Biology subjects

Brandt, R.

Publications and source records attributed to Brandt, R..

12 recordsLinked to original sources

bioRxiv2026

TomoSegNet: Augmented membrane segmentation for cryo-electron tomography by simulating the cellular context

Membrane segmentation is an essential task in the workflow for processing cryo-electron tomography data. Recently, machine learning algorithms have successfully been adopted to perform membrane segmentation. However, the performance of these approaches is limited by the training dataset, as models are trained from manual annotations, thereby hindering the models generalization and preventing the recovery of membranes that have vanished due to distortions. Here, we address these limitations by generating training data with a simulator. To provide a representative and realistic dataset, we have extended the current state-of-the-art in simulators for cryo-electron tomography by incorporating a biophysical model for membranes. We demonstrate that our machine learning model, trained solely from synthetic data, and thanks to the physical knowledge learned from the simulator, outperforms the current state-of-the-art for membrane segmentation in a diverse set of experimental data. This performance is particularly noteworthy in terms of recovering membranes lost due to imaging distortions.

bioinformatics↗

bioRxiv2025

Dissecting structural and functional determinants of microtubule stabilization through guided chemical modulation

Paclitaxel (PTX) is a widely used chemotherapeutic, but its efficacy is limited by peripheral neuropathy, likely due to its structural impact on neuronal microtubules (MTs). To decouple MT stabilization from adverse structural changes, we designed, synthetized and characterized several PTX analogues. Our compound 1b retains PTXs stabilizing activity in vitro and in cells, and the 1b-bound MTs preserve a native-like structure. These facts allowed us to investigate the influence of specific MT structural features on motor proteins behavior and interaction with MT-associated protein Tau. PTX-induced lattice expansion disrupted dynein-mediated retrograde transport and altered kinesin-1 motility. Additionally, PTX reduced Taus initial non-cooperative binding and envelopes growth rate while increasing dwell time and suppressing dynamic binding. In contrast, 1b preserved more physiological Tau dynamics. These findings reveal that MT stabilization and structural modulation can be separated and highlight the functional importance of MT heterogeneity in maintaining neuronal transport and MAP interactions.

molecular biology↗

bioRxiv2024

Application of modular isoxazoline-β2,2-amino acid-based peptidomimetic foldamers as a chemical model system for studying the tau misfolding mechanism.

Tauopathies are a group of neurodegenerative diseases characterized by the deposition of abnormal aggregates of tau protein into the brain. Tau is a microtubule-associated protein whose physiological role is related to the modulation of microtubule dynamics and the correct axonal transport in neurons. In tauopathies, the mechanism of conversion of the tau molecule from a physiological to a pathological form is still unclear. Nevertheless, two hexapeptide sequences, named PHF6 and PHF6*, located in the R3 and R2 repetition domains of tau, respectively, seem to be crucial in triggering the aggregation. In the native state, the hydrophobic residues of PHF6 and PHF6* are protected by a {beta}-hairpin-like structure. Contrary to that, the misfolded protein exposes hydrophobic residues, which can form hydrophobic interactions and drive the self-assembly process. Chemical model systems, other than being more straightforward and easily accessible than full-length proteins, can provide valuable insight, which is difficult to tease out by studying the natural systems directly, and can especially be employed to examine the structures and modes of folding of amyloid proteins. Here, we exploit the use of chemical systems to modulate and study the mechanism of tau misfolding and aggregation. By employing a new non-natural {beta}2,2-amino acid, we have induced the two hot spot sequences of tau (PHF6 and PHF6*) either to have a totally extended conformation or a {beta}-hairpin, according to the (S) or (R) stereochemistry of the scaffold, respectively. The aim of this work was to provide, through the interchange between {beta}-hairpin-like and extended conformations, a possible explanation for the mechanism of tau misfolding. We demonstrated that an extended conformation, exposing the hydrophobic residues of both sequences and driving away PHF6* from PHF6, can trigger the aggregation of tau and behaves as a seed-competent monomer model system. Conversely, a {beta}-hairpin imitates the favorable folding of tau, allowing the protein to maintain its soluble monomeric form. Furthermore, a {beta}-hairpin mimic, based on the chaperone protein Hsp90, demonstrated the further application of this type of peptidomimetic foldamers as both aggregation inhibitors and chaperone mimics for the corrective folding of tau in a neuronal environment.

