scholarly journals Nuclear transcriptomes at high resolution using retooled INTACT

2017 ◽  
Author(s):  
Mauricio A. Reynoso ◽  
Germain C. Pauluzzi ◽  
Kaisa Kajala ◽  
Sean Cabanlit ◽  
Joel Velasco ◽  
...  
Keyword(s):  
High Resolution ◽  
Nuclear Envelope ◽  
Affinity Purification ◽  
Cell Types ◽  
Specific Cell ◽  
Root Tips ◽  
E Coli ◽  
Dna Insertion ◽  
Improved Technology ◽  
Rrna Degradation

AbstractIsolated nuclei provide access to early steps in gene regulation involving chromatin as well as transcript production and processing. Here we describe transfer of the Isolation of Nuclei from TAgged specific Cell Types (INTACT) to the monocot rice (Oryza sativa L.). The purification of biotinylated nuclei was redesigned by replacing the outer nuclear envelope-targeting domain of the Nuclear Tagging Fusion (NTF) protein with an outer nuclear envelope-anchored domain. This modified NTF was combined with codon optimized E. coli BirA in a single T-DNA construct. We also developed inexpensive methods for INTACT, T-DNA insertion mapping and profiling of the complete nuclear transcriptome, including a rRNA degradation procedure that minimizes pre-rRNA transcripts. A high-resolution comparison of nuclear and steady-state poly (A)+ transcript populations of seedling root tips confirmed the capture of pre-mRNA and exposed distinctions in diversity and abundance of the nuclear and total transcriptomes. This retooled INTACT can enable high-resolution monitoring of the nuclear transcriptome and chromatin in specific cell-types of rice and other species.Summary:Improved technology and methodology for affinity purification of nuclei and analysis of nuclear transcriptomes, chromatin and other nuclear components.

scholarly journals Assembly of Caenorhabditis elegans acentrosomal spindles occurs without evident microtubule-organizing centers and requires microtubule sorting by KLP-18/kinesin-12 and MESP-1

2016 ◽  
Vol 27 (20) ◽  
pp. 3122-3131 ◽  
Author(s):  
Ian D. Wolff ◽  
Michael V. Tran ◽  
Timothy J. Mullen ◽  
Anne M. Villeneuve ◽  
Sarah M. Wignall
Keyword(s):  
Caenorhabditis Elegans ◽  
High Resolution ◽  
Nuclear Envelope ◽  
Spindle Assembly ◽  
Cell Types ◽  
High Resolution Imaging ◽  
Spindle Formation ◽  
Mixed Polarity ◽  
Resolution Imaging ◽  
Meiosis I

Although centrosomes contribute to spindle formation in most cell types, oocytes of many species are acentrosomal and must organize spindles in their absence. Here we investigate this process in Caenorhabditis elegans, detailing how acentrosomal spindles form and revealing mechanisms required to establish bipolarity. Using high-resolution imaging, we find that in meiosis I, microtubules initially form a “cage-like” structure inside the disassembling nuclear envelope. This structure reorganizes so that minus ends are sorted to the periphery of the array, forming multiple nascent poles that then coalesce until bipolarity is achieved. In meiosis II, microtubules nucleate in the vicinity of chromosomes but then undergo similar sorting and pole formation events. We further show that KLP-18/kinesin-12 and MESP-1, previously shown to be required for spindle bipolarity, likely contribute to bipolarity by sorting microtubules. After their depletion, minus ends are not sorted outward at the early stages of spindle assembly and instead converge. These proteins colocalize on microtubules, are interdependent for localization, and can interact, suggesting that they work together. We propose that KLP-18/kinesin-12 and MESP-1 form a complex that functions to sort microtubules of mixed polarity into a configuration in which minus ends are away from the chromosomes, enabling formation of nascent poles.

scholarly journals Cell-type specific visualization and biochemical isolation of endogenous synaptic proteins in mice

2018 ◽  
Author(s):  
Fei Zhu ◽  
Mark O. Collins ◽  
Johan Harmse ◽  
Jyoti S. Choudhary ◽  
Seth G. N. Grant ◽  
...  
Keyword(s):  
Fluorescent Proteins ◽  
Protein Complexes ◽  
Neurological Diseases ◽  
Affinity Purification ◽  
Cell Types ◽  
Mouse Genetics ◽  
Synaptic Proteins ◽  
Specific Cell ◽  
Cell Type Specific ◽  
Molecular Complexity

