The rapid development of advanced microscopy techniques over recent decades has significantly inc... more The rapid development of advanced microscopy techniques over recent decades has significantly increased the quality of imaging and our understanding of subcellular structures, such as the organization of the filaments of the cytoskeleton using fluorescence and electron microscopy. However, these recent improvements in imaging techniques have not been matched by similar development of techniques for computational analysis of the images of filament networks that can now be obtained. Hence, for a wide range of applications, reliable computational analysis of such two-dimensional (2D) methods remains challenging. Here, we present a new algorithm for tracing of filament networks. This software can extract many important parameters from grayscale images of filament networks, including the Mesh Hole Size, and Filament Length and Connectivity (also known as Coordination Number. In addition, the method allows sub-networks to be distinguished in 2D images using intensity thresholding. We show that the algorithm can be used to analyze images of cytoskeleton networks obtained using different advanced microscopy methods. We have thus developed a new improved method for computational analysis of 2D images of filamentous networks that has wide applications for existing imaging techniques. The algorithm is available as open-source software.
Natural killer (NK) cells play a vital role in eliminating tumorigenic cells. Efficient locating ... more Natural killer (NK) cells play a vital role in eliminating tumorigenic cells. Efficient locating and killing of target cells in complex three-dimensional (3D) environments is critical for their functions under physiological conditions. Recent studies have shown that NK cell activation is regulated by substrate stiffness. However, the role of mechanosensing in regulating NK cell killing efficiency in physiologically relevant scenarios is poorly understood. In this study, we report that the responsiveness of NK cells is regulated by tumor cell stiffness. NK cell killing efficiency in 3D is impaired against softened tumor cells, while it is enhanced against stiffened tumor cells. Notably, the durations required for NK cell killing and detachment are significantly shortened for stiffened tumor cells. Furthermore, we have identified PIEZO1 as the predominantly expressed mechanosensitive ion channel in NK cells. Perturbation of PIEZO1 by GsMTx4 abolishes stiffness-dependent NK cell respon...
Adhesion induces dramatic morphological and mechanical changes to cells, which are reflected by c... more Adhesion induces dramatic morphological and mechanical changes to cells, which are reflected by changes to the actin cortex. Among the many different proteins involved in this sub-membranous layer, motor proteins (e.g., nonmuscle myosin II [NMII]) and actin nucleators (e.g., Arp2/3, formins) are known to have significant influences on its dynamics and structure. The different roles of NMII, Arp2/3, and formins in the dynamics, structure, and mechanics of the actin cortex depend on the adhesion state of the cell. In this study, we unravel the interplay between the dynamics, structure, and mechanics of the actin cortex in adhered cells and in cells in suspension. We show that treatments with extrinsic cellular perturbants lead to alterations of all three properties that are correlated. However, intrinsic actin cortex variations between different cell adhesion states lead to unexpected correlations. Surprisingly, we find that NMII minifilaments have a minor influence on the actin corte...
Shape, dynamics, and viscoelastic properties of eukaryotic cells are primarily governed by a thin... more Shape, dynamics, and viscoelastic properties of eukaryotic cells are primarily governed by a thin, reversibly cross-linked actomyosin cortex located directly beneath the plasma membrane. We obtain time-dependent rheological responses of fibroblasts and MDCK II cells from deformation-relaxation curves using an atomic force microscope to access the dependence of cortex fluidity on pre-stress. We introduce a viscoelastic model that treats the cell as a composite shell and assumes that relaxation of the cortex follows a power law giving access to cortical pre-stress, area compressibility modulus, and the power law (fluidity) exponent. Cortex fluidity is modulated by interfering with myosin activity. We find that the power law exponent of the cell cortex decreases with increasing intrinsic pre-stress and area compressibility modulus, in accordance with previous finding for isolated actin networks subject to external stress. Extrapolation to zero tension returns the theoretically predicte...
Vimentin is a protein that has been linked to a large variety of pathophysiological conditions, i... more Vimentin is a protein that has been linked to a large variety of pathophysiological conditions, including cataracts, Crohn's disease, rheumatoid arthritis, HIV and cancer. Vimentin has also been shown to regulate a wide spectrum of basic cellular functions. In cells, vimentin assembles into a network of filaments that spans the cytoplasm. It can also be found in smaller, non-filamentous forms that can localise both within cells and within the extracellular microenvironment. The vimentin structure can be altered by subunit exchange, cleavage into different sizes, re-annealing, post-translational modifications and interacting proteins. Together with the observation that different domains of vimentin might have evolved under different selection pressures that defined distinct biological functions for different parts of the protein, the many diverse variants of vimentin might be the cause of its functional diversity. A number of review articles have focussed on the biology and medic...
