Biology subjects

Lautenschlaeger, F.

Publications and source records attributed to Lautenschlaeger, F..

10 recordsLinked to original sources

bioRxiv2025

The mechanism how Pretubulysin-induced microtubule disassembly improves T cell search efficiency

To clean tissue from tumorigenic and infected cells, cytotoxic T lymphocytes (CTLs) must navigate confined environments in vivo, locate the infected cells and eliminate them. Impaired CTL migration towards the tumor can limit the efficacy of immunotherapy. Microtubules (MTs) have emerged as promising targets, because destabilizing MTs enhances T-cell migration and subsequent killing, yet the underlying mechanisms are poorly understood. Here, we use pretubulysin, a potent MT depolymerizer, to uncover how MT dynamics regulate CTL motility. Complete MT disassembly markedly increased CTL infiltration and migration in 3D matrices. To investigate how altered migration affects target elimination, we employed a persistent random-walk model parameterized solely with experimental motility data. The model shows that the increase in speed and persistence induced by pretubulysin explains enhanced search efficiency increasing the encounter rate of CTLs with target cells. The simulations also predict how these gains in search efficiency scale with tissue thickness and CTL density. Mechanistically, MT depolymerization in activated CTLs triggers localized actomyosin accumulation at the uropod. This enhances rear contraction forces and promotes faster, more persistent migration and efficient search. Our findings clarify how MT dynamics influence CTL ability to eliminate targets in 3D environments and highlights the potential of MT-targeting agents such as pretubulysin to optimize T cell-based immunotherapies.

cell biology↗

bioRxiv2023

PIEZO1-mediated mechanosensing governs NK cell killing efficiency in 3D

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 are critical for their functions under physiological conditions. However, the role of mechanosensing in regulating NK cell killing efficiency in physiologically relevant scenarios is poorly understood. Here, 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 among the examined candidates in NK cells. Perturbation of PIEZO1 abolishes stiffness-dependent NK cell responsiveness, significantly impairs the killing efficiency of NK cells in 3D, and substantially reduces NK cell infiltration into 3D collagen matrices. Conversely, PIEZO1 activation enhances NK killing efficiency as well as infiltration. In conclusion, our findings demonstrate that PIEZO1-mediated mechanosensing is crucial for NK killing functions, highlighting the role of mechanosensing in NK cell killing efficiency under 3D physiological conditions and the influence of environmental physical cues on NK cell functions.

immunology↗

bioRxiv2022

Amoeboid Cell Migration through Regular Arrays of Micropillars under Confinement

Migrating cells often encounter a wide variety of topographic features--including the presence of obstacles--when navigating through crowded biological environments. Unravelling the impact of topography and crowding on the dynamics of cells is key to better understand many essential physiological processes such as the immune response. We study how migration and search efficiency of HL-60 cells differentiated into neutrophils in quasi two-dimensional environments are influenced by the lateral and vertical confinement and spatial arrangement of obstacles. A microfluidic device is designed to track the cells in confining geometries between two parallel plates with distance h, in which identical micropillars are arranged in regular pillar forests. We find that at each cell-pillar contact event, the cell spends a finite time near the pillar surface, which is independent of the height h and the interpillar spacing e. At low pillar density regime, the directional persistence of cells reduces with decreasing h or e, influencing their diffusivity and first-passage properties. The dynamics is strikingly different at high pillar density regime, where the cells are in simultaneous contact with more than one pillar; the cell velocity and persistence are distinctly higher compared to dilute pillar configurations with the same h. Our simulations reveal that the interplay between cell persistence and cell-pillar interactions can dramatically affect cell diffusivity and, thus, its first-passage properties.

biophysics↗

bioRxiv2021

The role of actin and myosin II in the cell cortex of adhered and suspended cells

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 cortex. Using new microscopy techniques, we show that NMII minifilaments are not localized within the actin cortex, as previously thought, but concentrated in a layer beneath it. Our treatments affecting Arp2/3 and formin reveal correlations between the actin cortex characteristics. Our data build towards a comprehensive understanding of the actin cortex. This understanding allows the prediction and control of cortical changes, which is essential for the study of general cellular processes, such as cell migration, metastasis, and differentiation.

biophysics↗

bioRxiv2021

A novel universal algorithm for filament network tracing and cytoskeleton analysis

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.

biophysics↗

bioRxiv2020

Vimentin provides target search efficiency and mechanical resilience for dendritic cell migration

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 migration in vitro in confined environments, and blocks lymph-node homing in mice in 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 specific mechano-dynamics required for dendritic cell migration and for efficient target searching. Summary statementVimentin contributes to the mechanical stiffness of cells required for amoeboid cell migration through confined spaces, and improves cell-search efficiency. Vimentin-deficient cells migrate more slowly and their migration speed is less coupled to persistence compared to control cells.

