Biology subjects

Herrmann, D.

Publications and source records attributed to Herrmann, D..

9 recordsLinked to original sources

bioRxiv2025

ROCK2 inhibition has a dual role in reducing ECM remodelling and cell growth, while impairing migration and invasion

The Rho-associated coiled-coil containing protein kinases 1/2 (ROCK1/2) are key signalling proteins involved in the regulation of the actin cytoskeleton and control a variety of cellular processes. This includes cell proliferation, stemness, cell migration and invasion as well as actomyosin contraction and extracellular matrix (ECM) remodelling. Due to a lack of ROCK2 specific inhibitors, previous pharmacological studies of ROCK function have relied on pan-ROCK1/2 inhibition. Here, we use the ROCK2 specific inhibitor, GV101, in the context of breast cancer (BC) models to uncouple the effects of ROCK2 pharmacological inhibition on both epithelial and stromal fibroblast cell populations. We demonstrate that ROCK2 inhibition with GV101 reduces fibroblast-mediated remodelling of pre-existing and de novo synthesised ECM resulting in a reduction in biomechanical stiffness, while also limiting epithelial cell growth in 2D and 3D settings. We also demonstrate that ROCK2 inhibition exposes epithelial cell vulnerability to fluid flow-induced shear stress. Furthermore, using 3D co-cultures we reveal that GV101 reduces single-cell migratory capacity, while the disruption of stromal ECM architecture is sufficient to impede collective cell migration and invasion. Finally, we assessed ROCK2 expression in human BC patient tumours revealing that ROCK2 expression is up-regulated during BC progression and that high ROCK2 expression correlates with poor patient outcome in the triple-negative subtype of BC. Together, these results demonstrate that ROCK2 specific inhibition disrupts key functions in both epithelial cell and stromal fibroblast compartments warranting further assessment of ROCK2 in highly proliferative, fibrotic or metastatic diseases, such as BC. HighlightsO_LIFibroblast-mediated remodelling of pre-existing and de novo synthesised extracellular matrix is reduced upon ROCK2 specific targeting. C_LIO_LIROCK2 inhibition impairs epithelial cancer cell growth and colony formation in 2D and 3D contexts, while also limiting single-cell migration and cell survival upon fluid flow-induced shear stress. C_LIO_LIROCK2 targeting during fibroblast-mediated ECM remodelling is sufficient to disrupt collective cell migration as well as 3D invasion into organotypic fibroblast-contracted collagen matrices. C_LIO_LIROCK2 expression is up-regulated during breast cancer progression and high expression correlates with poor patient outcome in triple-negative breast cancer. C_LI

cell biology↗

bioRxiv2025

Using rotational integration of oblique interferometric scattering (RO-iSCAT) to track axial spatiotemporal responses of membrane protrusions

Despite the crucial importance of dynamic membrane protrusions for understanding phagocytosis, cellular communication and mechanobiology, current imaging modalities struggle to quantitatively track their real-time, 3D spatiotemporal dynamics with sufficient molecular specificity and minimal perturbation. Many membrane protrusions studies still utilize confocal microscopy where its axial resolution and high phototoxicity remains a key limiting factor for live axial imaging. We discovered that multiple rotational oblique interference scattering (RO-iSCAT) leverages off-axis illumination to induce a larger lateral shift in out-of-focus iSCAT signals compared to in-focus signals. This phenomenon provides a foundation to generate speckle-free widefield interferometric signals with a 10-fold signal to noise ratio improvement, eliminating the need for any background subtraction. RO-iSCAT enables real-time, label-free, and minimally invasive imaging of diverse membrane protrusions within complex co-cultures. RO-iSCAT enables nanoscale-sensitive tracking of membrane protrusion dynamics along the axial direction. This allows for the construction of dynamic axial variance maps, facilitating quantitative measurements of membrane protrusion formation at tens to hundreds of nanometer displacements, without requiring 3D volumetric imaging. RO-iSCAT empowers real time quantitatively dissection of the axial spatiotemporal complexities of membrane protrusions and unlock future insights into fundamental processes like cell migration, durotaxis, and intercellular communication. Key pointsO_LIDiscovered that multiple integrated rotational oblique interference scattering (RO-iSCAT) generates speckle-free widefield interferometric signals with a 10-fold signal to noise ratio improvement, eliminating the need for any background subtraction. C_LIO_LIRemoved need for 3D volumetric imaging to quantified axial motion of membrane protrusion forming tethers, trails and bridge with within [~] tens of nanometer accuracy. C_LIO_LIEnabled classification of membrane protrusions that, despite possessing identical chemical compositions, are differentiated by their interactions, thus offering a qualitative comparison of membrane protrusions at the nanoscale in living cells. C_LI Summary Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/644841v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@b183a4org.highwire.dtl.DTLVardef@39a944org.highwire.dtl.DTLVardef@197f019org.highwire.dtl.DTLVardef@1438b3_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

