Magi Is Associated with the Par Complex and Functions Antagonistically with Bazooka to Regulate the Apical Polarity Complex

We would like to thank Drs. Dave Bilder, Chris Doe, Denise Montell, Gregory Longmore, Vivian Budnik, Herbert Jäckle and Andreas Wordarz for generously providing reagents, the Bloomington Drosophila Stock Center, and Dr. Jody Summers for the generous permission to use her Olympus FV1000 confocal microscope. We thank Kate O’Connor-Giles and Scott Gratz for advice on CRISPR/Cas9 and for providing their U6-3 vector. We thank the TRiP at Harvard Medical School (NIH/NIGMS R01-GM084947) for providing stocks and the Developmental Studies Hybridoma Bank (University of Iowa, Department of Biology, Iowa City) for antibodies. This research was supported by grants from the Canadian Institute of Health Research (MOP-82862 to VJA) and in part from the National Institutes of Health/National Institute of Neurological Disorders and Stroke (RO1-NS060878 to BZ) and by an internal fund from the University of Missouri (to BZ).The mammalian MAGI proteins play important roles in the maintenance of adherens and tight junctions. The MAGI family of proteins contains modular domains such as WW and PDZ domains necessary for scaffolding of membrane receptors and intracellular signaling components. Loss of MAGI leads to reduced junction stability while overexpression of MAGI can lead to increased adhesion and stabilization of epithelial morphology. However, how Magi regulates junction assembly in epithelia is largely unknown. We investigated the single Drosophila homologue of Magi to study the in vivo role of Magi in epithelial development. Magi is localized at the adherens junction and forms a complex with the polarity proteins, Par3/Bazooka and aPKC. We generated a Magi null mutant and found that Magi null mutants were viable with no detectable morphological defects even though the Magi protein is highly conserved with vertebrate Magi homologues. However, overexpression of Magi resulted in the displacement of Baz/Par3 and aPKC and lead to an increase in the level of PIP3. Interestingly, we found that Magi and Baz functioned in an antagonistic manner to regulate the localization of the apical polarity complex. Maintaining the balance between the level of Magi and Baz is an important determinant of the levels and localization of apical polarity complex.Yeshttp://www.plosone.org/static/editorial#pee

Kinase control of the tricellular junction protein Gliotactin, and Gliotactin-induced phenotypes in epithelia

This thesis investigates the kinase-mediated regulation of the tricellular junction protein, Gliotactin and signaling pathways involved in Gliotactin overexpression-induced detrimental phenotypes. Tricellular junctions (TCJ) are uniquely placed permeability barriers formed in polarized epithelia where tight junctions in vertebrates or septate junctions in invertebrates from three cells converge. Misregulation of TCJ specific proteins is detrimental to life. However, mechanisms of their localization, maintenance, and potential signaling are largely unknown. Gliotactin is a transmembrane protein unique to TCJ in Drosophila and is essential for the maturation and maintenance of both bicellular and tricellular septate junctions. However, overexpression of Gliotactin leads to the spread of Gliotactin away from the TCJ and disrupts epithelial architecture by signaling for overproliferation, delamination, migration and apoptosis. One mechanism to control Gliotactin is phosphorylation of two highly conserved tyrosine residues and subsequent endocytosis. However, Gliotactin tyrosine phosphorylation also elicits detrimental phenotypes when dysregulated. The kinases involved in Gliotactin phosphorylation had not been broadly investigated prior to this work. We carried out an RNAi screen for phospho-regulators (kinases and some kinase-associated proteins) to determine which could modify the detrimental phenotypes triggered by Gliotactin overexpression. Four suppressors, four partial suppressors, and 53 enhancers were identified by screening 275 RNAi lines covering 164 genes. We determined that Gliotactin overexpression phenotypes involved TNF-JNK, PI3K-Akt signaling pathways and Btk29A. C-terminal Src kinase (Csk), Ret, PI4KIII-α, Skittles and Pkaap were also identified as candidates for further studies. We focused our analysis on Csk and determined Csk is a regulator of Gliotactin endocytosis and plays a role in the regulation of Gliotactin at the TCJ. Although Csk is known as a negative regulator of Src kinases, we identified that the effect of Csk on Gliotactin is independent of Src, and likely occur through an AJ-associated complex. Taken together, this thesis provides novel insights on the function of Csk and identifys other candidate kinases that have the potential to regulate localization and/or signaling events associated with TCJ formation and function.Science, Faculty ofZoology, Department ofGraduat

A balance between the levels of Magi and Baz regulates aPKC membrane levels.

