Biology subjects

Muresan, L.

Publications and source records attributed to Muresan, L..

14 recordsLinked to original sources

bioRxiv2024

3D super-resolution imaging of PSD95 reveals an abundance of diffuse protein supercomplexes in the mouse brain

PSD95 is an abundant scaffolding protein that assembles multiprotein complexes controlling synaptic physiology and behavior. Confocal microscopy has previously shown that PSD95 is enriched in the postsynaptic terminals of excitatory synapses and 2D super-resolution microscopy further revealed that it forms nanoclusters. In this study, we utilized 3D super-resolution microscopy to examine the nanoarchitecture of PSD95 in the mouse brain, characterizing over 8 million molecules. While we were able to identify structural subtypes previously reported, imaging in 3D allowed us to classify these with higher accuracy. Furthermore, 3D super-resolution microscopy enabled the quantification of protein levels, revealing an abundance of PSD95 molecules existed outside of synapses as a diffuse population of supercomplexes, containing multiple copies of PSD95. Further analysis of the supercomplexes containing two units identified two populations: one that had PSD95 molecules separated by 39 {+/-} 2 nm, and a second with a separation of 94 {+/-} 27 nm. These results suggest that PSD95 supercomplexes containing multiple protein copies assemble outside the synapse and then integrate into the synapse to form a supramolecular nanocluster architecture.

biophysics↗

bioRxiv2024

Live imaging human embryos reveals mitotic errors and lineage specification prior to implantation

Meiotic and mitotic chromosome segregation errors in human development have been studied mostly prior to and at the time of fertilisation. Despite chromosomal errors being a leading cause of miscarriage and infertility, chromosome missegregation has not been extensively studied at later stages of human development. Here we optimised labelling, light-sheet live imaging and semi-quantitative analysis of human embryos and reveal chromosome segregation errors just prior to implantation. We found that human embryos exhibited a number of chromosome missegregation events including multipolar spindle formation, lagging chromosomes, misalignment and chromosome slippage. We found that the majority of lagging chromosomes were passively inherited by one of the daughter cells, instead of reincorporating into the nuclei, suggesting a distinct pattern of micronuclei inheritance. By semi-automated segmentation, we tracked the position of labelled cells in human embryos and observed that while most labelled cells remained segregated to the outside, and therefore restricted to a placental-progenitor fate, there was evidence of a rare cell migration to cells positioned on the inside, which suggests that there may be plasticity. Altogether, we found that mitotic chromosome segregation errors arise just prior to implantation, which has implications for our understanding of biological events that contribute to aneuploidy mosaicism.

developmental biology↗

bioRxiv2023

Transcriptionally active chromatin loops contain both active and inactive histone modifications that exhibit exclusivity at the level of nucleosome clusters

Chromatin state is thought to impart regulatory function to the underlying DNA sequence. This can be established through histone modifications, and chromatin organisation, but exactly how these factors relate to one another to regulate gene expression is unclear. In this study, we have used super-resolution microscopy to image the Y loops of Drosophila melanogaster primary spermatocytes, which are enormous transcriptionally active chromatin fibres, each representing single transcription units that are individually resolvable in the nuclear interior. We previously found that the Y loops consist of regular clusters of nucleosomes, with an estimated median of 54 nucleosomes per cluster with wide variation. In this study, we report that the histone modifications H3K4me3, H3K27me3, and H3K36me3 are also clustered along the Y loops, with H3K4me3 more associated with diffuse chromatin compared to H3K27me3. These histone modifications form domains that can be stretches of Y loop chromatin micrometres long, or can be in short alternating domains. The different histone modifications are associated with different sizes of chromatin clusters and unique morphologies. Strikingly, a single chromatin cluster almost always only contains only one type of the histone modifications that were labelled, suggesting exclusivity, and therefore regulation at the level of individual chromatin clusters. The active mark H3K36me3 is more associated with actively elongating RNA polymerase II than H3K27me3, with polymerase often appearing on what are assumed to be looping regions on the periphery of chromatin clusters. These results provide a foundation for understanding the relationship between chromatin state, chromatin organisation, and transcription regulation - with potential implications for pause-release dynamics, splicing complex organisation and chromatin dynamics during polymerase progression along a gene.

molecular biology↗

bioRxiv2023

Changes in searching behaviour of CSL transcription complexes in Notch active conditions

