Biology subjects

Kempf, A.

Publications and source records attributed to Kempf, A..

17 recordsLinked to original sources

bioRxiv2026

Tag-stabilized fluorescent protein binders for endogenous cell type-specific protein labelling

Protein labelling using genetically encoded fluorescent protein fusions has revolutionized the study of protein localization and function. However, visualizing endogenous proteins in specific cell types remains challenging, as existing approaches often require overexpression or laborious genetic engineering. To address these limitations, we developed a library of fluorescent protein binders using existing nanobodies and single-chain variable fragments that recognize fluorescent proteins and short peptide tags, allowing for modular, cell type-specific labelling of genetically tagged endogenous proteins. We found that fluorescent protein binders generate background fluorescence in the absence of the target protein, reducing signal specificity. To overcome this limitation, we generated destabilized fluorescent protein binders directed against GFP, ALFA, GCN4 peptide and V5. These binders display increased antigen-dependent stability and reduced background fluorescence, thereby improving the signal-to-noise ratio of endogenous protein visualization. We demonstrate the detection of endogenous proteins in drosophila larvae and embryos, zebrafish embryos, as well as in specific neuronal populations of the adult drosophila brain. In the brain, tissue-specific labelling revealed previously unknown protein expression and subcellular localization in sleep-regulatory neurons. Together, our panel of tag-stabilized fluorescent protein binders provides a versatile, ready-to-use toolkit for background-free, cell type-specific visualization of endogenous proteins.

cell biology↗

bioRxiv2026

Olfactory Loss Enhances Visual Learning in Drosophila through Structural and Functional Reorganisation

Loss of a sensory modality can enhance performance in the remaining senses. However, the circuit mechanisms by which such cross-modal compensation can improve cognitive functions, including learning, are unknown. Here, we show that compromising olfaction in both larval and adult Drosophila enhances visual associative learning. Using behavioural analysis, functional imaging, and comparative connectomics, we reveal the circuit mechanisms that underlie this improvement. Animals with improved learning ability have enhanced responses to visual stimuli in the higher-order learning circuit. The complementary circuit mechanisms that can enhance these responses are structural reweighting of inputs in the larva, resulting in an increased fraction of synaptic inputs from visual pathways onto neurons in the learning circuit, and a reduction in cross-modal inhibition in the adult. Together, these findings reveal synaptic and disinhibitory circuit mechanisms that enhance learning in higher-order associative networks following sensory loss.

neuroscience↗

bioRxiv2026

The IL-1 Family Controls Acute Mucosal Fungal Infection and Mucosal-Systemic Dissemination.

Candida albicans is a major opportunistic pathogen in humans that is capable of breaching mucosal barriers and causing severe systemic infections with high mortality. How the host controls mucosal infection and prevents dissemination remains unclear but is essential for improving disease outcomes. Here, we demonstrate that C. albicans induces specific IL-1 family members, which are critical for initiating mucosal protection by controlling antimicrobial peptides, IL-17, and neutrophil responses. Loss of combined IL-1 family signalling led to severe mucosal C. albicans infection, which was eventually resolved by a potent neutrophil response. However, in neutropenic conditions (a key risk patient factor) abolishing IL-1 family signalling resulted in C. albicans dissemination, predominantly to the liver, mirroring clinical disease and leading to mortality. This study highlights the IL-1 family as a key initiator of mucosal immunity, restricting mucosal invasion and cooperating with neutrophils to prevent life- threatening systemic infections.

microbiology↗

bioRxiv2025

Intestinal control of feeding initiation in Drosophila melanogaster

The interplay between feeding and excretion is essential for organismal nutrition and survival, yet their mechanistic coupling remains poorly understood. At the onset of life, feeding must be initiated while developmental waste products - the meconium - need to be eliminated. Using Drosophila as a model system, we explored the in vivo mechanisms coordinating these processes. We developed novel behavioral assays for newly eclosed flies and discovered that, similar to neonatal mammals, Drosophila excrete their meconium shortly after eclosion. Remarkably, feeding initiation occurs only after partial meconium elimination. We identified a cis-regulatory element associated with the apterous gene, which, when disrupted, prevents both meconium excretion and adult feeding initiation. These flies develop hindgut obstruction (ileus), avoid food, and exhibit increased proboscis extension sleep - a behaviour we found plays a functional role in waste clearance under normal conditions. Through experimental inhibition of meconium excretion, we established that this process is prerequisite for feeding initiation, suggesting a gut-to-brain signaling circuit that couples these fundamental physiological processes. The progression of phenotypes we observed parallels the hallmarks of mechanical ileus in humans. Our findings reveal previously unrecognized links between intestinal clearance, feeding behavior, and survival, with potential implications for understanding similar processes across species.

