Biology subjects

Hauke, L.

Publications and source records attributed to Hauke, L..

6 recordsLinked to original sources

bioRxiv2025

SarcAsM: AI-based multiscale analysis of sarcomere organization and contractility in cardiomyocytes

Cardiomyocyte function critically depends on sarcomere dynamics and their organization in myofibrils. To uncover how cardiomyocyte function emerges from individual sarcomere dynamics, comprehensive analysis of Z-bands (nanometer scale), sarcomeres ([~]2 {micro}m scale), and myofibrils ([~]10 to 100 {micro}m scale) is required. Tools for such multiscale analyses are presently lacking. Here we introduce the Sarcomere Analysis Multitool (SarcAsM), which combines deep learning and graph-based methods for automated, fast, and unbiased structural assessment of sarcomeric Z-bands, sarcomeres, and myofibrils as well as their organization in larger myofibril domains. SarcAsM features a generalist deep learning model, pre-trained on a broad range of experimental and published images, ensuring its versatile application and immediate usability across diverse datasets. Finally, we demonstrate the versatile utility of SarcAsM in analyzing sarcomere structure and dynamics under acute and chronic drug exposure in hiPSC-derived cardiomyocytes with fluorescently tagged Z-bands. SarcAsM is available as open-source Python package and stand-alone application.

bioinformatics↗

bioRxiv2024

Stochastic tug-of-war among sarcomeres mediates cardiomyocyte response to environmental stiffness

Cardiac contraction is driven by the collective action of cardiomyocytes, that contain parallel bundles of myofibrils consisting of linear chains of sarcomeres, the basic force-generating units. The dynamics of individual sarcomeres within intact cardiomyocytes remain incompletely understood. While most models assume uniform, synchronized contractions, recent studies hint at unexpected heterogeneity whose origins and significance are not yet clear. By combining the culture of fluorescent sarcomere-reporter hiPSC-derived cardiomyocytes on micropatterned soft gels of different stiffness (5 - 85 kPa) with AI-based tracking of sarcomere motion, we found that increasingly stiff substrates inhibited overall cardiomyocyte contraction, but, surprisingly, did not diminish individual sarcomere dynamics. Instead, sarcomeres competed in a tug-of-war causing increasing heterogeneity, including rapid length oscillations and overextensions (popping). Statistical analysis showed that the heterogeneous dynamics were not caused by static structural differences but were largely stochastic. Stochastic heterogeneity is thus an intrinsic property of cardiac sarcomeres and likely mediates the adaptation of cardiomyocyte contractility to mechanical constraints. A mesoscopic model of coupled sarcomeres shows that these phenomena can be explained by a non-monotonic force-velocity relationship and stochastic fluctuations, where dynamic instability at a critical yielding force creates heterogeneity. Stochastic heterogeneity compensates for structural disorder by randomizing yield events beat-to-beat, preventing damage to specific sarcomeres. Our findings recast cardiac sarcomeres as active, dynamically unstable, and stochastic units engaged in a stochastic tug-of-war, where transient, velocity-dependent forces dominate. We propose that pathological disorder in cardiomyopathy drives a transition from protective stochastic fluctuations to more deterministic, persistently overloaded sarcomeres.

biophysics↗

bioRxiv2024

Three-dimensional multi-target super-resolution microscopy of cells using Metal-Induced Energy Transfer and DNA-PAINT

AbsrtactAchieving nanometer precision in 3D remains a major challenge in super-resolution microscopy. DNA-PAINT offers excellent lateral resolution and versatile multiplexing, but its axial localization precision is typically 3-5 times poorer, limiting quantitative 3D imaging. Here, we present MIET-PAINT, which combines DNA-PAINT with Metal-Induced Energy Transfer (MIET) to overcome this limitation. We implement MIET-PAINT on both wide-field fluorescence lifetime and confocal TCSPC platforms. Wide-field MIET-PAINT enables robust, multiplexed imaging of focal adhesion proteins and actin in fixed cells. To address the lateral resolution limits of lifetime cameras, we further developed confocal MIET-PAINT, which leverages optical background rejection and high-efficiency SPAD detection. This modality achieves [~]12 nm lateral precision and resolves the 3D architecture of microtubules, vimentin, and actin with high fidelity. MIET-PAINT thus unites nanometer-scale axial accuracy with the multiplexing versatility of DNA-PAINT, establishing a powerful tool for quantitative 3D cell biology.