During development cells receive a variety of signals, which are of crucial importance to their fate determination. One such source of signal is the Notch signalling pathway, where Notch activity regulates expression of target genes through the core transcription factor CSL. To understand changes in transcription factor behaviour that lead to transcriptional changes in Notch active cells, we have probed CSL behaviours in real time, using in vivo Single Molecule Localisation Microscopy. Trajectory analysis reveals that Notch-On conditions increase the fraction of bound CSL molecules, but also the proportion of molecules with exploratory behaviours. These properties are shared by the co-activator Mastermind. Furthermore, both CSL and Mastermind, exhibit characteristics of local exploration near a Notch target locus. A similar behaviour is observed for CSL molecules diffusing in the vicinity of other bound CSL clusters. We suggest therefore that CSL acquires an exploratory behaviour when part of the activation complex, favouring local searching and retention close to its target enhancers. This change explains how CSL can efficiently increases its occupancy at target sites in Notch-ON conditions.

developmental biology↗

bioRxiv2023

Mechanical stress combines with planar polarised patterning during metaphase to orient embryonic epithelial cell divisions

The orientation of cell division (OCD) in the plane of epithelia drives tissue morphogenesis and relaxes stresses, with errors leading to pathologies. Stress anisotropy, cell elongation and planar polarisation can all contribute to the OCD, but it is unclear how these interact in vivo. In the planar polarised Drosophila embryonic ectoderm during axis elongation, planar OCD is highly variable. We show that both a temporary reversal of tissue stress anisotropy and local compression from neighbouring dividing cells re-orient mitotic spindles during metaphase, independently of interphase cell elongation. Isotropic cells align their OCD to the anterior-posterior (AP) embryonic axis, mediated by tissue-wide planar polarised Myosin II, while the spindle of elongated cells is sterically constrained to cell long axes. Thus AP-patterning ensures that cell division combines with cell rearrangement to extend the body axis, except when strong local stress anisotropy is dissipated by cells dividing according to their elongation.

biophysics↗

bioRxiv2022

The individual vulnerability to develop compulsive adjunctive behavior is associated with the recruitment of activity-regulated cytoskeleton-associated protein (Arc) within the Locus Coeruleus

Some compulsive disorders have been considered to stem from the loss of control over coping strategies, such as displacement. However, the cellular mechanisms involved in the acquisition of coping behaviors and their ensuing compulsive manifestation in vulnerable individuals have not been elucidated. Considering the role of the locus coeruleus (LC) noradrenaline dependent system in stress and related excessive behaviors, we hypothesised that neuroplastic changes in the LC may be involved in the acquisition of an adjunctive polydipsic water drinking, a prototypical displacement behavior, and the subsequent development of compulsion in vulnerable individuals. Thus, male Sprague Dawley rats were characterised for their tendency, or not, to develop compulsive polydipsic drinking in a schedule-induced polydipsia (SIP) procedure before their fresh brains were harvested. A new quantification tool for RNAscope assays revealed that the development of compulsive adjunctive behavior was associated with a low mRNA copy number of the plasticity marker Arc in the LC which appeared to be driven by specific adaptations in an ensemble of tyrosine hydroxylase (TH)+, zif268-neurons. This ensemble was specifically engaged by the expression of compulsive adjunctive behavior, not by stress, because its functional recruitment was not observed in individuals that no longer had access to the water bottle before sacrifice while it consistently correlated with the levels of polydipsic water drinking only when it had become compulsive. Together these findings suggest that downregulation of Arc mRNA levels in a population of a TH+zif268-LC neurons represents a signature of the tendency to develop compulsive coping behaviors.

neuroscience↗

bioRxiv2022

The anatomy of transcriptionally active chromatin loops in Drosophila primary spermatocytes using super-resolution microscopy

While the biochemistry of gene transcription has been well studied, our understanding of how this process is organised in 3D within the intact nucleus is less well understood. Here we investigate the structure of actively transcribed chromatin and the architecture of its interaction with active RNA polymerase. For this analysis, we have used super-resolution microscopy to image the Drosophila melanogaster Y loops which represent huge, several megabases long, single transcription units. The Y loops provide a particularly amenable model system for transcriptionally active chromatin. We find that, although these transcribed loops are decondensed they are not organised as extended 10nm fibres, but rather they largely consist of chains of nucleosome clusters. The average width of each cluster is around 50nm. We find that foci of active RNA polymerase are generally located off the main fibre axis on the periphery of the nucleosome clusters. Foci of RNA polymerase and nascent transcripts are distributed around the Y loops rather than being clustered in individual transcription factories. However, as the RNA polymerase foci are considerably less prevalent than the nucleosome clusters, the organisation of this active chromatin into chains of nucleosome clusters is unlikely to be determined by the activity of the polymerases transcribing the Y loops. These results provide a foundation for understanding the topological relationship between chromatin and the process of gene transcription.

