Influence of the 3D environment in collective cell migration

Motivation: Cell migration plays a key role in several biological processes, such as embryo development or immune system maintenance, and its alteration is involved in pathological conditions like cancer and metastasis. Cells can migrate individually or as a group, but in both cases, they migrate in vivo through a complex 3D environment, although the contribution of that environment to cell migration is still poorly understood. The migration of the border cells in the Drosophila melanogaster ovaries constitutes an ideal system to study collective cell migration in vivo. Border cells are a group of cells that detach from the follicular epithelium that surrounds the egg chamber and migrate through 15 germ cells, called nurse cells, until they reach the oocyte. In this project we use the border cell migration to study the influence of the 3D-environment over the collective migration of a group of cells.Methods: we analysed the border cell migration in both fixed and live samples in wild type and mutant conditions, which were generated either using mutants or expressing RNAis using the UAS-GAL4 system. Fixed samples were stained with some dyes and antibodies to visualize actin, DNA and the border cells, and in vivo analysis was performed using fly strains carrying fluorescently labelled proteins. In all cases, samples were analysed by confocal microscopy and images and movies were processed with ImageJ and Imaris software.Results: the aim of this project was to analyse the influence of the 3D environment (organization of the nurse cells) in border cell migration. For this purpose, the first step was the identification of two mutant alleles of a gene that, when combined, cause an abnormal organization of the nurse cells. Then, we found that in this mutant background border cell migration was affected. Furthermore, there was a correlation between border cell migration defects and aberrant distribution of nurse cells (especially in the most anterior part of the egg chamber). Conclusions: according to our findings, we can say that the organization of the 3D environment is a key factor in collective cell migration, since it is necessary to maintain the forces balance required by cells to migrate

Myosin light-chain phosphatase regulates basal actomyosin oscillations during morphogenesis

Contractile actomyosin networks generate forces that drive tissue morphogenesis. Actomyosin contractility is controlled primarily by reversible phosphorylation of the myosin-II regulatory light chain through the action of myosin kinases and phosphatases. While the role of myosin light-chain kinase in regulating contractility during morphogenesis has been largely characterized, there is surprisingly little information on myosin light-chain phosphatase (MLCP) function in this context. Here, we use live imaging of Drosophila follicle cells combined with mathematical modelling to demonstrate that the MLCP subunit flapwing (flw) is a key regulator of basal myosin oscillations and cell contractions underlying egg chamber elongation. Flw expression decreases specifically on the basal side of follicle cells at the onset of contraction and flw controls the initiation and periodicity of basal actomyosin oscillations. Contrary to previous reports, basal F-actin pulsates similarly to myosin. Finally, we propose a quantitative model in which periodic basal actomyosin oscillations arise in a cell-autonomous fashion from intrinsic properties of motor assembliesResearch in our laboratories is funded by the Spanish Ministerio de Economía y Competitividad and the FEDER programme (BFU2013-48988-C2-1-P to M.D.M.-B, BFU2012-35446 to A.G.-R., BFU2011-30303 and BFU2010-18959 to D.G.M.) and by the Junta de Andalucía (Proyecto de Excelencia P09-CVI-5058). I.G. was supported by a JAE-DOC (CSIC) and D.G.M. by a Ramon y Cajal Fellowship (Ministerio Español de Economía y Competitividad Ref. RYC-2010-07450) and a Marie Curie International Reintegration Grant (EU, Ref. 248346-NMSSBLS

Integrins regulate epithelial cell shape by controlling the architecture and mechanical properties of basal actomyosin networks.