developmental biology↗

bioRxiv2024

Identification of the Staphylococcus aureus Endothelial Cell Surface Interactome by Proximity Labeling

Virulence strategies of pathogens depend on interaction with host cells. Binding and activation of receptors located on the plasma membrane is crucial for the attachment to or pathogen internalization by host cells. Identification of host cell receptors often is difficult and hence the identity of many proteins that play important roles during host-pathogen interaction remains elusive. We developed a novel proximity labeling approach, in which we decorated the opportunistic pathogen Staphylococcus aureus with ascorbate peroxidase 2. Upon addition of hydrogen peroxide the peroxidase initiates proximity biotinylation of S. aureus host receptors, thereby enabling identification of these proteins. We here demonstrate an endothelial cell surface interactome of 306 proteins of which neuronal adhesion molecule, protein tyrosine kinase PTK7, melanotransferrin, protein-tyrosine kinase Met, CD109 and others constitute novel S. aureus co-receptors. Filtering the interactome for validated surface proteins resulted in a list of 89 receptor candidates of which a 53% were described to interact with S. aureus or other pathogens.

microbiology↗

bioRxiv2024

Impact of JN.1 booster vaccination on neutralisation of SARS-CoV-2 variants KP.3.1.1 and XEC

The SARS-CoV-2 KP.3.1.1 lineage is currently the dominating lineage on several continents. In parallel, the XEC lineage, a recombinant of KS.1.1 and KP.3.3, is on track of becoming the next dominant lineage in Europe and North America. Here we performed a rapid virological characterisation of the XEC lineage and studied the impact of JN.1 mRNA booster vaccination on KP.3.1.1 and XEC neutralisation.

molecular biology↗

bioRxiv2024

Virological traits of the SARS-CoV-2 BA.2.87.1 lineage

The highly mutated SARS-CoV-2 BA.2.87.1 lineage was recently detected in South Africa, but its transmissibility is unknown. Here, we report that BA.2.87.1 efficiently enters human cells but is more sensitive to antibody-mediated neutralization than the currently dominating JN.1 variant. Acquisition of adaptive mutations might thus be needed for high transmissibility.

molecular biology↗

bioRxiv2024

Sleep pressure accumulates in a voltage-gated lipid peroxidation memory

Voltage-gated potassium (KV) channels contain cytoplasmic {beta}-subunits whose aldo-keto reductase activity is required for the homeostatic regulation of sleep. Here we show that Hyperkinetic, the {beta}-subunit of the KV1 channel Shaker in Drosophila, forms a dynamic lipid peroxidation memory. Information is stored in the oxidation state of Hyperkinetics nicotinamide adenine dinucleotide phosphate (NADPH) cofactor, which changes when lipid-derived carbonyls, such as 4-oxo-2-nonenal or an endogenous analog generated by illuminating a membrane-bound photosensitizer, abstract an electron pair. NADP+ remains locked in the active site of KV{beta} until membrane depolarization permits its release and replacement with NADPH. Sleep-inducing neurons use this voltage-gated oxidoreductase cycle to encode their recent lipid peroxidation history in the collective binary states of their KV{beta}-subunits; this biochemical memory influences--and is erased by--spike discharges driving sleep. The presence of a lipid peroxidation sensor at the core of homeostatic sleep control suggests that sleep protects neuronal membranes against oxidative damage. Indeed, brain phospholipids are depleted of vulnerable polyunsaturated fatty acyl chains after enforced waking, and slowing the removal of their carbonylic breakdown products increases the demand for sleep.

neuroscience↗

bioRxiv2024

Mitochondrial origins of the pressure to sleep

To obtain a comprehensive, unbiased view of molecular changes in the brain that may underpin the need for sleep, we have characterized the transcriptomes of single cells isolated from rested and sleep-deprived flies. Transcripts upregulated after sleep deprivation, in sleep-control neurons projecting to the dorsal fan-shaped body (dFBNs) but not ubiquitously in the brain, encode almost exclusively proteins with roles in mitochondrial respiration and ATP synthesis. These gene expression changes are accompanied by mitochondrial fragmentation, enhanced mitophagy, and an increase in the number of contacts between mitochondria and the endoplasmic reticulum, creating conduits for the replenishment of peroxidized lipids. The morphological changes are reversible after recovery sleep and blunted by the installation of an electron overflow in the respiratory chain. Inducing or preventing mitochondrial fission or fusion in dFBNs alters sleep and the electrical properties of sleep-control cells in opposite directions: hyperfused mitochondria increase, whereas fragmented mitochondria decrease, neuronal excitability and sleep. ATP levels in dFBNs rise after enforced waking because of diminished ATP consumption during the arousal-mediated inhibition of these neurons, which predisposes them to heightened oxidative stress. Consistent with this view, uncoupling electron flux from ATP synthesis relieves the pressure to sleep, while exacerbating mismatches between electron supply and ATP demand (by powering ATP synthesis with a light-driven proton pump) promotes sleep. Sleep, like ageing, may be an inescapable consequence of aerobic metabolism.