molecular biology↗

bioRxiv2022

BigNeuron: A resource to benchmark and predict best-performing algorithms for automated reconstruction of neuronal morphology

BigNeuron is an open community bench-testing platform combining the expertise of neuroscientists and computer scientists toward the goal of setting open standards for accurate and fast automatic neuron reconstruction. The project gathered a diverse set of image volumes across several species representative of the data obtained in most neuroscience laboratories interested in neuron reconstruction. Here we report generated gold standard manual annotations for a selected subset of the available imaging datasets and quantified reconstruction quality for 35 automatic reconstruction algorithms. Together with image quality features, the data were pooled in an interactive web application that allows users and developers to perform principal component analysis, t-distributed stochastic neighbor embedding, correlation and clustering, visualization of imaging and reconstruction data, and benchmarking of automatic reconstruction algorithms in user-defined data subsets. Our results show that image quality metrics explain most of the variance in the data, followed by neuromorphological features related to neuron size. By benchmarking automatic reconstruction algorithms, we observed that diverse algorithms can provide complementary information toward obtaining accurate results and developed a novel algorithm to iteratively combine methods and generate consensus reconstructions. The consensus trees obtained provide estimates of the neuron structure ground truth that typically outperform single algorithms. Finally, to aid users in predicting the most accurate automatic reconstruction results without manual annotations for comparison, we used support vector machine regression to predict reconstruction quality given an image volume and a set of automatic reconstructions.

bioinformatics↗

bioRxiv2021

The zebrafish presomitic mesoderm elongates through compression-extension

In vertebrate embryos the presomitic mesoderm become progressively segmented into somites at the anterior end while extending along the anterior-posterior axis. A commonly adopted model to explain how this tissue elongates is that of posterior growth, driven in part by the addition of new cells from uncommitted progenitor populations in the tailbud. However, in zebrafish, much of somitogenesis is associated with an absence of overall volume increase and posterior progenitors do not contribute new cells until the final stages of somitogenesis. Here, we perform a comprehensive 3D morphometric analysis of the paraxial mesoderm and reveal that extension is linked to a volumetric decrease, compression in both dorsal-ventral and medio-lateral axes, and an increase in cell density. We also find that individual cells decrease in their cell volume over successive somite stages. Live cell tracking confirms that much of this tissue deformation occurs within the presomitic mesoderm progenitor zone and is associated with non-directional rearrangement. Furthermore, unlike the trunk somites that are laid down during gastrulation, tail somites develop from a tissue that can continue to elongate in the absence of functional PCP signalling. Taken together, we propose a compression-extension mechanism of tissue elongation that highlights the need to better understand the role of tissue intrinsic and extrinsic forces play in regulating morphogenesis.

developmental biology↗

bioRxiv2020

Single Molecule Light Field Microscopy

We introduce single molecule light field microscopy (SMLFM), a novel 3D single molecule localization technique that is capable of up to 20 nm isotropic precision across a 6 m depth of field. SMLFM can be readily implemented by installing a refractive microlens array into the conjugate back focal plane of any widefield single molecule localization system. We demonstrate that 3D localization can be performed by post-processing 2D localization data generated by common, widely-used, algorithms. In this work we benchmark the performance of SMLFM and finally showcase its capabilities by imaging fluorescently labeled membranes of fixed eukaryotic cells below the diffraction limit.

biophysics↗

bioRxiv2019

Super-resolving light fields in microscopy: depth from disparity

Single molecule localisation microscopy (SMLM) has opened a new window for imaging fluorescently labelled biological specimens. Common 3D SMLM techniques enable data collection across an axial range of 1 - 5m with high precision. Despite the success of 3D single molecule imaging there is a real need to image larger volumes. Here we demonstrate, through simulation and experiment, the potential of Single Molecule Light Field Microscopy (SMLFM) for extended depth-of-field super-resolution imaging, extracting 3D point source position by measuring the disparity between localizations of a point emitter in multiple perspective views.

