Biology subjects

Wolff, M.

Publications and source records attributed to Wolff, M..

12 recordsLinked to original sources

bioRxiv2025

Structural Acrobatics of IAA7: Exercising Fuzzy Regions to Aid Auxin-Mediated Catalysis

Auxin is the paragon of small molecules driving recognition of proteins for their ubiquitylation and degradation, enabling rapid decoding of external signals into transcriptional responses. Auxin functions as a molecular glue, enhancing interactions between the TRANSPORT INHIBITOR RESPONSE 1 (TIR1) and other TIR1-like proteins with various AUXIN INDUCIBLE/INDOLE-3-ACETIC ACID (AUX/IAA) transcriptional repressors. A Cullin RING E3 ubiquitin ligase, SCFTIR1/AFBs, mediates the ubiquitylation and degradation of AUX/IAA proteins by positioning them for ubiquitin transfer, leading to their rapid degradation by the 26S proteasome. AUX/IAAs contain a Phox Bem 1 (PB1) domain, a degron, and intrinsically disordered regions (IDRs) that influence auxin sensing and their polyubiquitylation by interacting with TIR1. However, the precise role of IDRs in AUX/IAA positioning for ubiquitylation, and the mechanism of multisite ubiquitylation within AUX/IAAs remain unclear. We used biophysical techniques and coarse-grained simulations (CGS) to study the structural behavior of IDRs in IAA7, a model AUX/IAA protein from Arabidopsis thaliana. Our findings show that IAA7 expands by approximately 2.6 nm upon binding to TIR1 in the presence of auxin, likely increasing the proximity between a lysine acceptor in IAA7 and the active site of the SCFTIR1 E3 ligase for ubiquitylation. Our data also suggest that IAA7 remains conformationally heterogeneous when bound to TIR1, forming numerous transient contacts along its IDRs and C-terminal PB1 with TIR1. We propose that these transient interactions outside the IAA7 degron interface enhance the TIR1{middle dot}IAA7 association, resulting in highly dynamic and heterogeneous complexes that facilitate IAA7 multisite ubiquitylation, targeting it for rapid proteasomal degradation. Significance StatementIn this study, we use biophysical techniques to elucidate the implications of AUX/IAA intrinsic disorder on their conformational ensemble and processing by the E3 ubiquitin ligase, SCFTIR1. Our findings demonstrate that IAA7 undergoes a structural expansion upon binding to TIR1 and remains highly dynamic, facilitating multisite ubiquitylation. These insights enhance our understanding of how ubiquitylation targets engage with their E3 receptor modules and underscore the structural advantages of intrinsic disorder in protein ubiquitylation targets.

biophysics↗

bioRxiv2023

Adaptive responding to stimulus-outcome associations requires noradrenergic transmission in the medial prefrontal cortex

A dynamic environment, such as the one we inhabit, requires organisms to continuously update their knowledge of the setting. While the prefrontal cortex is recognized for its pivotal role in regulating such adaptive behavior, the specific contributions of each prefrontal area remain elusive. In the current work, we investigated the direct involvement of two major prefrontal subregions, the medial prefrontal cortex (mPFC) and the ventrolateral orbitofrontal cortex (vlOFC), in updating Pavlovian stimulus-outcome (S-O) associations following contingency degradation. Specifically, animals had to learn that a specific cue, previously fully predicting the delivery of a specific reward, was no longer a reliable predictor. First, we found that chemogenetic inhibition of mPFC, but not of vlOFC, neurons altered the rats ability to adaptively respond to degraded and non-degraded cues. Next, given the growing evidence pointing at noradrenaline (NA) as a main neuromodulator of adaptive behavior, we decided to investigate the possible involvement of NA projections to the two subregions in this higher-order cognitive process. Employing a pair of novel retrograde vectors, we traced NA projections from the locus coeruleus (LC) to both structures and observed an equivalent yet relatively segregated amount of inputs. Then, we showed that chemogenetic inhibition of NA projections to the mPFC, but not to the vlOFC, also impaired the rats ability to adaptively respond to the degradation procedure. Altogether, our findings provide important evidence of functional parcellation within the prefrontal cortex and point at mPFC-NA as key for updating Pavlovian S-O associations. Significant statementThe ability to update stimulus-outcome (S-O) associations is a key adaptive behavior, essential for surviving in an ever-changing environment. The prefrontal cortex is well-known for playing a key role in this process. The discrete contribution of each prefrontal subregion and of different neurotransmitters, however, remains unclear. In the current study, we show that inhibiting medial prefrontal (mPFC), but not ventrolateral orbitofrontal cortex (vlOFC), neurons impairs the rats ability to update S-O associations following contingency degradation. Moreover, we demonstrate that discrete noradrenergic projections to the two subregions exist and that inhibiting the ones projecting to the mPFC, but not to the vlOFC, once again impairs the rats behavior, thereby implying a substantial contribution of noradrenaline in orchestrating this higher-order cognitive process.

