Biology subjects

Acosta, N.

Publications and source records attributed to Acosta, N..

7 recordsLinked to original sources

bioRxiv2024

mRNA expression is co-regulated by non-nucleolar RNA polymerase I

The relationship between gene transcription and chromatin organization is an area of active study. Due to its role in mRNA synthesis, many studies have focused on the regulaton of RNA polymerase II (Pol-II) function by supranucleosomal structure and vice-versa. In contrast, there is little work on the function of RNA polymerase I (Pol-I) in non-nucleolar chromatin. Prior work has shown that Pol-I engages with components of Pol-II on rDNA, but its role in global transcription and chromatin structure beyond the nucleolus has largely been ignored. By pairing auxin-inducible degron technology with nanoscopic imaging, RNA-Seq, and Hi-C, we found that Pol-I and Pol-II co-regulate conformationally defined chromatin domains and mRNA synthesis. Mechanistically, Pol-I maintains the positioning of intronic and intergenic chromatin within domains for the proper expression of exon elements. Consequently, Pol-I loss disrupts genome connectivity, in situ chromatin domains, and the expression of mRNA, genome-wide.

genomics↗

bioRxiv2024

Three-color single-molecule localization microscopy in chromatin

Super-resolution microscopy has revolutionized our ability to visualize structures below the diffraction limit of conventional optical microscopy and is particularly useful for investigating complex biological targets like chromatin. Chromatin exhibits a hierarchical organization with structural compartments and domains at different length scales, from nanometers to micrometers. Single molecule localization microscopy (SMLM) methods, such as STORM, are essential for studying chromatin at the supra-nucleosome level due to their ability to target epigenetic marks that determine chromatin organization. Multi-label imaging of chromatin is necessary to unpack its structural complexity. However, these efforts are challenged by the high-density nuclear environment, which can affect antibody binding affinities, diffusivity and non-specific interactions. Optimizing buffer conditions, fluorophore stability, and antibody specificity is crucial for achieving effective antibody conjugates. Here, we demonstrate a sequential immunolabeling protocol that reliably enables three-label studies within the dense nuclear environment. This protocol couples multiplexed localization datasets with a robust analysis algorithm, which utilizes localizations from one target as seed points for distance, density and multi-label joint affinity measurements to explore complex organization of all three targets. Applying this multiplexed algorithm to analyze distance and joint density reveals that heterochromatin and euchromatin are not-distinct territories, but that localization of transcription and euchromatin couple with the periphery of heterochromatic clusters. This work is a crucial step in molecular imaging of the dense nuclear environment as multi-label capacity enables for investigation of complex multi-component systems like chromatin with enhanced accuracy.

bioengineering↗

bioRxiv2024

Deep Learning-driven Automatic Nuclei Segmentation of Label-free Live Cell Chromatin-sensitive Partial Wave Spectroscopic Microscopy Imaging

Chromatin-sensitive Partial Wave Spectroscopic (csPWS) microscopy offers a non-invasive glimpse into the mass density distribution of cellular structures at the nanoscale, leveraging the spectroscopic information. Such capability allows us to analyze the chromatin structure and organization and the global transcriptional state of the cell nuclei for the study of its role in carcinogenesis. Accurate segmentation of the nuclei in csPWS microscopy images is an essential step in isolating them for further analysis. However, manual segmentation is error-prone, biased, time-consuming, and laborious, resulting in disrupted nuclear boundaries with partial or over-segmentation. Here, we present an innovative deep-learning-driven approach to automate the accurate nuclei segmentation of label-free live cell csPWS microscopy imaging data. Our approach, csPWS-seg, harnesses the Convolutional Neural Networks-based U-Net model with an attention mechanism to automate the accurate cell nuclei segmentation of csPWS microscopy images. We leveraged the structural, physical, and biological differences between the cytoplasm, nucleus, and nuclear periphery to construct three distinct csPWS feature images for nucleus segmentation. Using these images of HCT116 cells, csPWS-seg achieved superior performance with a median Intersection over Union (IoU) of 0.80 and a Dice Similarity Coefficient (DSC) score of 0.88. The csPWS-seg overcame the segmentation performance over the baseline U-Net model and another attention-based model, SE-U-Net, marking a significant improvement in segmentation accuracy. Further, we analyzed the performance of our proposed model with four loss functions: binary cross-entropy loss, focal loss, dice loss, and Jaccard loss. The csPWS-seg with focal loss provided the best results compared to other loss functions. The automatic and accurate nuclei segmentation offered by the csPWS-seg not only automates, accelerates, and streamlines csPWS data analysis but also enhances the reliability of subsequent chromatin analysis research, paving the way for more accurate diagnostics, treatment, and understanding of cellular mechanisms for carcinogenesis.

