Nuevos mecanismos de regulación de las funciones celulares de la proteína contráctil miosina II no muscular

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 10-07-2017Esta tesis tiene embargado el acceso al texto completo hasta el 10-01-2019La generación de fuerza mecánica es un proceso esencial en la morfogénesis de los tejidos, las interacciones intercelulares y los procesos de migración y diferenciación. A nivel celular, las fuerzas mecánicas dirigen la proliferación celular, controlan el movimiento y participan en los procesos de muerte celular programada. La mayor parte de la fuerza mecánica generada en una célula emerge de la actividad contráctil de motores moleculares de la superfamilia de las miosinas. En tejidos no musculares, el principal generador de fuerza mecánica es la miosina no muscular de clase II (NMII), que forma estructuras poliméricas (mini-filamentos), que se asocian a los microfilamentos y los desplazan, generando trabajo mecánico. Debido a la importancia de su función, la NMII tiene unos mecanismos muy sofisticados de regulación que controlan su asociación a los microfilamentos, su estado de polimerización en mini-filamentos para generar estructuras dinámicas y transitorias, o estables y resistentes, y su actividad contráctil propulsada por su capacidad para hidrolizar ATP. Durante los últimos 40 años, más de 7000 artículos han contribuido a descifrar muchos de estos mecanismos, incluyendo fosforilaciones en la cadena reguladora que controlan el estado conformacional de la molécula; asociaciones inter- e intracatenarias que regulan la formación de filamentos; y muchos otros. En este trabajo de Tesis doctoral, hemos estudiado aspectos de la regulación de la NMII poco caracterizados que sin embargo controlan críticamente su función a nivel celular. En la primera parte del trabajo, hemos caracterizado la función de un tipo de fosforilación de la cadena ligera (fosforilación en tirosina) descrito por el premio Nóbel Edwin Krebs hace más de 30 años, cuya función a nivel celular permanecía sin describir. Este trabajo establece un nuevo mecanismo de regulación de la función de NMII que depende de la fosforilación de Tyr155 y que regula no su función, sino su disponibilidad para ensamblarse de manera eficiente y formar estructuras estables que definen parámetros claves de la arquitectura celular. En la segunda parte, hemos separado las funciones celulares que dependen de la capacidad contráctil de la NMII de aquellas que dependen de su capacidad entrecruzadora de los microfilamentos. Esta parte del trabajo establece que los dos parálogos principales de la NMII expresados en la mayoría de tejidos, NMII-A y NMII-B, cooperan para transmitir el trabajo mecánico a la matriz extracelular mediante la transmisión de tensión mecánica a través de los haces de actomiosina. Este trabajo también establece que la arquitectura del entrecruzamiento de los microfilamentos determina la eficiencia de aplicación de la tracción y que existen funciones que dependen absolutamente de la actividad contráctil de la NMII-A, como es la retracción del polo posterior durante la migración y la segregación espacial de las isoformas en células polarizadas

Microfilament-coordinated adhesion dynamics drives single cell migration and shapes whole tissues [version 1; Referees:4:approved]

Cell adhesion to the substratum and/or other cells is a crucial step of cell migration. While essential in the case of solitary migrating cells (for example, immune cells), it becomes particularly important in collective cell migration, in which cells maintain contact with their neighbors while moving directionally. Adhesive coordination is paramount in physiological contexts (for example, during organogenesis) but also in pathology (for example, tumor metastasis). In this review, we address the need for a coordinated regulation of cell-cell and cell-matrix adhesions during collective cell migration. We emphasize the role of the actin cytoskeleton as an intracellular integrator of cadherin- and integrin-based adhesions and the emerging role of mechanics in the maintenance, reinforcement, and turnover of adhesive contacts. Recent advances in understanding the mechanical regulation of several components of cadherin and integrin adhesions allow us to revisit the adhesive clutch hypothesis that controls the degree of adhesive engagement during protrusion. Finally, we provide a brief overview of the major impact of these discoveries when using more physiological three-dimensional models of single and collective cell migration