Forces generated by the actomyosin cytoskeleton are key contributors to many morphogenetic processes. The actomyosin cytoskeleton organises in different types of networks depending on intracellular signals and on cell-cell and cell-extracellular matrix (ECM) interactions. However, actomyosin networks are not static and transitions between them have been proposed to drive morphogenesis. Still, little is known about the mechanisms that regulate the dynamics of actomyosin networks during morphogenesis. This work uses the Drosophila follicular epithelium, real-time imaging, laser ablation and quantitative analysis to study the role of integrins on the regulation of basal actomyosin networks organisation and dynamics and the potential contribution of this role to cell shape. We find that elimination of integrins from follicle cells impairs F-actin recruitment to basal medial actomyosin stress fibers. The available F-actin redistributes to the so-called whip-like structures, present at tricellular junctions, and into a new type of actin-rich protrusions that emanate from the basal cortex and project towards the medial region. These F-actin protrusions are dynamic and changes in total protrusion area correlate with periodic cycles of basal myosin accumulation and constriction pulses of the cell membrane. Finally, we find that follicle cells lacking integrin function show increased membrane tension and reduced basal surface. Furthermore, the actin-rich protrusions are responsible for these phenotypes as their elimination in integrin mutant follicle cells rescues both tension and basal surface defects. We thus propose that the role of integrins as regulators of stress fibers plays a key role on controlling epithelial cell shape, as integrin disruption promotes reorganisation into other types of actomyosin networks, in a manner that interferes with proper expansion of epithelial basal surfaces

Las integrinas regulan positivamente la supervivencia celular en el disco imaginal de ala en Drosophila melanogaster

Motivación: Las integrinas son una amplia familia de receptores transmembrana que se unen preferentemente a componentes de la matriz extracelular. Además de su importancia como conectores mecánicos, las integrinas también participan en la activación de diferentes cascadas de señalización y en el control de diferentes procesos celulares como adhesión, migración, proliferación, diferenciación y supervivencia celular en la cual se centra este trabajo. La disrupción de la función de interacción de las integrinas resulta en un tipo de apoptosis denominada anoikis. La anoikis es esencial no solo durante el desarrollo y el mantenimiento de la homeostasis durante la vida adulta, sino también como un importante mecanismo de supervivencia asegurando que toda célula que pierde su posición apropiada en un tejido es señalizada para sufrir apoptosis. Aunque existe abundante información sobre el papel de las integrinas como promotores de supervivencia celular, se conoce poco acerca de la significancia biológica de esta función de las integrinas y el mecanismo molecular que regula la anoikis durante el desarrollo. Por ello, nuestro objetivo principal en este proyecto es comprender los mecanismos moleculares a través de los cuales las integrinas regulan la supervivencia celular durante el desarrollo utilizando el disco imaginal de ala de Drosophila como sistema modelo.Métodos: Para alcanzar nuestro objetivo hemos reducido los niveles de integrinas en el disco de ala y en este contexto hemos analizado el posible papel de diferentes proteínas proapoptóticas en la muerte celular mediada por la falta de función de integrinas. Por otra parte, hemos estudiado el papel de la tensión de miosina en la muerte celular mediada por la falta de función de integrinas. Los fenotipos resultantes los hemos analizado a través de la tinción inmunohistoquímica de los discos imaginales previamente fijados.Resultados: Mostramos que la falta de función de integrinas en el disco de ala resulta en anoikis dependiente de caspasa debida a la activación de la ruta JNK la cual a su vez activa a la proteína proapoptótica Hid.Mostramos por otra parte que la falta de función de integrinas en el disco de ala resulta en un aumento de tensión por miosina.Conclusiones: Tras nuestros estudios hemos concluido que la falta de función de integrinas promueve la activación de la ruta JNK la cual media la muerte celular a través de la expresión de la proteína proapoptótica Hid

A coarse-grained approach to model the dynamics of the actomyosin cortex

Background: The dynamics of the actomyosin machinery is at the core of many important biological processes. Several relevant cellular responses such as the rhythmic compression of the cell cortex are governed, at a mesoscopic level, by the nonlinear interaction between actin monomers, actin crosslinkers, and myosin motors. Coarse-grained models are an optimal tool to study actomyosin systems, since they can include processes that occur at long time and space scales, while maintaining the most relevant features of the molecular interactions. Results: Here, we present a coarse-grained model of a two-dimensional actomyosin cortex, adjacent to a three-dimensional cytoplasm. Our simplified model incorporates only well-characterized interactions between actin monomers, actin crosslinkers and myosin, and it is able to reproduce many of the most important aspects of actin filament and actomyosin network formation, such as dynamics of polymerization and depolymerization, treadmilling, network formation, and the autonomous oscillatory dynamics of actomyosin. Conclusions: We believe that the present model can be used to study the in vivo response of actomyosin networks to changes in key parameters of the system, such as alterations in the attachment of actin filaments to the cell corte