biophysics↗

bioRxiv2020

Correlative Fluorescence and Electron Microscopy of Whole Breast Cancer Cells Reveals Differential Distribution of ErbB2 Dependent on Underlying Actin

Epidermal growth factor receptor 2 (ErbB2) is found overexpressed in several cancers, such as gastric, and breast cancer, and is, therefore, an important therapeutic target. ErbB2 plays a central role in cancer cell invasiveness, and is associated with cytoskeletal reorganization. In order to study the spatial correlation of single ErbB2 proteins and actin filaments, we applied correlative fluorescence microscopy, and scanning transmission electron microscopy (STEM) to image specifically labeled SKBR3 breast cancer cells. The breast cancer cells were grown on microchips, transformed to express an actin-green fluorescent protein (GFP) fusion protein, and labeled with quantum dot (QD) nanoparticles attached to specific anti-ErbB2 Affibodies. Fluorescence microscopy was performed to identify cellular regions with spatially correlated actin and ErbB2 expression. For STEM of the intact plasma membrane of whole cells, the cells were fixed and covered with graphene. Spatial distribution patterns of ErbB2 in the actin rich ruffled membrane regions were examined, and compared to adjacent actin-low regions of the same cell, revealing an association of putative signaling active ErbB2 homodimers with actin-rich regions. ErbB2 homodimers were found absent from actin-low membrane regions, as well as after treatment of cells with Cytochalasin D, which breaks up larger actin filaments. In both latter data sets, a significant inter-label distance of 36 nm was identified, possibly indicating an indirect attachment to helical actin filaments via the formation of heterodimers of ErbB2 with epidermal growth factor receptor (EGFR). The possible attachment to actin filaments was further explored by identifying linear QD-chains in actin-rich regions, which also showed an inter-label distance of 36 nm.

biophysics↗

bioRxiv2020

Highly coordinated mechanical motion mediated by the microtubule cytoskeletonis a pivotal element of de-novo symmetry breaking in hydraspheroids

The establishment of polarity in cells and tissues is one of the first steps in multicellular development. The eternal embryo hydra can 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 undergoes de-novo symmetry 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 regenerating hydra. Stabilized cytosolic actin structures disappear during the de-novo polarization, while polymerized microtubules remain. In our observations applied drugs that depolymerize actin filaments accelerate the symmetry breaking process, while drug-stabilized actin filaments prevent it. Nocodazole-depolymerized microtubules prevent symmetry breaking, but regeneration can be rescued by the microtubule-stabilizing drug paclitaxel at concentrations where microtubular structures start to reappear. We discuss the possibility that mechanical fluctuations induce the orientation and position of microtubules, which contribute to {beta}-catenin nuclear translocation, to increase the organizer-forming-potential of the cells. Our data suggest that in regenerating hydra spheroids, microtubules play a pivotal role in the cooperative polarization process of the self-organizing hydra spheroid.

biophysics↗

bioRxiv2020

Scanning electron microscopy preparation and analysis of the cell cortex ultrastructure

The cellular cortex is an approximately 200-nm-thick actin network that lies just beneath the cell membrane. It is responsible for the mechanical properties of cells, and as such, it is involved in many cellular processes, including cell migration and cellular interactions with the environment. To develop a clear view of this dense structure, high-resolution imaging is essential. As one such technique, electron microscopy, involves complex sample preparation procedures. The final drying of these samples has significant influence on potential artifacts, like cell shrinkage and the formation of artifactual holes in the actin cortex. In this study, we compared the three most used final sample drying procedures: critical-point drying (CPD), CPD with lens tissue (CPD-LT), and hexamethyldisilazane drying. We show that both hexamethyldisilazane and CPD-LT lead to fewer artifactual mesh holes within the actin cortex than CPD. Moreover, CPD-LT leads to significant reduction in cell height compared to hexamethyldisilazane and CPD. We conclude that the final drying procedure should be chosen according to the reduction in cell height, and so CPD-LT, or according to the spatial separation of the single layers of the actin cortex, and so hexamethyldisilazane.

biophysics↗

bioRxiv2019

Vimentin provides the mechanical resilience required for amoeboid migration and protection of the nucleus.

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 both to minimize resistance and to avoid DNA damage. Vimentin-deficiency also led to DNA double strand breaks in the compressed dendritic cells, pointing to a role of vimentin in nuclear positioning. Together, these observations show that vimentin IF-microfilament interactions provide both the specific mechano-dynamics required for dendritic cell migration and the protection the genome needs in compressed spaces.\n\nSummary statementVimentin -- in joint action with actin -- mediates the mechanical stiffness of cells required for amoeboid cell migration through confined spaces and protects the nucleus from DNA damage.

biophysics↗