biochemistry↗

bioRxiv2024

Synthetic chaperone based on Hsp90-Tau interaction inhibits pathological Tau aggregation and rescues physiological Tau-Microtubule interaction

The accumulation of intracellular aggregates of Tau protein is one main hallmark of Alzheimers disease (AD) and is the consequence of Tau conformational changes, increased phosphorylation, and self-association to form fibrillar aggregates. This pathological process prevents the physiological interaction of Tau with microtubules to the detriment of the structural integrity of neurons. In healthy cells, aberrant protein misfolding and aggregation are counteracted by chaperone proteins whose protective capacity decreases with age. The role of the chaperone Hsp90 and the mechanism by which it can prevent Tau aggregation toxicity are controversial. The innovative strategy of mimicking Hsp90 through the design of the {beta}-hairpin like peptidomimetic {beta}-Hsp90, inspired by two Hsp90/Tau interaction sequences, is presented here. {beta}-Hsp90 inhibits Tau aggregation both in vitro and in cells, restoring Taus physiological interaction with microtubules. {beta}-Hsp90, which interacts with the P1 region of Tau, is more effective than individual peptide sequences from the chaperone HSP90 and another {beta}-hairpin mimic based on Tau sequences. Moreover, {beta}-Hsp90 dramatically reduces AD-associated A{beta}1-42 aggregation, offering the development of a dual inhibitor. This work paves the way for the design of new drugs targeting devastating untreated amyloid diseases, by mimicking physiological chaperones with small synthetic peptide drugs.

pharmacology and toxicology↗

bioRxiv2024

DeepOrientation: Deep Orientation Estimation of Macromolecules in Cryo-electron tomography

Orientation estimation of macromolecules in cryo-electron tomography (cryo-ET) images is one of the fundamental steps in applying subtomogram averaging. The standard method in particle picking and orientation estimation is template matching (TM), which is computationally very expensive, with its performance depending linearly on the number of template orientations. In addition to conventional image processing methods like TM, the investigation of crowded cell environments using cryo-ET has also been attempted with deep learning (DL) methods. These attempts were restricted to macromolecule localization and identification while orientation estimation was not addressed due to a lack of a large enough dataset of ground truth annotations suitable for DL. To this end, we first generate a large-scale synthetic dataset of 450 tomograms containing almost 200K samples of two macromolecular structures using the PolNet simulator. Utilizing this synthetic dataset, we address the problem of particle orientation estimation as a regression problem by proposing a DL-based model based on multi-layer perceptron networks and a six-degree-of-freedom orientation representation. The iso-surface visualizations of the averaged subtomograms show that the predicted results by the network are overly similar to that of ground truth. Our work shows that orientation estimation of particles using DL methods is in principle possible provided that ground truth data is available. What remains to be solved is the gap between synthetic and experimental data. The source code is available at https://github.com/noushinha/DeepOrientation.

molecular biology↗

bioRxiv2024

Redox signaling by hydrogen peroxide modulates axonal microtubule organization and induces a specific phosphorylation signature of microtubule proteins distinct from distress

Many life processes are regulated by physiological redox signals, referred to as oxidative eustress. However, excessive oxidative stress can damage biomolecules and contribute to disease. The neuronal microtubule system is critically involved in axon homeostasis, regulation of axonal transport, and neurodegenerative processes. However, whether and how physiological redox signals affect axonal microtubules is largely unknown. Using live cell imaging and super- resolution microscopy, we show that subtoxic concentrations of the central redox metabolite hydrogen peroxide increase axonal microtubule dynamics, alter the structure of the axonal microtubule array, and affect the efficiency of axonal transport. We report that the mitochondria-targeting antioxidant SkQ1 and the microtubule stabilizer EpoD abolish the increase in microtubule dynamics. We found that oxidative eustress and distress specifically modulate the phosphorylation state of the microtubule system and induce a largely non- overlapping phosphorylation pattern of MAP1B as the main target. Cell-wide phosphoproteome analysis revealed that different signaling pathways are inversely activated by oxidative eustress and distress. Signaling via casein kinase (CK2) and pyruvate dehydrogenase kinases (PDK) is activated during eustress and signaling via mammalian target of rapamycin (mTOR) and serum/glucocorticoid-regulated protein kinase (SGK) is activated during distress. The results suggest that the redox metabolite and second messenger hydrogen peroxide induces rapid and local reorganization of the microtubule array in response to mitochondrial activity or as a messenger from neighboring cells by activating specific signaling cascades.

