote cell cycle progression in neuroblasts. The related vertebrate aPKC regulates cell proliferation in Xenopus oocytes, mouse fibroblasts and in human glioblastoma cell lines (Berra et al., 1993; Donson et al., 2000), indicating that aPKC promotes cell cycle progression in many cell types. Similarly, the vertebrate Dap160-related intersectin protein is sufficient to induce oncogenic transformation of rodent fibroblasts (Adams et al., 2000), indicating that intersectin can also promote cell cycle progression. It would be interesting to investigate the relationship between intersectin and PKC in this tumor model system.

Dap160 and aPKC maintain proliferating neuroblast pool size
We find that both dap160 and aPKC mutant larvae have a partial reduction in the number of proliferating neuroblasts (this work; Lee et al., 2006b). This may be due to neuroblasts differentiating or dying. We cannot exclude neuroblast death, although we see no difference in caspase 3 staining (a cell death marker) between wild-type and mutant larval brains (data not shown). Differentiation of the mutant neuroblasts is more likely, based on our observation that the Miranda differentiation factor scaffolding protein frequently fails to be properly segregated into the GMC during neuroblast asymmetric cell division in dap160 or aPKC mutants. Arguing against this model is the fact that dap160 or aPKC single neuroblast mutant clones maintain a single neuroblast in all cases examined. It is possible that the mutant single neuroblast clones may not fully deplete Dap160 or aPKC protein, e.g. owing to a long half-life of the mRNA or protein. Indeed, neuroblasts in dap160 mutant clones have a weaker cortical polarity phenotype than neuroblasts in dap160 organismal mutants, consistent with the presence of residual Dap160 protein in the clones. Alternatively, loss of Dap160 or aPKC outside of the neuroblast lineage may lead to the observed decrease in neuroblast numbers in the whole animal mutants, and this would not be seen in the single neuroblast mutant clones.

Misexpression of Dap160 produces a modest increase in the number of proliferating neuroblasts in second instar larvae, whereas misexpression of cortically tethered aPKC results in a dramatic expansion of neuroblast numbers at all larval stages (Lee et al., 2006). It is likely that activity levels of aPKC are limiting in Dap160 overexpression experiments: unknown proteins(s) may oppose Dap160 stimulation of aPKC (similar to Par-6) leading to a weakly active aPKC and thus causing only a modest increase in neuroblast numbers. Surprisingly, we found that prolonged misexpression of Dap160, into the third larval instar, resulted in loss of neuroblasts. This could be due to neuroblast cell death caused by continued exposure of the neuroblast to elevated levels of Dap160 protein.

	ACKNOWLEDGMENTS

 
We thank Graeme Davis, Bruno Marie, Hugo Bellen, Scott Atwood, Donald Ready and Mani Ramaswami for providing reagents. We thank Laurina Manning for technical assistance. We also thank members of the Doe laboratory, Bruce Bowerman and Kenneth Prehoda for comments on the manuscript. This work was supported by HHMI and AHA pre-doctoral fellowship (0615592Z).

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