The Dynamics of Viscous Droplets near Micro-Patterned Solid Surfaces in Creeping Flow

The interaction between droplets and solid surfaces is of great importance in industrial applications, biochemical processes, and fundamental materials research on surface wettability. In this work, a three-dimensional spectral boundary element method has been employed to investigate the dynamics of a viscous droplet moving under gravity influence normal and parallel to a micro-patterned solid surface. The dynamics of the droplet moving perpendicular to the substrate are investigated under the influence of Bond number, droplet size, and topological features of the substrate. We find that the droplet dynamics can be controlled by varying Bond number, droplet size, and pattern height and width; however, the pattern length has little effect. For a droplet moving parallel to the surface, the Bond number and pattern projection direction greatly change the droplet dynamics. However, after moving past the pattern, the droplet position, velocity, and deformation return to that of a flat-plate solution

The consequence of substrates of large- scale rigidity on actin network tension in adherent cells

International audienceThere is compelling evidence that substrate stiffness affects cell adhesion as well as cytoskeleton organization and contractile activity. This work was designed to study the cytoskeletal contractile activity of cells plated on microposts of different stiffness using a numerical model simulating the intracellular tension of individual cells. We allowed cells to adhere onto micropost substrates of various rigidities and used experimental traction force data to infer cell contractility using a numerical model. The model discriminates between the influence of substrate stiffness on cell tension and shows that higher substrate stiffness leads to an increase in intracellular tension. The strength of this model is its ability to calculate the mechanical state of each cell in accordance to its individual cytoskeletal structure. This is achieved by regenerating a numerical cytoskeleton base

TFPa/HADHA is required for fatty acid beta-oxidation and cardiolipin re-modeling in human cardiomyocytes

Mitochondrial trifunctional protein deficiency, due to mutations in hydratase subunit A (HADHA), results in sudden infant death syndrome with no cure. To reveal the disease etiology, we generated stem cell-derived cardiomyocytes from HADHA-deficient hiPSCs and accelerated their maturation via an engineered microRNA maturation cocktail that upregulated the epigenetic regulator, HOPX. Here we report, matured HADHA mutant cardiomyocytes treated with an endogenous mixture of fatty acids manifest the disease phenotype: defective calcium dynamics and repolarization kinetics which results in a pro-arrhythmic state. Single cell RNA-seq reveals a cardiomyocyte developmental intermediate, based on metabolic gene expression. This intermediate gives rise to mature-like cardiomyocytes in control cells but, mutant cells transition to a pathological state with reduced fatty acid beta-oxidation, reduced mitochondrial proton gradient, disrupted cristae structure and defective cardiolipin remodeling. This study reveals that HADHA (tri-functional protein alpha), a monolysocardiolipin acyltransferase-like enzyme, is required for fatty acid beta-oxidation and cardiolipin remodeling, essential for functional mitochondria in human cardiomyocytes.Peer reviewe

Black Dots: Reference-Free Traction Force Microscopy for Measuring Single-Cell Forces

Thesis (Ph.D.)--University of Washington, 2022Cells generate cytoskeletal forces for a many purposes including cell locomotion and tissue functions like heart beating and wound healing. The amount of force produced by a cell depends on many things including its cell type, size, and environment. Measuring the forces produced by cells can provide insight into their behavior and physiological function, or can be used to study the effect of drugs and disease. To isolate their behavior, forces are measured in single cells, typically on a 2D surface for simplicity, yielding "traction forces" as a cell pulls parallel to the 2D plane. This principle has been used to develop techniques such as membrane wrinkling, microposts, and traction force microscopy among many others. However, existing traction force methods have several drawbacks including the limited number of cells that can be measured per experiment or inadvertent impact on cell functions by strictly constraining cell size and shape. In this work, a novel reference-free traction force microscopy technique is developed to overcome limitations from existing methods. The technique, named "black dots", microcontact prints a fluorescent micropattern onto a flexible substrate to measure cellular traction forces without constraining cell shape or needing to detach the cells. Chapter 2 describes the theory and development behind the black dots approach. Several techniques are combined including microcontact printing and sacrificial films to deposit a fluorescent pattern on very soft PDMS with high fidelity. The pattern is characterized and an example data set is generated to study the sensitivity of the black dots approach. In Chapter 3 the technique is demonstrated by assessing forces in human platelets, which are the smallest cells in the human body and can generate very large forces for their size. Because the black dots approach is reference-free, fixation and immunofluorescent staining can be applied along with geometric measurements to accompany the traction force data. In this study, platelets that exert more force tend to have more spread area, are more circular, and have more uniformly distributed F-actin filaments. As a result of the high yield of data obtainable by the black dots approach, a clustering analysis and a multivariate mixed effects model with interaction terms can be used to identify clusters and trends between force, area, circularity, and F-actin dispersion. Finally, Chapter 4 explores the use of black dots in measuring forces in live, dynamically beating cardiomyocytes, including a protocol and special considerations for using black dots with cardiomyocytes to ultimately measure both isometric and twitch forces over time. These forces as well as the twitch dynamics are analyzed for a small set of example cells that showcase the potential for new discoveries enabled black dots

