Summer Undergraduate Research Program - Fred Hutchinson ...
Summer Undergraduate Research Program - Fred Hutchinson ...
Summer Undergraduate Research Program - Fred Hutchinson ...
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Role of Dlg5 in Lung Branching Morphogenesis<br />
Julia Rosenberg 1,2 , Tamilla Nechiporuk 2,3 , Olga Kezovitch 2 , and Valeri Vasioukhin 2<br />
1. Cornell University, Ithaca, NY 14853 2. <strong>Fred</strong> <strong>Hutchinson</strong> Cancer <strong>Research</strong> Center, Seattle, WA 98109 3. OHSU, Vollum Institute, Portland, OR 97239<br />
3. PDZ domains of Dlg5 are involved in interaction with aPKC:<br />
METHODS<br />
RhoA and Rac Protein Assays:<br />
Analysis of differential RhoA and/or Rac activity in wild-type and Dlg5<br />
knockout E15.5 lungs<br />
•Total protein extracts of E15.5 and wild-type were analyzed<br />
•Cytoskeleton Inc. Kits were used to determine activity<br />
INTRODUCTION<br />
The evolutionarily conserved Dlg5 protein plays a crucial role in<br />
epithelial cell adhesion and polarity in mammalian brain and kidneys.<br />
Our laboratory recently found that Dlg5 also affects lung morphogenesis.<br />
Dlg5 knockout embryos display lung branching morphogenesis defects,<br />
and mutant animals develop lung emphysema. Loss of apical localization<br />
of aPKC is one of the earliest phenotypes in Dlg5-/- lungs, and abnormal<br />
apical-basal polarity may explain the branching morphogenesis defect.<br />
Here we analyzed potential mechanisms responsible for lung<br />
morphogenesis abnormalities in Dlg5-/- mice.<br />
2009 Best Poster Award<br />
V5-Dlg5 Construct Flag-aPKC Pulled Down by aPKC?<br />
1. Mock Control - No<br />
2. + V5-Full-Length Dlg5 + Yes<br />
3. V5-Full Length Dlg5 - No<br />
4. V5-N-terminal + Weakly<br />
5. V5-N-terminus - No<br />
6. V5-SH3-MAGUK + No<br />
7. V5-SH3-MAGUK - No<br />
8. V5-PDZ1-PDZ2 + Weakly<br />
9. V5-PDZ1-PDZ2 - No<br />
10. V5-PDZ3-PDZ4 + Strongly<br />
11. V5-PDZ3-PDZ4 - No<br />
12. V5-PDZ1 + Yes<br />
13. V5-PDZ1 - No<br />
14. V5-PDZ2 + Yes<br />
15. V5-PDZ2 - No<br />
16. V5-PDZ3 + Unknown<br />
17. V5-PDZ3 - No<br />
18. V5-PDZ4 + Unkown<br />
19. V5-PDZ4 - No<br />
Co-Immunoprecipitation (Co-IP):<br />
Analysis of Dlg5–aPKC and Dlg5-Lgl1 Interactions<br />
•Wild-type embryonic mouse lungs (E15.5) were dissected<br />
•Total protein lysates underwent immunoprecipitation with either:<br />
•Protein-A sepharose beads preabsorbed with anti-Dlg5 serum<br />
•Protein-A sepharose beads preabsorbed with pre-immune serum<br />
•Results from IP were analyzed by Western blotting with:<br />
•Anti-nPKC, anti-Lgl1, and anti-Dlg5 antibodies<br />
Input After IP Input After IP Input After IP Input After IP Input After IP Input After IP Input After IP Input After IP Input After IP<br />
Input After IP<br />
WB:<br />
1 1 2 3 2 3 4 5 4 5 6 7 6 7 8 9 8 9 10 11 10 11 12 13 12 13 14 15 14 15 16 17 16 17 18 19 18 19<br />
Co-Transfection and Overexpression:<br />
Identification of domain of Dlg5 interaction with aPKC<br />
•Human Embryonic Kidney (HEK) 293 FT cells were transfected with<br />
V5-Dlg5 constructs alone with Flag-aPKC<br />
•Cells were lysed and immunoprecipitation was performed using antiflag<br />
agarose beads<br />
Figure 1. Dlg5 expression pattern (A) and its domain organization and protein conservation (B).<br />
1 2<br />
161<br />
Anti-<br />
V5<br />
1 1 2 3 2 3 4 5 4 5 6 7 6 7 8 9 8 9 10 11 10 11 12 13 12 13 14 15 14 15 16 17 16 17 18 19 18 19<br />
Anti-<br />
Flag<br />
RESULTS and DISCUSSION<br />
3<br />
Figure 5. Summary and the results of co-IP experiments. Indicated<br />
V5-Dlg5 and Flag-aPKC constructs (1-19) were expressed in HEK293FT<br />
cells, immunoprecipitated with anti-Flag and analyzed by Western<br />
blotting with anti-V5 and anti-Flag antibodies. Proteins were analyzed<br />
before (input) and after anti-Flag (after IP) immunoprecipitation.<br />
1. Dlg5 does not significantly affect RhoA or Rac activity:<br />
Figure 2. Lung emphysema and branching defects in Dlg5-/- mice. 1. Abnormal lung histology<br />
in mutant lungs 2. Loss of apical localization of aPKC in Dlg5-/- lungs. 3. Abnormal branching<br />
morphogenesis in E13.5 Dlg5-/- lungs.<br />
Figure 3. Activity assays indicated no significant difference in RhoA<br />
and/or Rac activity in wild-type and knockout Dlg5 E15.5 lungs.<br />
OBJECTIVES<br />
The objective of this study is to better characterize the role of Dlg5 in<br />
lung branching morphogenesis through the following aims:<br />
2. Interaction between endogenous Dlg5 and aPKC in<br />
developing mouse lungs:<br />
FUTURE DIRECTIONS<br />
•Elucidate if Dlg5 PDZ3 and/or PDZ4 domains interact with aPKC<br />
Pre-Immune Anti-Dlg5<br />
Input<br />
1.Determine if Dlg5 regulates RhoA and/or Rac1 activity. Rac and Rho<br />
are small GTPases that influence actin cytoskeleton. Actin dynamics are<br />
necessary for branching morphogenesis, and inhibition of RhoA results in<br />
branching morphogenesis defect.<br />
•Analyze Dlg5 localization in developing lungs<br />
•Perform lung-specific loss-of-function aPKC experiments to determine<br />
whether loss of aPKC is sufficient for branching morphogenesis defects.<br />
Figure 4. Co-IP experiments with proteins<br />
extracted from E15.5 embryonic lungs. Total<br />
protein (input) were pulled down with preimmune<br />
or anti-Dlg5 (immune) antibodies<br />
and analyzed by Western blotting with antiaPKC,<br />
anti-Lgl1, and anti-Dlg5 antibodies.<br />
WB:<br />
Anti-aPKC<br />
2.Analyze potential interaction between Dlg5 and aPKC in embryonic<br />
lungs. Determine domains of Dlg5 responsible for interaction.<br />
Anti-Lgl1<br />
Anti-Dlg5<br />
3.Analyze localization of aPKC, Dlg5, and Integrins in wild-type and Dlg5- /- lungs using immunofluorescence staining.<br />
Co-IP (Dlg5 Pull Down), Western Blot
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