Harnessing genome prediction in Brassica napus through a nested association mapping population

Genome prediction (GP) significantly enhances genetic gain by improving selection efficiency and shortening crop breeding cycles. Using a nested association mapping (NAM) population a set of diverse scenarios were assessed to evaluate GP for vital agronomic traits in B. napus. GP accuracy was examined by employing different models, marker sets, population sizes, marker densities, and incorporating genome-wide association (GWAS) markers. Eight models, including linear and semi-parametric approaches, were tested. The choice of model minimally impacted GP accuracy across traits. Notably, two models, rrBLUP and RKHS, consistently yielded the highest prediction accuracies. Employing a training population of 1500 lines or more resulted in increased prediction accuracies. Inclusion of single nucleotide absence polymorphism (SNaP) markers significantly improved prediction accuracy, with gains of up to 15%. Utilizing the Brassica 60K Illumina SNP array, our study effectively revealed the genetic potential of the B. napus NAM panel. It provided estimates of genomic predictions for crucial agronomic traits through varied prediction scenarios, shedding light on achievable genetic gains. These insights, coupled with marker application, can advance the breeding cycle acceleration in B. napus. Core ideasO_LIGenome prediction (GP) enhances genetic gains by improving selection efficiency and shortening breeding cycles. C_LIO_LIFactors influencing GP accuracy include model choice, marker types, and population size. C_LIO_LIInclusion of SNaP markers and highly significant GWAS markers improves prediction accuracy, shedding light on achievable genetic gains. C_LI Plain SummaryGenome prediction (GP) is a powerful tool that helps us improve crops more efficiently. In this study, we assessed how well GP works for predicting important traits in Brassica napus plants. We tested different models and marker sets to see which ones were most accurate. We found that two models, rrBLUP and RKHS, were consistently the best. Also, including certain types of genetic markers, like SNaP markers and highly significant GWAS markers, improved the predictions. Overall, our study shows that GP can help us understand the genetic potential of B. napus plants and improve breeding strategies, which can be exploited to develop better varieties more quickly, which is good news for farmers and the food supply.

genomics↗

bioRxiv2023

Fibroblast Growth Factor-induced lens fiber cell elongation is driven by the stepwise activity of Rho and Rac

The spheroidal shape of the eye lens is critical for precise light focusing onto the retina. This shape is determined by concentrically aligned, convexly elongated lens fiber cells along the anterior and posterior axis of the lens. Upon differentiation at the lens equator, the fiber cells increase in height as their apical and basal tips migrate towards the anterior and posterior poles, respectively. The forces driving this elongation and migration remain unclear. We found that membrane protrusions or lamellipodia are observed only in the maturing fibers undergoing cell curve conversion, indicating lamellipodium is not the primary driver of earlier fiber migration. We demonstrated that elevated levels of fibroblast growth factor (FGF) suppressed the extension of Rac-dependent protrusions, suggesting changes in the activity of FGF controling Rac activity, switching to lamellipodium-driven migration. Inhibitors of ROCK, myosin, and actin reduced the height of both early and later fibers, indicating elongation of these fibers relies on actomyosin contractility. Consistently, active RhoA was detected throughout these fibers. Given that FGF promotes fiber elongation, we propose it to do so through regulation of Rho activity. Summary statementFGF differentially regulates Rho- and Rac-activity at the lens equator to promote lens fiber cell elongation and migration.

