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Cellular polarity: from mechanisms to disease

Event

Starts:

April
152013

09:00

Ends:

April
162013

17:00

Location

The Royal Society, London, 6-9 Carlton House Terrace, London, SW1Y 5AG

Overview

Image by Claudia Stocker

Scientific discussion meeting organised by Dr Rafael Edgardo Carazo Salas, Dr Attila Csikasz-Nagy and Dr Masamitsu Sato

Event details

This event brought together an interdisciplinary group of scientists from the UK and abroad to speak about the latest progress in the field of cell polarity research, from basic principles to medical implications. It provided a unique opportunity to bring together basic and translational scientists and the public, to discuss this important subject at a high calibre meeting.

Biographies of the organisers and speakers are available below and you can also download the draft programme (PDF). Recorded audio of the presentations are now available below and the papers have been published in an issue of Philosophical Transactions B.

A related satellite meeting immediately followed this event.

Enquiries: Contact the events team.

Schedule of talks

Organisers

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Organiser

Dr Rafael Carazo Salas, University of Cambridge, UK

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Organiser

Dr Masamitsu Sato, University of Tokyo, Japan

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Organiser

Dr Attila Csikász-Nagy, King's College London, UK

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Session 1: Emergence of cell polarity

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Establishment and maintenance of cell polarity for asymmetric meiotic cell divisions in mouse oocytes

Professor Rong Li, Stowers Institute for Medical Research, US

Abstract

Mammalian oocytes undergo two rounds of asymmetric cell divisions during meiotic maturation. In in-vitro cultured oocytes, symmetry breaking occurs concurrent with migration of meiosis I spindle to a subcortical localization, whereby the meiotic chromatin induces the assembly of a polar actomyosin domain critical for the 1st polar body extrusion. Subsequently, meiosis II spindle forms at a nearby subcortical location and maintains oocyte cortical polarity in preparation for the second polar body extrusion upon fertilization. Recent studies have shown that, whereas the chromatin provides the inductive signal for oocyte cortical polarity, dynamic actin assembly at different cellular locations powers chromosome migration in meiosis I and the maintenance of spindle positioning in meiosis II. Recent progress on the mechanisms of actin-based force production and a mechanical model of cellular symmetry breaking will be presented.

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Modelling the roles of small GTPases in cell polarization, and cell shape

Professor Leah Edelstein-Keshet, University of British Columbia, US

Abstract

The small GTPAses Cdc42, Rac, and Rho are signalling proteins that coordinate the assembly, disassembly, and dynamics of the actin cytoskeleton. In this way, they regulate both cell polarization, cell shape, and motility in eukaryotic cells such as neutrophils. In my talk, I will describe spatio-temporal models of small GTPases, showing how their mutual interactions and feedback can affect the ability of the cell to respond to stimuli, polarize robustly, as well as its sensitivity to new stimuli. Both detailed and simplified (abstract) models have contributed to our understanding of these phenomena. Furthermore, in studying such models, we have utilized some recently developed mathematical methods that are of wider applicability in analysis of pattern formation. I will briefly describe these methods and our results.

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Positive feedback and mutual antagonism in cell polarity

Dr Barry Thompson, London Research Institute, Cancer Research UK, UK

Abstract

Epithelial tissues are composed of polarised cells with distinct apical and basolateral membrane domains. In the Drosophila ovarian follicle cell epithelium, apical membranes are specified by several apical determinants: the transmembrane protein Crumbs (Crb), its binding partner Stardust (Sdt), and the aPKC-Par6-cdc42 complex. Basolateral membranes are specified by the determinants Lgl, Dlg and Scrib. Apical and basolateral determinants are known to act in a mutually antagonistic fashion, but it remains unclear how this interaction generates polarity. We have built a computer model of apico-basal polarity which suggests that the combination of positive feedback among apical determinants plus mutual antagonism between apical and basal determinants is essential for polarisation. In agreement with this model, in vivo experiments define a positive feedback loop in which Crb self-recruits via Crb-Crb extracellular domain interactions, recruitment of Sdt and aPKC-Par6-cdc42 to the plasma membrane, aPKC phosphorylation, and recruitment of Expanded and Kibra to prevent endocytic removal of Crb from the plasma membrane. Ectopic activation of components of this loop can generate runaway positive feedback and ectopic spreading of apical determinants. Lgl antagonises the operation of this feedback loop, explaining why apical determinants do not normally spread into the basolateral domain. Once Crb is removed from the plasma membrane, it undergoes recycling via Rab11 endosomes. Our results provide a dynamic model for understanding how epithelial polarity is maintained in Drosophila follicle cells.

