Biology subjects

Stradal, T. E. B.

Publications and source records attributed to Stradal, T. E. B..

12 recordsLinked to original sources

bioRxiv2026

Arp2/3 complex-dependent actin remodeling is required for efficient RSV uncoating in A549 cells

Productive human respiratory syncytial virus (RSV) cell-entry requires coordinated interactions between viral proteins and host-cell factors at the plasma membrane-actin cortex interface. Branched actin networks remodel this interface, but their precise contribution to the early stages of RSV infection remains unclear. Here, we interfered with Arp2/3 complex-dependent actin filament branching by generating A549 cell lines disrupted for expression of the essential Arp2 subunit by CRISPR/Cas9. Permanent loss of Arp2 reduced the infection of the RSV long GFP reporter virus as quantified over the first 24 h post-infection. Compromised infection efficiency in Arp2 knockout cells persisted at later time points and also resulted in reduced syncytia formation. Notably, these infection phenotypes were not accompanied by obvious changes in viral host cell attachment. Moreover, photoactivated localization microscopy (PALM) studies revealed comparable receptor diffusion and clustering in cells stably expressing mEos3.2-tagged insulin-like growth factor I receptor (IGF1R). Although Arp2/3-deficient cells displayed fewer albeit larger macropinosomes as compared to WT cells, no changes were observed for internalized RSV genome levels. In contrast, Arp2-deficient cells appeared suppressed in viral uncoating efficiency. Consequently, viral mRNA expression and the cellular type III interferon response were reduced. Together, these data reveal that Arp2/3 complex-dependent, branched actin networks contribute to the efficiency of RSV uncoating. ImportanceHuman respiratory syncytial virus (RSV) is a major cause of severe respiratory disease. The infection initiates at the plasma membrane-actin cortex interface, yet the role of actin in productive RSV entry has remained unclear. Using CRISPR/Cas9 disruption of the essential Arp2/3 complex subunit Arp2 in A549 cells, we show that branched actin networks are required for efficient RSV infection. Despite actin network remodeling, photoactivated localization microscopy showed unchanged diffusion and clustering of the RSV receptor IGF1R. Although macropinocytosis was affected in Arp2-deficient cells, RSV attachment and internalization were not influenced. In contrast, a {beta}-lactamase virus-like-particle-based assay revealed a defect in uncoating, followed by reduced viral gene expression and a weaker type III interferon response. These findings define Arp2/3 complex-dependent branched actin networks as a host determinant of RSV uncoating and provide a practical approach to quantify uncoating without engineering the RSV genome.

microbiology↗

bioRxiv2025

Dynamic protrusions mediate unique crawling motility in Asgard Archaea (Promethearchaeota)

Crawling motility is a hallmark of eukaryotic cells and requires a dynamic actin cytoskeleton, regulated adhesion, and spatially organized signalling pathways1-3. Asgard archaea (phylum Promethearchaeota) which are considered the closest known prokaryotic relatives of eukaryotes potentially encode these functions within their large set of eukaryotic signature proteins4-9. The few cultivated members show a complex cell morphology, consisting of a central cell body from which several protrusions extend, filled with an actin-based cytoskeleton10,11. Here, live cell microscopy of two organisms of the Loki- and Hodarchaea lineages10,12 showed that they dynamically and drastically change their cell shape on a minute time scale and grow and retract their extensive protrusions with a speed of 1.5 to 5.3 {micro}m/min, respectively. After adhering to a glass surface, cells employ their protrusions to undergo active crawling motion. In the presence of selected actin inhibitors however, the observed dynamics were arrested, suggesting a central role of actin in these processes. The observed cellular plasticity and motility are unique features among prokaryotes and might have been crucial for the emergence of the first eukaryotic cells that are thought to have formed through the association of a member of the Promethearchaeota and an alphaproteobacterium, the ancestor of mitochondria.

microbiology↗

bioRxiv2025

Profilin promotes lamellipodium protrusion by tuning the antagonistic activities of CP and VASP

