Biology subjects

Grosse, R.

Publications and source records attributed to Grosse, R..

12 recordsLinked to original sources

bioRxiv2025

Regulation of YAP activity by nuclear G-actin binding

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/667612v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1d109c2org.highwire.dtl.DTLVardef@d7d6daorg.highwire.dtl.DTLVardef@5ab7fcorg.highwire.dtl.DTLVardef@1e51b37_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO YAP functions as a G-actin binding protein. Three high affinity actin binding sites, L65, L68 and W199 are identified in YAP that when mutated into YAPDDY attenuate YAP/actin interaction as well as YAP activity. Thus, nuclear actin binding to YAP is necessary for its transcriptional activity as a co-activator of TEAD. Incorporation of G-actin into the YAP/TEAD complex is predicted to induce TEAD conformational change involving a 4 interface. Therefore, the formation of a dynamic TEAD/YAP/actin ternary complex necessary for transcription is proposed. C_FIG The Yes-associated protein YAP belongs to the TEAD (TEA/ATTS domain) transcriptional co-activators that shuttle between cytoplasm and nuclear compartment. YAP and its paralog TAZ (transcriptional co-activator with PDZ-binding motif) play essential roles in the Hippo pathway to control tissue and organ size. In addition, YAP is critically involved in numerous cellular processes such as differentiation, proliferation, cell migration and cancer metastasis as well as mechanotransduction and cytoskeletal dynamics. The actin cytoskeleton controls YAP activity in multiple ways via tensile forces, cell density and cell-cell adhesion as well as shear stress or other biomechanical cues. Here we discover YAP as a novel G-actin binding protein. We identify three high affinity YAP actin binding sites involving critical residues within the N-terminus of YAP. Moreover, actin binding to YAP is necessary for its transcriptional activity and function such as during cell density control. Mutation of the three actin binding residues results in a loss of nuclear YAP co-activator function towards TEAD while actin binding to YAP is required for TEAD target gene regulation. Our data point towards the formation of a dynamic TEAD/YAP/actin ternary complex necessary for transcription.

cell biology↗

bioRxiv2024

Microcystin shapes the Microcystis phycosphere through community filtering and by influencing cross-feeding interactions

The cyanobacterium Microcystis causes harmful algal blooms (cyanoHABs) that pose a major threat to human health and ecosystem services, particularly due to the prevalence of the potent hepatotoxin microcystin. With their pronounced EPS layer, Microcystis colonies also serve as a hub for heterotrophic phycosphere bacteria. Here, we tested the hypothesis that the genotypic plasticity in its ability to produce microcystin influences the composition and assembly of the Microcystis phycosphere microbiome. In an analysis of individual colonies of a natural Microcystis bloom, we observed a significantly reduced richness of the community in the presence of microcystin biosynthesis genes. A subsequent synthetic community experiment with 21 heterotrophic strains in co-cultivation with either the wild-type strain M. aeruginosa PCC 7806 or the microcystin-free mutant {Delta}mcyB revealed not only a tug-of-war between phototrophic and heterotrophic bacteria, but also a reciprocal dominance of two isolates of the genus Sphingomonas and Flavobacterium. In contrast, an Agrobacterium isolate thrived equally well in both consortia. In substrate utilization tests, Sphingomonas showed the strongest dependence on Microcystis exudates with a clear preference for the wild-type strain. Genome sequencing revealed a high potential for complementary cross-feeding, particularly for the Agrobacterium and Sphingomonas isolates but no potential for microcystin degradation. We postulate that strain-specific functional traits, such as the ability to perform photorespiration and to produce vitamin B12, play a crucial role in the cross-feeding interactions, and that microcystin is one of the determining factors in the Microcystis phycosphere due to its interference with inorganic carbon metabolism.

microbiology↗

bioRxiv2024

Thrombospondin-1 inhibits alternative complement pathway activation in vasculitis synergistically to factor H