bioengineering↗

bioRxiv2024

Nuclear blebs are associated with destabilized chromatin packing domains

Disrupted nuclear shape is associated with multiple pathological processes including premature aging disorders, cancer-relevant chromosomal rearrangements, and DNA damage. Nuclear blebs (i.e., herniations of the nuclear envelope) have been induced by (1) nuclear compression, (2) nuclear migration (e.g., cancer metastasis), (3) actin contraction, (4) lamin mutation or depletion, and (5) heterochromatin enzyme inhibition. Recent work has shown that chromatin transformation is a hallmark of bleb formation, but the transformation of higher-order structures in blebs is not well understood. As higher-order chromatin has been shown to assemble into nanoscopic packing domains, we investigated if (1) packing domain organization is altered within nuclear blebs and (2) if alteration in packing domain structure contributed to bleb formation. Using Dual-Partial Wave Spectroscopic microscopy, we show that chromatin packing domains within blebs are transformed both by B-type lamin depletion and the inhibition of heterochromatin enzymes compared to the nuclear body. Pairing these results with single-molecule localization microscopy of constitutive heterochromatin, we show fragmentation of nanoscopic heterochromatin domains within bleb domains. Overall, these findings indicate that translocation into blebs results in a fragmented higher-order chromatin structure. SUMMARY STATEMENTNuclear blebs are linked to various pathologies, including cancer and premature aging disorders. We investigate alterations in higher-order chromatin structure within blebs, revealing fragmentation of nanoscopic heterochromatin domains.

bioengineering↗

bioRxiv2024

Formamide denaturation of double-stranded DNA for fluorescence in situ hybridization (FISH) distorts nanoscale chromatin structure

As imaging techniques rapidly evolve to probe nanoscale genome organization at higher resolution, it is critical to consider how the reagents and procedures involved in sample preparation affect chromatin at the relevant length scales. Here, we investigate the effects of fluorescent labeling of DNA sequences within chromatin using the gold standard technique of three-dimensional fluorescence in situ hybridization (3D FISH). The chemical reagents involved in the 3D FISH protocol, specifically formamide, cause significant alterations to the sub-200 nm (sub-Mbp) chromatin structure. Alternatively, two labeling methods that do not rely on formamide denaturation, resolution after single-strand exonuclease resection (RASER)-FISH and clustered regularly interspaced short palindromic repeats (CRISPR)-Sirius, had minimal impact on the three-dimensional organization of chromatin. We present a polymer physics-based analysis of these protocols with guidelines for their interpretation when assessing chromatin structure using currently available techniques.

biophysics↗

bioRxiv2023

The mitochondrial calcium uniporter is necessary for synaptic plasticity and proper mitochondrial morphology and distribution in the distal dendrites of CA2 neurons

