Biology subjects

Mark, C.

Publications and source records attributed to Mark, C..

11 recordsLinked to original sources

bioRxiv2026

Mechanochemical synchronization of force oscillations in connective tissue models

Cells in a tissue coordinate their behavior collectively by mechanical and chemical feedback with their surroundings. To study how such feedback mechanisms interact, we embed multicellular spheroids from mouse and primary human fibroblasts of different origin in 3D collagen matrices. Immediately after embedding, spheroids apply steadily increasing, radially symmetric, inward-directed forces to the surrounding matrix. Typically, within a few hours, collective force oscillations emerge with an average time interval of {approx}30 min. Similar force oscillations also arise in engineered, parallel-aligned microtissues attached to flexible pillars. Force oscillations propagate between neighboring cells as a mechanical wave, accompanied by calcium waves that travel at comparable speeds of several hundred m/min. Blocking gap junctions or mechanosensitive Piezo1 channels impairs these oscillations, indicating that calcium signaling contributes to their propagation. Their amplitude and frequency are regulated by integrin-mediated feedback in actin-rich protrusions and by ERK signaling, which also controls collective invasion into the surrounding matrix. Neighboring spheroids mechanically synchronize their force oscillations in phase and frequency over distances of up to {approx}2 mm without direct contact. Together, our results show that oscillatory mechanochemical feedback enables multicellular systems to coordinate force generation and invasive migration across cellular and millimeter length scales in 3D matrices.

biophysics↗

bioRxiv2024

Glioblastoma invasion relies on actomyosin contractility and metalloproteinase activity

PurposeGlioblastoma multiforme (GBM) is an aggressive brain tumor with a 5-year survival rate below 7%. Poor outcomes are driven in part by diffuse invasion into surrounding brain tissue, which limits complete surgical resection and promotes recurrence. Improved therapies require a quantitative understanding of the mechanics of GBM invasion. MethodsPatient-derived GBM neurospheres were embedded in Matrigel and analyzed using live imaging, three-dimensional traction force microscopy (3D TFM), and targeted cytoskeletal perturbations. ResultsInvading GBM cells adopted an elongated, protrusion-rich morphology, with F-actin enriched at the cell periphery and microtubules extending along the protrusion shaft, consistent with a mesenchymal-like invasion program. 3D TFM revealed sustained matrix engagement, including progressive bead clustering, increasing cumulative traction forces, and traction hotspots concentrated near protrusion tips. Perturbation studies separated cytoskeletal requirements for invasion and force transmission: actin polymerization was essential for invasion, myosin II activity was required for robust traction generation and efficient invasion, and microtubule polymerization supported directional persistence and maximal traction output. Notably, a low level of invasion persisted under myosin II inhibition despite minimal detectable traction forces, and this residual invasion was not suppressed by pan-MMP inhibition, indicating a traction-poor, MMP-independent invasion component in Matrigel. ConclusionsThese findings establish a quantitative mechanical framework for GBM neurosphere invasion in 3D Matrigel and define distinct contributions of actomyosin contractility and microtubules to invasive progression.

cancer biology↗

bioRxiv2023

Remote sensing of emperor penguin abundance and breeding success

Emperor penguins (Aptenodytes forsteri) are under increasing environmental pressure. Monitoring colony size and trends of this Antarctic seabird relies primarily on satellite imagery recorded near the end of the breeding season, when illumination levels are sufficient to capture images, but colony occupancy is highly variable. To correct population estimates for this variability, we develop a phenological model that accurately predicts the number of breeding pairs and fledging chicks, as well as key phenological events such as arrival, hatching and foraging times, from as few as six data points from a single season. The ability to extrapolate occupancy from sparse data makes the model particularly useful for monitoring remotely sensed animal colonies where ground-based population estimates are very rare or unavailable. TeaserThe Emperor penguin becomes the Southern Oceans canary in a coal mine through remote sensing its annual breeding success.

ecology↗

bioRxiv2023

Fiber alignment in 3D collagen networks as a biophysical marker for cell contractility

Cells cultured in 3D fibrous biopolymer matrices exert traction forces on their environment that induce deformations and remodeling of the fiber network. By measuring these deformations, the traction forces can be reconstructed if the mechanical properties of the matrix and the force-free matrix configuration are known. These requirements severely limit the applicability of traction force reconstruction in practice. In this study, we test whether force-induced matrix remodeling can instead be used as a proxy for cellular traction forces. We measure the traction forces of hepatic stellate cells and different glioblastoma cell lines and quantify matrix remodeling by measuring the fiber orientation and fiber density around these cells. In agreement with simulated fiber networks, we demonstrate that changes in local fiber orientation and density are directly related to cell forces. By resolving Rho-kinase (ROCK) Inhibitor-induced changes of traction forces and fiber alignment and density in hepatic stellate cells, we show that the method is suitable for drug screening assays. We conclude that differences in local fiber orientation and density, which are easily measurable, can be used as a qualitative proxy for changes in traction forces. The method is available as an open-source Python package with a graphical user interface.

