scholarly journals Kinetic principles underlying pioneer function of GAGA transcription factor in live cells

2021 ◽  
Author(s):  
Carl Wu ◽  
Xiaona Tang ◽  
Taibo Li ◽  
Sheng Liu ◽  
Jan Wisniewski ◽  
...  
Keyword(s):  
Dynamic Range ◽  
Single Particle Tracking ◽  
Live Cells ◽  
Kinetic Properties ◽  
Open Chromatin ◽  
Transcription Control ◽  
Transcriptional Responses ◽  
Chromatin Remodelers ◽  
Intrinsically Disordered

How pioneer factors interface with chromatin to promote accessibility for transcription control is poorly understood in vivo. Here, we directly visualize chromatin association by the prototypical GAGA pioneer factor (GAF) in live Drosophila hemocytes. Single-particle tracking reveals that the majority of GAF is chromatin-bound, with a stable-binding fraction showing nucleosome-like confinement residing on chromatin for over 2 minutes, far longer than the dynamic range of most transcription factors. These kinetic properties require the full complement of GAF's DNA-binding, multimerization and intrinsically disordered domains, and are autonomous from recruited chromatin remodelers NURF and PBAP, whose activities primarily benefit GAF's neighbors such as HSF. Evaluation of GAF kinetics together with its endogenous abundance indicates that despite on-off dynamics, GAF constitutively and fully occupies chromatin targets, thereby providing a temporal mechanism that sustains open chromatin for transcriptional responses to homeostatic, environmental, and developmental signals.

scholarly journals Mapping the native organization of the yeast nuclear pore complex using nuclear radial intensity measurements

2019 ◽  
Vol 116 (29) ◽  
pp. 14606-14613 ◽  
Author(s):  
Pascal Vallotton ◽  
Sasikumar Rajoo ◽  
Matthias Wojtynek ◽  
Evgeny Onischenko ◽  
Annemarie Kralt ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Nucleocytoplasmic Transport ◽  
Live Cells ◽  
Nuclear Pore ◽  
Intrinsically Disordered ◽  
Intensity Measurements ◽  
Composition And Structure ◽  
Associated Proteins ◽  
Multiple Copies

Selective transport across the nuclear envelope (NE) is mediated by the nuclear pore complex (NPC), a massive ∼100-MDa assembly composed of multiple copies of ∼30 nuclear pore proteins (Nups). Recent advances have shed light on the composition and structure of NPCs, but approaches that could map their organization in live cells are still lacking. Here, we introduce an in vivo method to perform nuclear radial intensity measurements (NuRIM) using fluorescence microscopy to determine the average position of NE-localized proteins along the nucleocytoplasmic transport axis. We apply NuRIM to study the organization of the NPC and the mobile transport machinery in budding yeast. This reveals a unique snapshot of the intact yeast NPC and identifies distinct steady-state localizations for various NE-associated proteins and nuclear transport factors. We find that the NPC architecture is robust against compositional changes and could also confirm that in contrast to Chlamydomonas reinhardtii, the scaffold Y complex is arranged symmetrically in the yeast NPC. Furthermore, NuRIM was applied to probe the orientation of intrinsically disordered FG-repeat segments, providing insight into their roles in selective NPC permeability and structure.

scholarly journals Transcription-dependent confined diffusion of enzymes within subcellular spaces of the bacterial cytoplasm

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Daniel A. O. Rotter ◽  
Christoph Heger ◽  
Luis M. Oviedo-Bocanegra ◽  
Peter L. Graumann
Keyword(s):  
Single Cells ◽  
Single Particle Tracking ◽  
Live Cells ◽  
Fast Diffusion ◽  
Bacterial Cells ◽  
Substrate Channeling ◽  
Active Transcription ◽  
Bacterial Cytoplasm

Abstract Background Knowledge on the localization and mobility of enzymes inside bacterial cells is scarce, but important for understanding spatial regulation of metabolism. The four central enzymes (Rib enzymes) of the riboflavin (RF) biosynthesis pathway in the Gram positive model bacterium Bacillus subtilis have been studied extensively in vitro, especially the heavy RF synthase, a large protein complex with a capsid structure formed by RibH and an encapsulated RibE homotrimer, which mediates substrate-channeling. However, little is known about the behavior and mobility of these enzymes in vivo. Results We have investigated the localization and diffusion of the Rib enzymes in the cytoplasm of B. subtilis. By characterizing the diffusion of Rib enzymes in live cells using single particle tracking (SPT) we provide evidence for confined diffusion at the cell poles and otherwise Brownian motion. A majority of RibH particles showed clear nucleoid occlusion and a high degree of confined motion, which is largely abolished after treatment with Rifampicin, revealing that confinement is dependent on active transcription. Contrarily, RibE is mostly diffusive within the cell, showing only 14% encapsulation by RibH nanocompartments. By localizing different diffusive populations within single cells, we find that fast diffusion occurs mostly across the nucleoids located in the cell centers, while the slower, confined subdiffusion occurs at the crowded cell poles. Conclusions Our results provide evidence for locally different motion of active enzymes within the bacterial cytoplasm, setting up metabolic compartmentalization mostly at the poles of cells.

