The Ballestrem Lab

It is remarkable how cells can develop from a single cell to a whole multicellular organism. For such healthy development cells need to continuously communicate with their environment enabling co-ordinated growth and secretion of substances, which is essential to form functional tissue, organs and the entire body. During the entire life of organisms, such cell communication with its environment sustains appropriate tissue function.

 

What is the cells’ environment?

The extracellular matrix (ECM) is a critical part of the cellular environment. It consists of a web of interlinked fibrous material which hosts growth and other factors that are important for cell viability, proliferation, movement and differentiation. The ECM is synthesised by the majority of the cells themselves; some of them (fibroblasts) are specialised in this function and can produce a lot of it. Cells adhere to the ECM, they can reorganise it or degrade/disassemble and assemble it to keep it in a homeostatic functional state. Perhaps the most prominent ECM components are collagens, laminins and fibronectin. Collagens and laminin form a membrane like structure, the basement membrane, to which epithelial cells attach to. Other types of collagen and fibronectin form a network of connective tissue throughout the body, that holds the body together. Examples for connective tissues with specialised functions are the bone, cartilage and adipose (fat) tissue.

 

Neighbouring cells can be part of the cells’ environment. When cells divide and depending on the tissue they form, cells will stay in touch with each other. For example, in the skin cells form a layer of neighbouring cells that stay in close contact with each other to seal the body protecting the tissue from foreign substances. Many other epithelial cells that cover internal body surfaces are arranged and act in the same way (e.g. those of the digestive system or the cardiovascular system). Communication between these cells is critical to maintain tissue integrity and function.

 

 

How do cells communicate with each other and sense their environment?

A major focus of our research lies in understanding how cells use the molecular machinery located in cell-matrix adhesion sites (CMAS) to sense the biochemical and biophysical properties of the ECM and how the sensing of the ECM feeds back into signals that regulate ECM synthesis and remodelling.  A second focus is on how cells use specialised cell-cell attachment sites called desmosomes to regulate the maintenance and function of epithelial cell layers.

 

Cell-matrix adhesions (CMAS) and their role in ECM sensing/remodelling:

The ECM of tissues alters enormously particularly during ageing, injury and certain diseases. For example, the mechanical properties of the ECM influences tumour progression, and stiffening of ECM causes fibrosis (excess matrix production), which in turn can lead to malfunctioning of the affected tissues.  Cells sense such changes and mechanistic understanding of how this works is particularly important to prevent diseases and induce regeneration processes that require specific cellular responses to changing ECM environments. Cellular responses include changes in motile behaviour (e.g. closing of wounds) and also active reorganisation of their ECM when forming new functional tissue.

 

Cells sense their environment by binding to and pulling on the ECM using receptors named integrins that span the cell membrane. These integrins not only bind to the ECM but also connect to the contractile actin cytoskeleton with their intracellular domains inside the cells. This link is not direct but is regulated by structural and signalling components that couple the two. We published a number of manuscripts showing that two of the proteins that connect integrins with actin, talin and vinculin, are central to sensing environmental changes (1-5). They are particularly important for measuring the mechanical properties (stiffness) of their surroundings and they control cell migration. We are currently focussing how these mechanosensory proteins are linked to the wider network of adhesion regulatory proteins that coordinate not only cell movements but also regulate ECM synthesis and remodelling.

 

Cell-cell adhesions are specialised adhesion sites that cells use to interact with each other. There are a variety of different types of such adhesion sites consisting of different protein complexes. Adherens junctions and tight junctions contain adhesion receptors and proteins that are linked intracellularly to the contractile actin cytoskeleton (reviewed in 6). A third type of complexes that mediate strong cell-cell adhesions are the so-called desmosomes (from Greek desmos meaning “bond, fastening, connection”), in which adhesion receptors (desmocollins and desmogleins) are linked through adapter proteins to intermediate filaments (reviewed in 7 and 8). These desmosomes are particularly resistant to mechanical stress and are mostly expressed in cells of tissues that are exposed to such stress (e.g. heart muscle and the coverings of body surfaces). Embryos lacking key desmosomal components die during development and aberrant desmosome function leads to skin blistering, heart disease, wound healing defects and cancer.

 

During normal development, epidermal wound healing and cancer, cells undergo a plastic change known as epithelial-mesenchymal transition (EMT) that enables them to move. This necessitates the dynamic process of adhesion formation and down-regulation of desmosomes. It is not known how these processes occur but it needs to be clarified in order to understand normal development and to formulate treatments to aid wound healing and prevent the spread of cancer.

 

An important factor contributing to the toughness of tissues is that desmosomes exhibit a highly adhesive state known as hyper-adhesion. Hyper-adhesion is important for tissue strength, but also locks cells together, thus restricting their movement. To shed new light into desmosome regulation we currently determine the composition of desmosomes during formation, down-regulation and the different adhesion states in normal and diseased conditions.

 

The methods we use to study cell adhesion mechanisms:

 

To address our research questions, we have established interdisciplinary approaches including:

​

1. Advanced fluorescence microscopy allows us to visualise the localisation of proteins in time and space (time-lapse video microscopy & ratiometric imaging) and analyse the dynamic behaviour proteins and their interactions in live cells (using FRET, FRAP, photoactivation). 

2. Mass spectrometry (BioID) enables us to analyse the molecular neighbourhood of specific proteins of interest. The information from detailed mass spec analysis help us to identify potential protein networks and how they relate molecular functions that direct cell behaviour.

3. Biochemical methods such as Co-IPs help us to identify/confirm molecular associations.

4. Molecular biology methods including CRISPR, siRNA knockdown, site directed mutagenesis and molecular cloning (e.g. to generate fluorescently labelled fusion proteins) enable detailed analysis of protein functions and interactions in cells.

5. Engineering is helpful to prepare ECM substrates that can be used to provoke cellular responses or mimic specific extracellular properties. For example, we prepare hydrogels coated with different ECM proteins to alter biophysical properties in the surrounding of cells and we established a method where we can generate micro-patterns to explore how cells respond to simple and more complex changes in ECM patterns 9.

 

 

References

1. Atherton, P. et al. Relief of talin autoinhibition triggers a force-independent association with vinculin. J Cell Biol 219 (2020).

2. Atherton, P. et al. Vinculin controls talin engagement with the actomyosin machinery. Nat Commun 6, 10038 (2015).

3. Carisey, A. et al. Vinculin regulates the recruitment and release of core focal adhesion proteins in a force-dependent manner. Current biology : CB 23, 271-281 (2013).

4. Humphries, J.D. et al. Vinculin controls focal adhesion formation by direct interactions with talin and actin. J Cell Biol 179, 1043-1057 (2007).

5. Stutchbury, B., Atherton, P., Tsang, R., Wang, D.Y. & Ballestrem, C. Distinct focal adhesion protein modules control different aspects of mechanotransduction. J Cell Sci 130, 1612-1624 (2017).

6. Hartsock, A. & Nelson, W.J. Adherens and tight junctions: structure, function and connections to the actin cytoskeleton. Biochim Biophys Acta 1778, 660-669 (2008).

7. Hatzfeld, M., Keil, R. & Magin, T.M. Desmosomes and Intermediate Filaments: Their Consequences for Tissue Mechanics. Cold Spring Harbor perspectives in biology 9 (2017).

8. Garrod, D. & Chidgey, M. Desmosome structure, composition and function. Biochim Biophys Acta 1778, 572-587 (2008).

9. Melero, C. et al. Light-Induced Molecular Adsorption of Proteins Using the PRIMO System for Micro-Patterning to Study Cell Responses to Extracellular Matrix Proteins. J Vis Exp (2019).

​