Cell migration on rigid surfaces employs flat protrusions termed lamellipodia, which constitute the prime model system for branched actin filament networks that generate pushing forces for membrane movement. Branched actin filaments are nucleated by the Arp2/3 complex and play vital roles in various cell biological processes, such as organelle trafficking, phagocytosis and autophagy. Here we utilize genome editing to explore the functional connections between the actin monomer-binding protein profilin (Pfn), the filament nucleating Arp2/3 complex, its co-factor heterodimeric capping protein (CP) and the Ena/VASP family of actin filament polymerases in lamellipodial actin assembly. Individual and combinations of knockouts show that Pfn counters Ena/VASP but promotes Arp2/3 complex activity, while Ena/VASP and CP mutually antagonize each other. Notably, while Pfn is important for Arp2/3 complex activity irrespective of Ena/VASP, sensitivity of CP to Pfn removal is lost in the absence of Ena/VASP. Our findings establish Pfn as master regulator of Arp2/3 complex-dependent actin network formation, which differentially regulates VASP and its antagonizer CP. Finally, mathematical modeling of our data suggest that Ena/VASP and CP compete for binding at the lamellipodial edge, likely contributing to their functional antagonism at this subcellular site. This work provides critical insights into the molecular logic of branched actin network assembly in membrane protrusion and cellular force generation.

cell biology↗

bioRxiv2024

Differential interference with actin-binding protein function by acute Cytochalasin B

Dynamic actin filament remodeling is crucial for a plethora of fundamental cell biological processes, ranging from cell division and migration to cell communication, intracellular trafficking or tissue development. Cytochalasin B and -D are fungal secondary metabolites frequently used for interference with such processes. Although generally assumed to block actin filament polymerization at their rapidly growing barbed ends and compete with regulators at these sites, our molecular understanding of their precise effects in dynamic actin structures is scarce. Here we combine live cell imaging and analysis of fluorescent actin-binding protein dynamics with acute treatment of lamellipodia in migrating cells with cytochalasin B. Our results show that in spite of an abrupt halt of lamellipodium protrusion, cytochalasin B affects various actin filament barbed end-binding proteins in a differential fashion. Cytochalasin B enhances instead of diminishes the accumulation of prominent barbed end-binding factors such as Ena/VASP family proteins and heterodimeric capping protein (CP) in the lamellipodium. Similar results were obtained with cytochalasin D. All these effects are highly specific, as cytochalasin-induced VASP accumulation requires the presence of CP, but not vice versa, and coincides with abrogation of both actin and VASP turnover. Cytochalasin B can also increase apparent barbed end interactions with the actin-binding {beta}-tentacle of CP and partially mimic its Arp2/3 complex-promoting activity in the lamellipodium. In conclusion, our results reveal a new spectrum of cytochalasin activities on barbed end-binding factors, with important implications for the interpretation of their effects on dynamic actin structures.

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↗

bioRxiv2022

Neurotrophic and immunomodulatory lanostane triterpenoids from wood-inhabiting Basidiomycota

Neurotrophins such as nerve growth factor (ngf) and brain derived neurotrophic factor (bdnf) play important roles in the central nervous system. They are potential therapeutic drugs for treatment of neurodegerative diseases including Alzheimers disease and Parkinsons disease. In this study, we investigated the neurotrophic properties of triterpenes isolated from fruiting bodies of Laetiporus sulphureus and a mycelial culture of Antrodia sp. MUCL 56049. The structures of the isolated compounds were elucidated based on nuclear magnetic resonance (NMR) spectroscopy in combination with high-resolution Electrospray mass spectrometry (HR-ESIMS). The secondary metabolites were tested for neurotrophin (ngf and bdnf) expression levels on human astrocytoma 1321N1 cells. Neurite outgrowth activity using rat pheochromocytoma (PC-12) cells was also determined. Twelve triterpenoids were isolated, of which several potently stimulated the expression of neurotrophic factors namely, ngf (sulphurenic acid, 15-dehydroxytrametenolic acid, fomefficinic acid D and 16-hydroxyeburicoic acid) and bdnf (sulphurenic acid and 15- dehydroxytrametenolic acid), respectively. The triterpenes also potentiated ngf-induced neurite outgrowth in PC-12 cells. This is, to the best of our knowledge, the first report on the compound class of lanostanes in direct relation to bdnf and ngf enhancement. These compounds are widespread in medicinal mushrooms; hence, they appear promising as starting point for development of drugs and mycopharmaceuticals to combat neurodegenerative diseases. Interestingly, they do not show any pronounced cytotoxicity and may therefore be better suited for therapy than many other neurotrophic compounds that were previously reported.

