Orchestrated control of filaggrin-actin scaffolds underpins cornification

Epidermal stratification critically depends on keratinocyte differentiation and programmed death by cornification, leading to formation of a protective skin barrier. Cornification is dynamically controlled by the protein filaggrin, rapidly released from keratohyalin granules (KHGs). However, the mechanisms of cornification largely remain elusive, partly due to limitations of the observation techniques employed to study filaggrin organization in keratinocytes. Moreover, while the abundance of keratins within KHGs has been well described, it is not clear whether actin also contributes to their formation or fate. We employed advanced (super-resolution) microscopy to examine filaggrin organization and dynamics in skin and human keratinocytes during differentiation. We found that filaggrin organization depends on the cytoplasmic actin cytoskeleton, including the role for α- and β-actin scaffolds. Filaggrin-containing KHGs displayed high mobility and migrated toward the nucleus during differentiation. Pharmacological disruption targeting actin networks resulted in granule disintegration and accelerated cornification. We identified the role of AKT serine/threonine kinase 1 (AKT1), which controls binding preference and function of heat shock protein B1 (HspB1), facilitating the switch from actin stabilization to filaggrin processing. Our results suggest an extended model of cornification in which filaggrin utilizes actins to effectively control keratinocyte differentiation and death, promoting epidermal stratification and formation of a fully functional skin barrier

Spatiotemporally Super-Resolved Volumetric Traction Force Microscopy

Quantification of mechanical forces is a major challenge across biomedical sciences. Yet such measurements are essential to understanding the role of biomechanics in cell regulation and function. Traction force microscopy remains the most broadly applied force probing technology but typically restricts itself to single-plane two-dimensional quantifications with limited spatiotemporal resolution. Here, we introduce an enhanced force measurement technique combining 3D super-resolution fluorescence structural illumination microscopy and traction force microscopy (3D-SIM-TFM) offering increased spatiotemporal resolution, opening-up unprecedented insights into physiological three-dimensional force production in living cells

Investigating the active role of mechanical force during T-cell activation

The role of mechanical force has gained increasing interest in the field of cell biology owing to the realisation that cells are continually subject to stresses and strains induced by the cellular environment. Cells are known to be able to sense and react to forces imposed on them by their local environment as well as being able to directly impart force during motility, adhesion and cell division. This is also true for cells of the adaptive immune system, specifically during the intimate cell-cell interaction occurring between the T-cell and Antigen Presenting Cell (APC), known as the Immunological Synapse (IS). This highly selective process by which a T cell is able to bind, recognise and react to only foreign antigens has been the focus of intense study due to its crucial importance in the adaptive immune response. The actin cytoskeleton is known to play an essential role in the formation and maintenance of the IS, but questions remain regarding the influence of forces generated by actin during this process. With the aim of measuring mechanical force generated at the IS, we present a novel method combining the super resolution imaging technique, Stimulated Emission Depletion (STED) microscopy and Traction Force Microscopy (TFM). Using the tunable kinetics of the 1G4 Jurkat T-cell system in combination with high spatial and temporal resolution microscopy we demonstrate that actin dynamics at the IS is antigen dependent and show by TFM that force generation occurs on two distinct time scales during activation, mediated by the actin cytoskeleton. Together, the results highlight the intimate links between the dynamics of the actin cytoskeleton, force generation and the antigen response of T cells during activation.</p

Investigating the active role of mechanical force during T-cell activation

The role of mechanical force has gained increasing interest in the field of cell biology owing to the realisation that cells are continually subject to stresses and strains induced by the cellular environment. Cells are known to be able to sense and react to forces imposed on them by their local environment as well as being able to directly impart force during motility, adhesion and cell division. This is also true for cells of the adaptive immune system, specifically during the intimate cell-cell interaction occurring between the T-cell and Antigen Presenting Cell (APC), known as the Immunological Synapse (IS). This highly selective process by which a T cell is able to bind, recognise and react to only foreign antigens has been the focus of intense study due to its crucial importance in the adaptive immune response. The actin cytoskeleton is known to play an essential role in the formation and maintenance of the IS, but questions remain regarding the influence of forces generated by actin during this process. With the aim of measuring mechanical force generated at the IS, we present a novel method combining the super resolution imaging technique, Stimulated Emission Depletion (STED) microscopy and Traction Force Microscopy (TFM). Using the tunable kinetics of the 1G4 Jurkat T-cell system in combination with high spatial and temporal resolution microscopy we demonstrate that actin dynamics at the IS is antigen dependent and show by TFM that force generation occurs on two distinct time scales during activation, mediated by the actin cytoskeleton. Together, the results highlight the intimate links between the dynamics of the actin cytoskeleton, force generation and the antigen response of T cells during activation.</p

