Super-resolution studies of the organisation of polarity regulators at the plasma membrane


Supervising PI

Joint supervision by Dr. Paul van Bergen en Henegouwen (Utrecht University) and U-Protein Express

Project Description

Background

The orientation of the spindle determines the correct organisation of epithelial tissue. Different molecular complexes have recently been described with essential roles in spindle orientation. These molecules include different inositol lipids and the Gα-LGN-NuMA and EGFR/IQGAP1 protein complexes. The aim of this project is to determine how these protein:lipid complexes function, and how they are regulated spatiotemporally by both external and intracellular polarity signals and cues.

Objectives

  • Characterization of protein/lipid complexes involved in spindle orientation in mammalian epithelial cells.
  • Generation of molecular tools for high resolution imaging of spindle formation in 3D cell cultures of epithelial cells.
  • Functional analysis of identified regulatory components for asymmetric spindle orientation.

Methodology

  • Purification of protein:lipid complexes from dividing epithelial cells using nanodisc technology
  • Molecular characterisation using protein and lipid mass spectrometry (proteomics and lipidomics).
  • Generation of nanobodies against identified components using phage display.
  • Super resolution imaging of these components using nanobodies during asymmetric cell division in 3D epithelial cell cultures (MDCK- and Caco2 cells).
  • Functional analysis of these components by loss of function studies using CRISPR/Cas9-mediated genome enginering

Nanodisc

Collaborators

Key publications

  1. Mikhaylova M, Cloin BM, Finan K, van den Berg R, Teeuw J, Kijanka MM, Sokolowski M, Katrukha EA, Maidorn M, Opazo F, Moutel S, Vantard M, Perez F, van Bergen en Henegouwen PM, Hoogenraad CC, Ewers H, Kapitein LC. Resolving bundled microtubules using anti-tubulin nanobodies. Nat Commun. 2015 Aug 11;6:7933.
  2. Heukers R, Vermeulen JF, Fereidouni F, Bader AN, Voortman J, Roovers RC, Gerritsen HC, van Bergen En Henegouwen PM. Endocytosis of EGFR requires its kinase activity and N-terminal transmembrane dimerization motif. J Cell Sci. 2013 Nov 1;126(Pt 21):4900-12.
  3. de Heus C, Kagie N, Heukers R, van Bergen en Henegouwen PM, Gerritsen HC. Analysis of EGF receptor oligomerization by homo-FRET. Methods Cell Biol. 2013;117:305-21.
  4. Kijanka M, Dorresteijn B, Oliveira S, van Bergen en Henegouwen PM. 2015 Nanobody-based cancer therapy of solid tumors. Nanomedicine (Lond). 10:161-74.
  5. Low-Nam ST, Lidke KA, Cutler PJ, Roovers RC, van Bergen en Henegouwen PMP, Wilson BS, and Lidke DS. (2011) ErbB1 dimerization is promoted by domain co-confinement and stabilized by ligand binding. Nat Struct Mol Biol. 18:1244-1249.