Biology subjects

Wolff, G.

Publications and source records attributed to Wolff, G..

11 recordsLinked to original sources

bioRxiv2026

Deinococcus radiodurans HU: a versatile architect of nucleoid structure and plasticity

Nucleoid-associated proteins are known to compact and organise bacterial genomes, but how their local DNA binding shapes higher-order chromatin architecture remains unclear. Using in vitro and in situ approaches, we investigated how Deinococcus radiodurans HU (DrHU) orchestrates nucleoid organisation. DrHU engages DNA through two synergistic binding modes: high-affinity {beta}-hairpin-mediated stabilisation of DNA loops and low-affinity, multivalent interaction via its N-terminal tail, driving parallel, uniformly spaced DNA alignment. In vitro, DrHU organises supercoiled plasmid DNA into regular 2D lattices of double-spirals, while in situ, UV-C light induces a nucleoid transition from a loose DNA mesh to a blue liquid crystalline phase characterised by DNA swirls. The striking structural similarity and shared inter-filament spacing of these two arrangements suggests that DrHU plays a pivotal role in genome organisation through its versatile binding properties that enable DNA loop stabilisation, protection and DNA bridging, linking molecular interactions to 3D genome ordering and stress-induced remodelling.

microbiology↗

bioRxiv2026

Double-membrane-spanning RNA export pores are a conserved feature in nidovirus replication organelles

Upon infection, arteriviruses, coronaviruses, and other nidoviruses transform endoplasmic reticulum membranes into viral replication organelles. These include large numbers of double-membrane vesicles (DMVs) whose interior is considered the primary site of viral RNA synthesis. Early studies characterized nidovirus DMVs as sealed compartments, leaving it unclear how newly synthesized viral RNA could be exported to the cytosol. The discovery of DMV-spanning pore complexes in coronavirus-infected cells provided a plausible solution for this topological challenge. However, their structural organization, functional features, and evolutionary conservation across the nidovirus order, have remained unclear. Here, we investigated the macromolecular architecture of DMVs induced by two prototypic arteriviruses using cellular cryo-electron tomography. Despite the substantial evolutionary distance separating arteriviruses and coronaviruses, we observed DMV-spanning pore complexes with striking structural similarities to those previously described in coronaviruses. These pores appear to facilitate both export and encapsidation of viral RNA. In the absence of viral RNA synthesis, ectopic expression of the arterivirus transmembrane nonstructural proteins nsp2 and nsp3 sufficed to induce the formation of pore-containing DMVs. Together, our findings reveal the conservation of key structural features of DMV pores across two distantly related nidovirus families and support a central role for these pores in nidovirus replication.

microbiology↗

bioRxiv2024

EasyGrid: A versatile platform for automated cryo-EM sample preparation and quality control

Imaging biological macromolecules in their native state with single-particle cryo-electron microscopy (cryo-EM) or in situ cryo-electron tomography (cryo-ET) requires optimized approaches for the preparation and vitrification of biological samples. Here, we describe EasyGrid, a versatile technology enabling systematic, tailored and advanced sample preparation for cellular and structural biology. This automated, standalone platform combines in-line plasma treatment, microfluidic dispensing, blot-less sample spreading, jet-based vitrification and on-the-fly grid quality control using light interferometry to streamline cryo-EM sample optimization. With EasyGrid, we optimized grid preparation for different purified macromolecular complexes and subsequently determined their structure with cryo-EM. We also demonstrated how the platform allows better vitrification of large, mammalian cells compared to standard plunge-freezing. Automated sample preparation with EasyGrid establishes an advanced, high-throughput platform for both single-particle cryo-EM and cellular cryo-ET sample preparation.

molecular biology↗

bioRxiv2022

AreTomo: An integrated software package for automated marker-free, motion-corrected cryo-electron tomographic alignment and reconstruction

