Organisation of basolateral microtubule attachment complexes in 3D epithelial cultures


Supervising PI

Dr. Anna Akhmanova (website)

Project Description

Background

3Dcell

In polarized epithelial cells, a significant proportion of microtubules is oriented with their plus ends towards the basolateral surface. Recent studies in our lab have shown that an important mechanism of basolateral cortical microtubule attachment relies on the microtubule plus-end tracking proteins EB1, CLASP1/2, MACF2/ACF7 as well as the cortical factors LL5β, ELKS, liprin-α1, β1, and KANK1. The aim of this project is to determine how this network connects to the major polarity complexes, such as Scribble, how it functions in 3D-cultured cells, how it is modified during epithelial-mesenchymal transition and how it is affected by microtubule–targeting agents used in cancer therapy.

Objectives

  • Quantitative description of the role of microtubule-attachment complexes in cortical polarization in 3D breast epithelia acini and their modification during epithelial-mesenchymal transition and invasive growth in 3D.
  • Characterization of the biochemical connection between microtubule attachment complexes and the Scribble complex.
  • Quantitative characterization of the role of microtubule dynamics in polarity maintenance in 3D-cultured epithelial cells and the effects of microtubule-targeting agents on this process.

Methodology

  • Loss-of function studies in mammalian cells through RNA interference.
  • CRISPR/Cas9-mediated genome engineering to generate deletion alleles, specific point mutants, and endogenously tagged fluorescent proteins.
  • High resolution live cell microscopy of 3D-cultured normal human breast epithelial cells and breast cancer cells.
  • Application of a switchable model of epithelial-mesenchymal transition.

Collaborators

  • Etienne-Manneville (Institut Pasteur, Paris, France)
  • Dale (Cardiff University, UK)
  • Cellesce Ltd (UK)

Key publications

  1. van der Vaart B, van Riel WE, Doodhi H, Kevenaar JT, Katrukha EA, Gumy L, Bouchet BP, Grigoriev I, Spangler SA, Yu KL, Wulf PS, Wu J, Lansbergen G, van Battum EY, Pasterkamp RJ, Mimori-Kiyosue Y, Demmers J, Olieric N, Maly IV, Hoogenraad CC, Akhmanova A. CFEOM1-associated kinesin KIF21A is a cortical microtubule growth inhibitor. Dev Cell. 2013 Oct 28;27(2):145-60.
  2. Jiang K, Toedt G, Montenegro Gouveia S, Davey NE, Hua S, van der Vaart B, Grigoriev I, Larsen J, Pedersen LB, Bezstarosti K, Lince-Faria M, Demmers J, Steinmetz MO, Gibson TJ, Akhmanova A Proteome-wide screen for mammalian SxIP motif-containing microtubule plus-end tracking proteins. Curr Biol. 2012 Oct 9;22(19):1800-7.
  3. Grigoriev I, Yu KL, Martinez-Sanchez E, Serra-Marques A, Smal I, Meijering E, Demmers J, Peränen J, Pasterkamp RJ, van der Sluijs P, Hoogenraad CC, Akhmanova Rab6, Rab8, and MICAL3 cooperate in controlling docking and fusion of exocytotic carriers.Curr Biol. 2011 Jun 7;21(11):967-74.
  4. Grigoriev I, Splinter D, Keijzer N, Wulf PS, Demmers J, Ohtsuka T, Modesti M, Maly IV, Grosveld F, Hoogenraad CC, Akhmanova Rab6 regulates transport and targeting of exocytotic carriers. Dev Cell. 2007 Aug;13(2):305-14.
  5. Lansbergen G, Grigoriev I, Mimori-Kiyosue Y, Ohtsuka T, Higa S, Kitajima I, Demmers J, Galjart N, Houtsmuller AB, Grosveld F, Akhmanova CLASPs attach microtubule plus ends to the cell cortex through a complex with LL5beta. Dev Cell. 2006 Jul;11(1):21-32.