neuroscience↗

bioRxiv2022

Emergence of a time-independent population code in auditory cortex enables sound categorization and discrimination learning

The temporal structure of sensory inputs contains essential information for their interpretation by the brain1-9. Sensory systems represent these temporal cues through two codes: the temporal sequences of neuronal activity and the spatial patterns of neuronal firing rate3,7,10-20. However, it is still unknown which of these two coexisting codes causally drives sensory decisions3,10,20,21. To separate their contributions, we designed an optogenetic stimulation paradigm in the mouse auditory cortex to generate neuronal activity patterns differing exclusively along their temporal or spatial dimensions. Training mice to discriminate these patterns shows that they efficiently learn to discriminate spatial but not temporal patterns, indicating that spatial representations are necessary for sensory learning. In line with this result, we observed, based on large-scale neuronal recordings of the auditory system, that the auditory cortex is the first region in which spatial patterns efficiently represent temporal auditory cues varying over several hundred milliseconds. This feature is shared by the deep layers of neural networks trained to categorise time-varying sounds. Therefore, the emergence of a spatial code for temporal sensory cues is a necessary condition to associate temporally structured stimuli to decisions. We expect this constraint to be crucial for re-engineering perception by cortical stimulation.

neuroscience↗

bioRxiv2021

The Omicron variant is highly resistant against antibody-mediated neutralization - implications for control of the COVID-19 pandemic

The rapid spread of the SARS-CoV-2 Omicron variant suggests that the virus might become globally dominant. Further, the high number of mutations in the viral spike-protein raised concerns that the virus might evade antibodies induced by infection or vaccination. Here, we report that the Omicron spike was resistant against most therapeutic antibodies but remained susceptible to inhibition by Sotrovimab. Similarly, the Omicron spike evaded neutralization by antibodies from convalescent or BNT162b2-vaccinated individuals with 10- to 44-fold higher efficiency than the spike of the Delta variant. Neutralization of the Omicron spike by antibodies induced upon heterologous ChAdOx1/BNT162b2-vaccination or vaccination with three doses of BNT162b2 was more efficient, but the Omicron spike still evaded neutralization more efficiently than the Delta spike. These findings indicate that most therapeutic antibodies will be ineffective against the Omicron variant and that double immunization with BNT162b2 might not adequately protect against severe disease induced by this variant.

microbiology↗

bioRxiv2021

Increased lung cell entry of B.1.617.2 and evasion of antibodies induced by infection and BNT162b2 vaccination

The delta variant of SARS-CoV-2, B.1.617.2, emerged in India and has subsequently spread to over 80 countries. B.1.617.2 rapidly replaced B.1.1.7 as the dominant virus in the United Kingdom, resulting in a steep increase in new infections, and a similar development is expected for other countries. Effective countermeasures require information on susceptibility of B.1.617.2 to control by antibodies elicited by vaccines and used for COVID-19 therapy. We show, using pseudotyping, that B.1.617.2 evades control by antibodies induced upon infection and BNT162b2 vaccination, although with lower efficiency as compared to B.1.351. Further, we found that B.1.617.2 is resistant against Bamlanivimab, a monoclonal antibody with emergency use authorization for COVID-19 therapy. Finally, we show increased Calu-3-lung cell entry and enhanced cell-to-cell fusion of B.1.617.2, which may contribute to augmented transmissibility and pathogenicity of this variant. These results identify B.1.617.2 as an immune evasion variant with increased capacity to enter and fuse lung cells.

molecular biology↗

bioRxiv2021

SARS-CoV-2 variant B.1.617 is resistant to Bamlanivimab and evades antibodies induced by infection and vaccination

