Integrin Binding Dynamics Modulate Ligand-Specific Mechanosensing in Mammary Gland Fibroblasts

The link between integrin activity regulation and cellular mechanosensing of tissue rigidity, especially on different extracellular matrix ligands, remains poorly understood. Here, we find that primary mouse mammary gland stromal fibroblasts (MSFs) are able to spread efficiently, generate high forces, and display nuclear YAP on soft collagen-coated substrates, resembling the soft mammary gland tissue. We describe that loss of the integrin inhibitor, SHARPIN, impedes MSF spreading specifically on soft type I collagen but not on fibronectin. Through quantitative experiments and computational modeling, we find that SHARPIN-deficient MSFs display faster force-induced unbinding of adhesions from collagen-coated beads. Faster unbinding, in turn, impairs force transmission in these cells, particularly, at the stiffness optimum observed for wild-type cells. Mechanistically, we link the impaired mechanotransduction of SHARPIN-deficient cells on collagen to reduced levels of collagen-binding integrin α11β1. Thus integrin activity regulation and α11β1 play a role in collagen-specific mechanosensing in MSFs.publishedVersio

The laminin-keratin link shields the nucleus from mechanical deformation and signalling

The mechanical properties of the extracellular matrix dictate tissue behaviour. In epithelial tissues, laminin is a very abundant extracellular matrix component and a key supporting element. Here we show that laminin hinders the mechanoresponses of breast epithelial cells by shielding the nucleus from mechanical deformation. Coating substrates with laminin-111-unlike fibronectin or collagen I-impairs cell response to substrate rigidity and YAP nuclear localization. Blocking the laminin-specific integrin β4 increases nuclear YAP ratios in a rigidity-dependent manner without affecting the cell forces or focal adhesions. By combining mechanical perturbations and mathematical modelling, we show that β4 integrins establish a mechanical linkage between the substrate and keratin cytoskeleton, which stiffens the network and shields the nucleus from actomyosin-mediated mechanical deformation. In turn, this affects the nuclear YAP mechanoresponses, chromatin methylation and cell invasion in three dimensions. Our results demonstrate a mechanism by which tissues can regulate their sensitivity to mechanical signals.© 2023. The Author(s)

The force loading rate drives cell mechanosensing through both reinforcement and cytoskeletal softening.

Cell response to force regulates essential processes in health and disease. However, the fundamental mechanical variables that cells sense and respond to remain unclear. Here we show that the rate of force application (loading rate) drives mechanosensing, as predicted by a molecular clutch model. By applying dynamic force regimes to cells through substrate stretching, optical tweezers, and atomic force microscopy, we find that increasing loading rates trigger talin-dependent mechanosensing, leading to adhesion growth and reinforcement, and YAP nuclear localization. However, above a given threshold the actin cytoskeleton softens, decreasing loading rates and preventing reinforcement. By stretching rat lungs in vivo, we show that a similar phenomenon may occur. Our results show that cell sensing of external forces and of passive mechanical parameters (like tissue stiffness) can be understood through the same mechanisms, driven by the properties under force of the mechanosensing molecules involved

The laminin–keratin link shields the nucleus from mechanical deformation and signalling