AbstractIn recent years, the remarkable molecular complexity of synapses has been revealed, with over 1000 proteins identified in the synapse proteome. Although it is known that different receptors and other synaptic proteins are present in different types of neurons and synapses, the extent of synapse diversity across the brain is largely unknown, mainly owing to technical limitations. Combining mouse genetics and proteomics we have previously reported highly efficient methods for purification of synaptic protein complexes under native conditions. In that approach, tandem affinity purification (TAP) tags were fused to the carboxyl terminus of PSD95 using gene targeting in mice. Here we report an approach that restricts tagging of endogenous PSD95 to cells expressing Cre recombinase. In addition, we developed a labelling strategy enabling visualization of endogenous PSD95 tagged by fluorescent proteins in Cre-expressing cells. We demonstrate the feasibility of proteomic characterisation of synapse proteomes and visualization of synapse proteins in specific cell types. We find that composition of PSD95 complexes purified from specific cell types differs from those extracted from tissues with diverse cellular composition. Therefore, these novel conditional PSD95 tagging lines will not only serve as powerful tools for precisely dissecting synapse diversity in specific subsets of regions/neuronal cells, but also provide an opportunity to better understand brain region-specific alterations associated with various psychiatric/neurological diseases. The newly developed conditional gene tagging methods can be applied to many different synaptic proteins and will thus facilitate research on the molecular complexity of synapses.

scholarly journals High-resolution contrast-enhanced microCT reveals the true three-dimensional morphology of the murine placenta

2019 ◽  
Vol 116 (28) ◽  
pp. 13927-13936 ◽  
Author(s):  
Katrien De Clercq ◽  
Eleonora Persoons ◽  
Tina Napso ◽  
Catherine Luyten ◽  
Tatjana N. Parac-Vogt ◽  
...  
Keyword(s):  
High Resolution ◽  
Volume Fraction ◽  
Three Dimensional ◽  
Cell Types ◽  
Mouse Strains ◽  
Specific Cell ◽  
Placental Development ◽  
Individual Gene ◽  
Contrast Enhanced ◽  
Late Stages

Genetic engineering of the mouse genome identified many genes that are essential for embryogenesis. Remarkably, the prevalence of concomitant placental defects in embryonic lethal mutants is highly underestimated and indicates the importance of detailed placental analysis when phenotyping new individual gene knockouts. Here we introduce high-resolution contrast-enhanced microfocus computed tomography (CE-CT) as a nondestructive, high-throughput technique to evaluate the 3D placental morphology. Using a contrast agent, zirconium-substituted Keggin polyoxometalate (Zr-POM), the soft tissue of the placenta (i.e., different layers and cell types and its vasculature) was imaged with a resolution of 3.5 µm voxel size. This approach allowed us to visualize and study early and late stages of placental development. Moreover, CE-CT provides a method to precisely quantify placental parameters (i.e., volumes, volume fraction, ratio of different placental layers, and volumes of specific cell populations) that are crucial for statistical comparison studies. The CE-CT assessment of the 3D morphology of the placentas was validated (i) by comparison with standard histological studies; (ii) by evaluating placentas from 2 different mouse strains, 129S6 and C57BL/6J mice; and (iii) by confirming the placental phenotype of mice lacking phosphoinositol 3-kinase (PI3K)-p110α. Finally, the Zr-POM–based CE-CT allowed for inspection of the vasculature structure in the entire placenta, as well as detecting placental defects in pathologies characterized by embryonic resorption and placental fusion. Taken together, Zr-POM–based CE-CT offers a quantitative 3D methodology to investigate placental development or pathologies.

scholarly journals Photoperiod-responsive changes in chromatin accessibility in phloem-companion and epidermis cells of Arabidopsis leaves

2021 ◽  
Author(s):  
Hao Tian ◽  
Yuru Li ◽  
Ce Wang ◽  
Xingwen Xu ◽  
Yajie Zhang ◽  
...  
Keyword(s):  
Cell Types ◽  
Chromatin Accessibility ◽  
Day Length ◽  
Specific Cell ◽  
Long Day ◽  
Cell Tissue ◽  
Companion Cells ◽  
Transgenic Lines ◽  
Dna Insertion ◽  
Accessible Chromatin