Scutoids are a geometrical solution to three-dimensional packing of epithelia

As animals develop, tissue bending contributes to shape the organs into complex three-dimensional structures. However, the architecture and packing of curved epithelia remains largely unknown. Here we show by means of mathematical modelling that cells in bent epithelia can undergo intercalations along the apico-basal axis. This phenomenon forces cells to have different neighbours in their basal and apical surfaces. As a consequence, epithelial cells adopt a novel shape that we term “scutoid”. The detailed analysis of diverse tissues confirms that generation of apico-basal intercalations between cells is a common feature during morphogenesis. Using biophysical arguments, we propose that scutoids make possible the minimization of the tissue energy and stabilize three-dimensional packing. Hence, we conclude that scutoids are one of nature's solutions to achieve epithelial bending. Our findings pave the way to understand the three-dimensional organization of epithelial organs

Two cell line models to study multiorganic metastasis and immunotherapy in lung squamous cell carcinoma

There is a paucity of adequate mouse models and cell lines available to study lung squamous cell carcinoma (LUSC). We have generated and characterized two models of phenotypically different transplantable LUSC cell lines, i.e. UN-SCC679 and UN-SCC680, derived from A/J mice that had been chemically induced with N-nitroso-tris-chloroethylurea (NTCU). Furthermore, we genetically characterized and compared both LUSC cell lines by performing whole-exome and RNA sequencing. These experiments revealed similar genetic and transcriptomic patterns that may correspond to the classic LUSC human subtype. In addition, we compared the immune landscape generated by both tumor cells lines in vivo and assessed their response to immune checkpoint inhibition. The differences between the two cell lines are a good model for the remarkable heterogeneity of human squamous cell carcinoma. Study of the metastatic potential of these models revealed that both cell lines represent the organotropism of LUSC in humans, i.e. affinity to the brain, bones, liver and adrenal glands. In summary, we have generated valuable cell line tools for LUSC research, which recapitulates the complexity of the human disease.This work was supported by FIMA, Centro de Investigacion Biomedica en Red de Cancer (CIBERONC) (grant number: CB16/12/00443), Fundacion Cientifica Asociacion Espanola Contra el Cancer (grant number: GCB14-2170), Fundacion Ramon Areces, Instituto de Salud Carlos III and the European Regional Development Fund (ERDF, A way to make Europe) (grant numbers: PI19/00098; PI19/00230; PI20/00419), Fundacion Roberto Arnal Planelles and an IASLC Fellowship funding (K.V.); D.S. was supported by the Juan de la Cierva-Incorporacion program, Spanish Ministry of Science and Innovation (grant number: IJCI-2016-27595); E.R. was supported by a FPU, Spanish Ministry of Education ( grant number: FPU17/01168); M.E. was supported by PFIS, Spanish Ministry of Health, M.L. was supported by a Junior Investigator grant from AECC

Identification of novel synthetic lethal vulnerability in non small cell lung cancer by co targeting TMPRSS4 and DDR1

Finding novel targets in non-small cell lung cancer (NSCLC) is highly needed and identification of synthetic lethality between two genes is a new approach to target NSCLC. We previously found that TMPRSS4 promotes NSCLC growth and constitutes a prognostic biomarker. Here, through large-scale analyses across 5 public databases we identified consistent co-expression between TMPRSS4 and DDR1. Similar to TMPRSS4, DDR1 promoter was hypomethylated in NSCLC in 3 independent cohorts and hypomethylation was an independent prognostic factor of disease-free survival. Treatment with 5-azacitidine increased DDR1 levels in cell lines, suggesting an epigenetic regulation. Cells lacking TMPRSS4 were highly sensitive to the cytotoxic effect of the DDR1 inhibitor dasatinib. TMPRSS4/DDR1 double knock-down (KD) cells, but not single KD cells suffered a G0/G1 cell cycle arrest with loss of E2F1 and cyclins A and B, increased p21 levels and a larger number of cells in apoptosis. Moreover, double KD cells were highly sensitized to cisplatin, which caused massive apoptosis (~40%). In vivo studies demonstrated tumor regression in double KD-injected mice. In conclusion, we have identified a novel vulnerability in NSCLC resulting from a synthetic lethal interaction between DDR1 and TMPRSS4

