Research in my lab is organised around three main themes: i) the cellular actin cortex; ii) mechanics of cells and tissues, and iii) cell migration in confined environments.
The first theme seeks to understand the dynamic molecular mechanisms responsible for homeostasis of the submembranous actin cortex in cells. Combining proteomics and siRNA screening, we have uncovered the actin nucleators responsible for generation of the cell cortex as well as its proteic composition (Bovellan et al, Current Biology, 2014). The ultimate goal of this research is to understand cortical mechanics from the bottom-up using Scanning Electron Microscopy, mechanical characterisation by Atomic Force Microscopy, and polymer physics theories.
At the cellular level, the second theme aims to understand the time-dependent mechanical properties of single cells (Moeendarbary et al, Nature Materials, 2013) and at the tissue level, we are trying to uncover how subcellular organisation and cellular mechanics govern tissue mechanical properties (Harris et al, PNAS, 2012). The cellular-scale work utilises Atomic Force Microscopy and informs our research on the tissue-scale work. At the tissue-level, we use monolayers devoid of a substrate to study the mechanics of load-bearing monolayers under well-controlled mechanical conditions while allowing imaging at subcellular, cellular and tissue resolutions. Our ultimate aim is to understand the biological determinants of monolayer mechanics and the biophysical processes that drive the individual cell behaviours participating in tissue morphogenesis.
The third theme utilises microfluidic devices to examine cell migration in confined three-dimensional environments. Indeed, it is becoming increasingly apparent that migration in 3D environments differs substantially from migration on 2D surfaces. Microfluidic devices offer a highly constrained environment that simplifies understanding of the physical processes underlying migration. In particular, our studies have highlighted a novel organisation for the cell leading edge during migration in confined environments (Wilson et al, Nature Communications, 2013).
In all of its research, my laboratory combines analytical and characterisation techniques from physics and engineering with molecular cell biology techniques and quantitative microscopy to study questions relevant to cell and developmental biology.
My laboratory is funded by the Biotechnology and Biological Sciences Research Council (BBSRC), the Human Frontier Science Program (HFSP), and the Rosetrees Trust.
There are often openings for PhD and post-doctoral positions in the lab, if you are interested please get in touch with me directly.
In this study, we identified the proteins that nucleate the submembranous actin cortex using a proteomic analysis and a localisation/shRNA screen. Two proteins generated the bulk of cortical actin: the formin mDia1 and the Arp2/3 complex. These two nucleators had radically different accumulation kinetics but contributed a similar amount of F-actin to the cortex. http://www.ncbi.nlm.nih.gov/pubmed/25017211
In this study, we used deep AFM indentation to probe the mechanics of epithelial monolayers reforming from dissociated cells. We showed that the formation of intercellular junctions coincided with an increase in the apparent stiffness of reforming monolayers that reflected the generation of a tissue-level tension. http://www.ncbi.nlm.nih.gov/pubmed/24659804
Here, we investigated actin turnover mechanisms during cell migration in small cross-section microfluidic channels. We show that the leading edge comprises two distinct F-actin networks: an adherent network that polymerizes perpendicular to cell-wall interfaces and a ‘free’ network that grows from the free membrane at the cell front. http://www.ncbi.nlm.nih.gov/pubmed/24305616
The cytoplasm is the largest part of the cell by volume and hence its rheology sets the rate at which cellular shape changes can occur. Here we examined the applicability of the “fluid-filled sponge” model to the cytoplasm using Atomic Force Microscopy combined with chemical and genetic treatments. http://www.ncbi.nlm.nih.gov/pubmed/23291707
Epithelial monolayers fulfil critical mechanical roles in development and normal physiology, yet little is known about their mechanics. Here, we characterised the mechanics of epithelial monolayers using a novel experimental device. We showed that monolayers are remarkably stiffer than individual cells and can withstand more than a doubling in length.http://www.ncbi.nlm.nih.gov/pubmed/22991459
Charras G.T., Hu C.-K., Coughlin M., and Mitchison T.J., “Re-assembly of a contractile actin cortex in cell blebs”, Journal of Cell Biology, 175(3), 477-490 (2006). [download PDF file]
The cortex is a 0.1 to 1 µm thick layer of actin and associated proteins that underlies the cell membrane and determines the shape of animal cells. Whereas the biology of some actin structures (lamellipodia, filopodia) is well understood, the events leading to the formation and subsequent regulation of a submembranous actin cortex are not due to the lack of a good model system. In this paper, we use blebs as model systems to study actin cortex assembly. We identify the proteins involved in cortex assembly as well as their dynamics and ultrastructural arrangement.
