Biology subjects

Herrmann, H.

Publications and source records attributed to Herrmann, H..

9 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↗

bioRxiv2026

Desmin Mutations Disrupt Filament Elongation and Drive Polymorphic Aggregation

In muscle cells, desmin intermediate filaments form a cytoskeletal network that maintains the structural integrity and mechanical coupling of myofibrils. Dominant missense mutations in desmin cause myopathies characterized by intracellular protein aggregation. To explore the process of aggregate formation, we investigate the assembly kinetics of wild-type desmin and four disease-associated variants (N342D, L345P, R350P, and R406W) using dual-wavelength stopped-flow spectroscopy, complemented by atomic force microscopy and molecular dynamics simulations. At low ionic strength, all proteins form uniform tetramers. Increasing the ionic strength initiates assembly byrapid lateral association of tetramers into unit-length filaments (ULFs), followed by longitudinal elongation and radial filament compaction. R406W forms ULFs with wild-type-like kinetics, whereas N342D, L345P, and R350P exhibit delayed lateral assembly. After short filaments have formed, all four mutants diverge from productive filament maturation, but through distinct pathways. Quantitative analysis of atomic force microscopy images together with kinetic modelling of the spectroscopic data shows that wild-type filaments elongate continuously, whereas R406W filaments progressively associate into fibrillar clusters and cease elongating. By contrast, N342D, L345P, and R350P rapidly collapse into globular complexes that subsequently coalesce into larger aggregates. Molecular dynamics simulations indicate that the mutations differentially destabilize coil 2, which leads to local structural perturbations and mutation-specific assembly defects. Together, these findings identify early filament maturation - when elongating ULF-derived filaments would normally undergo radial compaction to form stable, mature filaments - as a critical time point in desmin assembly. At this stage, pathogenic mutations redirect the internal reorganization of the filament from productive stabilization toward mutation-specific structural collapse and aggregation. SignificanceDominant mutations in the intermediate filament protein desmin cause myofibrillar myopathies characterized by protein aggregation and progressive muscle degeneration. Using in vitro filament-assembly experiments, we identify intrinsic assembly defects of disease-associated desmin mutants. Mutant proteins initially enter the normal assembly pathway, albeit with delayed kinetics. They subsequently fail at an early maturation step in which short filaments would normally reorganize to sustain productive elongation. Instead, mutant proteins form morphologically distinct aggregates. Similar structural abnormalities occur when mutant proteins co-assemble with wild-type desmin. Our findings identify the radial compaction phase during early longitudinal assembly as a critical checkpoint that determines whether desmin assembly yields functional intermediate filaments or is redirected into pathogenic aggregation pathways.

biophysics↗

bioRxiv2026

S-Adenosyl-D-methionine as a Non-Physiological Substrate for a Wide Range of SAM-Dependent Enzymes

The ability of SAM-dependent enzymes to accept S-adenosyl-D-methionine [D-SAM, (SS,RC)-SAM] instead of the native cofactor S-adenosyl-L-methionine [L-SAM, (SS,SC)-SAM] remains largely unexplored. Challenging the stereochemical preference of SAM-dependent enzymes, we investigated the ability of different enzyme classes to accept D-SAM. Contrary to common assumptions, the tested N- and O-methyl transferases (MTs), as well as one of the examined C-MTs accepted D-SAM. Docking studies suggest that acceptance of D-SAM by C-MTs may be influenced by the angle between the transferable methyl group of SAM and the nucleophilic carbon of the substrate, along with enzyme and substrate flexibility. In addition to conventional MTs, the radical SAM glutamine C-MT QCMT showed low but detectable methylation activity with D-SAM. Furthermore, the azetidine-2-carboxylic acid synthase AzeJ not only uses D-SAM but also incorporates the stereocentre of D-methionine into the cyclic amino acid product. The pyridoxal 5'-phosphate (PLP)-dependent enzyme 1-aminocyclopropyl-1-carboxylic acid synthase (ACCS) also showed detectable turnover with D-SAM. These findings broaden the understanding of enzyme stereoselectivity, provide an overview of D-SAM-utilising enzymes, and identify first enzyme systems that may serve as starting points for engineering efforts aimed at shifting cofactor preference towards D-SAM.

biochemistry↗

bioRxiv2022

Dual-wavelength stopped-flow analysis of the lateral and longitudinal assembly kinetics of vimentin