bioengineering↗

bioRxiv2018

MAVS polymers smaller than 80 nm induce mitochondrial membrane remodeling and interferon signaling

Double-stranded RNA (dsRNA) is a potent proinflammatory signature of viral infection. Oligomerization of RIG-I-like receptors on cytosolic dsRNA nucleates self-assembly of the mitochondrial antiviral signaling protein (MAVS). In the current signaling model, the caspase recruitment domains of MAVS form helical fibrils that self-propagate like prions to promote signaling complex assembly. However, there is no conclusive evidence that MAVS forms fibrils in cells or with the transmembrane anchor present. We show here with super-resolution light microscopy that MAVS activation by dsRNA induces mitochondrial membrane remodeling. Quantitative image analysis at imaging resolutions as high as 32 nm shows that in the cellular context MAVS signaling complexes and the fibrils within them are smaller than 80 nm. The transmembrane domain of MAVS is required for its membrane remodeling, interferon signaling and proapoptotic activities. We conclude that membrane tethering of MAVS restrains its polymerization and contributes to mitochondrial remodeling and apoptosis upon dsRNA sensing.

molecular biology↗

bioRxiv2018

Neuromesodermal Progenitors are a Conserved Source of Spinal Cord with Divergent Growth Dynamics

During gastrulation, embryonic cells become specified into distinct germ layers. In mouse, this continues throughout somitogenesis from a population of bipotent stem cells called neuromesodermal progenitors (NMps). However, the degree self-renewal is associated with NMps in the fast-developing zebrafish embryo is unclear. With a genetic clone tracing method, we labelled early embryonic progenitors and find a strong clonal similarity between spinal cord and mesoderm tissues. We then followed individual cell lineages by light-sheet imaging and reveal a common neuromesodermal lineage contribution to a subset of spinal cord tissue across the anterior-posterior body axis. An initial population subdivides at mid gastrula stages and is directly allocated to neural and mesodermal compartments during gastrulation. A second population in the tailbud undergoes delayed allocation to contribute to the neural and mesodermal compartment only at late somitogenesis. We suggest that NMps undergo vastly different rates of differentiation and growth in a species-specific manner.

developmental biology↗

bioRxiv2017

Par3/Baz levels control epithelial folding at actomyosin-enriched compartmental boundaries

Epithelial folding is crucial to shape embryos and tissues during development. Here we investigate the coupling between epithelial folding and actomyosin-rich boundaries. The mechanistic relationship between the two is unclear, since actomyosin-rich boundaries can be either associated with folds or not, while epithelial folding has been found to be either dependent or independent of actomyosin contractility. Here we investigate the shallow folds that form at compartmental parasegment boundaries (PSBs) in the early Drosophila embryo. First, we demonstrate that formation of these folds is dependent on the contractility of supracellular actomyosin cables. When the Myosin II phosphatase Flawing is depleted at the PSBs, actomyosin contractility increases, resulting in deeper folds. Conversely, in wingless mutants, actomyosin enrichment and increased contractility at PSBs are lost and this correlates with an absence of folding. Furthermore, when we make ectopic PSBs by expressing Wingless ubiquitously, the ectopic boundaries become enriched in actomyosin and epithelial folds form. Ectopic PSB folds, however, are much deeper than endogenous ones, indicating that epithelial folding is normally under inhibitory control. We present evidence that depletion of Bazooka/Par-3 levels at PSB cell-cell contacts, which is under Wingless signaling control, is responsible for this inhibition. Bazooka is found depleted at endogenous but not ectopic PSBs. In embryos overexpressing Bazooka, endogenous PSB folds form earlier and are much deeper. To ask how local signaling at the boundaries control Bazooka levels at cell-cell contacts, we examined embryos that ectopically expressed Wingless in an hedgehog mutant background. In these embryos, inhibition of folding is rescued, with ectopic PSBs now forming shallow folds as endogenous PSBs. Bazooka is depleted at these ectopic PSBs in absence of Hedgehog, suggesting an opposite effect of Wingless and Hedgehog signaling on Bazooka levels at PSB cell-cell contacts. This uncovers a new role of Bazooka in controlling fold formation at actomyosin-rich compartmental boundaries.

cell biology↗