AreTomo, an abbreviation for Alignment and Reconstruction for Electron Tomography, is a GPU accelerated software package that fully automates motion-corrected marker-free tomographic alignment and reconstruction in a single package. By correcting in-plane rotation, translation, and importantly, the local motion resulting from beam-induced motion from tilt to tilt, AreTomo can produce tomograms with sufficient accuracy to be directly used for subtomogram averaging. Another major application is the on-the-fly reconstruction of tomograms in parallel with tilt series collection to provide users with real-time feedback of sample quality allowing users to make any necessary adjustments of collection parameters. Here, the multiple alignment algorithms implemented in AreTomo are described and the local motions measured on a typical tilt series are analyzed. The residual local motion after correction for global motion was found in the range of {+/-}80 [A], indicating that the accurate correction of local motion is critical for high-resolution cryo-electron tomography (cryoET).

cell biology↗

bioRxiv2021

Hepatocyte-specific activity of TSC22D4 triggers progressive NAFLD by impairing mitochondrial function

ObjectiveFibrotic organ responses have recently been identified as long-term complication in diabetes. Indeed, insulin resistance and aberrant hepatic lipid accumulation represent driving features of progressive non-alcoholic fatty liver disease (NAFLD), ranging from simple steatosis and non-alcoholic steatohepatitis (NASH) to fibrosis. Effective pharmacological regimens to stop progressive liver disease are still lacking to-date. MethodsBased on our previous discovery of transforming growth factor beta-like stimulated clone (TSC)22D4 as a key driver of insulin resistance and glucose intolerance in obesity and type 2 diabetes, we generated a TSC22D4-hepatocyte specific knockout line (TSC22D4-HepaKO) and exposed mice to control or NASH diet models. Mechanistic insights were generated by metabolic phenotyping and single cell liver sequencing. ResultsHepatic TSC22D4 expression was significantly correlated with markers of liver disease progression and fibrosis in both murine and human livers. Indeed, hepatic TSC22D4 levels were elevated in human NASH patients as well as in several murine NASH models. Specific genetic deletion of TSC22D4 in hepatocytes led to reduced liver lipid accumulation, improvements in steatosis and inflammation scores and decreased apoptosis in mice. Single cell RNA sequencing revealed a distinct gene signature identifying an upregulation of mitochondrial-related processes. An enrichment of genes involved in the TCA cycle, mitochondrial organization, and triglyceride metabolism underscored the hepatocyte-protective phenotype and overall decreased liver damage as seen in mouse models. ConclusionsTogether, our data uncover a new connection between targeted depletion of TSC22D4 and intrinsic metabolic processes in progressive liver disease. Cell-specific reduction of TSC22D4 improves hepatic steatosis, inflammation and promotes hepatocyte survival thus paving the way for further preclinical therapy developments.

physiology↗

bioRxiv2021

TSC22D4 interacts with Akt1 in response metabolic and stress signals

Transforming Growth Factor {beta} 1 Stimulated Clone 22 D4 (TSC22D4) is an intrinsically disordered protein that regulates cellular and physiological processes such as cell proliferation, cellular senescence as well as hepatic glucose and lipid metabolism. The molecular mechanism of TSC22D4 action in these cellular and metabolic functions, however, remains largely elusive. Here, we identified TSC22D4 as a novel protein kinase B/Akt1 interacting protein, a critical mediator of insulin/PI3K signaling pathway implicated in diverse set of diseases including type 2 diabetes, obesity and cancer. TSC22D4 interacts with Akt1 not constitutively but rather in a regulatory manner. While glucose and insulin stimulation of cells or refeeding of mice impair the hepatic TSC22D4-Akt1 interaction, inhibition of mitochondria and oxidative stress, promote it; indicating that extra- and intra-cellular cues play a key role in controlling TSC22D4-Akt1 interaction. Our results also demonstrate that together with its dimerization domain, i.e. the TSC box, TSC22D4 requires its intrinsically disordered region (D2 domain) to interact with Akt1. To understand regulation of TSC22D4 function further, we employed tandem mass spectrometry and identified 15 novel phosphorylation sites on TSC22D4. Similar to TSC22D4-Akt1 interaction, TSC22D4 phosphorylation also responds to environmental signals such as starvation, mitochondrial inhibition and oxidative stress. Interestingly, 6 out of the 15 novel phosphorylation sites lie within the TSC22D4 D2 domain, which is required for TSC22D4-Akt1 interaction. Characterization of the regulation and function of these novel phosphorylation sites, in the future, will shed light on our understanding of the role of TSC22D4-Akt1 interaction in both cell biological and physiological functions. Overall, our findings postulate a model whereby TSC22D4 acts as an environmental sensor and interacts with Akt1 to regulate cell proliferation, cellular senescence as well as maintain metabolic homeostasis.