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Cell shape changes: quantitative role of membrane, cytoskeleton, and how they are attached

Professor Cécile Sykes, Institut Curie, France

Abstract

In order to unveil generic mechanisms of cell movements and shape changes, we design stripped-down experimental systems that reproduce cellular behaviours in simplified conditions, using liposome membranes on which the cytoskeleton is attached. Such stripped-down systems allow for a controlled study of the physical mechanisms that underlie cell movements and cell shape changes. Moreover, these experimental systems are used to address biological issues within a controlled, simplified environment.

We have reconstituted the actin cortex of cells inside liposomes, and used it as a simplified system to study endocytosis. We will present our work on reconstituted actin cortices at the membrane of liposomes, and a characterization of their mechanical properties measured by tube pulling, and liposome spreading, as done previously in cells. We will show how these cortices contract in the presence of myosin motors, and how such experiments shed light of the mechanisms of cell shape changes.

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Session 2: Interactions between cell polarity and other biological processes or machineries

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Interaction between bud-site-selection and polarity-establishment machineries in budding yeast

Professor Daniel Lew, Duke University, US

Abstract

Yeast cells polarize and form a single bud in each cell cycle. Genetic approaches have identified the molecular machinery responsible for positioning the bud site. Immobile landmark proteins, deposited at specific locations during bud formation, act after cytokinesis to promote activation of the conserved Rho-family GTPase, Cdc42, in their vicinity. Cdc42 accumulates further by positive feedback, creating a concentrated patch of GTP-Cdc42 at the bud site. Using high-resolution imaging and mathematical modeling, we examined the process of bud-site establishment. Polarity factors sometimes accumulated at more than one of the landmark-specified locations, and we suggest that competition between clusters of polarity factors determines the final bud location. Modeling indicated that competition would be impaired by continuing landmark-localized activity, and we suggest that polarity factors terminate landmark activity to preclude such interference. Imaging reveals unexpected effects of the bud-site-selection system on the dynamics of polarity establishment, raising new questions about how that system may operate.

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Evolution of Epithelial Organization and the Cadherin-Catenin Complex

Dr W. James Nelson, Stanford University, US

Abstract

A tube surrounded by a simple epithelium is the most basic tissue organization in metazoans. Epithelial tubes are formed by actomyosin-based constriction of the apical surface of epithelial sheets. In higher animals, epithelial organization is maintained by cadherin adhesion proteins which bind -catenin and -catenin that in turn organizes the actin cytoskeleton. I will discuss recent structure/function studies of the catenin complex in a variety of organisms. The results show that the catenin complex plays remarkably conserved functions in the formation and functional organization of epithelial structures from mammals to slime molds.

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Molecular organization of polarized domains of epithelial cells

Professor Norbert Perrimon, Harvard Medical School and Howard Hughes Medical Institute, USA

Abstract

Epithelial cells are polarized by the concerted activity of a conserved set of polarity proteins. To understand how these proteins establish and maintain distinct membrane domains we screened for downstream effectors using a library of shRNA constructs, which permit the analysis of nearly protein-null embryos by knockdown of a gene’s maternal and zygotic expression. One of our hits encodes a previously uncharacterized protein which contains a RhoGEF domain and interacts with the small GTPase Rac. This novel protein is localized to the apical cell cortex where it binds the polarity determinants Par-3 and aPKC, suggesting a role in directing Rac activity to the apical cortex. In addition, to define the molecular organization of the polarized domains in epithelial cells, we are developing a method for spatially resolved proteomics in Drosophila. Our approach uses a promiscuous labeling enzyme to catalyze the covalent labeling of all nearby proteins in a nanometer-scale radius. In this way, any subcellular region accessible to genetic targeting should become amenable to proteomic mapping.