neuroscience↗

bioRxiv2023

The pathogenic p.N1662D SCN2A mutation reveals an essential molecular interaction for Nav1.2 channel inactivation

Mutations in the SCN2A gene encoding the Nav1.2 sodium channel can lead to neurodevelopmental disorders. We studied the N1662D variant associated with severe early-onset developmental and epileptic encephalopathy (DEE). The N1662D mutation almost completely prevented fast inactivation without affecting activation. The comparison of wild-type and N1662D channel structures suggested that the ambifunctional hydrogen bond formation between residues N1662 and Q1494 is essential for fast inactivation. Fast inactivation could also be prevented with engineered Q1494A or Q1494L Nav1.2 channel variants, whereas Q1494E or Q1494K variants resulted in incomplete inactivation and persistent current. Molecular dynamics simulations revealed a reduced affinity of the hydrophobic IFM-motif to its receptor site with N1662D and Q1494L variants relative to wild-type. These results demonstrate that the interactions between N1662 and Q1494 underpin the stability and the orientation of the inactivation gate and are essential for the development of fast inactivation. Six DEE-associated Nav1.2 variants, with mutations mapped to channel segments known to be implicated in fast inactivation were also evaluated. Remarkably, the L1657P variant also prevented fast inactivation and produced biophysical characteristics similar to N1662D, whereas the M1501V, M1501T, F1651C, P1658S, and A1659V variants resulted in biophysical properties that were consistent with gain-of-function and enhanced action potential firing of hybrid neurons in dynamic action potential clamp experiments. Paradoxically, low densities of N1662D or L1657P currents potentiated action potential firing, whereas increased densities resulted in sustained depolarization. The contribution of non-inactivating Nav1.2 channels to neuronal excitability may constitute a novel cellular mechanism in the pathogenesis of SCN2A-related DEE. SIGNIFICANCE STATEMENTSCN2A gene-related early-onset developmental and epileptic encephalopathy (EO-DEE) is a rare and severe disorder that manifests in early infancy and childhood. SCN2A mutations affecting the fast inactivation gating mechanism can cause altered voltage dependence and incomplete inactivation of the encoded Nav1.2 channel, leading to abnormal neuronal excitability. In this biophysical and clinical study of neuronal Nav1.2 variants, we identified amino acid residues that are critical for the stability and orientation of the inactivation gate during fast inactivation. Mutations of these residues prevent fast inactivation and may lead to EO-DEE via a novel pathophysiological mechanism. The results provide novel structural insights into the molecular mechanism of Nav1.2 channel fast inactivation and inform treatment strategies for SCN2A-related EO-DEE.

biophysics↗

bioRxiv2022

The K+ exchange antiporter 3 senses the chloroplast energy status to synchronize photosynthesis