scholarly journals Mechanisms Governing Target Search and Binding Dynamics of Hypoxia-Inducible Factors

2021 ◽  
Author(s):  
Yu Chen ◽  
Claudia Cattoglio ◽  
Gina Dailey ◽  
Qiulin Zhu ◽  
Robert Tjian ◽  
...  
Keyword(s):  
Dna Binding ◽  
Transcription Initiation ◽  
Specific Inhibitor ◽  
Single Particle Tracking ◽  
Specific Gene ◽  
Hypoxia Inducible Factors ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions

Transcription factors (TFs) are classically attributed a modular construction, containing well-structured sequence specific DNA-binding domains (DBDs) paired with disordered activation domains (ADs) responsible for protein-protein interactions targeting cofactors or the core transcription initiation machinery. However, this simple division of labor model struggles to explain why TFs with identical DNA binding sequence specificity determined in vitro exhibit distinct non-overlapping binding profiles in vivo. The family of Hypoxia-Inducible Factors (HIFs) offer a stark example: aberrantly expressed in several cancer types, HIF-1α and HIF-2α subunit isoforms recognize the same DNA motif in vitro — the hypoxia response element (HRE) — but only share a subset of their target genes in vivo, while eliciting contrasting effects on cancer development and progression under certain circumstances. To probe the mechanisms mediating isoform-specific gene regulation, we used live cell single particle tracking (SPT) to investigate HIF nuclear dynamics and how they change upon genetic perturbation or drug treatment. We found that HIF-α subunits and their dimerization partner HIF-1β exhibit distinct diffusion and binding characteristics that are exquisitely sensitive to concentration and subunit stoichiometry. Using domain-swap variants, mutations, and a HIF-2α specific inhibitor, we found that although the DBD and dimerization domains are important, a major determinant of chromatin binding and diffusion behavior is dictated by the AD-containing intrinsically disordered regions. These findings reveal a previously unappreciated role of IDRs in regulating the TF search process that may play a role in selective functional target site binding on chromatin.

scholarly journals Visualizing long-term single-molecule dynamics in vivo by stochastic protein labeling

2017 ◽  
Vol 115 (2) ◽  
pp. 343-348 ◽  
Author(s):  
Hui Liu ◽  
Peng Dong ◽  
Maria S. Ioannou ◽  
Li Li ◽  
Jamien Shea ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Single Molecule ◽  
Dynamic Range ◽  
Live Cells ◽  
Resolution Limit ◽  
Anterograde Transport ◽  
Multiple Copies ◽  
Long Time

Our ability to unambiguously image and track individual molecules in live cells is limited by packing of multiple copies of labeled molecules within the resolution limit. Here we devise a universal genetic strategy to precisely control copy number of fluorescently labeled molecules in a cell. This system has a dynamic range of ∼10,000-fold, enabling sparse labeling of proteins expressed at different abundance levels. Combined with photostable labels, this system extends the duration of automated single-molecule tracking by two orders of magnitude. We demonstrate long-term imaging of synaptic vesicle dynamics in cultured neurons as well as in intact zebrafish. We found axon initial segment utilizes a “waterfall” mechanism gating synaptic vesicle transport polarity by promoting anterograde transport processivity. Long-time observation also reveals that transcription factor hops between clustered binding sites in spatially restricted subnuclear regions, suggesting that topological structures in the nucleus shape local gene activities by a sequestering mechanism. This strategy thus greatly expands the spatiotemporal length scales of live-cell single-molecule measurements, enabling new experiments to quantitatively understand complex control of molecular dynamics in vivo.

Multi-mode light microscopy of microtubule assembly dynamics and chromosome movement in vivo and In vitro

1996 ◽  
Vol 54 ◽  
pp. 730-731
Author(s):  
E. D. Salmon ◽  
J. C. Waters ◽  
C. Waterman-Storer
Keyword(s):  
Imaging System ◽  
Ccd Camera ◽  
Transmitted Light ◽  
Microtubule Assembly ◽  
Live Cells ◽  
Optical Efficiency ◽  
Multiple Band ◽  
Multi Mode

We have developed a multi-mode digital imaging system which acquires images with a cooled CCD camera (Figure 1). A multiple band pass dichromatic mirror and robotically controlled filter wheels provide wavelength selection for epi-fluorescence. Shutters select illumination either by epi-fluorescence or by transmitted light for phase contrast or DIC. Many of our experiments involve investigations of spindle assembly dynamics and chromosome movements in live cells or unfixed reconstituted preparations in vitro in which photodamage and phototoxicity are major concerns. As a consequence, a major factor in the design was optical efficiency: achieving the highest image quality with the least number of illumination photons. This principle applies to both epi-fluorescence and transmitted light imaging modes. In living cells and extracts, microtubules are visualized using X-rhodamine labeled tubulin. Photoactivation of C2CF-fluorescein labeled tubulin is used to locally mark microtubules in studies of microtubule dynamics and translocation. Chromosomes are labeled with DAPI or Hoechst DNA intercalating dyes.