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New tools for unlocking cell polarity

Professor Orion Weiner, University of California, San Francisco, US

Abstract

Neutrophils are innate immune cells that use directed migration to hunt and kill bacteria. This directed migration depends on several fundamental signaling capabilities. Neutrophils can migrate up chemotactic gradients spanning several orders of magnitude, requiring signaling adaptation so that cells respond to relative changes rather than steady-state concentrations of ligand. Neutrophils generate a consistent internal polarity that does not depend on the steepness of the external gradient, requiring positive feedback to amplify subtle signaling asymmetries and long-range inhibition so that protrusions can compete with one another to generate a dominant leading edge. Because the overall process of polarity is highly complex, we have developed (optogenetic and other) tools to isolate and dissect individual steps in the signaling cascade to better understand the overall signaling circuit. We find that plasma membrane tension orchestrates long-range inhibition, and actin dynamics are essential for adaptation.

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Session 3: Diseases where cell polarity is important

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Assembly of female meiotic spindles that mediate extreme asymmetric divisions

Professor Asako Sugimoto, Tohoku University, Japan

Abstract

Female meiosis is an extreme case of asymmetric cell division. A primary oocyte undergoes two rounds of meiotic divisions (meiosis I and II), which produce one large egg that inherits the majority of cytoplasm, while extruding two small polar bodies. These highly asymmetric divisions are mediated by meiotic spindles formed near the oocyte cortex. Unlike mitotic spindles that are formed with microtubules nucleated from centrosomes, female meiotic spindles in many animals are formed independently of centrosomes because centrosomes are eliminated during oogenesis. How and where microtubules are formed in oocytes and how they are assembled into meiotic spindles are not well understood. To elucidate the meiotic spindle assembly mechanism, we have been using C. elegans as a model system. We will discuss how distinct microtubule assembly pathways are used in mitosis and female meiosis.

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Breaking symmetry: Polarization of the C. elegans zygote

Professor Geraldine Seydoux, John Hopkins University, US

Abstract

We will discuss the molecular mechanisms that initiate and maintain polarity in the C. elegans zygote.

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Epithelial polarity and spindle orientation

Professor Daniel St Johnston, University of Cambridge, UK

Abstract

Simple epithelia are formed of a single layer of cells that adhere to each other to form barriers between compartments or the inside and outside of the organism. Epithelial cells divide with their mitotic spindles oriented in the plane of the epithelium, so that both daughters remain within the epithelial sheet, which is important for the maintenance of epithelial integrity. Furthermore, defects in spindle orientation have been proposed to contribute to tumorogenesis by producing daughter cells outside the epithelial sheet, leading to hypertrophy or metastasis. We have used the Drosophila follicular epithelium to investigate how the spindle is aligned perpendicular to the apical-basal axis of the cell. Our results reveal that spindle orientation does not require apical, junctional or basal cues, as previously proposed, and depends instead on a novel spindle orientation pathway. We have also disrupted spindle orientation to investigate the consequences of misoriented divisions on epithelial organisation.

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Polarity Proteins, Morphogenesis and Metastasis

Dr Ian Macara, Vanderbilt University, US

Abstract

Most human cancers arise from epithelial cells or their progenitors. Epithelial cells possess a distinctive apical-basal polarity, and loss of polarity is frequently assumed to be a common feature of cancer progression. However, there has been little experimental evidence for any role of the polarity machinery in tumor suppression. To address this issue we depleted the Par3 polarity gene by RNAi in combination with oncogenic Notch or Ras61L expression in the murine mammary gland. Par3 silencing dramatically reduced tumor latency in both models, increased tumor growth, and produced metastatic tumors that retained epithelial marker expression. Par3 depletion was associated with induction of MMP9, destruction of the extracellular matrix, and invasion, all mediated by atypical PKC-dependant JAK/Stat3 activation. Loss of Par3 also activated the Rac GTPase, which was essential for driving tumor growth. Importantly, Par3 expression is significantly reduced in human breast cancers, which correlates with active aPKC and Stat3. These data identify Par3 as a regulator of signaling pathways relevant to invasive breast cancer.

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Session 4: Towards better probes, diagnostics and treatment of polarity related diseases

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Polarized migration of leukocytes and cancer cells across the endothelium

Professor Anne Ridley, King's College London, UK

Abstract

In multicellular organisms, cells are constantly moving from one place to another. This cell migration is essential during development of the organism as well as maintenance of tissues in the adult. Cells of the immune system migrate out of the blood stream to fight infections and help repair wounds. Cell migration also contributes to the development of cancer. During cancer progression, cancer cells invade and migrate through the tissues and enter and then exit the blood stream to form secondary tumours, known as metastases. We are investigating how leukocytes and cancer cells attach to and cross the endothelial cells lining blood vessels. I will describe how we have identified specific proteins inside cells and on the surface of cells that are needed for leukocytes and cancer cells to interact with endothelial cells, and how reducing the expression of these proteins can decrease their transendothelial migration.

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Cell and tissue polarity in the intestinal tract

Professor Inke Näthke, University of Dundee, UK

Abstract

Cell and tissue polarity are tightly coupled and are vital for normal tissue homeostasis. Changes in cellular and tissue organisation are common to even early stages of disease, particularly cancer. The digestive tract is the site of the second most common cause of cancer deaths in the developed world. The epithelium that gives rise to tumours in this tissue displays a number of axes of cell and tissue polarity. Changes in cell and tissue polarity in response to genetic changes that are known to underpin disease progression provide clues about the link between molecular, cellular and tissue based mechanisms that accompany cancer. Mutations in APC are common to most colorectal cancers in humans and are sufficient to cause tumours in mouse intestine. Tissue organoids mimic many of features of whole tissue and permit identifying changes at different times after inactivation of APC. In gut tissue organoids polarity is lost very early during cancer progression while cell polarity, at least apical – basal polarity is maintained and only changes at later stages. These observations reflect the situation in tumours and validate tissue organoids as a useful system to investigate the relationship between cell polarity and tissue organisation.

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Dynamics of intercellular contacts during collective cell migration

Dr Sandrine Etienne-Manneville, Institut Pasteur, France

Abstract

Collective cell migration is essential during development as well as in adult organisms where it participates, for instance, in tissue renewal, wound healing or cancer invasion and metastasis. As cells migrate collectively, intercellular junctions maintain the integrity of the cell monolayer while allowing differential movement and rearrangements of adjacent cells. In astrocytes, intercellular contacts are mainly formed by N-cadherin-mediated adherens junctions. Downregulation of N-cadherin is frequently observed in astrocyte derived tumors, gliomas and lead to the perturbation of cell polarity and to an increased cell velocity. To understand how cells can maintain stable intercellular junctions and simultaneously rearrange them to accommodate cellular displacement, we have investigated N-cadherin dynamics during astrocyte collective migration. We show adherens junctions undergo a continuous retrograde movement compensated by a polarized recycling of N-cadherin from the rear to the leading edge. Such dynamics allows the cells to maintain stable contacts while permitting changes of cellular interactions. In glioma cells, N-cadherin dynamics and consequently the maintenance of cell-cell contacts are perturbed leading to loss of cell polarity and to increased migration.

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How Polarity Functions in Regulating Skin Stem Cell Behavior

Dr Elaine Fuchs, Rockefeller University, US

Abstract

Embryonic epidermis begins as a single layer of unspecified progenitors. During development, it receives external cues to undergo a series of morphogenetic events which culminate in the production of a stratified tissue replete with hair follicles. Postnatally, these tissues undergo self-renewal which requires stem cells. Stem cells exist both in the innermost (basal) layer of the epidermis, at the base of the sebaceous gland and in the hair follicle, in a region known as the bulge. How stem cells develop and how they balance self-renewal and differentiation is of fundamental importance to our understanding of normal tissue maintenance and wound repair. Using skin as our paradigm, we’ve been dissecting how extrinsic signaling to stem sets off a cascade of changes in transcription that governs the activation of stem cells during tissue development, homeostasis and hair cycling. Our findings have provided us with new insights into our understanding of the process of stem cell activation, and in so doing have revealed mechanisms which are also deregulated in a variety of different human cancers. In this talk, I will review some of our studies that implicate specific signaling pathways in regulating stem cell polarity and function.

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Cellular polarity: from mechanisms to disease The Royal Society, London 6-9 Carlton House Terrace London SW1Y 5AG UK