Biology subjects

Bastmeyer, M.

Publications and source records attributed to Bastmeyer, M..

7 recordsLinked to original sources

bioRxiv2025

Active and Probe-Free Intracellular Rheology via Phase-Sensitive Thermoviscous Flows

Determination of the rheological properties of cells is known to require active measurements, which largely depend on the internalisation of mechanical probes. Here we circumvent this problem via the introduction of Rheo-FLUCS: an active, yet probe-free approach that leverages light-induced flows to access the mechanics of complex systems. While Rheo-FLUCS is facilitated by thermoviscous expansion phenomena rather than external forces, we here show equivalence in its ability to measure canonical viscoelastic properties. Specifically, we demonstrate a phase-lag equivalence with probe-dependent active microrheology in a wide range of physically different, yet chemically identical materials. We exemplify the utility of Rheo-FLUCS in three distinctly different biological systems: compound-treated mouse fibroblasts (NIH-3T3), genetically modified human osteoblasts (U2OS) to elucidate the role of myosins in cytoplasmic mechanics, and early ascidian oocytes of Phallusia mammillata at fertilisation stage. This probe-free methodology represents a transformative advance in cellular rheology, enabling non-invasive and precise mechanical measurements across diverse biological systems without perturbing their natural state through probe internalisation.

biophysics↗

bioRxiv2023

Modelling Cell Shape in 3D Structured Environments: A Quantitative Comparison with Experiments

Cell shape plays a fundamental role in many biological processes, including migration, division and tissue morphogenesis, but it is not clear which shape model best predicts three-dimensional cell shape in structured environments. Here, we compare different modelling approaches with experimental data. 3D scaffolds for cell adhesion were manufactured using 3D laser nanoprinting and the shapes of single mesenchymal NIH/3T3 cells were compared by a Fourier method with surfaces that minimize area under the given adhesion and volume constraints. For the minimized surface model, we found marked differences to the experimentally observed cell shapes, which necessitated the use of more advanced shape models. We used different variants of the cellular Potts model, which effectively includes both surface and bulk contributions. The simulations revealed that the Hamiltonian with linear area energy outperformed the elastic area constraint in accurately modeling the 3D shapes of cells in structured environments. Explicit modeling the nucleus did not improve the accuracy of the simulated cell shapes. Overall, our work identifies effective methods for accurately modeling cellular shapes in complex environments, which in the future will enable the rational design of scaffolds for desired cell shapes. Author summaryCell shape and forces have emerged as important determinants of cell function and thus their prediction is essential to describe and control the behaviour of cells in complex environments. While there exist well-established models for the two-dimensional shape of cells on flat substrates, it is less clear how cell shape should be modeled in three dimensions. Different from the philosophy of the vertex model often used for epithelial sheets, we find that models based only on cortical tension as a constant geometrical surface tension are not sufficient to describe the shape of single cells in 3D. Therefore, we employ different variants of the cellular Potts model, where either a target area is prescribed by an elastic constraint or the area energy is described with a linear surface tension. By comparing the simulated shapes to experimental images of cells in 3D scaffolds manufactured with direct laser writing, we can identify parameters that accurately model the 3D cell shape.

biophysics↗

bioRxiv2023

S100A11 promotes focal adhesion disassembly via myosin II-driven contractility and Piezo1-mediated Ca2+ entry

S100A11 is a small Ca2+-activatable protein with an established role in different cellular processes involving actin cytoskeleton remodeling, such as cell migration, membrane protrusion formation, and plasma membrane repair. It also displays Ca2+-dependent F-actin binding activity and localizes to actin stress fibers (SFs), but its precise role in regulating these structures remains unclear. Analyzing endogenous S100A11 localization in HeLa and U2OS osteosarcoma cells confirmed SF association but in addition revealed steady localization to stable focal adhesions (FAs), typically at the end of dorsal stress fibers. In contrast, S100A11 levels at FAs increased sharply, but transiently, at the onset of peripheral FA disassembly. Elevating intracellular Ca2+ levels using the Ca2+ ionophore ionomycin reliably stimulated both S100A11 recruitment and subsequent FA disassembly. However, pre-incubation with the non-muscle myosin II (NM II) inhibitor blebbistatin, or with an inhibitor to the stretch-activatable Ca2+ channel Piezo1 effectively suppressed S100A11 recruitment, implicating S100A11 in an actomyosin contractility-driven FA disassembly mechanism involving Piezo1-dependent Ca2+ influx. Applying external mechanical forces on peripheral FAs via a micropipette likewise recruited S100A11 to FAs, even when NM II was inhibited by blebbistatin or in NM IIA knockout cells, corroborating the mechanosensitive recruitment mechanism of S100A11. However, extracellular Ca2+ and Piezo1 function was still indispensable, indicating that NM II-dependent contraction forces act upstream of Piezo1-mediated Ca2+ influx, in turn leading to S100A11 activation and FA recruitment. Moreover, S100A11 knockout cells feature enlarged FAs and display delayed FA disassembly during cell membrane retraction, consistent with impaired FA turnover in these cells. Our results thus demonstrate a novel mechano-sensitive function for S100A11 in promoting actomyosin contractility-driven FA disassembly.