Abstract Photoperiod plays a key role in controlling the phase transition from vegetative to reproductive growth in flowering plants. Leaves are the major organs perceiving day-length signals, but how specific leaf cell-types respond to photoperiod remains unknown. We integrated photoperiod-responsive chromatin accessibility and transcriptome data in leaf epidermis and vascular companion cells of Arabidopsis thaliana by combining INTACT (Isolation of Nuclei Tagged in specific Cell/Tissue types) with ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) and RNA-sequencing. Despite a large overlap, vasculature and epidermis cells responded differently. Long-day predominantly induced accessible chromatin regions (ACRs); in the vasculature, more ACRs were induced and these were located at more distal gene regions, compared with the epidermis. Vascular ACRs induced by long day were highly enriched in binding sites for flowering-related transcription factors. Among the highly ranked genes (based on chromatin and expression signatures in the vasculature), we identified TREHALOSE-6-PHOSPHATASE SYNTHASE 9 (TPS9) as a flowering activator, as shown by the late flowering phenotypes of T-DNA insertion mutants and transgenic lines with phloem-specific knockdown of TPS9. Our cell-type-specific analysis sheds light on how the long-day photoperiod stimulus impacts chromatin accessibility in a tissue-specific manner to regulate plant development.

scholarly journals Differential Protein Expression in Striatal D1- and D2-Dopamine Receptor-Expressing Medium Spiny Neurons

Proteomes ◽  
2020 ◽  
Vol 8 (4) ◽  
pp. 27
Author(s):  
M. Shahid Mansuri ◽  
Gang Peng ◽  
Rashaun S. Wilson ◽  
TuKiet T. Lam ◽  
Hongyu Zhao ◽  
...  
Keyword(s):  
Drugs Of Abuse ◽  
Affinity Purification ◽  
Neuronal Cell ◽  
Principal Component ◽  
Cell Types ◽  
Differentially Expressed ◽  
Cellular Heterogeneity ◽  
Specific Cell ◽  
Medium Spiny Neurons ◽  
Spiny Neurons

Many neurological disorders and diseases including drug addiction are associated with specific neuronal cell types in the brain. The striatum, a region that plays a critically important role in the development of addictive drug-related behavior, provides a good example of the cellular heterogeneity challenges associated with analyses of specific neuronal cell types. Such studies are needed to identify the adaptive changes in neuroproteomic signaling that occur in response to diseases such as addiction. The striatum contains two major cell types, D1 and D2 type dopaminoceptive medium spiny neurons (MSNs), whose cell bodies and processes are intermingled throughout this region. Since little is known about the proteomes of these two neuronal cell populations, we have begun to address this challenge by using fluorescence-activated nuclear sorting (FANS) to isolate nuclei-containing fractions from striatum from D1 and D2 “Translating Ribosome Affinity Purification” (TRAP) mice. This approach enabled us to devise and implement a robust and reproducible workflow for preparing samples from specific MSN cell types for mass spectrometry analyses. These analyses quantified at least 685 proteins in each of four biological replicates of 50 K sorted nuclei from two D1 mice/replicate and from each of four biological replicates of 50 K sorted nuclei from two D2 mice/replicate. Proteome analyses identified 87 proteins that were differentially expressed in D1 versus D2 MSN nuclei and principal component analysis (PCA) of these proteins separated the 8 biological replicates into specific cell types. Central network analysis of the 87 differentially expressed proteins identified Hnrnpd and Hnmpa2b1 in D1 and Cct2 and Cct7 in D2 as potential central interactors. This workflow can now be used to improve our understanding of many neurological diseases including characterizing the short and long-term impact of drugs of abuse on the proteomes of these two dopaminoceptive neuronal populations.

scholarly journals A split GFP system to enhance spatial and temporal sensitivity of translating ribosome affinity purification (TRAP)

2021 ◽  
Author(s):  
Kasia Dinkeloo ◽  
Zoe Pelly ◽  
John M. McDowell ◽  
Guillaume Pilot
Keyword(s):  
Ribosomal Protein ◽  
Temporal Resolution ◽  
Affinity Purification ◽  
Cell Types ◽  
Inducible Promoter ◽  
Infected Leaf ◽  
Growth Stages ◽  
Specific Cell ◽  
Trap System ◽  
Increased Sensitivity