The mechanical properties of the extracellular matrix dictate tissue behaviour. In epithelial tissues, laminin is a very abundant extracellular matrix component and a key supporting element. Here we show that laminin hinders the mechanoresponses of breast epithelial cells by shielding the nucleus from mechanical deformation. Coating substrates with laminin-111—unlike fibronectin or collagen I—impairs cell response to substrate rigidity and YAP nuclear localization. Blocking the laminin-specific integrin ß4 increases nuclear YAP ratios in a rigidity-dependent manner without affecting the cell forces or focal adhesions. By combining mechanical perturbations and mathematical modelling, we show that ß4 integrins establish a mechanical linkage between the substrate and keratin cytoskeleton, which stiffens the network and shields the nucleus from actomyosin-mediated mechanical deformation. In turn, this affects the nuclear YAP mechanoresponses, chromatin methylation and cell invasion in three dimensions. Our results demonstrate a mechanism by which tissues can regulate their sensitivity to mechanical signals.We thank A. Farré and the other members of IMPETUX OPTICS, S.L., for their help and expertise in the design and implementation of the optical tweezers experiments; R. Sunyer for help and advice with the microprinting experiments; S. Usieto, A. Menéndez, N. Castro, M. Purciolas and W. Haaksma for providing technical support; L. Rosetti and S. Saloustros for providing data analysis tools; and J. de Rooij, A. L. Le Roux, L. Faure, A. Labernadie, R. Oria and J. Abenza, as well as all the members of the groups of P.R.-C. and X.T. for helpful discussion. Finally, we thank G. Wiche, A. Sonnenberg and N. Montserrat for providing plasmids, antibodies or cell lines used for this work. We acknowledge funding from the Spanish Ministry of Science and Innovation (PID2021-128635NB-I00 MCIN/AEI/10.13039/501100011033 and ‘ERDF-EU A way of making Europe’ to X.T., PID2019-110949GB-I00 to M.A. and PID2019-110298GB-I00 to P.R.-C.), the European Commission (H2020-FETPROACT-01-2016-731957), the European Research Council (Adv-883739 to X.T.; CoG-681434 to M.A.; StG- 851055 to A.E.-A.), the Generalitat de Catalunya (2017-SGR-1602 to X.T. and P.R.-C.; 2017-SGR-1278 to M.A. and P.S.) and European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 797621 to M.G.-G. The prize ‘ICREA Academia’ for excellence in research to M.A. and P.R.-C., Fundació la Marató de TV3 (201936-30-31 and 201903-30-31-32), and ‘la Caixa’ Foundation (LCF/PR/HR20/52400004 and ID 100010434 under agreement LCF/PR/HR20/52400004). IBEC and CIMNE are recipients of a Severo Ochoa Award of Excellence from MINCIN. A.E.M.B. was supported by a Sir Henry Wellcome Fellowship (210887/Z/18/Z). A.E.-A. receives funding from the Francis Crick Institute, which receives its core funding from the Cancer Research UK (CC2214), the UK Medical Research Council (CC2214) and the Wellcome Trust (CC2214).Peer ReviewedPostprint (published version

The laminin-keratin link shields the nucleus from mechanical deformation and signalling

The mechanical properties of the extracellular matrix dictate tissue behaviour. In epithelial tissues, laminin is a very abundant extracellular matrix component and a key supporting element. Here we show that laminin hinders the mechanoresponses of breast epithelial cells by shielding the nucleus from mechanical deformation. Coating substrates with laminin-111-unlike fibronectin or collagen I-impairs cell response to substrate rigidity and YAP nuclear localization. Blocking the laminin-specific integrin β4 increases nuclear YAP ratios in a rigidity-dependent manner without affecting the cell forces or focal adhesions. By combining mechanical perturbations and mathematical modelling, we show that β4 integrins establish a mechanical linkage between the substrate and keratin cytoskeleton, which stiffens the network and shields the nucleus from actomyosin-mediated mechanical deformation. In turn, this affects the nuclear YAP mechanoresponses, chromatin methylation and cell invasion in three dimensions. Our results demonstrate a mechanism by which tissues can regulate their sensitivity to mechanical signals

Cancerous and Non-Cancerous Lung Extracellular Matrix: from a microstructure-mechanical property study to the development of a 3D Platform to unravel Cell-ECM interactions.