cell biology↗

bioRxiv2021

Nonmuscle myosin IIA dynamically guides regulatory light chain phosphorylation and assembly of nonmuscle myosin IIB

Nonmuscle myosin II minifilaments have emerged as central elements for force generation and mechanosensing by mammalian cells. Each minifilament can have a different composition and activity due to the existence of the three nonmuscle myosin II isoforms A, B and C and their respective phosphorylation pattern. We have used CRISPR/Cas9-based knockout cells, quantitative image analysis and mathematical modelling to dissect the dynamic processes that control the formation and activity of heterotypic minifilaments and found a strong asymmetry between isoforms A and B. Loss of NM IIA completely abrogates regulatory light chain phosphorylation and reduces the level of assembled NM IIB. Activated NM IIB preferentially co-assembles into pre-formed NM IIA minifilaments and stabilizes the filament in a force-dependent mechanism. NM IIC is only weakly coupled to these processes. We conclude that NM IIA and B play clearly defined complementary roles during assembly of functional minifilaments. NM IIA is responsible for the formation of nascent pioneer minifilaments. NM IIB incorporates into these and acts as a clutch that limits the force output to prevent excessive NM IIA activity. Together these two isoforms form a balanced system for regulated force generation.

cell biology↗

bioRxiv2020

Distinct roles of nonmuscle myosin II isoforms for establishing tension and elasticity during cell morphogenesis

Nonmuscle myosin II (NM II) is an integral part of essential cellular processes, including adhesion and migration. Mammalian cells express up to three isoforms termed NM IIA, B, and C. We used U2OS cells to create CRISPR/Cas9-based knockouts of all three isoforms and analyzed the phenotypes on homogeneous substrates, in collagen gels, and on micropatterned substrates. We find that NM IIA is essential to build up cellular tension during initial stages of force generation, while NM IIB is necessary to elastically stabilize NM IIA-generated tension. A scale-bridging mathematical model explains our observations by relating actin fiber stability to the molecular rates of the myosin crossbridge cycle. A dynamic cell stretch/release experiment confirms these predictions and in addition reveals a novel role for NM IIC, namely the ability to establish tensional homeostasis.

biophysics↗

bioRxiv2020

Phosphorylated paxillin and FAK constitute subregions within focal adhesions

Integrin-mediated adhesions are convergence points of multiple signaling pathways. Their inner structure and their diverse functions can be studied with super-resolution microscopy. We used structured illumination microscopy (SIM) to analyze spatial organization of paxillin phosphorylation (pPax) within adhesions. We found that pPax and focal adhesion kinase (FAK) form spot-like, spatially defined clusters within adhesions in several cell lines. In contrast, other adhesion proteins showed no consistent organization in such clusters. Live-cell super-resolution imaging revealed that pPax-FAK clusters persist over time but modify distance to each other dynamically. Moreover, we show that the distance between separate clusters of pPax is mechanosensitive. Thus, in this work we introduce a new structural organization within focal adhesions and demonstrate its regulation and dynamics.

cell biology↗

bioRxiv2017

Force-dependent ligand sensing enables αVβ3 integrin to differentiate between fibronectin and high-affinity ligands

V{beta}3 integrin recognizes multiple extracellular matrix proteins, including vitronectin (Vn) and fibronectin (Fn). However, cell experiments are frequently performed on homogenously coated substrates with only one integrin ligand present. Here, we employed binary-choice substrates of Fn and Vn to dissect V{beta}3 integrin-mediated binding to both ligands on the subcellular scale. Superresolution imaging revealed that V{beta}3 integrin preferred binding to Vn under various conditions. In contrast, binding to Fn required mechanical load on V{beta}3 integrin. Integrin mutations, structural analysis, and molecular dynamics simulations established a model where the extended-closed conformation of V{beta}3 integrin binds Vn but not Fn. Force-mediated hybrid domain swing-out characterizes the extended-open conformation needed for efficient Fn binding. Thus, force-dependent conformational changes in V{beta}3 integrin increase the number of available ligands and therefore the ligand promiscuity of this integrin. These findings for V{beta}3 integrin were shown to regulate cell migration and mechanotransduction differentially on Fn compared to Vn and therefore to regulate cell behavior.

cell biology↗