biophysics↗

bioRxiv2022

Metal-Induced Energy Transfer (MIET) for Live-Cell Imaging with Fluorescent Proteins

Metal-Induced Energy Transfer (MIET) imaging is an easy-to-implement super-resolution modality that achieves nanometer resolution along the optical axis of a microscope. Although its capability in numerous biological and biophysical studies has been demonstrated, its implementation for live-cell imaging with fluorescent proteins is still lacking. Here, we present its applicability and capabilities for live-cell imaging with fluorescent proteins in diverse cell types (adult human stem cells, human osteo-sarcoma cells, and Dictyostelium discoideum cells), and with various fluorescent proteins (GFP, mScarlet, RFP, YPet). We show that MIET imaging achieves nanometer axial mapping of living cellular and sub-cellular components across multiple timescales, from a few milliseconds to hours, with negligible phototoxic effects.

biophysics↗

bioRxiv2021

A DNA-based optical force sensor for live-cell applications

Mechanical forces are relevant for many biological processes, from wound healing or tumour formation to cell migration and differentiation. Cytoskeletal actin is largely responsible for responding to forces and transmitting them in cells, while also maintaining cell shape and integrity. Here, we describe a novel approach to employ a FRET-based DNA force sensor in vitro and in cellulo for non-invasive optical monitoring of intracellular mechanical forces. We use fluorescence lifetime imaging to determine the FRET efficiency of the sensor, which makes the measurement robust against intensity variations. We demonstrate the applicability of the sensor by monitoring cross-linking activity in in vitro actin networks by bulk rheology and confocal microscopy. We further demonstrate that the sensor readily attaches to stress fibers in living cells which opens up the possibility of live-cell force measurements.

biophysics↗

bioRxiv2021

A Focal Adhesion Filament Cross-correlation Kit for fast, automated segmentation and correlation of focal adhesions and actin stress fibers in cells

Focal adhesions (FAs) and associated actin stress fibers (SFs) form a complex mechanical system that mediates bidirectional interactions between cells and their environment. This linked network is essential for mechanosensing, force production and force transduction, thus directly governing cellular processes like polarization, migration and extracellular matrix remodeling. We introduce a tool for fast and robust coupled analysis of both FAs and SFs named the Focal Adhesion Filament Cross-correlation Kit (FAFCK). Our software can detect and record location, axes lengths, area, orientation, and aspect ratio of focal adhesion structures as well as the location, length, width and orientation of actin stress fibers. This enables users to automate analysis of the correlation of FAs and SFs and study the stress fiber system in a higher degree, pivotal to accurately evaluate transmission of mechanocellular forces between a cell and its surroundings. The FAFCK is particularly suited for unbiased and systematic quantitative analysis of FAs and SFs necessary for novel approaches of traction force microscopy that uses the additional data from the cellular side to calculate the stress distribution in the substrate. For validation and comparison with other tools, we provide datasets of cells of varying quality that are labelled by a human expert. Datasets and FAFCK are freely available as open source under the GNU General Public License. Author summaryOur novel Focal Adhesion Filament Cross-correlation Kit (FAFCK) allows for fast, reliable, unbiased, and systematic detection of focal adhesions and actin stress fibers in cells and their mutual correlation. Detailed analysis of these structures which are both key elements in mechano-sensing and force transduction will help tremendously to improve quantitative analysis of mechanocellular experiments, key to understanding the complex interplay between cells and the extracellular matrix. In particular, sophisticated analysis methods such as model-based traction force microscopy will benefit from correlating the detailed datasets of stress fibers and focal adhesions.

biophysics↗