Building synthetic cells to study mechanical processes in zebrafish development

Secondary Supervisor(s): Dr Michael Smutny

University of Registration: University of Warwick

BBSRC Research Themes: Understanding the Rules of Life (Stem Cells, Systems Biology)

Project Outline

A long-standing goal is to identify the roles of mechanical forces and cell material properties in regulating essential processes during development, including tissue morphogenesis, cell migration and cell fate specification [1]. We aim to design, build and control synthetic cellular systems with tuneable mechanical and adhesive properties that will allow us to quantitatively interrogate mechanical signalling in living systems. Combining biochemistry, engineering, biophysics and developmental biology we want to measure and manipulate force-driven biological processes in developing zebrafish embryos. Recent advances have shown the huge potential of biologically inspired synthetic cells as “smart systems” in opening new ways to control molecular, cellular and developmental processes in living systems [2]. Here we focus to use force generating artificial cells to emulate key mechanical signals necessary for tissue formation and remodelling.

The PhD project can cover the following aspects:

• Create giant unilamellar vesicles (GUV) containing minimal cytoskeletal networks that will allow us to control their material properties. This will generate a class of new soft, passive materials (passive system).
• Generate contractile GUVs by utilising reconstituted actomyosin networks and to link them to the lipid membrane of the vesicle. The outcome of this approach is the development of force-calibrated and tuneable contractile GUVs as an active synthetic cell capable of exerting forces to the microenvironment (active system).
• Incorporate membrane bound E-cadherin adhesion receptors into GUVs and to link receptors to the underlying actin cortex to allow force transduction from GUVs to neighbouring adhesive cells.
• Test the functionality of our synthetic cells in cultured biological tissues and in developing embryos where we aim to deliver mechanical signals to biological cells and obtain quantitative measurements of cellular and mechanical processes in vivo.

Cytoskeletal proteins will be expressed and purified in the lab or are commercially available. GUVs will be generated using continuous droplet interface crossing encapsulation (this will be done with support by Associate Prof. Allen Liu, University of Michigan)[2]. Material properties of our cytoskeleton GUVs will be probed by atomic force microscopy and micropipette aspiration. We will employ confocal fluorescence microscopy to study cytoskeleton GUV dynamics outside and after injection into zebrafish tissues.

These cytoskeleton GUVs will be of great value for cell and developmental biologists studying mechanical processes in biological tissues and developing organisms. Moreover, the project will provide new insights about the behaviour of biological cells at the interface with synthetic material which will be important to inform new potential therapies in regenerative medicine.

References

1. Heisenberg CP, Bellaiche Y. Forces in tissue morphogenesis and patterning. Cell 153, 948-962 (2013).

2. Bashirzadeh Y, Liu AP. Encapsulation of the cytoskeleton: towards mimicking the mechanics of a cell. Soft Matter 15, 8425-8436 (2019).

Investigating the interplay of cell and collagen matrix mechanics in the pathogenesis of the hypermobile Ehlers-Danlos Syndrome

Secondary Supervisor(s): Dr Wing Ying Chow

University of Registration: University of Warwick

BBSRC Strategic Research Priority: Integrated Understanding of Health (Ageing, Regenerative Biology)

Project Outline

Ehlers-Danlos Syndromes are a group of 13 hereditary connective tissue disorders characterized by tissue fragility and laxity. Hypermobile Ehlers-Danlos Syndrome (hEDS) is the most common form the EDS with an approximate incidence of 1 in 500. It primarily affects the joints causing joint instability and chronic pain. Little is known about the pathogenesis of hEDS. Fibroblast cells derived from patients with hEDS show an abnormal integrin expression, a class of transmembrane proteins that link the cell cytoskeleton to the extracellular matrix (ECM) (1). This alters the way fibroblasts bind to the ECM, how mechanical forces are transmitted and the mechano-signalling of cells. We propose that this change in cell-ECM interaction is an important factor in the pathogenesis of hEDS (1). Interestingly, hEDS fibroblasts were shown to express elevated levels of α-smooth muscle actin (α-SMA) compared to normal fibroblasts which is associated with a transition to the more contractile myofibroblasts. While trans-differentiation of fibroblast to myofibroblast is a complex process the mechanical properties of the ECM are an important influence on it.

Work in our lab established in our lab a system based on patient-derived cell lines that reflects these phenotypes, that can be used to grow ECM originating from wild-type or EDS patient cells and allows us to mechanically manipulate cells and their ECM. This system opens now several lines of investigation as part of a PhD project:

• We will test how the mechanical properties of the wildtype and hEDS ECM affect fibroblast contractility and vice versa, how wildtype or hEDS fibroblasts affect and remodel the ECM. To test the ultrastructure of the ECM, we will employ Nuclear Magnetic Resonance to investigate the crosslinking state of collagen at the amino-acid level (2) and electron microscopy to obtain information at the nanometre scale. We will use atomic force microscopy (AFM) to test the mechanical properties of the ECM.

• To study the dynamic interplay of cells and ECM we will use our state-of-the-art confocal microscope combined with an atomic force microscope (AFM). We will seed (wildtype or hEDS) fibroblasts within ECM (wildtype or hEDS) and visualise dynamics of cell cytoskeletal components (fluorescently labelled actin, microtubules, integrins) while testing the mechanical properties of the ECM-cell composite (this part would be in close collaboration with Prof. Anne Straube.

• Another interesting aspect is to test the idea that changes in the mechanical properties of the ECM-cell composite in hEDS causes mast cell activation syndrome, due to mechanical stimulation of mast cells leading to degranulation. Mast cells will be seeded together with wildtype and hEDS fibroblast ECM composites, and their activation monitored over time or before and after global mechanical stimulation by stretching the cell-ECM composite.

References

1. S. Malek, D. V. Köster, The Role of Cell Adhesion and Cytoskeleton Dynamics in the Pathogenesis of the Ehlers-Danlos Syndromes and Hypermobility Spectrum Disorders. Frontiers in Cell and Developmental Biology 9, 1-13 (2021).

2. W. Y. Chow et al., Proline provides site-specific flexibility for in vivo collagen. Scientific Reports 8, (2018).