Upload
arun-shipstone
View
42
Download
7
Embed Size (px)
Citation preview
Analysis of α-catenin mediated intercellular adhesion in Drosophila
Arun Shipstone, BSc.MSc. Candidate – Tepass LabDept of Cell & Systems Biology
University of Toronto
Adherens Junctions mediate intercellular adhesion
Importance of AJs- Essential for many cellular processes (polarity, tissue integrity, wound healing)- Disruption of AJs Heart dysfunction, tumorigenesis, metastasis
Project Goals
Aim 1 To generate and assess early embryonic phenotype(s) of α-Cat using an RNAi approach.
Aim 2To investigate if α-catenin function is conserved in metazoans and amoebozoans.
α-Catenin has strong maternal contribution in embryos
Sarpal et al. (2012). J. Cell Sci. 125, 233-245.
Magie et al. (2002). Development 129, 3771-3782.
Generating α-CatRNAi constructs for early embryonic knockdown
Transgenic Lines (attP2)Transformation Vector (WALIUM 20)
Mat-tub-Gal4
Maternal α-Cat knockdown embryos have reduced abdominal denticle belts
n = 100-300mat-tub-Gal4>α-CatRNAi1
Summary
• α-CatRNAi1 which targets the 5’UTR of a-Cat knocks down endogenous α-Cat when expressed in the female germline.
• Maternal α-CatRNAi1 expression alone produces an intermediate cuticle phenotype.
• Maternal expression of α-CatRNAi1 in combination with α-Cat1 produces a strong cuticle phenotype.
• Immunostaining experiments show that α-Cat levels are severely reduced or absent in α-CatRNAi1 expressing embryos.
Conclusions 1. Maternal α-Cat is indispensable for embryonic development.
2. aCatRNAi1 expression reduces α-Cat protein levels in embryos.
Project Goals
Aim 1 To generate and assess early embryonic phenotype(s) of α-Cat using an RNAi approach.
Aim 2To investigate if α-catenin function is conserved in metazoans and the amoebozoan Dictyostelium.
α-catenin constructs used to investigate functional conservation
Transgenic Lines (attP2) Recombine with α-Cat1Transformation Vector (pUASP)
Metazoan α-catenin proteins rescue embryonic lethality
ActDa-Gal4>UAS-α-X::HA
Input 40µg
ActDa-Gal4>UAS-α-X::HA in mutant background
n=100-200
Metazoan α-catenin proteins can support AJ integrity during Drosophila embryogenesis
aCat
1 mut
ants
exp
ress
ing
aCat
1 mut
ants
exp
ress
ing
α-Cat1, ActDa-Gal4>α-Cat1,UAS-α-X::HA
Summary
• All cross-species rescue constructs are expressed in Drosophila embryos.
• Metazoan α-catenin proteins can rescue α-Cat1 mutants to different stages of development.
• Dictyostelium discoideum α-catenin cannot rescue a-Cat1 mutants.
• Metazoan α-catenin proteins can localize to AJs, but Dictyostelium α-catenin localization is cytoplasmic.
Conclusions 1. Metazoan α-catenin proteins can support AJ integrity in Drosophila embryos.
2. Dictyostelium discoideum α-catenin cannot substitute for fly α-Cat during embryogenesis presumably because it cannot interact with Armadillo.
3. Seuquence conservation of α-catenin proteins does not play a significant role in α-catenin function in Drosophila.
Future Directions
α-CatRNAi Knockdown
• Perform staged embryo collections to investigate which morphogenetic processes are affected by maternal α-Cat knockdown.
• Perform live imaging on α-CatRNAi1 embryos to see how early the defects begin to occur.
• Express structure-function α-Cat constructs previously made by Dr. Ridhdhi Desai in α-CatRNAi1 embryos
Cross-species α-Cat1 rescue
• Assess rescue of constructs in other tissues ex. static FE cells and during BCM.
• Express a DE-cad::Ddα-cat::HA construct to see if Ddα-cat can function as an adhesive molecule since this construct can bypass Arm-dependent recruitment of α-catenin to AJs.
Acknowledgements
• Supervisor• Dr. Ulrich Tepass
• Committee• Dr. Tony Harris• Dr. Rudi Winklbauer• Dr. Ashley Bruce
• Lab Members• Kenana Al Kakouni• Dr. Ridhdhi Desai • Arman Draginov• Saba Haroon• Gayaanan Jeyanathan• Azadeh Laffafian• Milena Pellikka• Dr. Ritu Sarpal• Dr. Carol Schwartz• Luka Sheppard• Jordan Silver• Dr. Sergio Simoes• David ter Stal• Stefan Vujadinovic• Victoria Yan
• Imaging Facility• Henry Hong• Audrey Chong
• Other Labs• T. J. C. Harris Lab• W. James Nelson Lab• William I. Weis Lab