Arun Shipstone MSc Thesis Defense Presentation 2014

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

The cadherin-catenin-complex is a core component of AJs

α-catenin interacts with multiple actin binding proteins

α-catenin is a key regulator of intercellular adhesion

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)

Maternal α-CatRNAi expression is embryonic lethal

Maternal α-Cat knockdown produces variable embryonic phenotypes

Mat-tub-Gal4

Maternal α-Cat knockdown embryos have reduced abdominal denticle belts

n = 100-300mat-tub-Gal4>α-CatRNAi1

α-Cat protein levels are strongly reduced in α-CatRNAi embryos

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 protein sequence is conserved

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 localize to AJs

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