scholarly journals POS1388 ULTRASOUND FOR PANNICULITIS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 977.1-977
Author(s):  
A. Potapova ◽  
O. Egorova ◽  
O. Alekseeva ◽  
A. Volkov ◽  
S. Radenska-Lopovok
Keyword(s):  
Dynamic Range ◽  
Subcutaneous Fat ◽  
Diagnostic Value ◽  
High Energy ◽  
Contralateral Side ◽  
Imaging Method ◽  
Non Invasive ◽  
M 12

Background:Ultrasound (US) is a non-invasive and safe imaging method that allows in vivo differentiation of the morphological structures of subcutaneous fat (SCF) tissue in in normal and pathology.Objectives:Reveal features of ultrasound changes in SCF in panniculitis (Pn).Methods:57 patients (f – 45, m - 12) aged 18 - 67 years with an initial diagnosis of erythema nodosum and a disease duration of 3.6 ± 1.4 years were examined. In addition to the general clinical examination, a computed tomography of the chest organs and a pathomorphological examination of a skin biopsy from the site of the node were performed. Ultrasound was performed on a MyLabTwice apparatus (ESAOTE, Italy) using a multi-frequency linear transducer (10-18 MHz) with the PD technique, the parameters of which were adapted for recording low-speed flows (PRF 300-600 Hz, low filter, dynamic range - 20-40 dB), the presence of vascularization was assessed not only in the affected area, but also on the contralateral side using high-energy Doppler.Results:33 patients were diagnosed with septal Pn (SPn), 24 - lobular Pn (LPn). In all cases, the diagnosis was verified by histological examination. Ultrasound made it possible to assess the thickness, echoicity and vascularization of the SCF. In 35 patients, significant thickening of the SCF was revealed (as compared to the contralateral side), of which in 14 cases with SPn, in 21 - with LPn. Significant diffuse thickening of the SCF with the contralateral side was observed in 18 patients, incl. in 12 (66%) patients with LPn. Limited thickening was more typical for SPn (73%). A significant increase in the echoicity of the SCF was noted in all forms of Pn. A “lobular” echo pattern with an anechogenic environment was observed in 25 patients, of which 18 (72%) had LPn. An increase in vascularization compared to the contralateral side was recorded in 30 cases (SPn-17, LPn-13).Conclusion:The obtained preliminary results indicate the important role of ultrasound in assessing the depth and prevalence of the inflammatory process at Pn. To clarify the diagnostic value of this method, further studies are needed on a larger sample of patients.Disclosure of Interests:None declared

scholarly journals A paradigm shift in cell-free approach: the emerging role of MSCs-derived exosomes in regenerative medicine

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Soudeh Moghadasi ◽  
Marischa Elveny ◽  
Heshu Sulaiman Rahman ◽  
Wanich Suksatan ◽  
Abduladheem Turki Jalil ◽  
...  
Keyword(s):  
Regenerative Medicine ◽  
Ethical Issues ◽  
Therapeutic Potential ◽  
Kidney Diseases ◽  
Experimental Models ◽  
Live Cells ◽  
Target Cells ◽  
Intercellular Interaction ◽  
Micro Rnas

AbstractRecently, mesenchymal stem/stromal cells (MSCs) due to their pro-angiogenic, anti-apoptotic, and immunoregulatory competencies along with fewer ethical issues are presented as a rational strategy for regenerative medicine. Current reports have signified that the pleiotropic effects of MSCs are not related to their differentiation potentials, but rather are exerted through the release of soluble paracrine molecules. Being nano-sized, non-toxic, biocompatible, barely immunogenic, and owning targeting capability and organotropism, exosomes are considered nanocarriers for their possible use in diagnosis and therapy. Exosomes convey functional molecules such as long non-coding RNAs (lncRNAs) and micro-RNAs (miRNAs), proteins (e.g., chemokine and cytokine), and lipids from MSCs to the target cells. They participate in intercellular interaction procedures and enable the repair of damaged or diseased tissues and organs. Findings have evidenced that exosomes alone are liable for the beneficial influences of MSCs in a myriad of experimental models, suggesting that MSC- exosomes can be utilized to establish a novel cell-free therapeutic strategy for the treatment of varied human disorders, encompassing myocardial infarction (MI), CNS-related disorders, musculoskeletal disorders (e.g. arthritis), kidney diseases, liver diseases, lung diseases, as well as cutaneous wounds. Importantly, compared with MSCs, MSC- exosomes serve more steady entities and reduced safety risks concerning the injection of live cells, such as microvasculature occlusion risk. In the current review, we will discuss the therapeutic potential of MSC- exosomes as an innovative approach in the context of regenerative medicine and highlight the recent knowledge on MSC- exosomes in translational medicine, focusing on in vivo researches.

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