Quantitative Methodologies to Dissect Immune Cell Mechanobiology

Mechanobiology seeks to understand how cells integrate their biomechanics into their function and behavior. Unravelling the mechanisms underlying these mechanobiological processes is particularly important for immune cells in the context of the dynamic and complex tissue microenvironment. However, it remains largely unknown how cellular mechanical force generation and mechanical properties are regulated and integrated by immune cells, primarily due to a profound lack of technologies with sufficient sensitivity to quantify immune cell mechanics. In this review, we discuss the biological significance of mechanics for immune cells across length and time scales, and highlight several experimental methodologies for quantifying the mechanics of immune cells. Finally, we discuss the importance of quantifying the appropriate mechanical readout to accelerate insights into the mechanobiology of the immune response

Astigmatic traction force microscopy (aTFM)

Quantifying small, rapidly progressing three-dimensional forces generated by cells remains a major challenge towards a more complete understanding of mechanobiology. Traction force microscopy is one of the most broadly applied force probing technologies but ascertaining three-dimensional information typically necessitates slow, multi-frame z-stack acquisition with limited sensitivity. Here, by performing traction force microscopy using fast single-frame astigmatic imaging coupled with total internal reflection fluorescence microscopy we improve the temporal resolution of three-dimensional mechanical force quantification up to 10-fold compared to its related super-resolution modalities. 2.5D astigmatic traction force microscopy (aTFM) thus enables live-cell force measurements approaching physiological sensitivity

Single cell force profiling of human myofibroblasts reveals a biophysical spectrum of cell states

Mechanical force is a fundamental regulator of cell phenotype. Myofibroblasts are central mediators of fibrosis, a major unmet clinical need characterised by the deposition of excessive matrix proteins. Traction forces of myofibroblasts play a key role in remodelling the matrix and modulate the activities of embedded stromal cells. Here, we employ a combination of unsupervised computational analysis, cytoskeletal profiling and single cell traction force microscopy as a functional readout to uncover how the complex spatiotemporal dynamics and mechanics of living human myofibroblast shape sub-cellular profiling of traction forces in fibrosis. We resolve distinct biophysical communities of myofibroblasts, and our results provide a new paradigm for studying functional heterogeneity in human stromal cells.</p

Cytoskeletal Control of Antigen-Dependent T Cell Activation

Summary: Cytoskeletal actin dynamics is essential for T cell activation. Here, we show evidence that the binding kinetics of the antigen engaging the T cell receptor influences the nanoscale actin organization and mechanics of the immune synapse. Using an engineered T cell system expressing a specific T cell receptor and stimulated by a range of antigens, we found that the peak force experienced by the T cell receptor during activation was independent of the unbinding kinetics of the stimulating antigen. Conversely, quantification of the actin retrograde flow velocity at the synapse revealed a striking dependence on the antigen unbinding kinetics. These findings suggest that the dynamics of the actin cytoskeleton actively adjusted to normalize the force experienced by the T cell receptor in an antigen-specific manner. Consequently, tuning actin dynamics in response to antigen kinetics may thus be a mechanism that allows T cells to adjust the lengthscale and timescale of T cell receptor signaling. : T cell activation relies on a dynamic actin cytoskeleton. Here, Colin-York et al. show how the kinetics of the stimulating antigen influence the dynamics of actin. This feedback mechanism influences the mechanics at the immune synapse, allowing T cells to orchestrate the length scale and timescale of signaling. Keywords: TFM, actin dynamics, TCR cluster, immunological synapse, mechanosensation, mechanosensitivity, T cell activatio