Cell & Molecular Biophysics, Randall Division of

1. Riento, K., Totty, N., Garg, R., Villalonga, P., Ridley, A.J. (2005). RhoE function is regulated by ROCK I-mediated phosphorylation. EMBO J 24, 1170-1180.
2. Millan, J., Hewlett, L., Glyn, M., Toomre, D., Clark, P., Ridley, A.J. (2006) Lymphocyte transcellular migration occurs through recruitment of endothelial ICAM-1 to caveola- and F-actin-rich domains. Nature Cell Biol. 8, 113-123.
3. Wheeler, A.P., Wells, C.M., Smith, S.D., Vega, F., Henderson, R.B., Tybulewicz, V.L., Ridley, A.J. (2006) Rac1 and Rac2 regulate macrophage morphology but are not essential for migration. J. Cell Sci.119, 2749-2757.
4. Garg, R., Riento, K., Keep, N.H., Morris, J.D., Ridley AJ. (2008) N-terminus-mediated dimerization of ROCK-I is required for RhoE binding and actin reorganization. Biochem J. 411, 407-414.
5. Smith, S.D., Jaffer, Z.M., Chernoff, J., Ridley, A.J. (2008) PAK1-mediated activation of ERK1/2 regulates lamellipodial dynamics. J. Cell Sci. 121, 3729-3736.
6. Bright, M.D., Ridley, A.J. (2009) PAK1 and PAK2 have different roles in HGF-induced morphological responses. Cellular Signalling 21, 1738-1747.
7. Millán, J., Cain, R.J., Reglero-Real, N., Bigarella, C., Marcos-Ramiro, B., Fernandez-Martin, L., Correas, I., Ridley, A.J. (2010) Adherens junctions connect stress fibers between adjacent endothelial cells. BMC Biol. 8, 11.
8. Cain, R.J., Vanhaesebroeck, B., Ridley, A.J. (2010) The PI3K p110Ą isoform regulates endothelial adherens junctions via Pyk2 and Rac1. J. Cell Biol. 188, 863-876.
9. Takesono, A., Heasman, S.J., Wojciak-Stothard, B., Garg, R., Ridley, A.J. (2010) Microtubules regulate migratory polarity through Rho/ROCK signaling in T cells. PLoS ONE 5, e8774.
10. Heasman, S.J., Carlin, L.M., Ng, T., Ridley, A.J. (2010) Coordinated RhoA signaling at the leading edge and uropod is required for T cell transendothelial migration. J. Cell Biol. 190, 553-563.
11. Vega, F., Fruhwirth, G., Ng, T., Ridley, A.J. (2011) RhoA and RhoC have distinct roles in migration and invasion by acting through different targets. J. Cell Biol. 193, 655-665.

Cell adhesion and migration are critical processes during in normal development and wound healing, but can also contribute to pathological processes such as cancer metastasis. Research in my lab is focused around the study of cell adhesion receptors and how different receptor families control cytoskeleton remodelling and cell migration. We use advanced microscopy approaches coupled with biochemistry and molecular biology to study these events. Understanding the way in which these proteins signal and localise within the cell is key to understanding cell motility in any context.

Selected recent publications:
Morton PE and Parsons M. Dissecting cell adhesion architecture using advanced imaging techniques. Cell Adh Migrn. 2011 In Press.

Morton PE and Parsons M. Measuring FRET using time-resolved FLIM. Methods Mol Biol. 2011. 769:403-413.

1) Small molecules and cytokinesis: Cytokinesis has been difficult to study because it is a complex, rapid and dynamic process and many key proteins also perform important functions earlier in the cell cycle. Small molecules that act rapidly and can be added at specific time points for live imaging are ideal tools to dissect cytokinesis. We are in the process of creating a toolbox of small molecules that inhibit different proteins and pathways in cytokinesis, using technologies we developed that integrate chemical and genomic methods to target specific signalling pathways, with an emphasis on the pathway centred on Rho GTPase.

2) Cytokinesis and endocytosis: Although we know that endocytosis is essential for cytokinesis, there are many outstanding questions about how these two processes interface. We discovered a small molecule, XZ-1, that is both a potent inhibitor of cytokinesis and an inducer of endocytic tubulation. XZ-1 is thus an ideal tool to study how cytokinesis and endocytosis are connected. We are using a combination of genome-wide RNAi and imaging approaches to investigate how XZ-1 exerts its effects.

3) Cytokinesis and lipids: While it is known that cell membranes undergo dramatic structural rearrangements during cytokinesis, and it is obvious that membrane rearrangements are needed to seal daughter cells after severing, very little is known about whether (and how) specific lipids are involved in cytokinesis. We are well on our way to answering these questions, using mass spectrometry, imaging and small molecule perturbations.

Our lab uses a wide array of techniques such as Microarray analysis, RNAi, over-expression approaches, 3D invasion/imaging techniques and in vivo metastasis assays to understand the roles of these genes in determining modes of movement. The tumour microenvironment is a major focus of our research as it could potentially regulate these two migratory modes. Our group is funded by Cancer Research UK :

http://science.cancerresearchuk.org/funding/find-grant/all-funding-schemes/career-development-fellowship/

Gene Regulatory Networks in musculoskeleletal development
A central question in developmental biology is how the onset of gene expression results in the differentiation of a multi-potential progenitor cell pool into subsets of divergent cell fates. Progenitor cells in somites differentiate in response to extracellular signals to produce skeletal muscles, muscle stem cells, cartilage, tendon, endothelial and smooth muscle cells. We are investigating how signals are integrated at the level of gene expression through Gene Regulatory Networks to produce these differential outputs.