Author Correction: Scutoids are a geometrical solution to three-dimensional packing of epithelia.

The original version of this Article contained an error in ref. 39, which incorrectly cited 'Fristrom, D. & Fristrom, J. W. in The Development of Drosophila melanogaster (eds. Bate, M. & Martinez-Arias, A.) II, (Cold spring harbor laboratory press, 1993)'. The correct reference is 'Condic, M.L, Fristrom, D. & Fristrom, J.W. Apical cell shape changes during Drosophila imaginal leg disc elongation: a novel morphogenetic mechanism. Development 111: 23-33 (1991)'. Furthermore, the last sentence of the fourth paragraph of the introduction incorrectly omitted citation of work by Rupprecht et al. The correct citation is given below. These errors have now been corrected in both the PDF and HTML versions of the Article. Rupprecht, J.F., Ong, K.H., Yin, J., Huang, A., Dinh, H.H., Singh, A.P., Zhang, S., Yu, W. & Saunders, T.E. Geometric constraints alter cell arrangements within curved epithelial tissues. Mol. Biol. Cell 28, 3582-3594 (2017)

Regulación de la actividad de actomiosina y su papel en morfogénesis y homeostasis de epitelios

Programa de Doctorado en Biotecnología, Ingeniería y Tecnología QuímicaLínea de Investigación: Biología del DesarrolloClave Programa: DBICódigo Línea: 9Durante la morfogénesis de epitelios las capas celulares deben sufrir numerosos cambios morfológicos que permitan la adquisición de una anatomía concreta. Estos cambios normalmente están conducidos por reorganizaciones del citoesqueleto de actomiosina que permiten cambios de contractilidad celular requeridos para la adquisición de estructuras 3D de los tejidos. Estos procesos deben estar finamente regulados, siendo necesaria la activación local de contractilidad en grupos particulares de células y en momentos específicos del desarrollo. El objetivo de este trabajo ha consistido en analizar dos aspectos diferentes de la regulación de la actividad de actomiosina en morfogénesis. Así, se ha estudiado la dinámica de las estructuras basales de actina y miosina en el epitelio folicular durante la oogénesis de Drosophila. Por otra parte, se ha analizado el papel de la adhesión a la matriz extracelular mediada por integrinas en la morfogénesis y la homeostasis del epitelio del disco de ala. De esta manera en este trabajo de tesis se ha demostrado que: 1) El componente MLCP de Drosophila, Flw, es un regulador clave tanto del inicio como de la periodicidad de las oscilaciones basales de actomiosina que tienen lugar en las FCs. Por otra parte, al contrario de lo que se había publicado previamente, los niveles de F-actina basal oscilan con una amplitud y periodicidad similar a los de miosina. Esto ha permitido proponer un modelo simple para la emergencia de las oscilaciones de actomiosina en el cual, la combinación de la unión cooperativa de filamentos de actina en conjunción con la disociación de filamentos de actina del haz debido a la tensión inducida por miosina, es suficiente para generar oscilaciones autónomas celulares del contenido de F-actina y miosina. 2) En el epitelio del disco de ala, las integrinas han de mantenerse altas en las células que no van a evaginar, para así promover la supervivencia de las mismas. Sin embargo, en un determinado estadio del desarrollo, los niveles de integrinas deben reducirse de manera específica en las células del margen, las cuales sufren un proceso de evaginación y disminuyen y contraen su superficie basal.Universidad Pablo de Olavide de Sevilla. Escuela de DoctoradoPostprin