biochemistry↗

bioRxiv2021

SMER28 attenuates PI3K/mTOR signaling by direct inhibition of PI3K p110 delta

SMER28 (Small molecule enhancer of Rapamycin 28) is an autophagy-inducing compound functioning by a hitherto unknown mechanism. Here we confirm its autophagy-inducing effect by assessing classical autophagy-related parameters. Interestingly, we also discovered several additional effects of SMER28, including growth retardation and reduced G1 to S phase progression. Most strikingly, SMER28 treatment led to a complete arrest of receptor tyrosine kinase signaling, and consequently growth factor-induced cell scattering and dorsal ruffle formation. This coincided with a dramatic reduction of phosphorylation patterns of PI3K downstream effectors. Consistently, SMER28 directly inhibited PI3K{delta} and to a lesser extent p110{gamma}. The biological relevance of our observations was underscored by interference of SMER28 with InlB-mediated host cell entry of Listeria monocytogenes, which requires signaling through the prominent receptor typrosine kinase c-Met. This effect was signaling-specific, since entry of unrelated, gram-negative Salmonella Typhimurium was not inhibited.

cell biology↗

bioRxiv2021

Endocytic vacuole formation by WASH-mediated endocytosis

Endocytosis is a fundamental cellular process facilitated by diverse mechanisms. Remarkably, several distinct clathrin-independent endocytic processes were identified and characterized following virus uptake into cells. For some, however, mechanistic execution and biological function remain largely unclear. This includes an endocytic process exploited by human papillomavirus type 16 (HPV16). Using HPV16, we examined how vesicles are formed by combining systematic cellular perturbations with electron and video microscopy. Cargo uptake occurred by uncoated, inward-budding pits. Mechanistically, vesicle scission was facilitated by actin polymerization controlled through the actin nucleation promoting factor WASH. While WASH typically functions in conjunction with the retromer complex on endosomes during retrograde trafficking, endocytic vesicle formation was largely independent of retromer itself and the heterodimeric membrane-bending SNX-BAR retromer adaptor, thereby uncovering a role of WASH in endocytosis in addition to its canonical role in intracellular membrane trafficking.

cell biology↗

bioRxiv2021

Lamellipodia-like actin networks in cells lacking WAVE Regulatory Complex

Cell migration frequently involves the formation of lamellipodia induced by Rac GTPases mediating activation of WAVE Regulatory Complex (WRC) driving Arp2/3 complex-dependent actin assembly. Previous genome editing studies solidified the view of an essential, linear pathway employing aforementioned components. Using disruption of the WRC subunit Nap1 and its paralogue Hem1 followed by serum and growth factor stimulation or expression of active GTPases now revealed a pathway to formation of Arp2/3 complex-dependent, lamellipodia-like structures (LLS) that require both Rac and Cdc42, but not WRC. These observations were independent of WRC subunit eliminated and coincided with the lack of recruitment of Ena/VASP family actin polymerases. Moreover, aside from the latter, induced LLS contained all common lamellipodial regulators tested, including cortactin, the Ena/VASP ligand lamellipodin or FMNL subfamily formins. Our studies thus establish the existence of a signaling axis to Arp2/3 complex-dependent actin remodeling at the cell periphery operating without WRC and Ena/VASP.

cell biology↗

bioRxiv2020

Dendritic cell actin dynamics controls T cell priming efficiency at the immunological synapse