biophysics↗

bioRxiv2023

Immune cells employ traction forces to overcome steric hindrance in 3D biopolymer networks

To reach targets outside the bloodstream, immune cells can extravasate and migrate through connective tissue. During tissue infiltration, immune cells migrate in an amoeboid fashion, characterized by weak matrix adhesions and low traction forces, that allows them to achieve high migration speeds of up to 10 {micro}m/min. How immune cells reconcile amoeboid migration with the need to overcome steric hindrance in dense matrices is currently not understood. Here we show that NK92 (natural killer) cells can switch from their default amoeboid migration mode to a contractile, mesenchymal-like migration mode when moving through fibrous human amniotic membrane (HAM) tissue. We subsequently study immune cell migration in reconstituted 3D collagen networks with known mechanical properties and pore sizes and apply time-lapse confocal reflection microscopy to obtain simultaneous measurements of migration speed, directional persistence, and cell contractility. We find that NK92 cells are highly mechanoresponsive and exert substantial acto-myosin driven, integrin-mediated contractile forces of up to 100 nN on the extracellular matrix during short contractile phases. This burst-like contractile behavior is also found in primary B, T, NK cells, neutrophils, and monocytes, and is tightly related to the fraction of cells that appear to become stuck in narrow pores of the surrounding matrix. Our results demonstrate that steric hindrance guides the rapid regulation of integrin-mediated adhesion to the ECM in a large number of immune cell subtypes.

biophysics↗

bioRxiv2022

Dynamic traction force measurements of migrating immune cells in 3D matrices

Immune cells such as natural killer (NK) cells migrate with high speeds of several {micro}m/min through dense tissue, but the traction forces are unknown. We present a method to measure dynamic traction forces of fast migrating cells in non-linear biopolymer matrices. The method accounts for the mechanical non-linearity of the 3D tissue matrix and can be applied to time series of confocal or bright-field image stacks. The method is highly sensitive over a large range of forces and object sizes, from [~]1 nN for axon growth cones up to [~]10 {micro}N for mouse intestinal organoids. We find that NK cells display bursts of large traction forces that increase with matrix stiffness and facilitate migration through tight constrictions.

biophysics↗

bioRxiv2021

Timelapse viability assay to detect division and death of primary multiple myeloma cells in response to drug treatments with single cell resolution

Heterogeneity among cancer cells and in the tumor microenvironment (TME) is thought to be a significant contributor to the heterogeneity of clinical therapy response observed between patients and can evolve over time. A primary example of this is multiple myeloma (MM), a generally incurable cancer where such heterogeneity contributes to the persistent evolution of drug resistance. However, there is a paucity of functional assays for studying this heterogeneity in patient samples or for assessing the influence of the patient TME on therapy response. Indeed, the population-averaged data provided by traditional drug response assays and the large number of cells required for screening remain significant hurdles to advancement. To address these hurdles, we developed a suite of accessible technologies for quantifying functional drug response to a panel of therapies in ex vivo three-dimensional culture using small quantities of a patients own cancer and TME components. This suite includes tools for label-free single-cell identification and quantification of both cell division and death events with a standard brightfield microscope, an open-source software package for objective image analysis and feasible data management of multi-day timelapse experiments, and a new approach to fluorescent detection of cell death that is compatible with long-term imaging of primary cells. These new tools and capabilities are used to enable sensitive, objective, functional characterization of primary MM cell therapy response in the presence of TME components, laying the foundation for future studies and efforts to enable predictive assessment drug efficacy for individual patients. Insight BoxThe new tools and capabilities described here allow new insights into functional primary cell therapy response by (i) enabling more drugs to be tested on precious and limited numbers of patient cells ex vivo (ii) providing the ability to discriminate both cell division and death events over multiple days with single-cell resolution, and (iii) by incorporating the influences of a patients own cancer cells and TME components on drug response.

bioengineering↗

bioRxiv2020

BrainPhys neuronal medium optimized for imaging and optogenetics in vitro

The capabilities of imaging technologies, fluorescent sensors, and optogenetics tools for cell biology have improved exponentially in the last ten years. At the same time, advances in cellular reprogramming and organoid engineering have quickly expanded the use of human neuronal models in vitro. Altogether this creates an increasing need for tissue culture conditions better adapted to live-cell imaging. Here, we identified multiple caveats of traditional media when used for live imaging and functional assays on neuronal cultures (e.g., phototoxicity, suboptimal fluorescence signals, and unphysiological neuronal activity). To overcome these issues, we developed a new neuromedium, "BrainPhys Imaging", in which we adjusted fluorescent and phototoxic compounds. The new medium is based on the formulation of the original BrainPhys medium, which we designed to better support the neuronal activity of human neurons in vitro 1. We tested the new imaging-optimized formulation on human neurons cultured in monolayers or organoids, and rat primary neurons. BrainPhys Imaging enhanced fluorescence signals and reduced phototoxicity throughout the entire light spectrum. Importantly, consistent with standard BrainPhys, we showed that the new imaging medium optimally supports the electrical and synaptic activity of midbrain and human cortical neurons in culture. We also benchmarked the capacity of the new medium for functional calcium imaging and optogenetic control of human neurons. Altogether, our study shows that the new BrainPhys Imaging improves the quality of a wide range of fluorescence imaging applications with live neurons in vitro while supporting cell viability and neuronal functions.