Complement activation is a relevant driver in the pathomechanisms of vasculitis. The involved proteins in the interaction between endothelia, complement and platelets in these conditions are only partially understood. Thrombospondin-1 (TSP-1), found in platelet -granules and released from activated endothelial cells, interacts with factor H (FH) and von Willebrand factor (vWF). However, direct regulatory interaction with the complement cascade has not yet been described. We could show that TSP-1 is a potent, FH-independent inhibitor of the alternative complement pathway. TSP-1 binds to complement proteins, inhibits cleavage of C3 and C5 and the formation of the membrane attack complex. Complement-regulatory function is validated in blood samples from patients with primary complement defects. Physiological relevance of TSP-1 is demonstrated in ANCA-vasculitis patients by significantly enhanced TSP-1 staining in glomerular lesions and increased complement activity and NETosis following TSP-1 deficiency in an ANCA-vasculitis model. The newly described complement-inhibiting function of TSP-1 represents an important mechanism in the interaction of endothelia, complement and platelets. In particular, the interplay between released TSP-1, vWF and the complement system locally, especially on surfaces, influences the balance between complement activation and inhibition and may be relevant in various vascular diseases.

molecular biology↗

bioRxiv2024

The bat Influenza A virus subtype H18N11 induces nanoscale MHCII clustering upon host cell attachment

Prior to the discovery of the bat influenza A virus (IAV) subtypes H17N10 and H18N11, it was believed that all IAVs bind via the viral hemagglutinin (HA) to sialic acid residues to mediate attachment and subsequent viral entry. Both HA subtypes, H17 and H18, however, do not bind to sialic acids but instead engage a proteinaceous receptor: major histocompatibility complex class II (MHCII). The mechanistic details of this hitherto unknown protein-mediated entry are not understood. Since conventional IAVs require attachment to clusters of sialylated glycans to overcome the low affinity of the HA-sialic acid interaction, we hypothesized that bat HA would likewise interact with multiple MHCII molecules. Here, we used photoactivated localization microscopy (PALM) on fixed and live cells expressing MHCII fused to appropriate fluorescent reporters. We show that bat IAV particles attach to pre-existing MHCII clusters present on susceptible cells and that the local HA-MHCII interaction results in an increased cluster size. To measure the impact of viral attachment on the dynamics of MHCII, we utilized an experimental setup designated "inverse infection" where intact viral particles were immobilized on coverslips before live MHCII-expressing cells were seeded on top. Recording the trajectories of single MHCII molecules, this approach revealed that the mobility of MHCII was indeed slowed down in viral proximity leading to a local enrichment of MHCII molecules beneath the viral particle. Taken together, these data suggest that attachment of viral particles leads to clustering of MHCII, a process similar to the MHCII dynamics observed during the formation of an immunological synapse.

microbiology↗

bioRxiv2024

SUN2 mediates calcium-triggered nuclear actin polymerization to cluster active RNA polymerase II

The nucleoskeleton is essential for nuclear architecture as well as genome integrity and gene expression. In addition to lamins, titin or spectrins, dynamic actin filament polymerization has emerged as a potential intranuclear structural element but its functions are less well explored. Here we found that calcium elevations trigger rapid nuclear actin assembly requiring the nuclear membrane protein SUN2 independently of its function as a component of the LINC complex. Instead, SUN2 colocalized and associated with the formin and actin nucleator INF2 in the nuclear envelope in a calcium-regulated manner. Moreover, SUN2 is required for active RNA polymerase II (RNA Pol II) clustering in response to calcium elevations. Thus, our data uncover a SUN2-formin module linking the nuclear envelope to intranuclear actin assembly to promote signal-dependent spatial reorganization of active RNA Pol II.

cell biology↗

bioRxiv2024

Arp2/3 complex-dependent actin regulation protects the survival of tissue-resident mast cells

Actin network dynamics are pivotal in governing the motility and effector functions of immune cells. The Arp2/3 complex is a key regulator of actin filament branching, with mutations in its subunits being linked with human immunodeficiencies. While known for its role in phagocytosis and cell migration, our study uncovers a critical role of the Arp2/3 complex in safeguarding the tissue residency of mast cells (MCs), essential immune cells in allergies, venom detoxification and antigen-specific avoidance. Mechanistically, we show that MCs require Arp2/3-regulated actin filament assembly to resist their integrin-mediated mechano-coupling with their tissue niche. Arp2/3 complex depletion directs MCs into cell cycle arrest and death, which can be rescued by inhibiting their mechanical interactions with extracellular matrix. Our findings underscore the Arp2/3 complex as a mechano-protective element for maintaining MC survival and longevity in tissues, highlighting the importance of actin regulation in preserving the homeostasis of a tissue-resident immune cell population. One Sentence SummaryArp2/3 complex protects the tissue homeostasis of resident mast cell networks

immunology↗

bioRxiv2023

Replication fork plasticity upon replication stress requires rapid nuclear actin polymerization