The mesenchymal-amoeboid transition (MAT) was proposed as a mechanism for cancer cells to adapt t... more The mesenchymal-amoeboid transition (MAT) was proposed as a mechanism for cancer cells to adapt their migration mode to their environment. While the molecular pathways involved in this transition are well documented, the role of the microenvironment in the MAT is still poorly understood. Here, we investigated how confinement and adhesion affect this transition. We report that, in the absence of focal adhesions and under conditions of confinement, mesenchymal cells can spontaneously switch to a fast amoeboid migration phenotype. We identified two main types of fast migration--one involving a local protrusion and a second involving a myosin-II-dependent mechanical instability of the cell cortex that leads to a global cortical flow. Interestingly, transformed cells are more prone to adopt this fast migration mode. Finally, we propose a generic model that explains migration transitions and predicts a phase diagram of migration phenotypes based on three main control parameters: confineme...
Individual cells in their native physiological states face a dynamic multi-factorial environment.... more Individual cells in their native physiological states face a dynamic multi-factorial environment. This is true of both single-celled and multi-cellular organisms. A key challenge in cell biology is the design of experimental methods and specific assays to disentangle the contribution of each of the parameters governing cell behavior. After decades of studying cells cultured in Petri dishes or on glass coverslips, researchers can now benefit from a range of recent technological developments that allow them to study cells in a variety of contexts, with different levels of complexity and control over a range of environmental parameters. These technologies include new types of microscopy for detailed imaging of large cell aggregates or even whole tissues, and the development of cell culture substrates, such as 3D matrices. Here we will review the contribution of a third type of tool, collectively known as microfabricated tools. Derived from techniques originally developed for microelectronics, these tools range in size from hundreds of microns to hundreds of nanometers.
PhD – Thesis of Franziska Lautenschläger Title: Cell Compliance – Cytoskeletal Origin and Importa... more PhD – Thesis of Franziska Lautenschläger Title: Cell Compliance – Cytoskeletal Origin and Importance for
Dendritic cells use amoeboid migration to pass through confined tissues to reach the lymph nodes,... more Dendritic cells use amoeboid migration to pass through confined tissues to reach the lymph nodes, and this homing function is crucial for immune responses. The underlying mechanisms for this type of migration remain unknown. As vimentin intermediate filaments regulate adhesion-dependent migration, we analyzed whether they have a similar effect on amoeboid migration. We show that lack of vimentin impairs amoeboid migrationin vitroin confined environments, and blocks lymph-node homing in micein vivo. Importantly, we show that vimentin-deficient dendritic cells have a lower coupling factor between cell speed and persistence and reduced target search efficiency (e.g., finding a pathogen, or another cell). These data show that the characteristics of vimentin in its dynamic regulation of cell stiffness and load-bearing, and also elastic capacity, appear to explain the coupling between their migratory ability and search efficiency. Taken together, these data show that vimentin provides the...
The rapid development of advanced microscopy techniques over recent decades has significantly inc... more The rapid development of advanced microscopy techniques over recent decades has significantly increased the quality of imaging and our understanding of subcellular structures, such as the organization of the filaments of the cytoskeleton using fluorescence and electron microscopy. However, these recent improvements in imaging techniques have not been matched by similar development of techniques for computational analysis of the images of filament networks that can now be obtained. Hence, for a wide range of applications, reliable computational analysis of such two-dimensional (2D) methods remains challenging. Here, we present a new algorithm for tracing of filament networks. This software can extract many important parameters from grayscale images of filament networks, including the Mesh Hole Size, and Filament Length and Connectivity (also known as Coordination Number. In addition, the method allows sub-networks to be distinguished in 2D images using intensity thresholding. We show...
The establishment of polarity in cells and tissues is one of the first steps in multicellular dev... more The establishment of polarity in cells and tissues is one of the first steps in multicellular development. The ‘eternal embryo’hydracan completely regenerate from a disorganized cell cluster or a small fragment of tissue of about 10, 000 cells. During regeneration, the cells first form a hollow cell spheroid, which then undergoesde-novosymmetry breaking to irreversibly polarize. Here, we address the symmetry-related shape changes. Prior to axis establishment, the spheroid of regenerating cells presents inflation oscillations on several timescales that are isotropic in space. There are transient periods of fluctuations in defined arbitrary directions, until these undergo a clearly identified, irreversible transition to directed fluctuations along the future main axis of the regeneratinghydra. Stabilized cytosolic actin structures disappear during thede-novopolarization, while polymerized microtubules remain. In our observations applied drugs that depolymerize actin filaments accelera...