Somite development and differentiation
The skeletal muscles of the trunk and limbs and the axial skelelton (ribs and vertebrae) originate in somites, which are formed, as pairs on each side of the neural tube, as distinctive blocks of mesodermal cells. The somites give rise to the trunk and limb muscles and the asscouated muscle stem cells, the cartilage of the vertebrae and ribs, the tendons associated with the vertebral column, blood vessels of the limb and the dermis.

Muscle morphogenesis
The limb bud is an important embryological model for studying how the patterns of cells and tissues are established. Most studies, however, have focussed on the skeletal elements of the limb and there is a paucity of knowledge of the basis of muscle and muscle connective tissue (including tendons) patterning. We use transgenic models to investigate the formation of fully defined limb muscles and associated connective tissue and tendons to study reciprocal molecular and cellular interactions between these distinct cell types. Our transgenic models are used to research into muscle stem cell development,  and tendon fibroblasts and tendon stem cells recruitment and maintenance. We use a variety of molecular and cellular techniques, including: gene expression analysis, transgenesis, gene knockouts, microarrays, ChIP, gel shift assays, bioinformatics, explant cultures, and in vitro cell culture.

Recent publications:

• Kirilenko P, He G, Mankoo B, Mallo M, Jones R, Bobola N. (2011) Transient activation of Meox1 is an early component of the gene regulatory network downstream of Hoxa2. Mol Cell Biol. doi:10.1128/MCB.00705-10
• Perera S, Holt MR, Mankoo BS, Gautel M. (2011) Developmental regulation of MURF ubiquitin ligases and autophagy proteins nbr1, p62/SQSTM1 and LC3 during cardiac myofibril assembly and turnover. Dev Biol. 2011 351:46-61
• Otto A, Macharia R, Matsakas A, Valasek P, Mankoo BS, Patel K (2010) A hypoplastic model of skeletal muscle development displaying reduced foetal myoblast cell numbers, increased oxidative myofibres and improved specific tension capacity. Dev Biol. 343:51-62
• Reijntjes S, Francis-West P, Mankoo BS (2010) Retinoic acid is both necessary for and inhibits myogenic commitment and differentiation in the chick limb. Int. J. Dev. Biol. 54:125-34
• Skuntz S, Mankoo B, Nguyen M-T, Hustert E, Nakayama A, Tournier-Lasserve E, Wright CV, Pachnis V, Bharti K, Arnheiter H (2009) Lack of the mesodermal homeodomain protein MEOX1 disrupts sclerotome polarity and leads to a remodeling of the cranio-cervical joints of the axial skeleton. Dev. Biol. 332:383-95. 
• Plachez C, Andrews W, Liapi A, Knoell B, Drescher U, Mankoo B, Zhe L, Mambetisaeva E, Annan A, Bannister L, Parnavelas J, Richards L, and Sundaresan V (2008) Robos are required for the correct targeting of retinalganglioncell axons in the visual pathway of the brain. Mol Cell Neurosci. 37:719-30.
• Reijntjes S, Stricker S, Mankoo BS (2007) A comparative analysis of Meox1 and Meox2 in the developing somites and limbs of the chick embryo. International Journal of Developmental Biology. 51:753-9
• Jukkola T, Trokovic R, Maj P, Lamberg A, Mankoo B, Pachnis V, Savilahti H and Partanen J (2005) Meox1Cre - a mouse line expressing Cre recombinase in somitic mesoderm. Genesis 43:148-53
• Rodrigo I, Bovolenta P, Mankoo BS, Imai K (2004) Meox homeodomain proteins are required for Bapx1 expression in the sclerotome and activate its transcription by direct binding to its promoter. Mol Cell Biol. 24:2757-66.
• Mankoo BS, Skuntz S, Harrigan I, Grigorieva E, Candia A, Wright CV, Arnheiter H, Pachnis V (2003)The concerted action of Meox homeobox genes is required upstream of genetic pathways essential for the formation, patterning and differentiation of somites.. Development. 130:4655-64.

• How are myofibrils and intercalated disks assembled in heart cells during development?
• How and if are myofibrils and intercalated disks affected in the diseased heart?
• What is the functional basis for the adaptations of cardiomyocytes during development and disease?

• how giant ruler proteins called titin and obscurin control the assembly of many other structural, contractile and signalling proteins into ordered sarcomeres,
• how mechanical forces regulate sarcomere assembly, as well as controlled turnover by the autophagy and ubiquitin-proteasomal pathways,
• how mutations in sarcomeric proteins, especially in titin and obscurin, affect sarcomere and turnover functions.

We use biochemical, biophysical, advanced cell imaging and structural methods to elucidate these basic functions and to translate them to human disease.

A molecular level understanding requires structural knowledge, in the first place. However, knowledge of structure is not sufficient either. Structures provide a static description, while biomolecular systems are not static.  Protein interactions are often transient and they occur on a dynamic scale: proteins and nucleic acids constantly move, adapt themselves to different conditions, and assume different forms depending on their partners. The observed static structures simply represent one, most probable, conformation observed at the particular experimental conditions, among many, otherwise accessible.
We use computational biology to analyse and to simulate biomolecules in conditions often not accessible to experiments. We work in strict contact to experimentalists to verify their results and to challenge new experiments.