Lung cancer is the leading cause of cancer death among both women and men. It causes more deaths than colon, breast and prostate cancers combined. It is also the second most common cancer in both men and women, about 13% of all new cancers are lung cancer. Approximately 228,150 new cases are expected for the year 2020, which will cause about 142,670 deaths in the United States as the American Cancer Society expects. The mechanical properties of the Extracellular matrix (ECM) of many tissues, and specifically the lung, have been proven to affect cell and tissue functions. Moreover, it is well known that there is a dynamic reciprocity between cells and ECM mechanics, and this communication is affected during pathologies. However, the mechanisms by which cells stiffen the matrix remain understudied. The aim of this thesis is to characterize the mechanical behavior at local scale of healthy and pathological lung ECM and correlate it to its local microstructure. To achieve this goal, an Atomic Force Microscopy head has been mounted on top of an epifluorescence microscope to measure at the same locations the mechanical properties of the ECM and the microstructure of the three main fibrillar proteins of the lung ECM: collagen I, collagen III and elastin. Cancerous and non-cancerous lung ECM samples from 7 patients were obtained. The samples were sliced in 7 µm thick samples and the collagen I, collagen III and elastin were immunostained following a primary/secondary antibody protocol. Then 400 AFM indentations of 500 nm were performed in a 100*100µm area while each protein map was imaged using the epifluorescence microscope. Considering all the patients, the mean value of the effective elastic modulus measured by AFM was of 6.33 ±1.13 kPa for non-cancerous lung ECM and of 15.65±4.04 kPa. Therefore, there is a 2.5 fold increase of stiffness in cancerous lung ECM compared to non-cancerous lung ECM. For all the samples, the Young’s modulus showed a Gaussian stiffness distribution. When all the indentation tests performed for each patient were plotted together, that is tests performed on the cancerous and non-cancerous regions of the same slice, the distribution obtained was a bimodal for all the patients. The first peak of the distribution was related to the non-cancerous ECM and the second peak to the cancerous ECM. The mean values obtained from the peaks of the bimodal distribution overestimated the measured mean of both cancerous and non-cancerous ECM. Then, the correlation between the composition and the stiffness of the ECM was studied. First, the volume fraction of the fibrillary proteins in the samples was calculated using two different references, one relative to the maximum intensity of all the samples and the other one relative to the maximum intensity of each sample. Both showed an increment of the collagen I between the non-cancerous and cancerous samples with a mean increase of 1.7 folds and 1.5 folds, respectively. A positive correlation between the Collagen I volume fraction and measured stiffness was found for each sample. When the comparison was made between samples, a higher correlation was found for the second volume fraction, with an R2=0.60. Then, a microstructure-mechanical property relationship was studied. For that, a model based on Eshelby´s inclusion problem was used to predict the mechanical behavior of the lung cancerous and non-cancerous ECM. This model can estimate the elastic modulus of a matrix with ellipsoidal inclusions inside, that would resemble the ECM with the Collagen I fibers as the inclusions. Two different fiber distributions were considered. The first one assumes that the Collagen I fibers are oriented in 3D. Using an elastic modulus for the collagen I of 100 MPa, in the range reported in literature, the values of the elastic modulus of the ECM were overestimated by two orders of magnitude. A new value of the elastic modulus of collagen I fibers was calculated using the model and the measures obtained at the 10 points with the highest volume fraction of collagen I in all the samples. This calculation was done separately for non-cancerous and cancerous samples obtaining an elastic modulus of the collagen I fibers of 390 kPa for non-cancerous samples and of 1050 kPa for cancerous samples, well below the values reported in literature. The