Plant photosynthesis contains two functional modules, the light-driven reactions in the thylakoid membrane and the carbon-fixing reactions in the chloroplast stroma. In nature, light availability for photosynthesis often undergoes massive and rapid fluctuations. Efficient and productive use of such variable light supply requires an instant crosstalk and rapid synchronization of both functional modules. Here, we show that this communication involves the stromal exposed regulatory C-terminus (RCT) of the thylakoid K+-exchange antiporter KEA3. RCT-mediated control of KEA3 contributes to the balance between light capture and photoprotection. By combining in silico, in vitro, and in vivo approaches, we demonstrate that the RCT senses the energy state of the chloroplast in form of both, phosphorylation and redox potential, in a pH-dependent manner and regulates KEA3 activity in response. Together our data pinpoint a regulatory feedback loop by which the stromal energy state orchestrates light capture and photoprotection via KEA3.

plant biology↗

bioRxiv2022

Concurrent maintenance of both veridical and transformed working memory representations

In the dynamic environment we live in, the already limited information that human working memory can maintain needs to be constantly updated to optimally guide behaviour. Indeed, previous studies showed that leading up to a response, representations maintained in working memory representations are transformed continuously. This goes hand-in-hand with the removal of task-irrelevant items. However, does such removal also include the representations of stimuli as they were originally, prior to transformation? Here we assessed the neural representation of task-relevant transformed representations, and the no-longer-relevant veridical representations they originated from. We applied multivariate pattern analysis to electroencephalographic data during maintenance of orientation gratings with and without mental rotation. During maintenance, we perturbed the representational network by means of a visual impulse stimulus, and were thus able to successfully decode veridical as well as imaginary, transformed orientation gratings from impulse-driven activity. The impulse response reflected only task-relevant (cued), but not task-irrelevant (uncued) items, suggesting that the latter were quickly discarded from working memory. By contrast, even though the original cued orientation gratings were also no longer task-relevant after mental rotation, these items continued to be represented next to the rotated ones, in different representational formats. This seemingly inefficient use of scarce working memory capacity was associated with reduced probe response times and may thus serve to increase precision and flexibility in guiding behaviour in dynamic environments.

neuroscience↗

bioRxiv2022

The mediodorsal thalamus supports adaptive responding based on stimulus-outcome associations

The ability to engage into flexible behaviors is crucial in dynamic environments. We recently showed that in addition to the well described role of the orbitofrontal cortex (OFC), its thalamic input from the submedius thalamic nucleus (Sub) also contributes to adaptive responding during Pavlovian degradation. In the present study, we examined the role of the mediodorsal thalamus (MD) which is the other main thalamic input to the OFC. To this end, we assessed the effect of both pre- and post-training MD lesions in rats performing a Pavlovian contingency degradation task. Pre-training lesions mildly impeded the establishment of stimulus-outcome associations during the initial training of Pavlovian conditioning without interfering with Pavlovian degradation training when the sensory feedback provided by the outcome rewards were available to animals. However, we found that both pre- and post-training MD lesions produced a selective impairment during a test conducted under extinction conditions, during which only current mental representation could guide behavior. Altogether, these data suggest a role for the MD in the successful encoding and representation of Pavlovian associations.

animal behavior and cognition↗

bioRxiv2022

Structural Characterisation of Nanoparticle-Supported Lipid Bilayers by Grazing Incidence X-ray and Neutron Scattering

The structure of supported lipid bilayers formed on a monolayer of nanoparticles was determined using a combination of grazing incidence X-ray and neutron scattering techniques. Ordered nanoparticle arrays assembled on a silicon crystal using a Langmuir-Schaefer deposition were shown to be suitable and stable substrates for the formation of curved and fluid lipid bilayers that retained lateral mobility, as shown by fluorescence recovery after photobleaching. A comparison between the structure of the curved bilayer assembled around the nanoparticles with the planar lipid membrane formed on the flat underlying silicon oxide surface revealed a [~]5 [A] thinner bilayer on the curved interface, resolving the effects of curvature on the lipid packing and overall bilayer structure. The combination of neutron scattering techniques, which grant access to sub-nanometre scale structural information at buried interfaces, and nanoparticle-supported lipid bilayers, offers a novel approach to investigate the effects of membrane curvature on lipid bilayers.

biophysics↗

bioRxiv2022

Noradrenergic signaling in the rodent orbitofrontal cortex is required to update goal-directed actions.