neuroscience↗

bioRxiv2024

Cell surface RNA expression modulates alveolar epithelial function

Glycosylated RNA (glycoRNA) has recently emerged as a novel constituent of the glycocalyx on cell surfaces, yet its biological functions remain largely unexplored. In this report, we present the first analysis of glycoRNA expression and functionality in alveolar epithelial cells. To this end, we optimized new techniques for the detection of glycoRNA on living cell surfaces and in cell membrane-associated RNA samples through in-gel imaging after labeling with fluorescent dye conjugates. Specifically, we used conjugation of Cy5-hydrazide following mild oxidation with sodium periodate for detection of the entire cell surface sialoglycoRNA pool. Conjugation of dibenzocyclooctyne-sulfo-Cy5 (DBCO-Sulfo-Cy5) in cells fed with tetraacetylated N-azidoacetyl-mannosamine (Ac4ManNAz) or 6-azido-L-fucose (FucAz) detected de novo formed sialoglycoRNA or fucoglycoRNA. Finally, biotinylated lectins in combination with infrared dye-conjugated streptavidin was used to differentiate between specific glycosidic linkages. Comparisons across primary alveolar epithelial cells and different alveolar-epithelial like cell lines revealed a cell-type specific variation in glycoRNA abundance. Treatment of primary alveolar epithelial cells with an RNAse cocktail reduced epithelial surface glycoRNA and was associated with a reduction in trans-epithelial electrical resistance and influenza A viral particle abundance. As such, the present work identifies glycoRNA as a novel component of the alveolar epithelial glycocalyx, suggesting its potential relevance in epithelial barrier regulation and viral infection.

physiology↗

bioRxiv2022

Quantitative live cell imaging of a tauopathy model enables the identification of a polypharmacological drug candidate that restores physiological microtubule regulation

Tauopathies such as Alzheimers disease are characterized by the aggregation and increased phosphorylation of the microtubule-associated protein tau. The pathological changes in tau are closely linked to neurodegeneration, making tau a prime candidate for intervention. However, the multiple facets of tau function and the lack of cellular tauopathy models that could support mechanism-based drug development hampers progress. Here we report the development of a live-cell imaging approach to quantitatively monitor pathological changes of human tau as it interacts with axonal microtubules. We show that a full-length aggregation-prone tau construct exhibits reduced interaction with microtubules as it increasingly aggregates. Through chemoinformatic analyses, we identified 2-phenyloxazole (PHOX) derivatives as putative polypharmacological small molecules that inhibit tau aggregation and modulate tau phosphorylation. We found that PHOX15 restores the physiological microtubule interaction of aggregation-prone tau in neurons and inhibits the first phase of tau aggregation in vitro. Furthermore, we report that PHOX15 inhibits the tau kinases GSK3{beta} and Cdk5, alters the kinome activity of model neurons, and reduces tau phosphorylation at disease-relevant sites. Molecular dynamics simulations highlight cryptic channel-like pockets crossing tau protofilaments and indicate that the binding of PHOX15 in one of the channels reduces the protofilaments ability to adopt a PHF-like conformation. The data show that our imaging approach provides a useful tool for identifying compounds that modulate tau-microtubule interaction in axons. We demonstrate that a polypharmacological approach to simultaneously treat tau aggregation and tau phosphorylation is able to restore physiological microtubule regulation, identifying PHOX15 as a promising drug candidate to counteract tau-induced neurodegeneration.

neuroscience↗

bioRxiv2022

Tau and α-synuclein shape microtubule organization and microtubule-dependent transport in neuronal dendrites