model predicted the E of the non-cancerous and cancerous lung ECM with a mean absolute error of 25.08% and 32.74% respectively, and an R2=0.6155 was obtained when a linear regression was fitted for the predicted versus measured values. The second approach assumes that Collagen I fibers are oriented in 2D. In this case, the elastic modulus of collagen I fibers is assumed to be of 100 MPa, in the range reported in literature. The elastic modulus of the matrix was tuned in order to minimize the absolute average error between the measured and predicted elastic modulus of the ECM. This was done separately for the non-cancerous and cancerous samples, mainly because cross-linking was not measured in this work. The best results were obtained for an elastic modulus of the matrix of 0.12 kPa for the cancerous ECM and of 0.05 kPa for the non-cancerous ECM, and calculating the Collagen I volume fraction with the maximum intensity value of each sample as reference. The prediction showed a mean absolute error of 14.48% for the non-cancerous lung ECM and of 11.15% for the cancerous ECM, with a correlation of R2=0.944 when a linear regression is fitted for the predicted versus measured stiffness. Finally, a functional platform with tunable stiffness for the study of 3D single cell-ECM interactions based on Methacrylate Hyaluronic Acid hydrogels was developed. First, Hyaluronic Acid Methacrylate was synthesized, which when crosslinked with dithiothreitol gave a range of stiffnesses ranging from 0.2 to 19 kPa. This range comprehends both the mean values of the cancerous and non-cancerous ECM. Then, proof of concept 3D cell migration assays were performed for A549 and H1299 cells inside of three hydrogel with different stiffnesses.El cáncer de pulmón es la segunda causa más común de cáncer en mujeres y hombres, alrededor del 13% de todos los nuevos cánceres diagnosticados. Es la causa primaria de muerte por cáncer en mujeres y hombres. Causa más muertes al año que el cáncer de colon, mama y próstata combinados. Alrededor de 228,150 nuevos casos se esperan para el año 2020 que causaran un total de 142,670 muertes en los Estados Unidos tal y como anuncia La Sociedad Americana del Cáncer. Las propiedades mecánicas de la Matriz Extracelular (ECM) de muchos tejidos, y específicamente del pulmón, han demostrado afectar las funciones tanto celular como a nivel tisular. De hecho, se sabe que existe una reciprocidad dinámica entre las células y la mecánica de la ECM, y esta comunicación se ve afectada durante estados patológicos. El objetivo de esta tesis es caracterizar el comportamiento mecánico a escala local de la ECM de pulmón y correlacionarlo con su microestructura. Para realizar este estudio, se ha montado una cabeza de un Microscopio de Fuerza Atómica (AFM) sobre la base de un Microscopio de epifluorescencia, de tal forma que se pueda obtener información de la microestructura de las principales proteínas fibrilares de la ECM (Colágeno I, Colágeno III y Elastina) y de las propiedades mecánicas de la ECM de forma simultánea en la misma posición. Se obtuvieron 7 muestras de paciente de cáncer de pulmón, se hicieron cortes de 7 µm, se identificaron regiones cancerosas y no cancerosas en cada muestra, se descelularizaron y se realizaron tinciones de inmunofluorescencia siguiendo un protocolo de anticuerpo primario/secundario para las principales proteínas fibrilares de la ECM: colágeno I, colágeno III y elastina. Después, se realizaron 400 indentaciones de 500 nm de profundidad en un área de 100 µm *100 µm mientras se obtenían mapas de proteínas de la zona indentada. Considerando las muestras de los 7 pacientes, se midió un módulo elástico efectivo medio para las regiones no cancerosas de la ECM de pulmón de 6.33 ±1.13 kPa, mientras que el módulo elástico medio en las regiones cancerosas fue de 15.65±4.04 kPa. Es decir, la rigidez es 2,5 veces superior en las zonas cancerosas. Para todas las muestras, el módulo de Young mostró una distribución Gaussiana de rigideces. Cuando todos los ensayos de indentación fueron graficados de forma conjunta para una misma muestra, se obtuvo una distribución bimodal para todos los pacientes. El primer pico de la distribución correspondía a la ECM no cancerosa y el segundo pico de la distribución a la ECM cancerosa. Los valores medios obtenidos de