Dendritic cells use amoeboid migration through constricted passages to reach the lymph nodes, and... more Dendritic cells use amoeboid migration through constricted passages to reach the lymph nodes, and this homing function is crucial for immune responses. Amoeboid migration requires mechanical resilience, however, the underlying molecular mechanisms for this type of migration remain unknown. Because vimentin intermediate filaments (IFs) and microfilaments regulate adhesion-dependent migration in a bidirectional manner, we analyzed if they exert a similar control on amoeboid migration. Vimentin was required for cellular resilience, via a joint interaction between vimentin IFs and F-actin. Reduced actin mobility in the cell cortex of vimentin-reduced cells indicated that vimentin promotes Factin subunit exchange and dynamics. These mechano-dynamic alterations in vimentin-deficient dendritic cells impaired amoeboid migration in confined environments in vitro and blocked lymph node homing in mouse experiments in vivo. Correct nuclear positioning is important in confined amoeboid migration...
Metastasizing tumor cells show increased expression of the intermediate filament (IF) protein vim... more Metastasizing tumor cells show increased expression of the intermediate filament (IF) protein vimentin, which has been used to diagnose invasive tumors for decades. Recent observations indicate that vimentin is not only a passive marker for carcinoma, but may also induce tumor cell invasion. To clarify how vimentin IFs control cell adhesions and migration, we analyzed the nanoscale (30-50 nm) spatial organization of vimentin IFs and cell-matrix adhesions in metastatic fibroblast cells, using three-color stimulated emission depletion (STED) microscopy. We also studied whether wild-type and phospho-deficient or -mimicking mutants of vimentin changed the size and lifetime of focal adhesions (FAs), cell shape, and cell migration, using live-cell total internal reflection imaging and confocal microscopy. We observed that vimentin exists in fragments of different lengths. Short fragments were mostly the size of a unit-length filament and were mainly localized close to small cell-matrix ad...
The rapid development of advanced microscopy techniques over recent decades has significantly inc... more The rapid development of advanced microscopy techniques over recent decades has significantly increased the quality of imaging and our understanding of subcellular structures, such as the organization of the filaments of the cytoskeleton using fluorescence and electron microscopy. However, these recent improvements in imaging techniques have not been matched by similar development of techniques for computational analysis of the images of filament networks that can now be obtained. Hence, for a wide range of applications, reliable computational analysis of such two-dimensional (2D) methods remains challenging. Here, we present a new algorithm for tracing of filament networks. This software can extract many important parameters from grayscale images of filament networks, including the Mesh Hole Size, and Filament Length and Connectivity (also known as Coordination Number. In addition, the method allows sub-networks to be distinguished in 2D images using intensity thresholding. We show that the algorithm can be used to analyze images of cytoskeleton networks obtained using different advanced microscopy methods. We have thus developed a new improved method for computational analysis of 2D images of filamentous networks that has wide applications for existing imaging techniques. The algorithm is available as open-source software.
Natural killer (NK) cells play a vital role in eliminating tumorigenic cells. Efficient locating ... more Natural killer (NK) cells play a vital role in eliminating tumorigenic cells. Efficient locating and killing of target cells in complex three-dimensional (3D) environments is critical for their functions under physiological conditions. Recent studies have shown that NK cell activation is regulated by substrate stiffness. However, the role of mechanosensing in regulating NK cell killing efficiency in physiologically relevant scenarios is poorly understood. In this study, we report that the responsiveness of NK cells is regulated by tumor cell stiffness. NK cell killing efficiency in 3D is impaired against softened tumor cells, while it is enhanced against stiffened tumor cells. Notably, the durations required for NK cell killing and detachment are significantly shortened for stiffened tumor cells. Furthermore, we have identified PIEZO1 as the predominantly expressed mechanosensitive ion channel in NK cells. Perturbation of PIEZO1 by GsMTx4 abolishes stiffness-dependent NK cell respon...