In silico CDM model sheds light on force transmission in cell from focal adhesions to nucleus

International audienceCell adhesion is crucial for many types of cell, conditioning differentiation, proliferation, and protein synthesis. As a mechanical process, cell adhesion involves forces exerted by the cytoskeleton and transmitted by focal adhesions to extracellular matrix. These forces constitute signals that infer specific biological responses. Therefore, analyzing mechanotransduction during cell adhesion could lead to a better understanding of the mechanobiology of adherent cells. For instance this may explain how, the shape of adherent stem cells influences their differentiation or how the stiffness of the extracellular matrix affects adhesion strength. To assess the mechanical signals involved in cell adhesion, we computed intracellular forces using the Cytoskeleton Divided Medium model in endothelial cells adherent on micropost arrays of different stiffnesses. For each cell, focal adhesion location and forces measured by micropost deflection were used as an input for the model. The cytoskeleton and the nucleoskeleton were computed as systems of multiple tensile and compressive interactions. At the end of computation, the systems respected mechanical equilibrium while exerting the exact same traction force intensities on focal adhesions as the observed cell. The results indicate that not only the level of adhesion forces, but also the shape of the cell has an influence on intracellular tension and on nucleus strain. The combination of experimental micropost technology with the present CDM model constitutes a tool able to estimate the intracellular forces. (C) 2016 Published by Elsevier Ltd

Black Dots: Microcontact-Printed, Reference-Free Traction Force Microscopy

Measuring the traction forces produced by cells provides insight into their behavior and physiological function. Here, we developed a technique (dubbed ‘black dots’) that microcontact prints a fluorescent micropattern onto a flexible substrate to measure cellular traction forces without constraining cell shape or needing to detach the cells. To demonstrate our technique, we assessed human platelets, which can generate a large range of forces within a population. We find platelets that exert more force have more spread area, are more circular, and have more uniformly distributed F-actin filaments. As a result of the high yield of data obtainable by this technique, we were able to evaluate multivariate mixed effects models with interaction terms and conduct a clustering analysis to identify clusters within our data. These statistical techniques demonstrated a complex relationship between spread area, circularity, F-actin dispersion, and platelet force, including cooperative effects that significantly associate with platelet traction forces

Black Dots: High-Yield Traction Force Microscopy Reveals Structural Factors Contributing to Platelet Forces

Measuring the traction forces produced by cells provides insight into their behavior and physiological function. Here, we developed a technique (dubbed \u27black dots\u27) that microcontact prints a fluorescent micropattern onto a flexible substrate to measure cellular traction forces without constraining cell shape or needing to detach the cells. To demonstrate our technique, we assessed human platelets, which can generate a large range of forces within a population. We find platelets that exert more force have more spread area, are more circular, and have more uniformly distributed F-actin filaments. As a result of the high yield of data obtainable by this technique, we were able to evaluate multivariate mixed effects models with interaction terms and conduct a clustering analysis to identify clusters within our data. These statistical techniques demonstrated a complex relationship between spread area, circularity, F-actin dispersion, and platelet force, including cooperative effects that significantly associate with platelet traction forces. STATEMENT OF SIGNIFICANCE: Cells produce contractile forces during division, migration, or wound healing. Measuring cellular forces provides insight into their health, behavior, and function. We developed a technique that calculates cellular forces by seeding cells onto a pattern and quantifying how much each cell displaces the pattern. This technique is capable of measuring hundreds of cells without needing to detach them. Using this technique to evaluate human platelets, we find that platelets exerting more force tend to have more spread area, are more circular in shape, and have more uniformly distributed cytoskeletal filaments. Due to our high yield of data, we were able to apply statistical techniques that revealed combinatorial effects between these factors

TFPa/HADHA is required for fatty acid beta-oxidation and cardiolipin re-modeling in human cardiomyocytes.

Mitochondrial trifunctional protein deficiency, due to mutations in hydratase subunit A (HADHA), results in sudden infant death syndrome with no cure. To reveal the disease etiology, we generated stem cell-derived cardiomyocytes from HADHA-deficient hiPSCs and accelerated their maturation via an engineered microRNA maturation cocktail that upregulated the epigenetic regulator, HOPX.  Here we report, matured HADHA mutant cardiomyocytes treated with an endogenous mixture of fatty acids manifest the disease phenotype: defective calcium dynamics and repolarization kinetics which results in a pro-arrhythmic state. Single cell RNA-seq reveals a cardiomyocyte developmental intermediate, based on metabolic gene expression. This intermediate gives rise to mature-like cardiomyocytes in control cells but, mutant cells transition to a pathological state with reduced fatty acid beta-oxidation, reduced mitochondrial proton gradient, disrupted cristae structure and defective cardiolipin remodeling. This study reveals that HADHA (tri-functional protein alpha), a monolysocardiolipin acyltransferase-like enzyme, is required for fatty acid beta-oxidation and cardiolipin remodeling, essential for functional mitochondria in human cardiomyocytes