In a constantly changing environment, organisms must track the current relationship between actions and their specific consequences and use this information to guide decision-making. Such goal-directed behavior relies on circuits involving cortical and subcortical structures. Notably, a functional heterogeneity exists within the medial prefrontal, insular, and orbitofrontal cortices (OFC) in rodents. The role of the latter in goal-directed behavior has been debated, but recent data indicate that the ventral and lateral subregions of the OFC are needed to integrate changes in the relationships between actions and their outcomes. Neuromodulatory agents are also crucial components of prefrontal functions and behavioral flexibility might depend upon the noradrenergic modulation of prefrontal cortex. Therefore, we assessed whether noradrenergic innervation of the OFC plays a role in updating action-outcome relationships. We used an identity-based reversal task and found that depletion or chemogenetic silencing of noradrenergic inputs within the OFC rendered rats unable to associate new outcomes with previously acquired actions. Silencing of noradrenergic inputs in the medial prefrontal cortex or depletion of dopaminergic inputs in the OFC did not reproduce this deficit. Together, our results indicate that noradrenergic projections to the OFC are required to update goal-directed actions. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/498245v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@afc586org.highwire.dtl.DTLVardef@1d10ff4org.highwire.dtl.DTLVardef@6e6863org.highwire.dtl.DTLVardef@874b30_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIRats learn initial action-outcome associations in an instrumental task C_LIO_LINoradrenergic depletion in the OFC prevents the encoding and expression of these associations following reversal learning C_LIO_LIDopaminergic depletion in the OFC does not result in behavioral deficits C_LIO_LILC:OFC noradrenergic projections are required to update action-outcome associations C_LI IN BRIEFCerpa et al. investigate whether noradrenergic projections from the locus coeruleus (LC) to the orbitofrontal cortex are involved in updating previously established goal-directed actions following environmental change. They find that these LC projections are required to both encode and express reversed action-outcome associations in rats.

neuroscience↗

bioRxiv2021

Alpha-hemolysin of Staphylococcus aureus impairs thrombus formation

Toxins are key virulence determinants of pathogens and can impair the function of host immune cells including platelets. Insights into pathogen toxin interference with platelets will be pivotal to improve treatment of patients with bacterial bloodstream infections. In this study, we deciphered the effects of Staphylococcus aureus toxins -hemolysin, LukAB, LukDE and LukSF on human platelets and compared the effects with the pore forming toxin pneumolysin of Streptococcus pneumoniae. In contrast to pneumolysin, -hemolysin initially activates platelets as indicated by CD62P and IIb{beta}3 integrin expression, but the resulting pores also induce alterations in the phenotype of platelets and induce apoptosis of platelets. The presence of small amounts of -hemolysin (0.2 {micro}g/mL) in whole blood abrogates thrombus formation indicating that in systemic infections with S. aureus the stability of formed thrombi is impaired. This might be of high clinical relevance for S. aureus induced endocarditis of the aortic valves. Stabilizing the thrombi by inhibiting -hemolysin induced impairment of platelets likely reduces the risk for septic (micro-)embolization. However, in contrast to pneumolysin, -hemolysin induced platelets damage could not be neutralized by intravenous immune globulins. In contrast to -hemolysin, S. aureus bi-component pore forming leukocidins LukAB, LukED and LukSF do not bind to platelets and had no significant effect on platelet activation and viability. Main point 1: -hemolysin forms pores in platelets, which first activate but then result in apoptosis and impairs thrombus formation and stability Main point 2: Polyvalent immunoglobulins do not neutralize the mode of action of the toxin