la distribución bimodal sobreestimaban los valores medios medidos de la ECM cancerosa y no cancerosa. Después se calculó la correlación entre las medidas de fracción volumétrica de proteína obtenidas en las muestras y el módulo elástico medido. La fracción volumétrica se calculó de dos maneras, una de ellas relativa a la intensidad máxima de todas las muestras y la otra relativa a la intensidad máxima de cada muestra. Ambas mostraron un incremento en la fracción volumétrica del colágeno I de 1.7 y .1.5 veces mayor respectivamente para la ECM cancerosa frente a la no cancerosa. Ambas mostraron una correlación positiva entre la fracción volumétrica obtenida y el módulo elástico medido para todos los puntos de cada muestra. Entre muestras, la segunda mostró una correlación entre la fracción volumétrica media y el módulo elástico medio de cada muestra con R2=0.60. Se implementó el modelo de Eshelby para predecir el comportamiento mecánico de la matriz extracelular del pulmón canceroso y sano. El modelo puede estimar el módulo elástico de una matriz con inclusiones elipsoidales, que representarían a las fibras de colágeno dentro de la ECM. Se consideraron dos posibles distribuciones para la orientación de las fibras. La primera asume que las fibras de colágeno I están orientadas en 3D. Utilizando un módulo elástico para el colágeno I de 100 MPa, dentro del rango reportado en literatura, los valores del módulo elástico de la ECM se sobreestimaban por dos órdenes de magnitud. Se calculó un nuevo valor del módulo elástico de las fibras de colágeno I utilizando el modelo y las medidas obtenidas de los 10 puntos con mayor fracción volumétrica de colágeno I en todas las muestras. Este cálculo se hizo de manera separada para las muestras no cancerosas y cancerosas obteniendo un módulo elástico para las fibras de colágeno I de 390 kPa para las muestras no cancerosas y 1050 kPa para las muestras cancerosas, muy por debajo de los valores reportados en literatura. El modelo fue capaz de predecir el módulo elástico de la ECM no cancerosa y cancerosa con un error medio del 25.08% y 32.74% respectivamente con un ajuste a una regresión lineal de R2=0.6155 frente a los valores medidos en AFM. La segunda aproximación supone que las fibras de colágeno I están orientadas en el plano 2D. En este caso, se asume que el módulo elástico de las fibras de colágeno es de 100 MPa, dentro del rango reportado en literatura. El módulo elástico de la matriz fue ajustado para minimizar el error absoluto entre el valor medido y el módulo elástico predicho de la ECM. Esto se hizo de manera separada para las muestras cancerosas y no cancerosas, sobre todo porque el efecto del crosslinking no se midió en este trabajo. Los mejores resultados se obtuvieron para Ematrix = 0.12 kPa para las muestras cancerosas y Ematrix = 0.05 kPa para las no cancerosas y calculando la fracción volumétrica del colágeno I usando como referencia el máximo de intensidad medido en cada muestra. El modelo fue capaz de predecir el módulo elástico de la ECM con un error de 14.48% para las muestras no cancerosas y un error de 11.15% para las muestras cancerosas con un ajuste a una regresión lineal de R2=0.94 frente a los valores medidos en AFM. Finalmente, se desarrolló una plataforma para el estudio de las interacciones célula-ECM en tres dimensiones basada en hidrogeles de Ácido Hialurónico. Se sintetizó Ácido hialurónico con grupos metacrilato que permitían el crosslinking mediante dithiothreithiol, esto permitía a los hidrogeles alcanzar rigideces en un rango entre 0,2 y 19 kPa, rango que incluye las medias de los módulos de Young efectivos de la matriz cancerosa y no cancerosa. Resumidamente, la gelificación de los hidrogeles se realizaba con las células embebidas en ellos mediante el uso de un motor rotatorio que mantenía las células suspendidas en el espacio tridimensional en todo momento, a 37ºC dentro de una incubadora. El protocolo no sólo permitía una distribución homogénea de las células en los hidrogeles, sino que además permite evitar los efectos de durotaxis que puedan ser provocados por la plataforma. Se realizaron ensayos de migración en 3D dentro de la plataforma para las líneas celulares A549 y H1299 a diferentes niveles de rigidez, las cuales mostraron para la línea H1299 una mayor capacidad invasiva y migratoria