developmental biology↗

bioRxiv2021

Control of subunit stoichiometry in single-chain MspA nanopores

Transmembrane protein channels enable fast and highly sensitive electrical detection of single molecules. Nanopore sequencing of DNA was achieved using an engineered Mycobacterium smegmatis porin A (MspA) in combination with a motor enzyme. Due to its favorable channel geometry, the octameric MspA pore exhibits the highest current level as compared to other pore proteins. To date, MspA is the only protein nanopore with a published record of DNA sequencing. While widely used in commercial devices, nanopore sequencing of DNA suffers from significant base-calling errors due to stochastic events of the complex DNA-motor-pore combination and the contribution of up to five nucleotides to the signal at each position. Asymmetric mutations within subunits of the channel protein offer an enormous potential to improve nucleotide resolution and sequencing accuracy. However, random subunit assembly does not allow control of the channel composition of MspA and other oligomeric protein pores. In this study, we showed that it is feasible to convert octameric MspA into a single-chain pore by connecting eight subunits using peptide linkers. We constructed single-chain MspA trimers, pentamers, hexamers and heptamers to demonstrate that it is feasible to alter the subunit stoichiometry and the MspA pore diameter. All single-chain MspA proteins formed functional channels in lipid bilayer experiments. Importantly, we demonstrated that single-chain MspA discriminated all four nucleotides identical to MspA produced from monomers. Thus, single-chain MspA constitutes a new milestone in its development and adaptation as a biosensor for DNA sequencing and many other applications. STATEMENT OF SIGNFICANCENanopore sequencing of DNA is a fast and cheap technology that uniquely delivers multi-kilobase reads. It is currently used world-wide in many applications such as genome sequencing, epigenetics, and surveillance of viral and bacterial pathogens and has started to revolutionize human lives in medicine, agriculture and environmental studies. However, the high base-calling error rates prevent nanopore DNA sequencing from reaching its full potential. In this study, we converted octameric MspA into a single-chain pore enabling asymmetric mutations to fine-tune the pore geometry and chemistry and address the shortcomings of nanopores. Thus, single-chain MspA constitutes a new milestone in its development and adaptation as a biosensor for DNA sequencing and many other applications.

biophysics↗

bioRxiv2021

All-optical interrogation of neural circuits in behaving mice

Recent advances combining two-photon calcium imaging and two-photon optogenetics with digital holography now allow us to read and write neural activity in vivo at cellular resolution with millisecond temporal precision. Such "all-optical" techniques enable experimenters to probe the impact of functionally defined neurons on neural circuit function and behavioural output with new levels of precision. This protocol describes the experimental strategy and workflow for successful completion of typical all-optical interrogation experiments in awake, behaving head-fixed mice. We describe modular procedures for the setup and calibration of an all-optical system, the preparation of an indicator and opsin-expressing and task-performing animal, the characterization of functional and photostimulation responses and the design and implementation of an all-optical experiment. We discuss optimizations for efficiently selecting and targeting neuronal ensembles for photostimulation sequences, as well as generating photostimulation response maps from the imaging data that can be used to examine the impact of photostimulation on the local circuit. We demonstrate the utility of this strategy using all-optical experiments in three different brain areas - barrel cortex, visual cortex and hippocampus - using different experimental setups. This approach can in principle be adapted to any brain area for all-optical interrogation experiments to probe functional connectivity in neural circuits and for investigating the relationship between neural circuit activity and behaviour.

neuroscience↗

bioRxiv2021

Structural basis for human T-cell leukemia virus type 1 Gag targeting to the plasma membrane for assembly