Adhesion induces dramatic morphological and mechanical changes to cells, which are reflected by c... more Adhesion induces dramatic morphological and mechanical changes to cells, which are reflected by changes to the actin cortex. Among the many different proteins involved in this sub-membranous layer, motor proteins (e.g., nonmuscle myosin II [NMII]) and actin nucleators (e.g., Arp2/3, formins) are known to have significant influences on its dynamics and structure. The different roles of NMII, Arp2/3, and formins in the dynamics, structure, and mechanics of the actin cortex depend on the adhesion state of the cell. In this study, we unravel the interplay between the dynamics, structure, and mechanics of the actin cortex in adhered cells and in cells in suspension. We show that treatments with extrinsic cellular perturbants lead to alterations of all three properties that are correlated. However, intrinsic actin cortex variations between different cell adhesion states lead to unexpected correlations. Surprisingly, we find that NMII minifilaments have a minor influence on the actin corte...
Shape, dynamics, and viscoelastic properties of eukaryotic cells are primarily governed by a thin... more Shape, dynamics, and viscoelastic properties of eukaryotic cells are primarily governed by a thin, reversibly cross-linked actomyosin cortex located directly beneath the plasma membrane. We obtain time-dependent rheological responses of fibroblasts and MDCK II cells from deformation-relaxation curves using an atomic force microscope to access the dependence of cortex fluidity on pre-stress. We introduce a viscoelastic model that treats the cell as a composite shell and assumes that relaxation of the cortex follows a power law giving access to cortical pre-stress, area compressibility modulus, and the power law (fluidity) exponent. Cortex fluidity is modulated by interfering with myosin activity. We find that the power law exponent of the cell cortex decreases with increasing intrinsic pre-stress and area compressibility modulus, in accordance with previous finding for isolated actin networks subject to external stress. Extrapolation to zero tension returns the theoretically predicte...
Vimentin is a protein that has been linked to a large variety of pathophysiological conditions, i... more Vimentin is a protein that has been linked to a large variety of pathophysiological conditions, including cataracts, Crohn's disease, rheumatoid arthritis, HIV and cancer. Vimentin has also been shown to regulate a wide spectrum of basic cellular functions. In cells, vimentin assembles into a network of filaments that spans the cytoplasm. It can also be found in smaller, non-filamentous forms that can localise both within cells and within the extracellular microenvironment. The vimentin structure can be altered by subunit exchange, cleavage into different sizes, re-annealing, post-translational modifications and interacting proteins. Together with the observation that different domains of vimentin might have evolved under different selection pressures that defined distinct biological functions for different parts of the protein, the many diverse variants of vimentin might be the cause of its functional diversity. A number of review articles have focussed on the biology and medic...
The mesenchymal-amoeboid transition (MAT) was proposed as a mechanism for cancer cells to adapt t... more The mesenchymal-amoeboid transition (MAT) was proposed as a mechanism for cancer cells to adapt their migration mode to their environment. While the molecular pathways involved in this transition are well documented, the role of the microenvironment in the MAT is still poorly understood. Here, we investigated how confinement and adhesion affect this transition. We report that, in the absence of focal adhesions and under conditions of confinement, mesenchymal cells can spontaneously switch to a fast amoeboid migration phenotype. We identified two main types of fast migration--one involving a local protrusion and a second involving a myosin-II-dependent mechanical instability of the cell cortex that leads to a global cortical flow. Interestingly, transformed cells are more prone to adopt this fast migration mode. Finally, we propose a generic model that explains migration transitions and predicts a phase diagram of migration phenotypes based on three main control parameters: confineme...
Individual cells in their native physiological states face a dynamic multi-factorial environment.... more Individual cells in their native physiological states face a dynamic multi-factorial environment. This is true of both single-celled and multi-cellular organisms. A key challenge in cell biology is the design of experimental methods and specific assays to disentangle the contribution of each of the parameters governing cell behavior. After decades of studying cells cultured in Petri dishes or on glass coverslips, researchers can now benefit from a range of recent technological developments that allow them to study cells in a variety of contexts, with different levels of complexity and control over a range of environmental parameters. These technologies include new types of microscopy for detailed imaging of large cell aggregates or even whole tissues, and the development of cell culture substrates, such as 3D matrices. Here we will review the contribution of a third type of tool, collectively known as microfabricated tools. Derived from techniques originally developed for microelectronics, these tools range in size from hundreds of microns to hundreds of nanometers.