molecular biology↗

bioRxiv2021

Semaphorin 3C exacerbates liver fibrosis

Background & AimsChronic liver disease is a growing epidemic leading to fibrosis and cirrhosis. TGF-{beta} is the pivotal pro-fibrogenic cytokine which activates hepatic stellate cells (HSC), yet, other molecules can substantially modulate TGF-{beta} signaling in the course of liver fibrosis. Expression of the axon guidance molecules Semaphorins (SEMAs), which signal through Plexins and Neuropilins (NRPs), have been associated with liver fibrosis in HBV-induced chronic hepatitis. This study aims at determining their function in the regulation of HSCs. Approach & ResultsWe analyzed publicly available patient databases and liver biopsies. We employed transgenic mice where genes are deleted only in activated HSCs to perform ex vivo analysis and animal models. SEMA3C is the most enriched member of the Semaphorin family in liver samples from cirrhotic patients. Higher expression of SEMA3C in patients with NASH, alcoholic hepatitis or HBV-induced hepatitis discriminates those with a more pro-fibrotic transcriptomic profile. SEMA3C expression is also elevated in different mouse models of liver fibrosis and in isolated HSCs upon activation. In keeping with this, deletion of SEMA3C in activated HSCs reduces myofibroblast marker expression. Conversely, SEMA3C overexpression exacerbates TGF-{beta}-mediated myofibroblast activation, as shown by increased SMAD2 phosphorylation and target gene expression. Among SEMA3C receptors, only NRP2 expression is maintained upon activation of isolated HSCs. Interestingly, lack of NRP2 in those cells reduces myofibroblast marker expression. Finally, deletion of either SEMA3C or NRP2, specifically in activated HSCs, reduces liver fibrosis in mice. ConclusionSEMA3C is a novel marker for activated HSCs that plays a fundamental role in the acquisition of the myofibroblastic phenotype and liver fibrosis.

pathology↗

bioRxiv2020

A molecular pore spans the double membrane of the coronavirus replication organelle

Coronavirus genome replication is associated with virus-induced cytosolic double-membrane vesicles, which may provide a tailored micro-environment for viral RNA synthesis in the infected cell. However, it is unclear how newly synthesized genomes and mRNAs can travel from these sealed replication compartments to the cytosol to ensure their translation and the assembly of progeny virions. Here, using cellular electron cryo-microscopy, we unveiled a molecular pore complex that spans both membranes of the double-membrane vesicle and would allow export of RNA to the cytosol. A hexameric assembly of a large viral transmembrane protein was found to form the core of the crown-shaped complex. This coronavirus-specific structure likely plays a critical role in coronavirus replication and thus constitutes a novel drug target

microbiology↗

bioRxiv2019

Mind the gap: micro-expansion joints drastically decrease the bending of FIB-milled cryo-lamellae