<p>apterous-GAL4 was used to overexpress different combinations of tagged proteins in the wing imaginal disc. The average membrane intensity of each protein was measured and compared between the apterous (black bars) and non-apterous (white bars) side of the wing imaginal disc. The white lines mark the apterous dorsal/ventral boundary. (A-H) Overexpression of aPKC::GFP (green) had no effect on the membrane localization or levels of Magi (red) (A-D) or Baz (red) (E-H). (I-L) Co-expression of Magi::Cherry (blue) and Baz::GFP (green) attenuated the changes in aPKC (red) caused by high levels of Baz or Magi alone. aPKC levels were not increased by overexpression of Baz::GFP when Magi::Cherry was co-expressed. (M-P) Coexpression of Magi::Cherry (blue) and aPKC::GFP (green) lead to a reduction in the Baz membrane levels (red) and increased accumulation in the Magi vesicles. Overexpression of wildtype aPKC did not block the Magi induced reduction in Baz. (Q-X) Higher resolution image of the large accumulations seen with Magi::Cherry expression at the basolateral region of the epithelial cells. Each panel was digitally magnified 200%. (Q-T) Co-expression of Magi::Cherry (blue) and Baz::GFP (green) lead to the accumulation of Baz and Magi within the large internal accumulations. aPKC (red) was only weakly recruited (arrows). (U-X) Co-expression of Magi::Cherry (blue) and aPKC::GFP (green) lead to an increased accumulation of Magi vesicles that were also positive for Baz (red) (arrows). *** p<0.001; * p<0.05; ns not significant. Error bars indicate SEM. n = 5 discs for each experiment. Scale bars indicate 5μm for A-O and 2μm for Q-X.</p

Magi localization requires aPKC but not the other polarity proteins, Baz, Sdt or Crb.

<p>(A-I) Wing imaginal discs showing mitotic clones mutant for apical polarity protein genes, <i>baz</i>, <i>sdt</i> and <i>crb</i> (clone boundaries are indicated by white lines). Mutant clones for <i>baz</i>, <i>sdt</i> and <i>crb</i> survive and no polarity defects are seen. Somatic clones are marked by the loss of a cellular marker (GFP or RFP). Loss of these polarity proteins had no effect on membrane recruitment of Magi (red). In <i>crb</i> mutant clones, the continuous localization of Magi on the plasma membrane was altered. (J-L) Wing imaginal disc showing mitotic clones mutant for <i>aPKC</i>. Only small <i>aPKC</i> mutant clones were recovered and frequently twin spots were not associated with mutant clones. Magi (red) did not appear to be affected in the small <i>aPKC</i> mutant clones. (M-O) Wing imaginal discs co-expressing aPKC-RNAi and p35 to block cell death (<i>ap>aPKC-RNAi</i>, <i>p35</i>). Temporal control of expression was controlled using Gal80ts. Blocking cell death resulted in tissue overgrowth and loss of apicobasal polarity as Ecad (green) localization was impaired. Magi (red) localization to the plasma membrane was also disrupted. Scale bars indicate 5μm in A-L and 10μm in M-O.</p

Somatic null clones of <i>Magi</i> are viable with no loss of polarity or AJ.