neuroscience↗

bioRxiv2019

Cryopreservation impairs cytotoxicity and migration of NK cells in 3-D tissue: Implications for cancer immunotherapy

Natural killer (NK) cells are important effector cells in the immune response to cancer. Clinical trials on adoptively transferred NK cells in patients with solid tumors, however, have thus far been unsuccessful. As NK cells need to pass stringent safety evaluation for clinical use, the cells are cryopreserved to bridge the necessary evaluation time. While a degranulation assay confirms the ability of cryopreserved NK cells to kill target cells, we find a significant decrease of cytotoxicity after cryopreservation in a chromium release assay. We complement these standard assays with measurements of NK cell motility and cytotoxicity in 3-dimensional (3-D) collagen gels that serve as a substitute for connective tissue. We find a 5.6 fold decrease of cytotoxicity after cryopreservation and establish that this is mainly caused by a 6-fold decrease in the fraction of motile NK cells. These findings may explain the persistent failure of NK cell therapy in patients with solid tumors and highlight the crucial role of a 3-D environment for testing NK cell function.\n\nSynopsisCryopreservation of natural killer (NK) cells dramatically impairs their motility and cytotoxicity in tissue. This finding may explain the persistent failure of clinical trials in which NK cell therapy is used for treating solid tumors.

immunology↗

bioRxiv2019

Measurement of skeletal muscle fiber contractility with high-speed traction microscopy

We describe a technique for simultaneous quantification of the contractile forces and cytosolic calcium dynamics of muscle fibers embedded in three-dimensional biopolymer gels. We derive a scaling law for linear elastic matrices such as basement membrane extract hydrogels (Matrigel) that allows us to measure contractile force from the shape of the relaxed and contracted muscle cell and the Youngs modulus of the matrix, without further knowledge of the matrix deformations surrounding the cell and without performing computationally intensive inverse force reconstruction algorithms. We apply our method to isolated mouse flexor digitorum brevis (FDB) fibers that are embedded in 10 mg/ml Matrigel. Upon electrical stimulation, individual FDB fibers show twitch forces of 0.37 {micro}N {+/-} 0.15 {micro}N and tetanic forces (100 Hz stimulation frequency) of 2.38 {micro}N {+/-} 0.71 {micro}N, corresponding to a tension of 0.44 kPa {+/-} 0.25 kPa and 2.53 kPa {+/-} 1.17 kPa, respectively. Contractile forces of FDB fibers increase in response to caffeine and the troponin-calcium-stabilizer Tirasemtiv, similar to responses measured in whole muscle. From simultaneous high-speed measurements of cell length changes and cytosolic calcium concentration using confocal line scanning at a frequency of 2048 Hz, we show that twitch and tetanic force responses to electric pulses follow the low-pass filtered calcium signal. In summary, we present a technically simple high speed and high throughput method for measuring contractile forces and cytosolic calcium dynamics of single muscle fibers. We expect that our method will help to reduce preparation time, costs, and the number of sacrificed animals needed for experiments such as drug testing.\n\nStatement of significanceWe describe a high speed, high throughput method for the simultaneous measurement of contractile force and cytoplasmic calcium dynamics following electrical pulse stimulation of muscle fibers embedded in a 3-dimensional biopolymer matrix. In contrast to the classical approach of attaching muscle fibers to a force-transducer, our method allows for a highly efficient, parallel analysis of large numbers of fibers under different treatment conditions.

bioengineering↗

bioRxiv2019

Collective forces of tumor spheroids in three-dimensional biopolymer networks

We describe a method for quantifying the contractile forces that tumor spheroids collectively exert on highly nonlinear three-dimensional collagen networks. While three-dimensional traction force microscopy for single cells in a nonlinear matrix is computationally complex due to the variable cell shape, here we exploit the spherical symmetry of tumor spheroids to derive a scale-invariant relationship between spheroid contractility and the surrounding matrix deformations. This relationship allows us to directly translate the magnitude of matrix deformations to the total contractility of arbitrarily sized spheroids. We show that collective forces of tumor spheroids reflect the contractility of individual cells for up to 1h after seeding, while collective forces on longer time-scales are guided by mechanical feedback from the extracellular matrix.

biophysics↗