Principal Supervisor: Professor Andrew Fry - Department of Molecular and Cell Biology
Co-supervisor: Dr Mohammed El-Mezgueldi (Leicester) - expert in biochemical methods
Collaborator: Prof John Schwabe (Leicester) - expert in acetylation
Collaborator: Prof Anne Straube (Warwick) - expert in cell migration
PhD project title: Exploring the role of cortactin phosphorylation in cell division and migration
University of Registration: Leicester
Background: Cortactin is an F-actin binding protein that stimulates actin polymerization. It is frequently overexpressed in human cancer and promotes migration and invasion . Cortactin also has a role in regulating cell proliferation and can promote disassembly of the microtubule-based primary cilium upon re-entry of quiescent cells into the cell cycle . The primary cilium is an antennae-like structure that transduces external signals to control proliferation and differentiation, but which must be disassembled for cell division .
Preliminary work: We have discovered that cortactin is phosphorylated by the cell cycle-regulated Nek6 kinase at four positions in its actin-binding repeats (Figure 1). We therefore wish to first test whether this regulates the ability of cortactin to promote F-actin polymerization [4,5]. Second, as these phosphorylation sites lie close to sites that undergo acetylation and deacetylation of cortactin contributes to cilia disassembly by enhancing cortactin binding to F-actin [6,7], we will investigate potential cross-talk between phosphorylation and acetylation in regulating cortactin function. Finally, we will explore how phosphorylation of cortactin by Nek6 might promote proliferation, migration and invasion of human cancer cells in vivo.
Figure 1. Domain organization of human cortactin
Hypothesis and project objectives: We propose that Nek6 cooperates with cortactin overexpression in human cancer cells to enhance actin polymerization and promote proliferation and metastasis.
This hypothesis will be tested by addressing three mechanistic questions:
- Does Nek6 phosphorylation regulate actin binding, activity or localization of cortactin?
- Is Nek6 regulation of cortactin function dependent on acetylation status of cortactin?
- Does Nek6 cooperate with cortactin to regulate cilia disassembly, cell division, migration and invasion?
Objective 1: Does Nek6 phosphorylation regulate actin binding, activity or localization of cortactin?
- Establish a fluorescence-based F-actin polymerization assay using pyrene-labeled actin and stopped-flow kinetics.
- Generate phosphomimetic mutants of cortactin in bacterial and mammalian expression vectors and use chemical biology to generate non-hydrolysable phosphorylated cortactin .
- Examine the actin polymerization rates in the presence of purified cortactin proteins and binding to known cortactin partners, with and without Nek6.
- Examine cortactin localization in cells by confocal and super-resolution microscopy, with and without activation of depletion of Nek6.
Objective 2: Is Nek6 regulation of cortactin function dependent on acetylation status of cortactin?
- Generate combinations of phosphorylation and acetylation site mutants of cortactin.
- Measure actin binding, polymerization and localization of combination cortactin mutants.
- Characterise phospho-cortactin antibodies and use to assess expression and localization of phosphorylated cortactin in cells with and without acetylation activators and inhibitors.
Objective 3: Does Nek6 cooperate with cortactin to regulate cilia disassembly, cell division, migration and invasion?
- Generate stable cell lines expressing wild-type and mutant cortactin and analyse ciliary growth and resorption by confocal microscopy.
- Measure cell cycle status and proliferation rates of stable cell lines by flow cytometry.
- Analyse morphology, cortical actin distribution, migration and invasion of the stable cell lines using scratch wound, transwell migration, zebrafish invasion and single cell image-based tracking assays.
- MacGrath & Koleske (2012) J. Cell Sci. 125: 1621-1626.
- Bershteyn et al. (2010) Dev. Cell 19: 270-283
- Fry et al. (2014) Organogenesis 10: 62-68.
- Uruno et al. (2001) Nat. Cell Biol. 3: 259-266
- Weaver et al. (2001). Curr. Biol. 11: 370-374.
- Ran et al. (2015). Sci. Reports 5, 12917.
- Zhang et al. (2007) Mol. Cell 27: 197-213.
- Rogerson et al. (2015) Nat. Chem. Biol. 11, 496-503.
BBSRC Strategic Research Priority: Molecules, cells and systems
Techniques that will be undertaken during the project:
- Cell culture and stable cell line generation
- Expression and purification of recombinant proteins from bacteria
- Generation of engineered proteins with non-hydrolysable modifications
- Biochemical stopped-flow kinetics experiments
- Fixed and time-lapse quantitative confocal microscopy
Contact: Professor Andrew Fry, University of Leicester