Cells rapidly respond to replication stress actively slowing fork progression and inducing fork reversal. How replication fork plasticity is achieved in the context of nuclear organization is currently unknown. Using nuclear actin probes in living and fixed cells, we visualized nuclear actin filaments in unperturbed S phase, rapidly extending in number and thickness upon genotoxic treatments, and taking frequent contact with replication factories. Chemically or genetically impairing nuclear actin polymerization shortly before these treatments prevents active fork slowing and abolishes fork reversal. Defective fork plasticity is linked to reduced recruitment of RAD51 and SMARCAL1 to nascent DNA. Conversely, PRIMPOL gains access to replicating chromatin, promoting unrestrained and discontinuous DNA synthesis, which is associated with increased chromosomal instability and decreased cellular resistance to replication stress. Hence, nuclear F-actin orchestrates replication fork plasticity and is a key molecular determinant in the rapid cellular response to genotoxic treatments.

cancer biology↗

bioRxiv2022

Quantitative real-time in-cell imaging reveals heterogeneous clusters of proteins prior to condensation

The formation of biomolecular condensates underpins many cellular processes; however, our current understanding of condensate formation within cells is largely based on observing the final near-equilibrium condensate state. It is less clear how proteins behave before condensates form or at concentrations at which condensation does not occur in cells. Here, we use a combination of fluorescence microscopy and photobleaching analysis to quantify phase separation of negative elongation factor (NELF) in living and stressed cells. We use the recently reported system of stress-induced condensation of NELF in human nuclei as a model to study the behaviour of proteins before condensation. We find that pre-condensate heterogeneous clusters both grow and shrink and are not freely diffusing. Unexpectedly, we also find such small dynamic clusters in unstressed cells in which condensates do not form. We provide a categorisation of small and large clusters based on their dynamics and their response to p38 kinase inhibition. Overall, our data are best explained as non-classical nucleation with a flat free-energy landscape for clusters of a range of sizes and an inhibition of condensation.

biophysics↗

bioRxiv2020

Cyclase-associated protein 2 (CAP2) controls MRTF-A localization and SRF activity in mouse embryonic fibroblasts

Recent studies identified cyclase-associated proteins (CAPs) as important regulators of actin dynamics that control assembly and disassembly of actin filaments (F-actin). While these studies significantly advanced our knowledge of their molecular functions, the physiological relevance of CAPs largely remained elusive. Gene targeting in mice implicated CAP2 in heart physiology and skeletal muscle development. Heart defects in CAP2 mutant mice were associated with altered activity of serum response factor (SRF), a transcription factor involved in multiple biological processes including heart function, but also skeletal muscle development. By exploiting mouse embryonic fibroblasts (MEFs) from CAP2 mutant mice, we aimed at deciphering the CAP2-dependent mechanism relevant for SRF activity. Reporter assays and mRNA quantification by qPCR revealed reduced SRF-dependent gene expression in mutant MEFs. Reduced SRF activity was associated with moderately increased G-actin levels and reduced nuclear levels of MRTF-A, a transcriptional SRF coactivator that is shuttled out of the nucleus and, hence, inhibited upon G-actin binding. Moreover, pharmacological actin manipulation restored MRTF-A distribution in mutant MEFs. Our data are in line with a model in which CAP2 controls the MRTF-SRF pathway in an actin-dependent manner. While MRTF-A localization and SRF activity was impaired under basal conditions, serum stimulation induced nuclear MRTF-A translocation and SRF activity in mutant MEFs similar to controls. In summary, our data revealed that in MEFs CAP2 controls basal MRTF-A localization and SRF activity, while it was dispensable for serum-induced nuclear MRTF-A translocation and SRF stimulation.

cell biology↗

bioRxiv2020

Glycosaminoglycans are specific endosomal receptors for Yersinia pseudotuberculosis Cytotoxic Necrotizing Factor