Tau and -synuclein are major players in neurodegenerative diseases, but their physiological role, particularly in dendrites, is poorly understood. Here we show that, surprisingly, lack of tau protein induces the development of a more elaborate dendritic arbor of hippocampal pyramidal cells in organotypic tissue. Using high-speed volumetric lattice light-sheet microscopy and single particle tracking, we found a more directional KIF1A-mediated transport in dendrites of Tau KO cells. Increased transport processivity correlated with longer and straighter dendritic microtubules as revealed by three-dimensional super-resolution microscopy of cultured hippocampal neurons. Unbiased mass spectrometric analysis of tissue showed highly increased expression of -synuclein in Tau KO hippocampi. Overexpression of -synuclein mimicked the transport characteristics observed in Tau KO cells. Our data indicate that tau and -synuclein shape microtubule-dependent transport in neuronal dendrites, thereby promoting dendritic arborization during maturation. Furthermore, the data demonstrate that transport efficiency and length and straightness of microtubules are correlated.

neuroscience↗

bioRxiv2020

COMPOSITUM 1 (COM1) contributes to the architectural simplification of barley inflorescence via cell wall-mediated and meristem identity signals

Grasses have varying inflorescence shapes; however, little is known about the genetic mechanisms specifying such shapes among tribes. We identified the grass-specific TCP transcription factor COMPOSITUM 1 (COM1) expressed in inflorescence meristematic boundaries of different grasses. COM1 specifies branch-inhibition in Triticeae (barley) versus branch-formation in non-Triticeae grasses. Analyses of cell size, cell walls and transcripts revealed barley COM1 regulates cell growth, affecting cell wall properties and signaling specifically in meristematic boundaries to establish identity of adjacent meristems. COM1 acts upstream of the boundary gene Liguleless1 and confers meristem identity partially independent of the COM2 pathway. Furthermore, COM1 is subject to purifying natural selection, thereby contributing to specification of the spike inflorescence shape. This meristem identity pathway has conceptual implications for both inflorescence evolution and molecular breeding in Triticeae.

plant biology↗

bioRxiv2018

Linked-read sequencing of gametes allows efficient genome-wide analysis of meiotic recombination

Meiotic crossovers (COs) ensure proper chromosome segregation and redistribute the genetic variation that is transmitted to the next generation. Existing methods for CO identification are challenged by large populations and the demand for genome-wide and fine-scale resolution. Taking advantage of linked-read sequencing, we developed a highly efficient method for genome-wide identification of COs at kilobase resolution in pooled recombinants. We first tested this method using a pool of Arabidopsis F2 recombinants, and obtained results that recapitulated those identified from the same plants using individual whole-genome sequencing. By applying this method to a pool of pollen DNA from a single F1 plant, we established a highly accurate CO landscape without generating or sequencing a single recombinant plant. The simplicity of this approach now enables the simultaneous generation and analysis of multiple CO landscapes and thereby allows for efficient comparison of genotypic and environmental effects on recombination, accelerating the pace at which the mechanisms for the regulation of recombination can be elucidated.

bioinformatics↗

bioRxiv2018

Cell type-dependent differential activation of ERK by oncogenic KRAS or BRAF in the mouse intestinal epithelium

Mutations activating the KRAS GTPase or the BRAF kinase are frequent in colorectal cancer and are thought to constitutively activate the terminal mitogen-activated protein kinase, ERK. Using mass cytometry, we found graded phosphorylation of ERK anti-correlated with cell differentiation in patient-derived colorectal cancer organoids, independent of KRAS mutational status. Reporter, single cell transcriptome and mass cytometry analyses showed that transgenic KRASG12V activated ERK in a cell type-specific pattern in mouse intestinal organoids. In contrast, transgenic BRAFV600E triggered high ERK activity and downstream gene expression in all intestinal cell types, followed by epithelial disorganisation. Quantitative network modelling from perturbation data revealed that activation of ERK is shaped by cell type-specific MEK to ERK feed forward and negative feedback signalling. We identified dual-specificity phosphatases as candidate modulators of ERK activity between intestinal cell types. Furthermore, we found that oncogenic KRAS, together with {beta}-Catenin, favoured expansion of crypt cells with high ERK activity. Our experiments highlight key differences between ERK activity elicited by the BRAF or KRAS oncogenes in colorectal cancer and find unexpected heterogeneity in a signalling pathway with fundamental relevance for cancer therapy.

cell biology↗