Cancerous and Non-Cancerous Lung Extracellular Matrix: from a microstructure-mechanical property study to the development of a 3D Platform to unravel Cell-ECM interactions.

Lung cancer is the leading cause of cancer death among both women and men. It causes more deaths than colon, breast and prostate cancers combined. It is also the second most common cancer in both men and women, about 13% of all new cancers are lung cancer. Approximately 228,150 new cases are expected for the year 2020, which will cause about 142,670 deaths in the United States as the American Cancer Society expects. The mechanical properties of the Extracellular matrix (ECM) of many tissues, and specifically the lung, have been proven to affect cell and tissue functions. Moreover, it is well known that there is a dynamic reciprocity between cells and ECM mechanics, and this communication is affected during pathologies. However, the mechanisms by which cells stiffen the matrix remain understudied. The aim of this thesis is to characterize the mechanical behavior at local scale of healthy and pathological lung ECM and correlate it to its local microstructure. To achieve this goal, an Atomic Force Microscopy head has been mounted on top of an epifluorescence microscope to measure at the same locations the mechanical properties of the ECM and the microstructure of the three main fibrillar proteins of the lung ECM: collagen I, collagen III and elastin. Cancerous and non-cancerous lung ECM samples from 7 patients were obtained. The samples were sliced in 7 µm thick samples and the collagen I, collagen III and elastin were immunostained following a primary/secondary antibody protocol. Then 400 AFM indentations of 500 nm were performed in a 100*100µm area while each protein map was imaged using the epifluorescence microscope. Considering all the patients, the mean value of the effective elastic modulus measured by AFM was of 6.33 ±1.13 kPa for non-cancerous lung ECM and of 15.65±4.04 kPa. Therefore, there is a 2.5 fold increase of stiffness in cancerous lung ECM compared to non-cancerous lung ECM. For all the samples, the Young’s modulus showed a Gaussian stiffness distribution. When all the indentation tests performed for each patient were plotted together, that is tests performed on the cancerous and non-cancerous regions of the same slice, the distribution obtained was a bimodal for all the patients. The first peak of the distribution was related to the non-cancerous ECM and the second peak to the cancerous ECM. The mean values obtained from the peaks of the bimodal distribution overestimated the measured mean of both cancerous and non-cancerous ECM. Then, the correlation between the composition and the stiffness of the ECM was studied. First, the volume fraction of the fibrillary proteins in the samples was calculated using two different references, one relative to the maximum intensity of all the samples and the other one relative to the maximum intensity of each sample. Both showed an increment of the collagen I between the non-cancerous and cancerous samples with a mean increase of 1.7 folds and 1.5 folds, respectively. A positive correlation between the Collagen I volume fraction and measured stiffness was found for each sample. When the comparison was made between samples, a higher correlation was found for the second volume fraction, with an R2=0.60. Then, a microstructure-mechanical property relationship was studied. For that, a model based on Eshelby´s inclusion problem was used to predict the mechanical behavior of the lung cancerous and non-cancerous ECM. This model can estimate the elastic modulus of a matrix with ellipsoidal inclusions inside, that would resemble the ECM with the Collagen I fibers as the inclusions. Two different fiber distributions were considered. The first one assumes that the Collagen I fibers are oriented in 3D. Using an elastic modulus for the collagen I of 100 MPa, in the range reported in literature, the values of the elastic modulus of the ECM were overestimated by two orders of magnitude. A new value of the elastic modulus of collagen I fibers was calculated using the model and the measures obtained at the 10 points with the highest volume fraction of collagen I in all the samples. This calculation was done separately for non-cancerous and cancerous samples obtaining an elastic modulus of the collagen I fibers of 390 kPa for non-cancerous samples and of 1050 kPa for cancerous samples, well below the values reported in literature. The model predicted the E of the non-cancerous and cancerous lung ECM with a mean absolute error of 25.08% and 32.74% respectively, and an R2=0.6155 was obtained when a linear regression was fitted for the predicted