Naheed Ali, MD, PhD
The apical-basal axis of epithelial cells is polarised by the small GTPase Cdc42. It also activates Pak1, a second kinase, to define the identity of the apical domain in mammalian and Drosophila epithelia. The epithelial polarity and morphology are completely lost when the Pak1 and aPKC kinases are inactive. Pak1 serves a purpose downstream of Cdc42. Our findings identify a dual-kinase mechanism that is conserved for controlling the identity of the apical membrane in epithelia.
A group of cell polarity determinants has been discovered through ground-breaking studies of genetics in model organisms like the yeast Saccharomyces cerevisiae and Drosophila melanogaster (fruit fly). These determinants are necessary for the polarization of all other organelles, molecules, and cytoskeletal components in the cell (Thompson, 2013). In many species, the small (GTPase) GTP-binding protein, Cdc42 in particular plays a crucial role in controlling cell polarity. Cdc42 joins the kinase aPKC and Par6 to form a complex in epithelial cells (Genova et al., 2000, Garrard et al., 2003, Ohno, 2001, Yamanaka et al., 2001, Hutterer et al., 2004, Wodarz et al., 2000, Joberty et al., 2000, Peterson et al., 2004, Petronczki and Knoblich, 2001) that either Crumbs (Crb) complex (“PALS1-associated tight junction [PATJ]” or Crb-Sdt/PALS1) or the Bazooka (Baz/Par3) recruits to the plasma membrane to define the membrane of apical domain(Tanentzapf and Tepass, 2003, Fletcher et al., 2012, Penkert et al., 2004, Hurd et al., 2003, Benton and St Johnston, 2003, Joberty et al., 2000).
In Drosophila epithelia, null mutants for cdc42, par6, or aPKC cause a total loss of the apical domain (Fletcher et al., 2012, Hutterer et al., 2004, Petronczki and Knoblich, 2001, Rolls et al., 2003, Wodarz et al., 2000); however, recent research showed that polarity of apical-basal in Drosophila epithelia was not entirely disrupted by kinase-impaired mutants in aPKC (Kim et al., 2009) (Figure S1). This unexpected finding suggests that while aPKC's kinase activity is not necessary, its scaffold function is.
A study has shown that the identity of apical membrane also necessitates kinase activity Pak1, Cdc42 downstream. Pak1 now serves a function distinct from its previously reported functions as a regulator of integrins or E-cadherin. (Santiago-Medina et al., 2013, del Pozo et al., 2000, Dummler et al., 2009, Tomar and Schlaepfer, 2010, Harden et al., 1996, Lucanic and Cheng, 2008, Pirraglia et al., 2010, Conder et al., 2007). By phosphorylating a set of targets that overlap and taking actions in a genetically semi-redundant manner, Pak1 appears to work similarly to aPKC. These results define the identity of apical domain in epithelial cells.
Mitotic clones were generated in follicle cells using the FRT (“Flipase recombination target”)/FLP (Flipase) system and were either positive (presence of MARCM; GFP) or negative (absence of GFP; MARCM) (GFP absence). Heat shock was given to the third instar larvae for 1 hour at 37°C and dissected 3 days later. The tj.Gal4 line, the MARCM system, and the actin "flip-out" system were used to express upstream activation sequence (UAS) transgenic lines. Third instar larvae were subjected to heat shock at 37°C for 20 minutes and dissected three days after eclosion to create "flip-out" clones.
After being dissected in PBS, ovaries were fixed for 20 minutes in PBS in 4% paraformaldehyde, washed for 30 minutes in” 0.1% Triton X-100 (PBT)/PBS”, and were blocked for 15 minutes in normal goat serum 5% (NGS)/PBT. Samples were incubated with diluted primary antibodies in NGS/PBT for an overnight period at 4°C. After using secondary antibodies for two hours at room temperature, slides were mounted with Vectashield (Vector Laboratories). A “Leica SP5 confocal microscope” was used to capture the images, which were then edited in Adobe Photoshop.
The antibodies used primarily were “rat anti-E-cadherin (DCAD2) (1:100, DSHB)”, rabbit anti-PKCζ (C-20) (1:250), rabbit anti-pBazS980 (1:100, DSHB), mouse anti-Dlg (1:250, DSHB), and mouse anti-βPS. Rabbit anti-Baz (1:250), rabbit anti-BazS98 (1:100, DSHB).
The antibodies that were used secondarily are goat Alexa Fluor 546, 488, or 647 (1:500, Invitrogen), phalloidin (2.5:250, Life Technologies), and “4′,6-diamidino-2-phenylindole (DAPI”; 1 μg/mL, Life Technologies) to stain F-actin and nuclei.
Egg chambers of wild-type were cultured for 1 or 2 hours at room temperature in imaging media containing insulin (Sigma), Schneider's Media (Invitrogen), Ecdysone (Sigma), adenosine deaminase (Roche), “heat-inactivated fetal calf serum” (FCS; GE Healthcare), Methoprene, and trehalose (Sigma), with aPKC inhibitor (0.01 mM). Following treatment, the samples were fixed and imaged as usual.
In vitro kinase assays were performed using (HPLC) “high-pressure liquid chromatography” purified peptide substrates incubated with either 0.1 μM recombinant human aPKCiota kinase domain or 150 pg recombinant human Pak1 kinase domain (AbNOVA) at 30°C for 30 minutes in “kinase reaction buffer” (250 mM HEPES (PerkinElmer). Samples were blotted on Millipore “P81 phosphocellulose paper” and washed three times for 10 minutes in 1% phosphoric acid and once in acetone. The Y-P32 incorporation was measured in counts per min using scintillation (Beckman LS 6500).
The following peptides were employed in this study: