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Remodeling of adhesion and modulation of mechanical tensile forces during apoptosis in Drosophila epithelium
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Abstracts
PS1.65 The Regulation of Mitosis in Complex Epithelial Structures Natalie Kirkland, Yanlan Mao MRC Laboratory of Molecular Cell Biology, University College London, United Kingdom The development of a single-celled zygote to a multicellular organism requires the rapid yet controlled proliferation of cells. Both cell number and cell size are meticulously regulated in order to form complex tissues and organs. Cell division rate is fundamental for determining cell number and size. Mitosis is a critical process in tissue growth and cell biological research that remains to be fully understood, especially within a multicellular tissue environment. This project aims to investigate how mitosis is regulated in complex epithelial structures found during organ development and how mitosis is influenced by changing tissue structure. Mitotic divisions will be studied within the highly proliferative epithelial tissue, the Drosophila wing disc. The wing disc is an intermediatelength pseudostratified epithelium (PSE). PSE is found widely in early organ development, and notably during neural tube to neocortex formation. To undergo mitosis within PSE, a nucleus must translocate from the basal to the apical surface prior to mitotic cell rounding, a process called Inter-kinetic Nuclear Migration (IKNM). The machinery that drives IKNM differs depending on the height of the epithelium. Short epithelia depend on actomyosin, whilst tall epithelia utilise microtubule transport and motor proteins. These mechanisms may cooperate in intermediate PSE but the regulation of IKNM remains to be elucidated. How tissue architecture affects IKNM is the main focus of this project. The developmental increase in cell compaction and cell height within the Drosophila wing disc provides an ideal model system to study this question. Varying cell and tissue morphology may influence aspects of IKNM to regulate tissue growth. The characteristic folds of the wing disc provide regions of positive and negative curvature to observe the effects of opposing apical surface on IKNM. Furthermore, the powerful genetic tools provided by Drosophila will allow novel regulators of IKNM to be identified.
competition, whereby suboptimal but viable cells are eliminated by more fit cells, and suggest that such mechanical competition may promote tumoral cell expansion. We will present our current understanding of this mechanism and our attempt to identify new mechanosensing pathways responsible for crowding sensing and caspase activation. doi:10.1016/j.mod.2017.04.073
PS1.67 The architectural balance of the Ventral Nerve Cord depends on the level of JNK signaling activity Katerina Karkalia, Ignasi Jorbab, Anand Shingc, Daniel Navajasb, Timothy Saundersc, George Panayotoud, Enrique Martin-Blancoa a
IBMB (CSIC), Barcelona, Spain Biophysics (Universitat de Barcelona) - IBEC, Barcelona, Spain c MBI (NUS), Singapore, Singapore d BSRC Fleming, Athens, Greece b
The segmented nervous system of bilaterians is organized in structural and functional modules. Modules share across species a robust structural stability. How this robustness is acquired during development is currently unknown. Here, we investigate the sequence of events involved in the establishment of the architectural balance of the nervous system. We demonstrate that a unique robustness pattern is common to the arthropods nervous system plan. In Drosophila, this pattern depends on the fine control of JNK signaling activity in a subset of neurons. JNK controls the level of expression of cell adhesion molecules (Fas 2) and axonal fasciculation. Remarkably, the Drosophila embryonic central nervous system compliance, measured by Atomic Force Microscopy (AFM) is affected in JNK defective embryos. Failure to fasciculate affects both architectural robustness and tensional balance, ultimately impeding nervous system condensation. doi:10.1016/j.mod.2017.04.074
doi:10.1016/j.mod.2017.04.072
PS1.66 Tissue crowding drives caspase dependent competition for space Romain Levayer Institut Pasteur, Paris, France
PS1.68 Remodeling of adhesion and modulation of mechanical tensile forces during apoptosis in Drosophila epithelium Xiang Tenga, Lei Qina, Roland Le Borgneb,c, Yusuke Toyamaa,d,e a
Mechanobiology Institute, National University of Singapore, Singapore CNRS, Rennes, France c Université Rennes, France d National University of Singapore, Singapore e Temasek Life Sciences Laboratory, Singapore b
Developing tissues have an amazing plasticity which is mostly based on the capacity of single cells to adapt their behavior to local and tissue scale information. While the pathways regulating cell survival and proliferation are well known, we still known very little about how survival and proliferation can be adapted to tissue scale information (tissue size, tissue density). Few years back, it was shown tissue crowding could lead to live cell extrusion from the epithelial layer. By analyzing a similar process in the Drosophila pupal notum, we recently showed that tissue crowding was necessary and sufficient to drive cell elimination (Levayer et al., Current Biology 2016) and that caspase activation was preceding and necessary for every cell delamination event. This was suggesting that cells could have a differential sensitivity to crowding depending on their sensitivity to apoptosis. Indeed, inducing fast growth in clones resistant for apoptosis (activation of the oncogene RasV12) was sufficient to induce ectopic compaction and elimination of WT cells. This mechanism is reminiscent of the concept of cell
Apoptosis is a mechanism of eliminating damaged or unnecessary cells during development and tissue homeostasis. During apoptosis within a tissue, the adhesions between dying and neighboring nondying cells need to be remodeled so that the apoptotic cell is expelled. In parallel, contraction of actomyosin cables formed in apoptotic and neighboring cells drives cell extrusion. To date, the coordination between the dynamics of cell adhesion and the progressive changes in tissue tension around an apoptotic cell is not fully understood. Live imaging of histoblast expansion, which is a coordinated tissue replacement process during Drosophila metamorphosis, shows disengagemnt of adherens junctions (AJs) between apoptotic and non-dying cells, including E-cadherin. Concurrently, surrounding tissue tension is transiently released. Contraction of a
Abstracts
supra-cellular actomyosin cable, which forms in neighboring cells, brings neighboring cells together and further reshapes tissue tension toward the completion of extrusion. We propose a model in which modulation of tissue tension represents a mechanism of apoptotic cell extrusion.
S45
result in a reduction of the entire follicle cell migration, suggesting centripetal FCs push the cell in the front and pull the cells in the back. We further find the direction of the migration of centripetal FCs is determined by PVR/EGFR signaling. Our results highlight the active contribution of follower cell in follicle posterior migration, which could be applicable to general collective cell migration.
doi:10.1016/j.mod.2017.04.075 doi:10.1016/j.mod.2017.04.077 PS1.69 Desmosomal coupling influences junctional actomyosin contractility in steady state and apoptotic junctions Minnah Thomasa, Yusuke Toyamaa,b,c a
Mechanobiology Institute, Singapore National University of Singapore, Singapore c Temasek Lifescience Laboratory, Singapore b
Apoptotic extrusion is a process, where junctional contractility is spatially regulated at a supracellular scale to expel a dying cell from the epithelial sheet. During apoptotic extrusion, E-cadherin based cell coupling transduces forces from the constricting actomyosin cable through the apoptotic cell interface. Since epithelial turnover is very prevalent in intestine and other simple epithelia, we wanted to address the consequence of apoptotic extrusion on the other intercellular adhesion, the desmosome. In this study, the dynamics of the neighboring tissue during extrusion is studied by subjecting cells to an ultraviolet laser based apoptotic stimulus. We show that desmosome exhibits dynamics and retains its junctional presence during extrusion. The intensity of the junctional desmoglein-2 increased during the constriction phase of extrusion. Depletion of desmoplakin by RNA interference resulted in constriction defects, indicating its importance in facilitating extrusion. A concomitant recruitment of cytokeratin-18 filaments to the apoptotic cell interface was observed during constriction. The recruited keratin filaments were proximate to the actomyosin cable at the interface. We further show evidence that cytokeratin-18 and desmosome influence junctional contractility in steady state and extruding junctions. doi:10.1016/j.mod.2017.04.076
PS1.70 Active role of follower cells in collective cell migration during Drosophila mid-oogenesis Lei Qin, Xiang Teng, Yusuke Toyama Mechanobiology Institute, Singapore National University of Singapore, Singapore Temasek Life Sciences Laboratory, Singapore Collective cell migration is a crucial multi-cellular process widely observed from animal development, wound healing and pathological condition, including tumor metastasis. Compare to our current understanding of the role of leader cells, which exert a large traction force and pull the follower cells, the contributions of follower cells in collective cell migration is still illusive. Here we characterize the migration of a cohort of cells in the middle of a tissue, centripetal follicle cells (FCs), in follicle cell posterior migration during Drosophila mid-oogenesis. We find that centripetal FCs show actinrich lamellipodia-like protrusions and high migration speed with high Rac activities compared to the neighboring FCs. Inactivation of Rac by photo-activating DN-Rac specifically in centripetal FCs,
PS1.71 Cortical Tension Drives Pluripotent Inner Mass Formation in the Mammalian Embryo Melanie White, Jenny Zenker, Maxime Gasnier, Grace Lim, Stephanie Bissiere, Nicolas Plachta A*star, Singapore Every cell in our body originates from the pluripotent inner mass of the embryo, yet the mechanical forces forming this structure are unknown. We developed new quantitative imaging technologies to track changes in cell shape and position in living mouse embryos. We characterized the main morphogenetic mechanisms forming the pluripotent inner mass and showed how mechanical forces acting within the embryo control cell-positioning events. Using high temporal resolution live imaging we show that the apical domain, previously proposed to determine lineage segregation, is disassembled before cell division. Instead, we find that anisotropies in cortical tension play a key role in determining which cells undergo internalization to form the inner mass. We characterize the underlying mechanisms and explain how the directionality of tensile forces coordinates cell rearrangements during early mammalian development.
doi:10.1016/j.mod.2017.04.078
PS1.72 Spatial localisation of YAP during skeletal development implicates the Hippo pathway in skeletal mechanotransduction and morphogenesis Claire Shea, Rebecca Rolfe, Paula Murphy Trinity College Dublin, Ireland The Hippo-YAP/TAZ cell signalling pathway regulates cell proliferation and organ size and can facilitate the interpretation and response of cells to their mechanical environment by influencing the expression of downstream genes important to differentiation (Dupont et al., 2011). First characterized in Drosophila, it is known to be important to cellular differentiation in the early mouse embryo and developing kidney. During skeletal development, the formation of functional bones and joints depends on appropriate embryonic mechanical stimulation: in absent or reduced movement, misshapen and brittle bones and fused joints result (reviewed in Shea et al., 2015). Our group has previously characterized the complex genetic changes that occur in the humerus and associated joints of muscleless (Splotch delayed) mouse embryos (Rolfe et al., 2014). Here, we will present the expression of multiple components of the Hippo pathway during normal skeletal development, and identify changes occurring in the absence of muscle-driven limb movement. YAP, the primary co-activator of the pathway, is differentially expressed in the developing forelimb rudiments at key stages of ossification and
Abstracts
PS1.65 The Regulation of Mitosis in Complex Epithelial Structures Natalie Kirkland, Yanlan Mao MRC Laboratory of Molecular Cell Biology, University College London, United Kingdom The development of a single-celled zygote to a multicellular organism requires the rapid yet controlled proliferation of cells. Both cell number and cell size are meticulously regulated in order to form complex tissues and organs. Cell division rate is fundamental for determining cell number and size. Mitosis is a critical process in tissue growth and cell biological research that remains to be fully understood, especially within a multicellular tissue environment. This project aims to investigate how mitosis is regulated in complex epithelial structures found during organ development and how mitosis is influenced by changing tissue structure. Mitotic divisions will be studied within the highly proliferative epithelial tissue, the Drosophila wing disc. The wing disc is an intermediatelength pseudostratified epithelium (PSE). PSE is found widely in early organ development, and notably during neural tube to neocortex formation. To undergo mitosis within PSE, a nucleus must translocate from the basal to the apical surface prior to mitotic cell rounding, a process called Inter-kinetic Nuclear Migration (IKNM). The machinery that drives IKNM differs depending on the height of the epithelium. Short epithelia depend on actomyosin, whilst tall epithelia utilise microtubule transport and motor proteins. These mechanisms may cooperate in intermediate PSE but the regulation of IKNM remains to be elucidated. How tissue architecture affects IKNM is the main focus of this project. The developmental increase in cell compaction and cell height within the Drosophila wing disc provides an ideal model system to study this question. Varying cell and tissue morphology may influence aspects of IKNM to regulate tissue growth. The characteristic folds of the wing disc provide regions of positive and negative curvature to observe the effects of opposing apical surface on IKNM. Furthermore, the powerful genetic tools provided by Drosophila will allow novel regulators of IKNM to be identified.
competition, whereby suboptimal but viable cells are eliminated by more fit cells, and suggest that such mechanical competition may promote tumoral cell expansion. We will present our current understanding of this mechanism and our attempt to identify new mechanosensing pathways responsible for crowding sensing and caspase activation. doi:10.1016/j.mod.2017.04.073
PS1.67 The architectural balance of the Ventral Nerve Cord depends on the level of JNK signaling activity Katerina Karkalia, Ignasi Jorbab, Anand Shingc, Daniel Navajasb, Timothy Saundersc, George Panayotoud, Enrique Martin-Blancoa a
IBMB (CSIC), Barcelona, Spain Biophysics (Universitat de Barcelona) - IBEC, Barcelona, Spain c MBI (NUS), Singapore, Singapore d BSRC Fleming, Athens, Greece b
The segmented nervous system of bilaterians is organized in structural and functional modules. Modules share across species a robust structural stability. How this robustness is acquired during development is currently unknown. Here, we investigate the sequence of events involved in the establishment of the architectural balance of the nervous system. We demonstrate that a unique robustness pattern is common to the arthropods nervous system plan. In Drosophila, this pattern depends on the fine control of JNK signaling activity in a subset of neurons. JNK controls the level of expression of cell adhesion molecules (Fas 2) and axonal fasciculation. Remarkably, the Drosophila embryonic central nervous system compliance, measured by Atomic Force Microscopy (AFM) is affected in JNK defective embryos. Failure to fasciculate affects both architectural robustness and tensional balance, ultimately impeding nervous system condensation. doi:10.1016/j.mod.2017.04.074
doi:10.1016/j.mod.2017.04.072
PS1.66 Tissue crowding drives caspase dependent competition for space Romain Levayer Institut Pasteur, Paris, France
PS1.68 Remodeling of adhesion and modulation of mechanical tensile forces during apoptosis in Drosophila epithelium Xiang Tenga, Lei Qina, Roland Le Borgneb,c, Yusuke Toyamaa,d,e a
Mechanobiology Institute, National University of Singapore, Singapore CNRS, Rennes, France c Université Rennes, France d National University of Singapore, Singapore e Temasek Life Sciences Laboratory, Singapore b
Developing tissues have an amazing plasticity which is mostly based on the capacity of single cells to adapt their behavior to local and tissue scale information. While the pathways regulating cell survival and proliferation are well known, we still known very little about how survival and proliferation can be adapted to tissue scale information (tissue size, tissue density). Few years back, it was shown tissue crowding could lead to live cell extrusion from the epithelial layer. By analyzing a similar process in the Drosophila pupal notum, we recently showed that tissue crowding was necessary and sufficient to drive cell elimination (Levayer et al., Current Biology 2016) and that caspase activation was preceding and necessary for every cell delamination event. This was suggesting that cells could have a differential sensitivity to crowding depending on their sensitivity to apoptosis. Indeed, inducing fast growth in clones resistant for apoptosis (activation of the oncogene RasV12) was sufficient to induce ectopic compaction and elimination of WT cells. This mechanism is reminiscent of the concept of cell
Apoptosis is a mechanism of eliminating damaged or unnecessary cells during development and tissue homeostasis. During apoptosis within a tissue, the adhesions between dying and neighboring nondying cells need to be remodeled so that the apoptotic cell is expelled. In parallel, contraction of actomyosin cables formed in apoptotic and neighboring cells drives cell extrusion. To date, the coordination between the dynamics of cell adhesion and the progressive changes in tissue tension around an apoptotic cell is not fully understood. Live imaging of histoblast expansion, which is a coordinated tissue replacement process during Drosophila metamorphosis, shows disengagemnt of adherens junctions (AJs) between apoptotic and non-dying cells, including E-cadherin. Concurrently, surrounding tissue tension is transiently released. Contraction of a
Abstracts
supra-cellular actomyosin cable, which forms in neighboring cells, brings neighboring cells together and further reshapes tissue tension toward the completion of extrusion. We propose a model in which modulation of tissue tension represents a mechanism of apoptotic cell extrusion.
S45
result in a reduction of the entire follicle cell migration, suggesting centripetal FCs push the cell in the front and pull the cells in the back. We further find the direction of the migration of centripetal FCs is determined by PVR/EGFR signaling. Our results highlight the active contribution of follower cell in follicle posterior migration, which could be applicable to general collective cell migration.
doi:10.1016/j.mod.2017.04.075 doi:10.1016/j.mod.2017.04.077 PS1.69 Desmosomal coupling influences junctional actomyosin contractility in steady state and apoptotic junctions Minnah Thomasa, Yusuke Toyamaa,b,c a
Mechanobiology Institute, Singapore National University of Singapore, Singapore c Temasek Lifescience Laboratory, Singapore b
Apoptotic extrusion is a process, where junctional contractility is spatially regulated at a supracellular scale to expel a dying cell from the epithelial sheet. During apoptotic extrusion, E-cadherin based cell coupling transduces forces from the constricting actomyosin cable through the apoptotic cell interface. Since epithelial turnover is very prevalent in intestine and other simple epithelia, we wanted to address the consequence of apoptotic extrusion on the other intercellular adhesion, the desmosome. In this study, the dynamics of the neighboring tissue during extrusion is studied by subjecting cells to an ultraviolet laser based apoptotic stimulus. We show that desmosome exhibits dynamics and retains its junctional presence during extrusion. The intensity of the junctional desmoglein-2 increased during the constriction phase of extrusion. Depletion of desmoplakin by RNA interference resulted in constriction defects, indicating its importance in facilitating extrusion. A concomitant recruitment of cytokeratin-18 filaments to the apoptotic cell interface was observed during constriction. The recruited keratin filaments were proximate to the actomyosin cable at the interface. We further show evidence that cytokeratin-18 and desmosome influence junctional contractility in steady state and extruding junctions. doi:10.1016/j.mod.2017.04.076
PS1.70 Active role of follower cells in collective cell migration during Drosophila mid-oogenesis Lei Qin, Xiang Teng, Yusuke Toyama Mechanobiology Institute, Singapore National University of Singapore, Singapore Temasek Life Sciences Laboratory, Singapore Collective cell migration is a crucial multi-cellular process widely observed from animal development, wound healing and pathological condition, including tumor metastasis. Compare to our current understanding of the role of leader cells, which exert a large traction force and pull the follower cells, the contributions of follower cells in collective cell migration is still illusive. Here we characterize the migration of a cohort of cells in the middle of a tissue, centripetal follicle cells (FCs), in follicle cell posterior migration during Drosophila mid-oogenesis. We find that centripetal FCs show actinrich lamellipodia-like protrusions and high migration speed with high Rac activities compared to the neighboring FCs. Inactivation of Rac by photo-activating DN-Rac specifically in centripetal FCs,
PS1.71 Cortical Tension Drives Pluripotent Inner Mass Formation in the Mammalian Embryo Melanie White, Jenny Zenker, Maxime Gasnier, Grace Lim, Stephanie Bissiere, Nicolas Plachta A*star, Singapore Every cell in our body originates from the pluripotent inner mass of the embryo, yet the mechanical forces forming this structure are unknown. We developed new quantitative imaging technologies to track changes in cell shape and position in living mouse embryos. We characterized the main morphogenetic mechanisms forming the pluripotent inner mass and showed how mechanical forces acting within the embryo control cell-positioning events. Using high temporal resolution live imaging we show that the apical domain, previously proposed to determine lineage segregation, is disassembled before cell division. Instead, we find that anisotropies in cortical tension play a key role in determining which cells undergo internalization to form the inner mass. We characterize the underlying mechanisms and explain how the directionality of tensile forces coordinates cell rearrangements during early mammalian development.
doi:10.1016/j.mod.2017.04.078
PS1.72 Spatial localisation of YAP during skeletal development implicates the Hippo pathway in skeletal mechanotransduction and morphogenesis Claire Shea, Rebecca Rolfe, Paula Murphy Trinity College Dublin, Ireland The Hippo-YAP/TAZ cell signalling pathway regulates cell proliferation and organ size and can facilitate the interpretation and response of cells to their mechanical environment by influencing the expression of downstream genes important to differentiation (Dupont et al., 2011). First characterized in Drosophila, it is known to be important to cellular differentiation in the early mouse embryo and developing kidney. During skeletal development, the formation of functional bones and joints depends on appropriate embryonic mechanical stimulation: in absent or reduced movement, misshapen and brittle bones and fused joints result (reviewed in Shea et al., 2015). Our group has previously characterized the complex genetic changes that occur in the humerus and associated joints of muscleless (Splotch delayed) mouse embryos (Rolfe et al., 2014). Here, we will present the expression of multiple components of the Hippo pathway during normal skeletal development, and identify changes occurring in the absence of muscle-driven limb movement. YAP, the primary co-activator of the pathway, is differentially expressed in the developing forelimb rudiments at key stages of ossification and