versus measured values. The second approach assumes that Collagen I fibers are oriented in 2D. In this case, the elastic modulus of collagen I fibers is assumed to be of 100 MPa, in the range reported in literature. The elastic modulus of the matrix was tuned in order to minimize the absolute average error between the measured and predicted elastic modulus of the ECM. This was done separately for the non-cancerous and cancerous samples, mainly because cross-linking was not measured in this work. The best results were obtained for an elastic modulus of the matrix of 0.12 kPa for the cancerous ECM and of 0.05 kPa for the non-cancerous ECM, and calculating the Collagen I volume fraction with the maximum intensity value of each sample as reference. The prediction showed a mean absolute error of 14.48% for the non-cancerous lung ECM and of 11.15% for the cancerous ECM, with a correlation of R2=0.944 when a linear regression is fitted for the predicted versus measured stiffness. Finally, a functional platform with tunable stiffness for the study of 3D single cell-ECM interactions based on Methacrylate Hyaluronic Acid hydrogels was developed. First, Hyaluronic Acid Methacrylate was synthesized, which when crosslinked with dithiothreitol gave a range of stiffnesses ranging from 0.2 to 19 kPa. This range comprehends both the mean values of the cancerous and non-cancerous ECM. Then, proof of concept 3D cell migration assays were performed for A549 and H1299 cells inside of three hydrogel with different stiffnesses.El cáncer de pulmón es la segunda causa más común de cáncer en mujeres y hombres, alrededor del 13% de todos los nuevos cánceres diagnosticados. Es la causa primaria de muerte por cáncer en mujeres y hombres. Causa más muertes al año que el cáncer de colon, mama y próstata combinados. Alrededor de 228,150 nuevos casos se esperan para el año 2020 que causaran un total de 142,670 muertes en los Estados Unidos tal y como anuncia La Sociedad Americana del Cáncer. Las propiedades mecánicas de la Matriz Extracelular (ECM) de muchos tejidos, y específicamente del pulmón, han demostrado afectar las funciones tanto celular como a nivel tisular. De hecho, se sabe que existe una reciprocidad dinámica entre las células y la mecánica de la ECM, y esta comunicación se ve afectada durante estados patológicos. El objetivo de esta tesis es caracterizar el comportamiento mecánico a escala local de la ECM de pulmón y correlacionarlo con su microestructura. Para realizar este estudio, se ha montado una cabeza de un Microscopio de Fuerza Atómica (AFM) sobre la base de un Microscopio de epifluorescencia, de tal forma que se pueda obtener información de la microestructura de las principales proteínas fibrilares de la ECM (Colágeno I, Colágeno III y Elastina) y de las propiedades mecánicas de la ECM de forma simultánea en la misma posición. Se obtuvieron 7 muestras de paciente de cáncer de pulmón, se hicieron cortes de 7 µm, se identificaron regiones cancerosas y no cancerosas en cada muestra, se descelularizaron y se realizaron tinciones de inmunofluorescencia siguiendo un protocolo de anticuerpo primario/secundario para las principales proteínas fibrilares de la ECM: colágeno I, colágeno III y elastina. Después, se realizaron 400 indentaciones de 500 nm de profundidad en un área de 100 µm *100 µm mientras se obtenían mapas de proteínas de la zona indentada. Considerando las muestras de los 7 pacientes, se midió un módulo elástico efectivo medio para las regiones no cancerosas de la ECM de pulmón de 6.33 ±1.13 kPa, mientras que el módulo elástico medio en las regiones cancerosas fue de 15.65±4.04 kPa. Es decir, la rigidez es 2,5 veces superior en las zonas cancerosas. Para todas las muestras, el módulo de Young mostró una distribución Gaussiana de rigideces. Cuando todos los ensayos de indentación fueron graficados de forma conjunta para una misma muestra, se obtuvo una distribución bimodal para todos los pacientes. El primer pico de la distribución correspondía a la ECM no cancerosa y el segundo pico de la distribución a la ECM cancerosa. Los valores medios obtenidos de la distribución bimodal sobreestimaban los valores medios medidos de la ECM cancerosa y no cancerosa. Después se calculó la correlación entre las medidas de fracción volumétrica de proteína obtenidas en las muestras y el módulo elástico medido. La fracción volumétrica se calculó de dos maneras, una de ellas relativa a la intensidad máxima de todas las muestras y la otra relativa a la intensidad máxima de cada muestra. Ambas mostraron un incremento en la fracción volumétrica del colágeno I de 1.7 y .1.5 veces mayor respectivamente para la ECM cancerosa frente a la no cancerosa. Ambas mostraron una