Vimentin is a highly charged intermediate filament protein that inherently forms extended dimeric coiled-coils, which serve as the basic building blocks of intermediate filaments. Under low ionic strength conditions, vimentin filaments dissociate into uniform tetrameric complexes of two anti-parallel oriented, half-staggered coiled-coil dimers. By addition of salt, vimentin tetramers spontaneously reassemble into filaments in a time-dependent process: i) lateral assembly of tetramers into unit-length filaments (ULFs); ii) longitudinal annealing of ULFs; iii) longitudinal assembly of filaments coupled with subsequent radial compaction. To independently determine the lateral and longitudinal assembly kinetics, we measure with a stopped-flow instrument the static light scattering signal at two different wavelengths (405 and 594 nm) with a temporal resolution of 3 ms, and analyze the signals based on Rayleigh-Gans theory. This theory considers that the intensity of the scattered light depends not only on the molecular weight of the scattering object but also on its shape. This shape-dependence is more pronounced at shorter wavelengths, allowing us to decompose the scattered light signal into its components arising from lateral and longitudinal filament assembly. We demonstrate that both the lateral and longitudinal filament assembly kinetics increase with salt concentration. Significance statementThe proper formation of intermediate filament (IF) networks in the cytoplasm is important for numerous cell functions. Here, we present a stopped-flow method for measuring the in-vitro assembly kinetics of intermediate filaments with a temporal resolution of 3 ms using static light scattering at two different wavelengths. This allows us to compute the shape factor of the assembly products based on Rayleigh-Gans light scattering theory. From the shape factor, we can separately measure the lateral assembly of tetramers into unit-length filaments (ULFs), and the longitudinal annealing of ULFs and longer filaments. For the IF protein vimentin, we find that with increasing salt concentrations, both the lateral and longitudinal assembly rates increase, and unstable, hyper-aggregated assembly complexes emerge.

biophysics↗

bioRxiv2022

Viscoelastic properties of suspended cells measured with shear flow deformation cytometry

Numerous cell functions are accompanied by phenotypic changes in viscoelastic properties, and measuring them can help elucidate higher-level cellular functions in health and disease. We present a high-throughput, simple and low-cost microfluidic method for quantitatively measuring the elastic (storage) and viscous (loss) modulus of individual cells. Cells are suspended in a high-viscosity fluid and are pumped with high pressure through a 5.8 cm long and 200 {micro}m wide microfluidic channel. The fluid shear stress induces large, near ellipsoidal cell deformations. In addition, the flow profile in the channel causes the cells to rotate in a tank-treading manner. From the cell deformation and tank treading frequency, we extract the frequency-dependent viscoelastic cell properties based on a theoretical framework developed by R. Roscoe1 that describes the deformation of a viscoelastic sphere in a viscous fluid under steady laminar flow. We confirm the accuracy of the method using atomic force microscopy-calibrated polyacrylamide beads and cells. Our measurements demonstrate that suspended cells exhibit power-law, soft glassy rheological behavior that is cell cycle-dependent and mediated by the physical interplay between the actin filament and intermediate filament networks.

biophysics↗

bioRxiv2021

The desmin mutation R349P increases contractility and fragility of stem cell-generated muscle micro-tissues

Desminopathies comprise hereditary myopathies and cardiomyopathies caused by mutations in the intermediate filament protein desmin that lead to severe and often lethal degeneration of striated muscle tissue. Animal and single cell studies hinted that this degeneration process is associated with massive ultrastructural defects correlating with increased susceptibility of the muscle to acute mechanical stress. The underlying mechanism of mechanical susceptibility, and how muscle degeneration develops over time, however, has remained elusive. Here, we investigated the effect of a desmin mutation on the formation, differentiation, and contractile function of in vitro-engineered three-dimensional micro-tissues grown from muscle stem cells (satellite cells) isolated from heterozygous R349P desmin knock-in mice. Micro-tissues grown from desmin-mutated cells exhibited spontaneous unsynchronized contractions, higher contractile forces in response to electrical stimulation, and faster force recovery compared to tissues grown from wild-type cells. Within one week of culture, the majority of R349P desmin-mutated tissues disintegrated, whereas wild-type tissues remained intact over at least three weeks. Moreover, under tetanic stimulation lasting less than five seconds, desmin-mutated tissues partially or completely ruptured, whereas wild-type tissues did not display signs of damage. Our results demonstrate that the progressive degeneration of desmin-mutated micro-tissues is closely linked to extracellular matrix fiber breakage associated with increased contractile forces and unevenly distributed tensile stress. This suggests that the age-related degeneration of skeletal and cardiac muscle in patients suffering from desminopathies may be similarly exacerbated by mechanical damage from high-intensity muscle contractions. We conclude that micro-tissues may provide a valuable tool for studying the organization of myocytes and the pathogenic mechanisms of myopathies.

biophysics↗

bioRxiv2020

Keratins determine network stress responsiveness in reconstituted actin-keratin filament systems