<p>Mutant clones of <i>Magi</i>^<i>bst</i> in the wing imaginal disc were generated by FRT mediated recombination. Nuclear-localized GFP driven by the ubiquitin promoter (Ubi-nlGFP, green) labeled wildtype and heterozygous cells, and <i>Magi</i>^<i>bst</i> clones are indicated by the lack of GFP. The large GFP positive nuclei are from the overlying peripodial cells. <i>Magi</i>^<i>bst</i> clones lack Magi immunolabeling (A-D) but had no effect on the levels and localization of Baz (E-H, red), aPKC (I-L, red) or the adherens junction protein Armadillo (Arm, red) (M-P). There was no effect on cell survival, which is shown by lack of apoptotic marker cleaved Caspase-3 (Cas3, red) (Q-S). Panels D,H,L,P,T were digitally magnified 200% and the clonal boundaries are marked with white lines. Scale bars indicate 10μm.</p

Overexpression of Magi reduces Baz at the plasma membrane.

<p>Cherry-tagged Magi was overexpressed in the wing imaginal disc using apterous-GAL4 (ap>Magi::Cherry). The average membrane intensity of Baz, and aPKC was measured and compared between the apterous (ap-GAL4, black bars) and non-apterous side (WT, white bars) of the wing imaginal disc (X). The apterous boundary is indicated with a white line and in all panels the apterous side is to the left. All panels represent a single Z slice within the apical or basal domain of the columnar epithelia (H). (A-D) Wing imaginal disc overexpressing Magi::Cherry (red) immunolabeled for Baz (green). High levels of Magi result in mislocalization of Baz to the cytoplasm and a significant reduction on the plasma membrane (D). (E-G) At the basolateral region of the epithelia (H), Baz (green) was detected in large puncta (arrows) with Magi::Cherry (red). (I-L) Wing imaginal discs overexpressing Magi::Cherry (red) immunolabeled for aPKC (green). aPKC was significantly reduced at the plasma membrane (I) and present in small cytosolic puncta. (M-P) Wing imaginal disc expressing a Magi transgene lacking the PDZ domains (MagiΔPDZ) tagged with the FLAG epitope (red). Expression of this transgene had no effect on the membrane localization of Baz (green)(P) and the Magi protein appeared to be cytosolic. (Q-T) Wing imaginal disc expressing a Magi transgene lacking the two WW domains (MagiΔWW) tagged with the FLAG epitope (red). Expression of this transgene resulted in a significant reduction in Baz (green)(T). (U-W) MagiΔWW was found at the membrane but did not form large intracellular accumulations in basal regions. *** p<0.001; ns p>0.05. n = 5 discs for each experiment and error bars indicate SEM. Panels (E-G, U-W) were digitally magnified 200%. Scale bars indicate 5μm.</p

Magi localizes to the apicolateral region and adherens junctions in epithelia.

<p>(A-C). Enface view of the cellularizing embryo showing Magi (green) localization to the furrow canal where Baz (red) is present. (D-F). Enface view of stage 11 embryo showing Magi (green) present in the same membrane domain as Baz (red). (G-I). Enface view of stage 13 embryo showing Magi (green) is present in the same membrane domain as Baz (red) in both epithelial and amnioserosa cells. (J-L). Side view of the salivary gland showing Magi (green) and Baz (red) colocalizing in the same membrane region at the apicolateral domain. (M-O). Side view of the hindgut showing Magi (green) and Baz (red) colocalization at the apicolateral membrane. (P-R). Side view of the salivary gland showing Magi (green) is below the apical membrane domain where Crumbs (Crb, red) is present. (S-V’). Enface and side views of wing imaginal disc epithelia showing that Magi (green) predominantly colocalizes with Baz (Baz::GFP, red) but not with Dlg (blue). (W-Y’). Enface and side view of the wing imaginal disc epithelia showing that Magi (green) is present at the adherens junction, marked with Ecad (red) immunolabeling. Scale bars indicate 10μm. Each enface view represents a single Z slice.</p

Magi is associated with lipid subdomains and alters PIP3 levels.