The emergence of SARS-CoV-2 variants threatens efforts to contain the COVID-19 pandemic. The number of COVID-19 cases and deaths in India has risen steeply in recent weeks and a novel SARS-CoV-2 variant, B.1.617, is believed to be responsible for many of these cases. The spike protein of B.1.617 harbors two mutations in the receptor binding domain, which interacts with the ACE2 receptor and constitutes the main target of neutralizing antibodies. Therefore, we analyzed whether B.1.617 is more adept in entering cells and/or evades antibody responses. B.1.617 entered two out of eight cell lines tested with slightly increased efficiency and was blocked by entry inhibitors. In contrast, B.1.617 was resistant against Bamlanivimab, an antibody used for COVID-19 treatment. Finally, B.1.617 evaded antibodies induced by infection or vaccination, although with moderate efficiency. Collectively, our study reveals that antibody evasion of B.1.617 may contribute to the rapid spread of this variant.

molecular biology↗

bioRxiv2021

SARS-CoV-2 mutations acquired in mink reduce antibody-mediated neutralization

Transmission of SARS-CoV-2 from humans to farmed mink was observed in Europe and the US. In the infected animals viral variants arose that harbored mutations in the spike (S) protein, the target of neutralizing antibodies, and these variants were transmitted back to humans. This raised concerns that mink might become a constant source of human infection with SARS-CoV-2 variants associated with an increased threat to human health and resulted in mass culling of mink. Here, we report that mutations frequently found in the S proteins of SARS-CoV-2 from mink were mostly compatible with efficient entry into human cells and its inhibition by soluble ACE2. In contrast, mutation Y453F reduced neutralization by an antibody with emergency use authorization for COVID-19 therapy and by sera/plasma from COVID-19 patients. These results suggest that antibody responses induced upon infection or certain antibodies used for treatment might offer insufficient protection against SARS-CoV-2 variants from mink.

molecular biology↗

bioRxiv2021

SARS-CoV-2 variants B.1.351 and B.1.1.248: Escape from therapeutic antibodies and antibodies induced by infection and vaccination

The global spread of SARS-CoV-2/COVID-19 is devastating health systems and economies worldwide. Recombinant or vaccine-induced neutralizing antibodies are used to combat the COVID-19 pandemic. However, recently emerged SARS-CoV-2 variants B.1.1.7 (UK), B.1.351 (South Africa) and B.1.1.248 (Brazil) harbor mutations in the viral spike (S) protein that may alter virus-host cell interactions and confer resistance to inhibitors and antibodies. Here, using pseudoparticles, we show that entry of UK, South Africa and Brazil variant into human cells is susceptible to blockade by entry inhibitors. In contrast, entry of the South Africa and Brazil variant was partially (Casirivimab) or fully (Bamlanivimab) resistant to antibodies used for COVID-19 treatment and was less efficiently inhibited by serum/plasma from convalescent or BNT162b2 vaccinated individuals. These results suggest that SARS-CoV-2 may escape antibody responses, which has important implications for efforts to contain the pandemic.

molecular biology↗

bioRxiv2018

Cortical recruitment determines learning dynamics and strategy

Salience is a broad and widely used concept in neuroscience whose neuronal correlates, however, remain elusive. In behavioral conditioning, salience is used to explain various effects, such as stimulus overshadowing, and refers to how fast and strongly a stimulus can be associated with a conditioned event. Here, we show that sounds of diverse quality, but equal intensity and perceptual detectability, can recruit different levels of population activity in mouse auditory cortex. When using these sounds as cues in a Go/NoGo discrimination task, the degree of cortical recruitment matches the salience parameter of a reinforcement learning model used to analyze learning speed. We test an essential prediction of this model by training mice to discriminate light-sculpted optogenetic activity patterns in auditory cortex, and verify that cortical recruitment causally determines association or overshadowing of the stimulus components. This demonstrates that cortical recruitment underlies major aspects of stimulus salience during reinforcement learning.

neuroscience↗

bioRxiv2018

Context-dependent signaling of coincident auditory and visual events in primary visual cortex

Detecting rapid coincident changes across sensory modalities is essential to recognize sudden threats and events. Using two-photon calcium imaging in identified cell types in awake mice, we show that auditory cortex (AC) neurons projecting to primary visual cortex (V1) preferentially encode the abrupt onsets of sounds. In V1, a sub-population of layer 1 interneurons gates this selective cross-modal information by a suppression specific to the absence of visual inputs. However, when large auditory onsets coincide with visual stimuli, visual responses are strongly boosted in V1. Thus, a dynamic asymmetric circuit across AC and V1 specifically identifies visual events starting simultaneously to sudden sounds, potentially catalyzing localization of new sound sources in the visual field.

neuroscience↗