Dendritic cells (DCs) are crucial for the priming of naive T cells and the initiation of adaptive immunity. Priming is initiated at a heterologous cell-cell contact, the immunological synapse (IS). While it is established that actin dynamics regulates signalling at the T cell side of the contact, little is known about the cytoskeletal contribution on the DC side. We show that that the DC cytoskeleton is decisive for the formation of a multifocal synaptic structure, which correlates with T cell priming efficiency. We demonstrate that DC actin appears in transient foci at the IS and that these foci are dynamized by the WAVE complex. Absence of WAVE in DCs leads to stabilized contacts with T cells, caused by an increase in ICAM1-integrin mediated cell-cell adhesions. This results in a lower number of activated and proliferating T cells. Our results reveal an important role of DC actin in the regulation of synaptic contacts with crucial relevance for full T cell expansion.

cell biology↗

bioRxiv2020

Loss of Hem1 disrupts macrophage function and impacts on migration, phagocytosis and integrin-mediated adhesion

The hematopoietic-specific protein 1 (Hem1) comprises an essential subunit of the WAVE Regulatory Complex (WRC) in immune cells. WRC has a fundamental role in Arp2/3 complex activation and the protrusion of branched actin networks in motile cells. Hem1 deficiency leads to suppression of the entire WRC in immune cells. Defective WRC function in macrophages results in loss of lamellipodia and migration defects. Moreover, phagocytosis, commonly accompanied by lamellipodium protrusion during cup formation, is altered in Hem1 null cells concerning frequency and efficacy. When analyzing cell spreading, adhesion and podosome formation, we found that Hem1 null cells are capable, in principle, of podosome formation and consequently, do not show any quantitative differences in extracellular matrix degradation. Their adhesive behavior, however, was significantly altered. Specifically, adhesion as well as de-adhesion of Hem1 null cells was strongly compromised, likely contributing to the observed reduced efficiency of phagocytosis. In line with this, phosphorylation of the prominent adhesion component paxillin was diminished. Non-hematopoietic somatic cells disrupted in expression for both Hem1 and its ubiquitous orthologue Nck-associated protein 1 (Nap1) or the essential WRC components Sra-1/PIR121 did not only confirm defective paxillin phosphorylation, but also revealed that paxillin turnover in focal adhesions is accelerated in the absence of WRC. Finally, adhesion assays using platelets lacking functional WRC as model system unmasked radically decreased IIb{beta}3 integrin activation. Our results thus demonstrate that WRC-driven actin networks impact on integrin-dependent processes controlling formation and dismantling of different types of cell-substratum adhesion. One sentence summaryInterference of Hem1 function in mice and cells uncovers a hitherto unrecognized role in integrin-mediated cell adhesion that is crucial for macrophage function and connects to recently discovered immunodeficiencies in patients carrying Hem1 mutations.

cell biology↗

bioRxiv2019

Lamellipodin tunes cell migration by stabilizing protrusions and promoting adhesion formation

Efficient migration on adhesive surfaces involves the protrusion of lamellipodial actin networks and their subsequent stabilization by nascent adhesions. The actin binding protein lamellipodin (Lpd) is thought to play a critical role in lamellipodium protrusion, by delivering Ena/VASP proteins onto the growing plus ends of actin filaments and by interacting with the WAVE regulatory complex (WRC), an activator of the Arp2/3 complex, at the leading edge. Using B16-F1 melanoma cell lines, we demonstrate that genetic ablation of Lpd compromises protrusion efficiency and coincident cell migration without altering essential parameters of lamellipodia, including their maximal rate of forward advancement and actin polymerization. We also confirmed lamellipodia and migration phenotypes with CRISPR/Cas9-mediated Lpd knockout Rat2 fibroblasts, excluding cell type-specific effects. Moreover, computer-aided analysis of cell edge morphodynamics on B16-F1 cell lamellipodia revealed that loss of Lpd correlates with reduced temporal protrusion maintenance as a prerequisite of nascent adhesion formation. We conclude that Lpd optimizes protrusion and nascent adhesion formation by counteracting frequent, chaotic retraction and membrane ruffling. Summary statementWe describe how genetic ablation of the prominent actin- and VASP-binding protein lamellipodin combined with software-aided protrusion analysis uncovers mechanistic insights into its cellular function during cell migration.

cell biology↗