Cryo-focussed ion beam (FIB)-milling of biological samples can be used to generate thin electron-transparent slices from cells grown or deposited on EM grids. These so called cryo-lamellae allow high-resolution structural studies of the natural cellular environment by in situ cryo-electron tomography. However, the cryo-lamella workflow is a low-throughput technique and can easily be obstructed by technical issues like the bending of the lamellae during the final cryo-FIB-milling steps. The severity of lamella bending seems to correlate with shrinkage of the EM grid support film at cryogenic temperatures, which could generate tensions that may be transferred onto the thin lamella, leading to its bending and breakage. To protect the lamellae from these forces, we milled \"micro-expansion joints\" alongside the lamellae, creating gaps in the support that can act as physical buffers to safely absorb material motion. We demonstrate that the presence of such micro-expansion joints drastically decreases lamella bending. Furthermore, we show that this adaptation does not create instabilities that could constrain subsequent parts of the cryo-lamella workflow, as we obtained high-quality Volta phase plate tomograms revealing macromolecules in their natural structural context. The minimal additional effort required to implement micro-expansion joints in the cryo-FIB-milling workflow makes them an easy solution against cryo-lamella bending in any biological sample milled on EM grids.

microbiology↗

bioRxiv2019

The olfactory basis of orchid pollination by mosquitoes

Mosquitoes are important vectors of disease and require sources of carbohydrates for reproduction and survival. Unlike host-related behaviors of mosquitoes, comparatively less is understood about the mechanisms involved in nectar-feeding decisions, or how this sensory information is processed in the mosquito brain. Here we show that Aedes spp. mosquitoes, including Aedes aegypti, are effective pollinators of the Platanthera obtusata orchid, and demonstrate this mutualism is mediated by the orchids scent and the balance of excitation and inhibition in the mosquitos antennal lobe (AL). The P. obtusata orchid emits an attractive, nonanal-rich scent, whereas related Platanthera species - not visited by mosquitoes - emit scents dominated by lilac aldehyde. Calcium imaging experiments in the mosquito AL revealed that nonanal and lilac aldehyde each respectively activate the LC2 and AM2 glomerulus, and remarkably, the AM2 glomerulus is also sensitive to DEET, a mosquito repellent. Lateral inhibition between these two glomeruli reflects the level of attraction to the orchid scents: whereas the enriched nonanal scent of P. obtusata activates the LC2 and suppresses AM2, the high level of lilac aldehyde in the other orchid scents inverts this pattern of glomerular activity, and behavioral attraction is lost. These results demonstrate the ecological importance of mosquitoes beyond operating as disease vectors and open the door towards understanding the neural basis of mosquito nectar-seeking behaviors.\n\nSignificance StatementNectar-feeding by mosquitoes is important for survival and reproduction, and hence disease transmission. However, we know little about the sensory mechanisms that mediate mosquito attraction to sources of nectar, like those of flowers, or how this information is processed in the mosquito brain. Using a unique mutualism between Aedes mosquitoes and Platanthera obtusata orchids, we reveal that this mutualism is mediated by the orchids scent. Furthermore, lateral inhibition in the mosquitos antennal (olfactory) lobe - via the neurotransmitter GABA - is critical for the representation of the scent. These results have implications toward understanding the olfactory basis of mosquito-nectar-seeking behaviors.

neuroscience↗

bioRxiv2017

Modulation of host learning in Aedes aegypti mosquitoes

How mosquitoes determine which individuals to bite has important epidemiological consequences. This choice is not random; most mosquitoes specialize in one or a few vertebrate host species, and some individuals in a host population are preferred over others. Here we show that aversive olfactory learning contributes to mosquito preference both between and within host species. Combined electrophysiological and behavioural recordings from tethered flying mosquitoes demonstrated that these odours evoke changes in both behaviour and antennal lobe (AL) neuronal responses. Using electrophysiological and behavioural approaches, and CRISPR gene editing, we demonstrate that dopamine plays a critical role in aversive olfactory learning and modulating odour-evoked responses in AL neurons. Collectively, these results provide the first experimental evidence that olfactory learning in mosquitoes can play an epidemiological role.

neuroscience↗