Recruitment of the Par complex protein atypical Protein Kinase C (aPKC) to a specific membrane domain is a key step in the polarization of animal cells. While numerous proteins and phospholipids interact with aPKC, how these interactions cooperate to control its membrane recruitment has been unknown. Here we identify aPKC’s C1 domain as a phospholipid interaction module that targets aPKC to the membrane of Drosophila neural stem cells (NSCs). The isolated C1 binds the NSC membrane in an unpolarized manner during interphase and mitosis and is uniquely sufficient among aPKC domains for targeting. Other domains, including the catalytic module and those that bind the upstream regulators Par-6 and Bazooka, restrict C1’s membrane targeting activity – spatially and temporally – to the apical NSC membrane during mitosis. Our results suggest that aPKC polarity results from cooperative activation of autoinhibited C1-mediated membrane binding activity.

Drosophila larval brains were dissected, and the tissue was incubated in 4% PFA fixative for 20’ within 20’ of dissection. This and all subsequent wash steps involved agitation by placing on a nutator. After fixation, brains were rinsed and washed three times for 15’ each in PBST (1xPBS with 0.3% Triton-X). Brains were stored for up to three days at 4 ̊C before staining. Before staining, brains were blocked for 30’ in PBSBT (PBST with 1% BSA) and then incubated with primary antibody solution overnight at 4°C. Brains were subsequently rinsed and washed three times for 15’ each in PBSBT and incubated with secondary antibody (conjugated to Cy3, AlexaFluor 647, AlexaFluor 488, or AlexaFluor 405 as appropriate) for 2 hours in a vessel that protected the sample from light. Brains were subsequently rinsed and washed three times for 15’ each in PBST followed by storage in SlowFade w/DAPI at least overnight before imaging. Brains were imaged at room temperature on an upright Leica TCS SPE confocal using an ACS APO 40x 1.15 NA Oil CS objective or an Olympus FluoView FV1000 upright laser scanning confocal with PlanApo N 60x 1.42 NA oil objective. Acquisition was controlled with Leica LAS X or FluoView software, respectively.

Files are in ImageJ TIFF (.tif) or Olympus confocal (.oib) format.