microbiology↗

bioRxiv2021

BIFURCATING NEURONS IN THE ANTERIOR THALAMIC NUCLEI

The anterior thalamic nuclei (ATN) form a nodal point within a distributed memory network. The conventional view of ATN function describes segregated efferents to different terminal regions. By contrast, we found that bifurcating neurons are common within the anteromedial nucleus (AM) of the ATN. A substantial proportion of AM neurons ([~]36% within a region of interest) showed collateral projections when one of two retrograde neurotracers, Cholera Toxin Subunit B (CTB) conjugated to either Alexa Fluor(R) 488 or 594, was placed in the medial prefrontal cortex (mPFC) or dorsal subiculum (dSub). A marked degree of collateralization ([~]20% AM neurons) was also found when neurotracers were placed in mPFC and caudal retrosplenial cortex (cRSC); about 10% showed collaterals when the cRSC was paired with either dSub or vHF; the fewest (6%) was found for mPFC paired with the ventral hippocampal formation (vHF). A generally similar range of percentages of bifurcating neurons was found in the adjacent nucleus reuniens (Re). Evidence that AM neurons project simultaneously to many distantly-located structures provides a new perspective on ATN function. These neurons would facilitate direct coordination among key neural structures to support memory and may explain the strong association between the ATN and diencephalic amnesia.

neuroscience↗

bioRxiv2020

Trait-based responses to forestry and grazing modify long-term changes in biodiversity hot-spots of boreal forests

AimLand use is the foremost cause of global biodiversity decline, but species do not respond equally to land-use practices. Instead, it is suggested that responses vary with species traits, but long-term data on the trait-mediated effects of land-use on communities is scarce. Here we study how forest understory communities have been affected by two land-use practices during 4-5 decades, and whether changes in plant diversity are related to changes in functional composition. LocationFinland Time period1968-2019 Major taxa studiedVascular plants MethodsWe resurveyed 245 vegetation plots in boreal herb-rich forest understories, and used hierarchical Bayesian linear models to relate changes in diversity, species composition, average plant size, and leaf economic traits to reindeer abundance, forest management intensity, and changes in climate, canopy cover and composition. ResultsIntensively managed forests decreased in species richness and had increased turnover, but management did not affect functional composition. Increased reindeer densities corresponded with increased leaf dry matter content, evenness and diversity, and decreased height and specific leaf area. Successional development in the canopy was associated with increased specific leaf area and decreased leaf dry matter content and height in the understory over the study period. Effects of reindeer abundance and canopy density on diversity were partially mediated by vegetation height, which had a negative relationship with evenness. Observed changes in climate had no discernible effect on any variable. Main conclusionsFunctional traits are useful in connecting vegetation changes to the mechanisms that drive them, and provide unique information compared to turnover and diversity metrics. These trait-dependent selection effects could inform whether species benefit or suffer from land use changes and explain observed biodiversity changes under global change.

ecology↗

bioRxiv2018

Emergence of robust nucleosome patterns from an interplay of positioning mechanisms

Proper positioning of nucleosomes in eukaryotic cells is determined by a complex interplay of factors, including nucleosome-nucleosome interactions, DNA sequence, and active chromatin remodeling. Yet, characteristic features of nucleosome positioning, such as gene-averaged nucleosome patterns, are surprisingly robust across perturbations, conditions, and species. Here, we explore how this robustness arises despite the underlying complexity. We leverage mathematical models to show that a large class of positioning mechanisms merely affects the quantitative characteristics of qualitatively robust positioning patterns. We demonstrate how statistical positioning emerges as an effective description from the complex interplay of different positioning mechanisms, which ultimately only renormalize the model parameter quantifying the effective softness of nucleosomes. This renormalization can be species-specific, rationalizing a puzzling discrepancy between the effective nucleosome softness of S. pombe and S. cerevisiae. More generally, we establish a quantitative framework for dissecting the interplay of different nucleosome positioning determinants.

biophysics↗