Current models for protrusive motility in animal cells focus on cytoskeleton-based mechanisms, where localized protrusion is driven by local regulation of actin biochemistry. In plants and fungi, protrusion is driven primarily by hydrostatic pressure. For hydrostatic pressure to drive localized protrusion in animal cells, it would have to be locally regulated, but current models treating cytoplasm as an incompressible viscoelastic continuum or viscous liquid require that hydrostatic pressure equilibrates essentially instantaneously over the whole cell. Here, we use cell blebs as reporters of local pressure in the cytoplasm. When we locally perfuse blebbing cells with cortex-relaxing drugs to dissipate pressure on one side, blebbing continues on the untreated side, implying non-equilibration of pressure on scales of ~10um and ~10sec. We can account for localization of pressure by considering the cytoplasm as a contractile, elastic network infiltrated by cytosol. Motion of the fluid relative to the network generates spatially heterogenous transients in the pressure field, and can be described in the framework of poroelasticity.
Neutrophils are the primary cells of the immune system responsible for detecting and preventing bacterial infections, as well as driving inflammation. Neutrophils circulate freely in the bloodstream, and when passing through an inflamed region attach the blood vessel wall, traverse the endothelium (transendothelial migration), and migrate through the connective tissue to the site of infection (chemotaxis). Here a neutrophil (in red) is shown migrating through a 10x3µm microfluidic channel towards a source of chemoattractant (in blue). Scale bar = 5µm. In collaboration with Dr Irimia, Massachusetts General Hospital
Figure 1: Neutrophil ChemotaxisFigure 1: Neutrophil Chemotaxis
Dissociated cells of the animal pole of xenopus embryos display characteristic dynamics of the cell membrane, known as circus movements. In these cells, a local delamination of the cell membrane from the cytoskeleton (a bleb) can propagate around the cell as a traveling wave which circles the cell periphery multiple times. This wave progresses through cycles of delamination. In this image, the blastomere has been injected with RNA encoding the PH domain of phospholipase C _ tagged with GFP, which highlights the cell membrane. Three separate time points showing the progression of the traveling wave have been superimposed in the following colours: red t=0s, green t=5s, blue t=10s. Scale bar=10µm.
Figure 2: Circus movements in xenopus blastomeres
Given appropriate culture conditions, some cells can form structures akin to a football with each patch of the football being a cell. These structures are known as cysts and are a good model of multicellular structure as they are amenable to genetic manipulation, mechanical testing, and comprise sufficiently few cells (~100) that they can be modelled computationally. In this figure, a phase contrast image of a MDCK cell cyst is shown. The thickness of the rim of this cyst is approximately 10 µm and comprises only one cell. In the top left corner, the shadow of a micropipette used to deform the cyst can be distinguished.
Figure 3: MDCK cell cyst
Blebs are spherical cellular protrusions that occur in many physiological situations. Two distinct phases make up the life of a bleb each of which have their own biology and physics: expansion, which lasts ~30s, and retraction, which lasts ~2min. During expansion, the cell membrane delaminates from the actin cortex and fills with cytosol. Growth stalls as an actin cortex reforms under the bleb membrane, and retraction starts, driven by myosin-II. In this figure, the cell membrane has been removed with detergent and the actin cytoskeleton stabilized with phalloidin. The cell has been imaged using scanning electron microscopy revealing the cage-like structure of actin filaments within retracting blebs.
Figure 4: Actin cytoskeleton of a blebbing cell