Full L-1-regularized Traction Force Microscopy over whole cells

Background Traction Force Microscopy (TFM) is a widespread technique to estimate the tractions that cells exert on the surrounding substrate. To recover the tractions, it is necessary to solve an inverse problem, which is ill-posed and needs regularization to make the solution stable. The typical regularization scheme is given by the minimization of a cost functional, which is divided in two terms: the error present in the data or data fidelity term; and the regularization or penalty term. The classical approach is to use zero-order Tikhonov or L2-regularization, which uses the L2-norm for both terms in the cost function. Recently, some studies have demonstrated an improved performance using L1-regularization (L1-norm in the penalty term) related to an increase in the spatial resolution and sensitivity of the recovered traction field. In this manuscript, we present a comparison between the previous two regularization schemes (relying in the L2-norm for the data fidelity term) and the full L1-regularization (using the L1-norm for both terms in the cost function) for synthetic and real data. Results Our results reveal that L1-regularizations give an improved spatial resolution (more important for full L1-regularization) and a reduction in the background noise with respect to the classical zero-order Tikhonov regularization. In addition, we present an approximation, which makes feasible the recovery of cellular tractions over whole cells on typical full-size microscope images when working in the spatial domain. Conclusions The proposed full L1-regularization improves the sensitivity to recover small stress footprints. Moreover, the proposed method has been validated to work on full-field microscopy images of real cells, what certainly demonstrates it is a promising tool for biological applications.This work was partially supported by the Spanish Ministry of Economy and Competitiveness (TEC2013-48552-C2-1-R, TEC2015-73064-EXP and TEC2016-78052-R) (AMB, ASA) and (SAF2014-54705-R) (MVM, RAC), the European Research Council (ERC) under the EU-FP7/2007-2013 through ERC Grant Agreement n° 308,223 (HVO, AJP). ASA is supported by an FPI grant of the Spanish Ministry of Economy and Competitiveness. MVM is supported by a Marie Curie Grant (CIG293719) and a Ramon y Cajal fellowship (RYC2010-06094) from the Spanish Ministry of Economy and Competitiveness

Microfilament-coordinated adhesion dynamics drives single cell migration and shapes whole tissues [version 1; Referees:4:approved]

Cell adhesion to the substratum and/or other cells is a crucial step of cell migration. While essential in the case of solitary migrating cells (for example, immune cells), it becomes particularly important in collective cell migration, in which cells maintain contact with their neighbors while moving directionally. Adhesive coordination is paramount in physiological contexts (for example, during organogenesis) but also in pathology (for example, tumor metastasis). In this review, we address the need for a coordinated regulation of cell-cell and cell-matrix adhesions during collective cell migration. We emphasize the role of the actin cytoskeleton as an intracellular integrator of cadherin- and integrin-based adhesions and the emerging role of mechanics in the maintenance, reinforcement, and turnover of adhesive contacts. Recent advances in understanding the mechanical regulation of several components of cadherin and integrin adhesions allow us to revisit the adhesive clutch hypothesis that controls the degree of adhesive engagement during protrusion. Finally, we provide a brief overview of the major impact of these discoveries when using more physiological three-dimensional models of single and collective cell migration.Ministerio de Economía y Competitividad (MINECO)Asociación Española Contra el Cáncer (AECC)Programa Ramón y Cajal (España)Fac. de Ciencias BiológicasTRUEpu

Full L1-regularized Traction Force Microscopy over whole cells

Abstract Background Traction Force Microscopy (TFM) is a widespread technique to estimate the tractions that cells exert on the surrounding substrate. To recover the tractions, it is necessary to solve an inverse problem, which is ill-posed and needs regularization to make the solution stable. The typical regularization scheme is given by the minimization of a cost functional, which is divided in two terms: the error present in the data or data fidelity term; and the regularization or penalty term. The classical approach is to use zero-order Tikhonov or L2-regularization, which uses the L2-norm for both terms in the cost function. Recently, some studies have demonstrated an improved performance using L1-regularization (L1-norm in the penalty term) related to an increase in the spatial resolution and sensitivity of the recovered traction field. In this manuscript, we present a comparison between the previous two regularization schemes (relying in the L2-norm for the data fidelity term) and the full L1-regularization (using the L1-norm for both terms in the cost function) for synthetic and real data. Results Our results reveal that L1-regularizations give an improved spatial resolution (more important for full L1-regularization) and a reduction in the background noise with respect to the classical zero-order Tikhonov regularization. In addition, we present an approximation, which makes feasible the recovery of cellular tractions over whole cells on typical full-size microscope images when working in the spatial domain. Conclusions The proposed full L1-regularization improves the sensitivity to recover small stress footprints. Moreover, the proposed method has been validated to work on full-field microscopy images of real cells, what certainly demonstrates it is a promising tool for biological applications