The cytoskeleton is a major determinant of cell mechanics, a property that is altered during many pathological situations. To understand these alterations, it is essential to investigate the interplay between the main filament systems of the cytoskeleton in the form of composite networks. Here, we investigate the role of keratin intermediate filaments (IFs) in network strength by studying in vitro reconstituted actin and keratin 8/18 composite networks via bulk shear rheology. We co-polymerized these structural proteins in varying ratios and recorded how their relative content affects the overall mechanical response of the various composites. For relatively small deformations, we found that all composites exhibited an intermediate linear viscoelastic behavior compared to that of the pure networks. In stark contrast, the composites displayed increasing strain stiffening behavior as a result of increased keratin content when larger deformations were imposed. This strain stiffening behavior is fundamentally different from behavior encountered with vimentin IF as a composite network partner for actin. Our results provide new insights into the mechanical interplay between actin and keratin in which keratin provides reinforcement to actin. This interplay may contribute to the overall integrity of cells, providing an explanation for the stability of stressed epithelial tissues due to their high keratin contents. Additionally, this helps us to understand the physiological necessity to exchange IF systems during epithelial-mesenchymal transition (EMT) in order to suppress strain stiffening of the network, making cells more elastic and, thus, facilitating their migration through dense tissues.

biophysics↗

bioRxiv2019

Effect of the divalent cations zinc and calcium on the structure and mechanics of reconstituted vimentin intermediate filaments

Divalent cations in a concentration-dependent manner behave as effective crosslinkers of intermediate filaments (IFs) such as vimentin IF (VIF). These interactions have been mostly attributed to their multivalency. However, ion-protein interactions often depend on the ion species, and these effects have not been widely studied in IFs. Here we investigate the effects of two biologically important divalent cations, Zn2+ and Ca2+, on VIF network structure and mechanics in vitro. We find that the network structure is unperturbed at micromolar Zn2+ concentrations, but strong bundle formation is observed at a concentration of 100 M. Microrheological measurements show that network stiffness increases with cation concentration. However, bundling of filaments softens the network. This trend also holds for VIF networks formed in the presence of Ca2+, but remarkably, a concentration of Ca2+ that is two orders higher is needed to achieve the same effect as with Zn2+, which suggests the importance of salt-protein interactions as described by the Hofmeister effect. Furthermore, we find evidence of competitive binding between the two divalent ion species. Hence, specific interactions between VIFs and divalent cations are likely to be an important mechanism by which cells can control their cytoplasmic mechanics. SignificanceIntermediate filaments are key structural elements within cells; they are known to form networks that can be crosslinked by divalent cations, but the interactions between the ions and the filaments are not well understood. By measuring the effects that two divalent cations, zinc and calcium, have on the structure and mechanics of vimentin intermediate filaments (VIFs), we show that although both have concentration-dependent effects on VIFs, much more calcium is needed to achieve the same effect as a small amount of zinc. Furthermore, when mixtures of the ions are present, the results suggest that there is binding competition. Thus, cells may use the presence of different cation species to precisely control their internal mechanical properties.

biophysics↗

bioRxiv2017

Modeling The Consequences Of Heterogeneity In Microbial Population Dynamics

Chapter 1The rate at which unicellular micro-organisms progress through the cell cycle is a major component of their evolutionary fitness. Measuring fitness phenotypes in a given environment or genetic background forms the basis of most quantitative assays of drug sensitivity or genetic interaction, including genome-wide assays. Growth rate is typically measured in bulk cell populations, inoculated with anything from hundreds to millions of cells sampled from purified, isogenic colonies. High-throughput microscopy reveals that striking levels of growth rate heterogeneity arise between isogenic cell lineages (Levy et al., 2012). Using published Saccharomyces cerevisiae data, I examine the implications for interpreting bulk, population scale growth rate observations, given observed levels of growth rate heterogeneity at the lineage level. I demonstrate that selection between cell lineages with a range of growth rates can give rise to an apparent lag phase at the population level, even in the absence of evidence for a lag phase at the lineage level. My simulations further predict that, given observed levels of heterogeneity, final populations should be dominated by one or a few lineages.\n\nChapter 2In order to validate and further explore the conclusions from Chapter 1, I re-analyzed high-throughput microscopy experiments carried out on Quantitative Fitness Analysis (QFA) S. cerevisiae cultures (Addinall et al., 2011), an approach referred to as QFA. To allow for precise observation of purely clonal lineages including very fast-growing lineages and non-dividing cells, I re-designed an existing image analysis tool for QFA, now available as an open source Python package. Fast-growing outliers in particular influence the extent of the lag phase apparent at the population level, making the precision of growth rate estimation a key ingredient for successfully simulating population observations. QFA data include population observations which I used to validate the population simulations generated from individual lineage data. I explored various options for modeling lineage growth curves and for carrying out growth rate parameter inference, and included the full workflow in an open source R package.\n\nContacthelena.herrmann@postgrad.manchester.ac.uk

microbiology↗