The Cytotoxic Necrotizing Factor Y (CNFY) is produced by the gram-negative, enteric pathogen Yersinia pseudotuberculosis. The bacterial toxin belongs to a family of deamidases, which constitutively activate Rho GTPases, thereby balancing inflammatory processes. We identified heparan sulfate proteoglycans as essential host cell factors for intoxication with CNFY. Using flow cytometry, microscopy, knockout cell lines, pulsed electron–electron double resonance and bio-layer interferometry, we studied the role of glucosaminoglycans in the intoxication process of CNFY. To analyze toxin-glucosaminoglycan interaction we utilized a truncated CNFY (CNFY709-1014). Especially this C-terminal part of CNFY, which encompasses the catalytic activity, binds with high affinity to heparan sulfates. CNFY binding with the N-terminal domain to its protein receptor seems to induce a first conformational change supporting the interaction between the C-terminal domain and heparan sulfates, which seems sterically hindered in the full toxin. A second conformational change occurs by acidification of the endosome, probably allowing insertion of the hydrophobic regions of the toxin into the endosomal membrane. Our findings suggest that heparan sulfates play a major role for intoxication within the endosome, rather than being relevant for an interaction at the cell surface. Lastly, cleavage of heparin sulfate chains by heparanase is likely required for efficient uptake of the toxic enzyme into the cytosol of mammalian cells.Author Summary The RhoA deamidating Cytotoxic Necrotizing Factor Y (CNFY) from Yersinia pseudotuberculosis is a crucial virulence factor that is important for successful infection of mammalian cells by the pathogen. The mode of action by which CNFY is able to intoxicate cells can be divided into the following steps: Binding to the cell surface, internalization, translocation from the endosome to the cytosol and deamidation of RhoA. We show, that CNFY uses heparan sulfates to maximize the amount of molecules entering the cytosol. While not being necessary for toxin binding and uptake, the sugars hold a key role in the intoxication process. We show that CNFY undergoes a conformational change at a low endosomal pH, allowing the C-terminal domain to be released from the endosomal membrane by the action of heparanase. This study reveals new insights into the CNFY-host interaction and promotes understanding of the complex intoxication process of bacterial toxins.View Full Text

pharmacology and toxicology↗

bioRxiv2020

Post-mitotic expansion of cell nuclei requires ACTN4-mediated nuclear actin filament bundling

The actin cytoskeleton operates in a multitude of cellular processes including cell shape and migration, mechanoregulation, as well as membrane or organelle dynamics. However, its filamentous properties and functions inside the mammalian cell nucleus are less well explored. We previously described transient actin assembly at mitotic exit that promotes nuclear expansion during chromatin decondensation. Here, we identify non-muscle ACTN4 as a critical regulator to facilitate F-actin formation, reorganization and bundling during postmitotic nuclear expansion. ACTN4 binds to nuclear actin filaments and ACTN4 clusters associate with nuclear F-actin in a highly dynamic fashion. ACTN4 but not ACTN1 is required for proper postmitotic nuclear volume expansion, mediated by its actin binding domain. Using super-resolution imaging to quantify actin filament numbers and widths in individual nuclei we find that ACTN4 is necessary for postmitotic nuclear actin assembly and actin filament bundling. Our findings uncover a nuclear cytoskeletal function for ACTN4 to control nuclear size during mitotic cell division.

cell biology↗

bioRxiv2019

Actin chromobody imaging reveals sub-organellar actin dynamics

The actin cytoskeleton plays multiple critical roles in cells, from cell migration to organelle dynamics. The small and transient actin structures regulating organelle dynamics are difficult to detect with fluorescence microscopy. We developed an approach using fluorescent protein-tagged actin nanobodies targeted to organelle membranes to enable live cell imaging of sub-organellar actin dynamics with unprecedented spatiotemporal resolution. These probes reveal that ER-associated actin drives fission of multiple organelles including mitochondria, endosomes, lysosomes, peroxisomes, and the Golgi.\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC=\"FIGDIR/small/639278v2_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (46K):\norg.highwire.dtl.DTLVardef@18851e1org.highwire.dtl.DTLVardef@b7c608org.highwire.dtl.DTLVardef@12a7629org.highwire.dtl.DTLVardef@b39a0a_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