During the late phase of retroviral replication, the virally encoded Gag polyprotein is targeted to the plasma membrane (PM) for assembly. Gag-PM binding is mediated by the N-terminal matrix (MA) domain of Gag. For many retroviruses, Gag binding to the PM was found to be dependent on phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. However, it was shown that for human T-cell leukemia virus type 1 (HTLV-1), Gag binding to membranes is less dependent on PI(4,5)P2, suggesting that other factors may modulate Gag assembly. To elucidate the mechanism by which HTLV-1 Gag binds to the PM, we employed NMR techniques to solve the structure of unmyristoylated MA (myr(-)MA) and to characterize its interactions with lipids and liposomes. The MA structure consists of four -helices and unstructured N- and C-termini. We show that myr(-)MA binds to PI(4,5)P2 via the polar head and that myr(-)MA binding to inositol phosphates (IPs) is significantly enhanced by increasing the number of phosphate groups on the inositol ring, indicating that the MA-IP binding is governed by charge-charge interactions. The IP binding site was mapped to a well-defined basic patch formed by lysine and arginine residues. Using a sensitive NMR-based liposome binding assay, we show that myr(-)MA binding to membranes is significantly enhanced by phosphatidylserine (PS). Confocal microscopy data show that Gag is localized to the inner leaflet of the PM of infected cells, while the Gag G2A mutant, lacking myristoylation, is diffuse and cytoplasmic. These findings advance our understanding of a key mechanism in retroviral assembly.

microbiology↗

bioRxiv2018

Molecular Classification and Comparative Taxonomics of Foveal and Peripheral Cells in Primate Retina

High acuity vision in primates, including humans, is mediated by a small central retinal region called the fovea. As more accessible model organisms lack a fovea, its specialized function and dysfunction in ocular diseases remain poorly understood. We used 165,000 single-cell RNA-seq profiles to generate and validate comprehensive cellular taxonomies of macaque fovea and peripheral retina. More than 80% of >65 cell types match between the two regions, but exhibit substantial differences in proportions and gene expression, some of which we relate to functional differences. Comparison of macaque retinal types with those of mice reveals that interneuron types are tightly conserved, but that projection neuron types and programs diverge, despite conserved transcription factor codes. Key macaque types are conserved in humans, allowing mapping of cell-type and region-specific expression of >190 genes associated with 6 human retinal diseases. Our work provides a framework for comparative single-cell analysis across tissue regions and species.

neuroscience↗

bioRxiv2017

Targeting stromal remodeling and cancer stem cell plasticity to overcome chemoresistance in triple negative breast cancer

The cellular and molecular basis of stromal cell recruitment, activation and crosstalk in carcinomas is poorly understood, limiting the development of targeted anti-stromal therapies. In mouse models of triple negative breast cancer (TNBC), Hh ligand produced by neoplastic cells reprogrammed cancer-associated fibroblast (CAF) gene expression, driving tumor growth and metastasis. Hh-activated CAFs upregulated expression of FGF5 and production of fibrillar collagen, leading to FGFR and FAK activation in adjacent neoplastic cells, which then acquired a stem-like, drug-resistant phenotype. Treatment with smoothened inhibitors (SMOi) reversed these phenotypes. Stromal treatment of TNBC patient-derived xenograft (PDX) models with SMOi downregulated the expression of cancer stem cell markers and sensitized tumors to docetaxel, leading to markedly improved survival and reduced metastatic burden. In the phase I clinical trial EDALINE, 3 of 12 patients with metastatic TNBC derived clinical benefit from combination therapy with the SMOi Sonidegib and docetaxel chemotherapy, with one patient experiencing a complete response. Markers of pathway activity correlated with response. These studies identify Hh signaling to CAFs as a novel mediator of cancer stem cell plasticity and an exciting new therapeutic target in TNBC.\n\nSIGNIFICANCECompared to other breast cancer subtypes, TNBCs are associated with significantly worse patient outcomes. Standard of care systemic treatment for patients with non-BRCA1/2 positive TNBC is cytotoxic chemotherapy. However, the failure of 70% of treated TNBCs to attain complete pathological response reflects the relative chemoresistance of these tumors. New therapeutic strategies are needed to improve patient survival and quality of life. Here, we provide new insights into the dynamic interactions between heterotypic cells within a tumor. Specifically, we establish the mechanisms by which CAFs define cancer cell phenotype and demonstrate that the bidirectional CAF-cancer cell crosstalk can be successfully targeted in mice and humans using anti-stromal therapy.

cancer biology↗