SummaryTranslating ribosome affinity purification (TRAP) utilizes transgenic plants expressing a ribosomal protein fused to a tag for affinity purification of ribosomes and the mRNAs that they are translating. These actively translated mRNAs (translatome) can be interrogated by qPCR or RNAseq. Condition- or cell-specific promoters can be utilized to isolate the translatome of specific cell types, at different growth stages and/or in response to environmental variables. While advantageous for revealing differential expression, this approach may not provide sufficient sensitivity when activity of the condition/cell-specific promoter is weak, when ribosome turnover is low in the cells of interest, or when the targeted cells are ephemeral. In these situations, expressing tagged ribosomes under the control of these specific promoters may not yield sufficient polysomes for downstream analysis. Here, we describe a new TRAP system that employs two transgenes: one is constitutively expressed and encodes a ribosomal protein fused to one fragment of a split GFP; the second is controlled by a stimulus-specific promoter and encodes the second GFP fragment fused to an affinity purification tag. In cells where both transgenes are active, the purification tag is attached to ribosomes by bi-molecular folding and assembly of the split GFP fragments. This approach provides increased sensitivity and better temporal resolution because it labels pre-existing ribosomes and does not depend on rapid ribosome turnover. We describe the optimization and key parameters of this system, and then apply it to a plant-pathogen interaction in which spatial and temporal resolution are difficult to achieve with current technologies.SignificanceTranslating ribosome affinity purification (TRAP) has been modified to allow with increased sensitivity the isolation of RNA from sets of cells in which the activity of condition/cell-specific promoters is weak, ribosome turnover is low, or cells whose nature is ephemeral. Based on the use of a split linker constituted of the GFP driven by a pathogen-inducible promoter, this new TRAP system enabled efficient isolation of translated RNA from pathogen-infected leaf cells.

Correlative transmission and high-resolution scanning electron microscopic studies on pituitary cells

1990 ◽  
Vol 48 (3) ◽  
pp. 20-21
Author(s):  
S. Tai
Keyword(s):  
Cell Types ◽  
Depth Of Field ◽  
Secretory Cells ◽  
Specific Cell ◽  
Pituitary Cells ◽  
Electron Microscopic ◽  
3 Dimensional ◽  
Microscopic Studies ◽  
Structure And Activity ◽  
Intracellular Structure

Extensive cytological and histological research, correlated with physiological experimental analysis, have been done on the anterior pituitaries of many different vertebrates which have provided the knowledge to create the concept that specific cell types synthesize, store and release their specific hormones. These hormones are stored in or associated with granules. Nevertheless, there are still many doubts - that need further studies, specially on the ultrastructure and physiology of these endocrine cells during the process of synthesis, transport and secretion, whereas some new methods may provide the information about the intracellular structure and activity in detail.In the present work, ultrastructural study of the hormone-secretory cells of chicken pituitaries have been done by using TEM as well as HR-SEM, to correlate the informations obtained from 2-dimensional TEM micrography with the 3-dimensional SEM topographic images, which have a continous surface with larger depth of field that - offers the adventage to interpretate some intracellular structures which were not possible to see using TEM.

Nanotheranostic agents for neurodegenerative diseases

2020 ◽  
Vol 4 (6) ◽  
pp. 645-675
Author(s):  
Parasuraman Padmanabhan ◽  
Mathangi Palanivel ◽  
Ajay Kumar ◽  
Domokos Máthé ◽  
George K. Radda ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Neurodegenerative Diseases ◽  
Cell Types ◽  
Specific Cell ◽  
Ageing Population ◽  
Target Tissue ◽  
Therapeutic Approaches ◽  
Surface Receptors ◽  
Theranostic Agents ◽  
Preclinical Animal Models

Neurodegenerative diseases (NDDs), including Alzheimer's disease (AD) and Parkinson's disease (PD), affect the ageing population worldwide and while severely impairing the quality of life of millions, they also cause a massive economic burden to countries with progressively ageing populations. Parallel with the search for biomarkers for early detection and prediction, the pursuit for therapeutic approaches has become growingly intensive in recent years. Various prospective therapeutic approaches have been explored with an emphasis on early prevention and protection, including, but not limited to, gene therapy, stem cell therapy, immunotherapy and radiotherapy. Many pharmacological interventions have proved to be promising novel avenues, but successful applications are often hampered by the poor delivery of the therapeutics across the blood-brain-barrier (BBB). To overcome this challenge, nanoparticle (NP)-mediated drug delivery has been considered as a promising option, as NP-based drug delivery systems can be functionalized to target specific cell surface receptors and to achieve controlled and long-term release of therapeutics to the target tissue. The usefulness of NPs for loading and delivering of drugs has been extensively studied in the context of NDDs, and their biological efficacy has been demonstrated in numerous preclinical animal models. Efforts have also been made towards the development of NPs which can be used for targeting the BBB and various cell types in the brain. The main focus of this review is to briefly discuss the advantages of functionalized NPs as promising theranostic agents for the diagnosis and therapy of NDDs. We also summarize the results of diverse studies that specifically investigated the usage of different NPs for the treatment of NDDs, with a specific emphasis on AD and PD, and the associated pathophysiological changes. Finally, we offer perspectives on the existing challenges of using NPs as theranostic agents and possible futuristic approaches to improve them.

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