correlación positiva entre la fracción volumétrica obtenida y el módulo elástico medido para todos los puntos de cada muestra. Entre muestras, la segunda mostró una correlación entre la fracción volumétrica media y el módulo elástico medio de cada muestra con R2=0.60. Se implementó el modelo de Eshelby para predecir el comportamiento mecánico de la matriz extracelular del pulmón canceroso y sano. El modelo puede estimar el módulo elástico de una matriz con inclusiones elipsoidales, que representarían a las fibras de colágeno dentro de la ECM. Se consideraron dos posibles distribuciones para la orientación de las fibras. La primera asume que las fibras de colágeno I están orientadas en 3D. Utilizando un módulo elástico para el colágeno I de 100 MPa, dentro del rango reportado en literatura, los valores del módulo elástico de la ECM se sobreestimaban por dos órdenes de magnitud. Se calculó un nuevo valor del módulo elástico de las fibras de colágeno I utilizando el modelo y las medidas obtenidas de los 10 puntos con mayor fracción volumétrica de colágeno I en todas las muestras. Este cálculo se hizo de manera separada para las muestras no cancerosas y cancerosas obteniendo un módulo elástico para las fibras de colágeno I de 390 kPa para las muestras no cancerosas y 1050 kPa para las muestras cancerosas, muy por debajo de los valores reportados en literatura. El modelo fue capaz de predecir el módulo elástico de la ECM no cancerosa y cancerosa con un error medio del 25.08% y 32.74% respectivamente con un ajuste a una regresión lineal de R2=0.6155 frente a los valores medidos en AFM. La segunda aproximación supone que las fibras de colágeno I están orientadas en el plano 2D. En este caso, se asume que el módulo elástico de las fibras de colágeno es de 100 MPa, dentro del rango reportado en literatura. El módulo elástico de la matriz fue ajustado para minimizar el error absoluto entre el valor medido y el módulo elástico predicho de la ECM. Esto se hizo de manera separada para las muestras cancerosas y no cancerosas, sobre todo porque el efecto del crosslinking no se midió en este trabajo. Los mejores resultados se obtuvieron para Ematrix = 0.12 kPa para las muestras cancerosas y Ematrix = 0.05 kPa para las no cancerosas y calculando la fracción volumétrica del colágeno I usando como referencia el máximo de intensidad medido en cada muestra. El modelo fue capaz de predecir el módulo elástico de la ECM con un error de 14.48% para las muestras no cancerosas y un error de 11.15% para las muestras cancerosas con un ajuste a una regresión lineal de R2=0.94 frente a los valores medidos en AFM. Finalmente, se desarrolló una plataforma para el estudio de las interacciones célula-ECM en tres dimensiones basada en hidrogeles de Ácido Hialurónico. Se sintetizó Ácido hialurónico con grupos metacrilato que permitían el crosslinking mediante dithiothreithiol, esto permitía a los hidrogeles alcanzar rigideces en un rango entre 0,2 y 19 kPa, rango que incluye las medias de los módulos de Young efectivos de la matriz cancerosa y no cancerosa. Resumidamente, la gelificación de los hidrogeles se realizaba con las células embebidas en ellos mediante el uso de un motor rotatorio que mantenía las células suspendidas en el espacio tridimensional en todo momento, a 37ºC dentro de una incubadora. El protocolo no sólo permitía una distribución homogénea de las células en los hidrogeles, sino que además permite evitar los efectos de durotaxis que puedan ser provocados por la plataforma. Se realizaron ensayos de migración en 3D dentro de la plataforma para las líneas celulares A549 y H1299 a diferentes niveles de rigidez, las cuales mostraron para la línea H1299 una mayor capacidad invasiva y migratoria

Understanding the role of mechanics in nucleocytoplasmic transport

Cell nuclei are submitted to mechanical forces, which in turn affect nuclear and cell functions. Recent evidence shows that a crucial mechanically regulated nuclear function is nucleocytoplasmic transport, mediated by nuclear pore complexes (NPCs). Mechanical regulation occurs at two levels: first, by force application to the nucleus, which increases NPC permeability likely through NPC stretch. Second, by the mechanical properties of the transported proteins themselves, as mechanically labile proteins translocate through NPCs faster than mechanically stiff ones. In this perspective, we discuss this evidence and the associated mechanisms by which mechanics can regulate the nucleo-cytoplasmic partitioning of proteins. Finally, we analyze how mechanical regulation of nucleocytoplasmic transport can provide a systematic approach to the study of mechanobiology and open new avenues both in fundamental and applied research