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Interconnecting activin a signalling and endocytic trafficking in human embryonic stem cells

Principal Supervisor: Dr. Carol Murphy - School of Biosciences

Co-supervisor: Professor Tim Dafforn - School of Biosciences

PhD project title: Interconnecting activin a signalling and endocytic trafficking in human embryonic stem cells

University of Registration: University of Birmingham

Project outline:

Background: Activin A, a secreted ligand of the TGFβ superfamily, at low doses, maintains human embryonic stem cells (hESCs) in pluripotency by regulating the transcription protein NANOG1, whereas, at high doses, induces differentiation of hESCs into mesendoderm2,3,4.

Murphy

Moreover, Activin A is involved in the regulation of a remarkably wide range of processes such as apoptosis, inflammation, fibrosis and cell growth5, in a cell type-dependent manner.

Τhe complexity of the actions of Activin A contrast with the simple core SMAD machinery. Indeed, Activin A triggers heteromeric complex formation between specific transmembrane type I (ALK4) and type II (ACTRII/ACTRIIB) Ser/Thr kinase receptors, in which ACTRII/ACTRIIB transphosphorylates and activates ALK4. ALK4 then phosphorylates SMAD2/3 proteins which oligomerise with SMAD4 forming complexes that accumulate in the nucleus and exert transcriptional activity6 (Figure 1). TGF also activates SMAD2/3 further complicating the picture. Activin A activates non-SMAD pathways that also contribute to the biological activities of Activin A7.

It is not yet fully understood how SMAD2/3 mediate the plethora of activities of Activin A following ALK4 activation. Could intracellular trafficking of the ligand/receptor complex be partly responsible for this complexity? Following ligand binding, ligand/receptor complexes can be internalised via clathrin mediated endocytosis (CME), caveolae, macropinocytosis, the APPL8, the non-clathrin and non-caveolar pathway9, the FEME pathway10 and others. Cargo entering via these different routes is transported to a series of intracellular compartments, from where it signals or is recycled to the cell surface or directed to degradative compartments and this has an effect on the signalling outcome.

Hypothesis: Our working hypothesis is that Activin A/receptor complexes interact with different effectors localised along the endocytic compartment to tune the duration, amplitude and specificity of the signalling process. We have recently begun to address this by carrying out high throughput approaches to identify i) ALK4-interacting proteins using proteomics and a yeast 2-hybrid screen ii) ACTRIIB-interacting proteins using a yeast 2-hybrid screen. Each of these proteins is by definition a potential member of the Activin A/receptor trafficking complex as it interacts with one of the key components of the ligand /receptor complex. Systematic investigation of these proteins will generate a wealth of knowledge regarding Activin A signalling and will provide an answer as to how activation of SMAD2/3 proteins can become so versatile.

Aims of the project:

  • To address whether the complexity of Activin A signalling is dictated by interaction with effectors distributed along the endocytic pathway
  • To modify this pathway using a series of KO or over-expression studies of Activin A/Receptor complex-interacting proteins
  • To dissect this mechanism in hESCs which will allow us the address the role of trafficking regulators during differentiation
  • To simulate a more physiological method to investigate protein-protein interactions, of Activin Receptor complexes, to examine proteins purified from their native membrane in the absence of detergents using styrene maleic acid lipid particles (SMALPs)11.

Methods / Experiments: 

  • Generate CRISPR KO hESCs for one of the Activin A receptor interacting proteins already identified in our lab from high throughput screens
  • Define the role of the interactor in Activin A trafficking in hESCs. This will be carried out using Alexa 488 Activin A uptake in control hESCs and interacting protein KO hESCs. Analyses will be carried out by confocal microscopy using markers of intracellular compartments and quantitative analysis of the results using MotionTracking.
  • Address the role of the interacting protein in Activin A signalling responses.
  • Address whether the differentiation potential of interacting protein KO hESCs cells is compromised by differentiating hESCs to endoderm, mesoderm and ectoderm.
  • Activin A receptors are transmembrane serine/threonine kinases. We will express the receptors and use styrene maleic acid to form SMALPs11. SMALPs are stable, self-assembling, water-soluble particles consisting of SMA and lipids and in our case will contain Activin A receptor. Using this system we will address the characteristics of the receptor-interacting protein complex.

References:

  1. Vallier, L. et al. Activin/Nodal signalling maintains pluripotency by controlling Nanog expression. Development (Cambridge, England) 136, 1339-1349 (2009).
  2. D'Amour, K.A. et al. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nature biotechnology 23, 1534-1541 (2005).
  3. McLean, A.B. et al. Activin a efficiently specifies definitive endoderm from human embryonic stem cells only when phosphatidylinositol 3-kinase signaling is suppressed. Stem cells (Dayton, Ohio) 25, 29-38 (2007).
  4. Vallier, L. et al. Early cell fate decisions of human embryonic stem cells and mouse epiblast stem cells are controlled by the same signalling pathways. PLoS One 4, e6082 (2009).
  5. Wijayarathna, R. & de Kretser, D.M. Activins in reproductive biology and beyond. Hum Reprod Update (2016).
  6. Shi, Y. & Massague, J. Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell 113, 685-700 (2003).
  7. Moustakas, A. & Heldin, C.H. Non-Smad TGF-beta signals. Journal of cell science 118, 3573-3584 (2005).
  8. Miaczynska, M. et al. APPL proteins link Rab5 to nuclear signal transduction via an endosomal compartment. Cell 116, 445-456 (2004).
  9. Sabharanjak, S., Sharma, P., Parton, R. & Mayor, S. GPI-Anchored Proteins Are Delivered to Recycling Endosomes via a Distinct cdc42-Regulated, Clathrin-Independent Pinocytic Pathway. Developmental cell 2, 411-423 (2002).
  10. Boucrot, E. et al. Endophilin marks and controls a clathrin-independent endocytic pathway. Nature 517, 460-465 (2015).
  11. Knowles, T.J. et al. Membrane proteins solubilized intact in lipid containing nanoparticles bounded by styrene maleic acid copolymer. J Am Chem Soc 131, 7484-7485 (2009).

BBSRC Strategic Research Priority: Molecules, cells and systems

Techniques that will be undertaken during the project:

Culturing human embryonic stem cells & induced pluripotent stem cells, differentiation to various lineages, ligand/receptor trafficking assays, signalling assays, advanced microscopy, CRISPR genome editing in human embryonic stem cells, induced pluripotent stem cells and other cell types, all basic method associated with cell biology & molecular biology including western blot analysis, protein interactions, immunoprecipitations etc cloning, plasmid preparation, quantitative real time PCR, RNA extraction, protein expression, SMALP generation and characterisation.

Contact: Dr Carol Murphy, University of Birmingham