<p>(A-I) Cherry-tagged Magi (red) was expressed using apterous-GAL4 and accumulations within the basolateral region. (A-C) Magi::Cherry accumulations were positive for the ER marker Calnexin (green), arrows. (D-F) PIP3 (PH::GFP; green) was co-localized in the large accumulations (arrows) with Magi::Cherry. (G-I) Magi::Cherry accumulations were positive for Cholera toxin B (CTB, green)(arrows). (J-L) Endogenous Magi (red) colocalized with cholera toxin B (CTB, green) in wildtype discs at the plasma membrane. (M-S) Cherry-tagged Magi was overexpressed using apterous-GAL4 (ap>Magi::Cherry). The apterous boundary is indicated with a white line. (M-P) Wing imaginal discs overexpressing Magi::Cherry (red) with a PIP3 indicator (PH::GFP, green) under the control of the tubulin promoter. High levels of Magi resulted in an increase in of PIP3 at the plasma membrane. *** p<0.001; n = 5 discs. Error bars indicate SEM. (Q-S) Side projections showing the increase in PIP3 is within the apical domain (arrow) and within the large Magi accumulations (arrowhead). (T-Y) PTEN was expressed using apterous-GAL4 with Magi::Cherry (T-V) or without (W-Y). (T-V) Baz (green) was still displaced from the membrane when PTEN (blue) and Magi::Cherry (red) were coexpressed. (W-Y) Expression of PTEN alone had no effect on Magi recruitment to the membrane (arrowhead). Panels (A-L) were digitally magnified 200%. Scale bars indicate 5μm</p

Overexpression of Baz results in loss of Magi from the plasma membrane.

<p>GFP-tagged Baz was overexpressed in the wing imaginal disc using apterous-GAL4 (ap>Baz::GFP). The average membrane intensity of each protein was measured and compared between the apterous (black bars) and non-apterous side (white bars) of the wing imaginal disc. The white lines mark the apterous dorsal/ventral boundary. All images showing the apical region of the epithelia. (A-D) Overexpression of Baz::GFP (green) immunolabeled for Magi (red). Cells containing high levels of Baz show a significant reduction in the membrane levels of Magi. (E-P) Overexpression of Baz::GFP (green) resulted in a significant increase in the membrane levels of aPKC (red) (E-H), Crb (red) (I-L) and Par-6 (red) (M-P). (Q-V) Overexpression of Baz::GFP (green) lead to membrane ruffling (arrows) but not the accumulation of Magi (red) or aPKC (red) in intracellular puncta. *** p<0.001; ** p<0.01. n = 5 discs for each experiment and error bars indicate SEM. Panels (Q-V) were digitally magnified 200%. Scale bars indicate 5μm</p

Data from: Spatial genetic structure of the mountain pine beetle (Dendroctonus ponderosae) outbreak in western Canada: historical patterns and contemporary dispersal

Environmental change has a wide range of ecological consequences, including species extinction and range expansion. Many studies have shown that insect species respond rapidly to climatic change. A mountain pine beetle epidemic of record size in North America has led to unprecedented mortality of lodgepole pine, and a significant range expansion to the northeast of its historic range. Our goal was to determine the spatial genetic variation found among outbreak population from which genetic structure, and dispersal patterns may be inferred. Beetles from 49 sampling locations throughout the outbreak area in western Canada were analysed at 13 microsatellite loci. We found significant north-south population structure as evidenced by: (i) Bayesian-based analyses, (ii) north-south genetic relationships and diversity gradients; and (iii) a lack of isolation-by-distance in the northernmost cluster. The north-south structure is proposed to have arisen from the processes of postglacial colonization as well as recent climate-driven changes in population dynamics. Our data support the hypothesis of multiple sources of origin for the outbreak and point to the need for population specific information to improve our understanding and management of outbreaks. The recent range expansion across the Rocky Mountains into the jack/lodgepole hybrid and pure jack pine zones of northern Alberta is consistent with a northern British Columbia origin. We detected no loss of genetic variability in these populations, indicating that the evolutionary potential of mountain pine beetle to adapt has not been reduced by founder events. This study illustrates a rapid range-wide response to the removal of climatic constraints, and the potential for range expansion of a regional population