Mitochondria are dynamic organelles that are morphologically and functionally diverse across cell types and subcellular compartments in order to meet unique energy demands. Mitochondrial dysfunction has been implicated in a wide variety of neurological disorders, including psychiatric disorders like schizophrenia and bipolar disorder. Despite it being well known that mitochondria are essential for synaptic transmission and synaptic plasticity, the mechanisms regulating mitochondria in support of normal synapse function are incompletely understood. The mitochondrial calcium uniporter (MCU) regulates calcium entry into the mitochondria, which in turn regulates the bioenergetics and distribution of mitochondria to active synapses. Evidence suggests that calcium influx via MCU couples neuronal activity to mitochondrial metabolism and ATP production, which would allow neurons to rapidly adapt to changing energy demands. Intriguingly, MCU is uniquely enriched in hippocampal CA2 distal dendrites relative to neighboring hippocampal CA1 or CA3 distal dendrites, however, the functional significance of this enrichment is not clear. Synapses from the entorhinal cortex layer II (ECII) onto CA2 distal dendrites readily express long term potentiation (LTP), unlike the LTP- resistant synapses from CA3 onto CA2 proximal dendrites, but the mechanisms underlying these different plasticity profiles are unknown. We hypothesized that enrichment of MCU near ECII-CA2 synapses promotes LTP in an otherwise plasticity-restricted cell type. Using a CA2-specific MCU knockout (cKO) mouse, we found that MCU is required for LTP at distal dendrite synapses but does not affect the lack of LTP at proximal dendrite synapses. Loss of LTP at ECII-CA2 synapses correlated with a trend for decreased spine density in CA2 distal dendrites of cKO mice compared to control (CTL) mice, which was predominantly seen in immature spines. Moreover, mitochondria were significantly smaller and more numerous across all dendritic layers of CA2 in cKO mice compared to CTL mice, suggesting an overall increase in mitochondrial fragmentation. Fragmented mitochondria might have functional changes, such as altered ATP production, that might explain a deficit in synaptic plasticity. Collectively, our data reveal that MCU regulates layer-specific forms of plasticity in CA2 dendrites, potentially by maintaining proper mitochondria morphology and distribution within dendrites. Differences in MCU expression across different cell types and circuits might be a general mechanism to tune the sensitivity of mitochondria to cytoplasmic calcium levels to power synaptic plasticity. MAIN TAKE HOME POINTSO_LIThe mitochondrial calcium uniporter (MCU) regulates plasticity selectively at synapses in CA2 distal dendrites. C_LIO_LIThe MCU-cKO induced LTP deficit correlates with a trending reduction in spine density in CA2 distal dendrites. C_LIO_LILoss of MCU in CA2 results in ultrastructural changes in dendritic mitochondria that suggest an increase in mitochondrial fragmentation. These ultrastructural changes could result in functional consequences, such as decreased ATP production, that could underlie the plasticity deficit. C_LIO_LIDendritic mitochondrial fragmentation in MCU cKO occurred throughout the dendritic laminae, suggesting that MCU is dispensable for establishing layer-specific mitochondrial structural diversity. C_LI

neuroscience↗

bioRxiv2023

Depletion of lamins B1 and B2 alters chromatin mobility and induces differential gene expression by a mesoscale-motion dependent mechanism

BACKGROUNDB-type lamins are critical nuclear envelope proteins that interact with the 3D genomic architecture. However, identifying the direct roles of B-lamins on dynamic genome organization has been challenging as their joint depletion severely impacts cell viability. To overcome this, we engineered mammalian cells to rapidly and completely degrade endogenous B-type lamins using Auxin-inducible degron (AID) technology. RESULTSPaired with a suite of novel technologies, live-cell Dual Partial Wave Spectroscopic (Dual-PWS) microscopy, in situ Hi-C, and CRISPR-Sirius, we demonstrate that lamin B1 and lamin B2 depletion transforms chromatin mobility, heterochromatin positioning, gene expression, and loci-positioning with minimal disruption to mesoscale chromatin folding. Using the AID system, we show that the disruption of B-lamins alters gene expression both within and outside lamin associated domains, with distinct mechanistic patterns depending on their localization. Critically, we demonstrate that chromatin dynamics, positioning of constitutive and facultative heterochromatic markers, and chromosome positioning near the nuclear periphery are significantly altered, indicating that the mechanism of action of B-type lamins is derived from their role in maintaining chromatin dynamics and spatial positioning. CONCLUSIONSOur findings suggest that the mechanistic role of B-type lamins is stabilization of heterochromatin and chromosomal positioning along the nuclear periphery. We conclude that degrading lamin B1 and lamin B2 has several functional consequences related to both structural disease and cancer.

bioengineering↗