Additional file 3: of Full L1-regularized Traction Force Microscopy over whole cells

Ten samples frames illustrating the whole TFM experiment and comparing the traction recovery with the different regularization methods. For each frame: (Top row, left) CHO cell expressing Lifeact-GFP; (Top row, center) Pseudo-color image showing the fluorescent beads at the hydrogel surface. The beads of the unstressed and stressed hydrogels have been superposed and pseudo-colored in red and green, respectively; therefore, beads are colored in yellow when not displaced. The contrast of the pseudo-color images has been modified to highlight the areas with bead displacements; (Top row, right) Magnitude (in μm) and direction (arrows) of in-plane displacements estimated from the bead images. (Bottom row) Recovered traction magnitude (in Pa) and direction (arrows) using: (Left) Tikhonov regularization; (Center) L1-norm regularization; (Right) full L1-norm regularization. The outline of the mask used for traction recovery is shown in red (Top row, right) and white (for the rest). The scale bar represents 30 μm. Frame #5 corresponds to Fig. 7 in the main manuscript. (GIF 10461 kb

Additional file 2: of Full L1-regularized Traction Force Microscopy over whole cells

Quantitative results from synthetic data. Table with the different error metrics (mean±standard deviation) obtained by the different regularization methods on the synthetic data. Ten different traction maps have been considered and ten realizations for each one of them. The best results for each metric are highlighted in red. For all cases, the differences between the realizations are statistically significant (p < 0.001) as computed by a Student’s test. (TIFF 431 kb

Additional file 4: of Full L1-regularized Traction Force Microscopy over whole cells

Force balance over real cells. Table with the mean (in Pa and in percentage of the maximum traction magnitude) and the standard deviation (in Pa) of the sum of forces over the whole cell for each regularization scheme and for all real dataset. (TIFF 110 kb

Prediction of week 4 virological response in hepatitis C for making decision on triple therapy : the Optim study

Background: Virological response to peginterferon + ribavirin (P+R) at week 4 can predict sustained virological response (SVR). While patients with rapid virological response (RVR) do not require triple therapy, patients with a decline <1log10 IU/ml HCVRNA (D1L) should have treatment discontinued due to low SVR rate. Aim: To develop a tool to predict first 4 weeks' viral response in patients with hepatitis C genotype 1&4 treated with P+R. Methods: In this prospective and multicenter study, HCV mono-infected (n=538) and HCV/HIV co-infected (n=186) patients were included. To develop and validate a prognostic tool to detect RVR and D1L, we segregated the patients as an estimation cohort (to construct the model) and a validation cohort (to validate the model). Results: D1L was reached in 509 (80.2%) and RVR in 148 (22.5%) patients. Multivariate analyses demonstrated that HIV co-infection, Forns' index, LVL, IL28B-CC and Genotype-1 were independently related to RVR as well as D1L. Diagnostic accuracy (AUROC) for D1L was: 0.81 (95%CI: 0.76 ̶ 0.86) in the estimation cohort and 0.71 (95%CI: 0.62 ̶ 0.79) in the validation cohort; RVR prediction: AUROC 0.83 (95%CI: 0.78 ̶ 0.88) in the estimation cohort and 0.82 (95%CI: 0.76 ̶ 0.88) in the validation cohort. Cost-analysis of standard 48-week treatment indicated a saving of 30.3% if the prognostic tool is implemented. Conclusions: The combination of genetic (IL28B polymorphism) and viral genotype together with viral load, HIV co-infection and fibrosis stage defined a tool able to predict RVR and D1L at week 4. Using this tool would be a cost-saving strategy compared to universal triple therapy for hepatitis C