PhD – Thesis of Franziska Lautenschläger Title: Cell Compliance – Cytoskeletal Origin and Importa... more PhD – Thesis of Franziska Lautenschläger Title: Cell Compliance – Cytoskeletal Origin and Importance for
Dendritic cells use amoeboid migration to pass through confined tissues to reach the lymph nodes,... more Dendritic cells use amoeboid migration to pass through confined tissues to reach the lymph nodes, and this homing function is crucial for immune responses. The underlying mechanisms for this type of migration remain unknown. As vimentin intermediate filaments regulate adhesion-dependent migration, we analyzed whether they have a similar effect on amoeboid migration. We show that lack of vimentin impairs amoeboid migrationin vitroin confined environments, and blocks lymph-node homing in micein vivo. Importantly, we show that vimentin-deficient dendritic cells have a lower coupling factor between cell speed and persistence and reduced target search efficiency (e.g., finding a pathogen, or another cell). These data show that the characteristics of vimentin in its dynamic regulation of cell stiffness and load-bearing, and also elastic capacity, appear to explain the coupling between their migratory ability and search efficiency. Taken together, these data show that vimentin provides the...
The rapid development of advanced microscopy techniques over recent decades has significantly inc... more The rapid development of advanced microscopy techniques over recent decades has significantly increased the quality of imaging and our understanding of subcellular structures, such as the organization of the filaments of the cytoskeleton using fluorescence and electron microscopy. However, these recent improvements in imaging techniques have not been matched by similar development of techniques for computational analysis of the images of filament networks that can now be obtained. Hence, for a wide range of applications, reliable computational analysis of such two-dimensional (2D) methods remains challenging. Here, we present a new algorithm for tracing of filament networks. This software can extract many important parameters from grayscale images of filament networks, including the Mesh Hole Size, and Filament Length and Connectivity (also known as Coordination Number. In addition, the method allows sub-networks to be distinguished in 2D images using intensity thresholding. We show...
The establishment of polarity in cells and tissues is one of the first steps in multicellular dev... more The establishment of polarity in cells and tissues is one of the first steps in multicellular development. The ‘eternal embryo’hydracan completely regenerate from a disorganized cell cluster or a small fragment of tissue of about 10, 000 cells. During regeneration, the cells first form a hollow cell spheroid, which then undergoesde-novosymmetry breaking to irreversibly polarize. Here, we address the symmetry-related shape changes. Prior to axis establishment, the spheroid of regenerating cells presents inflation oscillations on several timescales that are isotropic in space. There are transient periods of fluctuations in defined arbitrary directions, until these undergo a clearly identified, irreversible transition to directed fluctuations along the future main axis of the regeneratinghydra. Stabilized cytosolic actin structures disappear during thede-novopolarization, while polymerized microtubules remain. In our observations applied drugs that depolymerize actin filaments accelera...
Dendritic cells use amoeboid migration through constricted passages to reach the lymph nodes, and... more Dendritic cells use amoeboid migration through constricted passages to reach the lymph nodes, and this homing function is crucial for immune responses. Amoeboid migration requires mechanical resilience, however, the underlying molecular mechanisms for this type of migration remain unknown. Because vimentin intermediate filaments (IFs) and microfilaments regulate adhesion-dependent migration in a bidirectional manner, we analyzed if they exert a similar control on amoeboid migration. Vimentin was required for cellular resilience, via a joint interaction between vimentin IFs and F-actin. Reduced actin mobility in the cell cortex of vimentin-reduced cells indicated that vimentin promotes Factin subunit exchange and dynamics. These mechano-dynamic alterations in vimentin-deficient dendritic cells impaired amoeboid migration in confined environments in vitro and blocked lymph node homing in mouse experiments in vivo. Correct nuclear positioning is important in confined amoeboid migration...
Metastasizing tumor cells show increased expression of the intermediate filament (IF) protein vim... more Metastasizing tumor cells show increased expression of the intermediate filament (IF) protein vimentin, which has been used to diagnose invasive tumors for decades. Recent observations indicate that vimentin is not only a passive marker for carcinoma, but may also induce tumor cell invasion. To clarify how vimentin IFs control cell adhesions and migration, we analyzed the nanoscale (30-50 nm) spatial organization of vimentin IFs and cell-matrix adhesions in metastatic fibroblast cells, using three-color stimulated emission depletion (STED) microscopy. We also studied whether wild-type and phospho-deficient or -mimicking mutants of vimentin changed the size and lifetime of focal adhesions (FAs), cell shape, and cell migration, using live-cell total internal reflection imaging and confocal microscopy. We observed that vimentin exists in fragments of different lengths. Short fragments were mostly the size of a unit-length filament and were mainly localized close to small cell-matrix ad...
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Papers by Franziska Lautenschläger