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Single-molecule imaging of the transcription factor SRF reveals prolonged chromatin-binding kinetics upon cell stimulation
摘要： Serum response factor (SRF) mediates immediate early gene (IEG) and cytoskeletal gene expression programs in almost any cell type. So far, SRF transcriptional dynamics have not been investigated at single-molecule resolution. We provide a study of single Halo-tagged SRF molecules in fibroblasts and primary neurons. In both cell types, individual binding events of SRF molecules segregated into three chromatin residence time regimes, short, intermediate, and long binding, indicating a cell type-independent SRF property. The chromatin residence time of the long bound fraction was up to 1 min in quiescent cells and significantly increased upon stimulation. Stimulation also enhanced the long bound SRF fraction at specific timepoints (20 and 60 min) in both cell types. These peaks correlated with activation of the SRF cofactors MRTF-A and MRTF-B (myocardin-related transcription factors). Interference with signaling pathways and cofactors demonstrated modulation of SRF chromatin occupancy by actin signaling, MAP kinases, and MRTFs.
关键词： SRF,single molecule,neuron,transcription,HaloTag
更新于2025-09-23 15:22:29
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Single-molecule imaging of DNA gyrase activity in living <i>Escherichia coli</i>
摘要： Bacterial DNA gyrase introduces negative supercoils into chromosomal DNA and relaxes positive supercoils introduced by replication and transiently by transcription. Removal of these positive supercoils is essential for replication fork progression and for the overall unlinking of the two duplex DNA strands, as well as for ongoing transcription. To address how gyrase copes with these topological challenges, we used high-speed single-molecule fluorescence imaging in live Escherichia coli cells. We demonstrate that at least 300 gyrase molecules are stably bound to the chromosome at any time, with ～12 enzymes enriched near each replication fork. Trapping of reaction intermediates with ciprofloxacin revealed complexes undergoing catalysis. Dwell times of ～2 s were observed for the dispersed gyrase molecules, which we propose maintain steady-state levels of negative supercoiling of the chromosome. In contrast, the dwell time of replisome-proximal molecules was ～8 s, consistent with these catalyzing processive positive supercoil relaxation in front of the progressing replisome.
关键词： Escherichia coli,transcription,DNA gyrase,replication,supercoiling,single-molecule imaging
更新于2025-09-23 15:21:21
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Simultaneous Nano-Texturing onto a CVD-Diamond Coated Piercing Punch with Femtosecond Laser Trimming
摘要： In this study, a CVD (Chemical Vapor Deposition)-diamond coated tungsten carbide cobalt (WC (Co)) punch was trimmed to adjust its surface roughness and to significantly reduce its edge curvature for fine piercing by femtosecond laser processing. Through this laser trimming, the surface quality of the diamond coating and the punch edge profile were improved to less than 0.5 μm at the maximum roughness and 2 μm in the edge width, respectively. In parallel with this improvement of surface quality, the side surface of the diamond coating was modified to include nano-textures via the LIPSS (Laser Induced Periodic Surface Structuring) process. Through the fine piercing process, this nanotexture was transcribed onto the pierced hole surface together with fine shearing of the hole by piercing. WLI (White-Light Interferometry) and SEM (Scanning Electron Microscopy) were utilized to describe this transcription of nanotextures during the piercing process. These semi-regular nanotextures with an LIPSS period of 300 nm on the pierced hole surface induced a blue colored surface plasmon.
关键词： diamond-coated WC (Co) punch,concurrent transcription of nanotextures,surface plasmon,simultaneous nanotexturing,femtosecond laser trimming,fine piercing
更新于2025-09-23 15:21:01
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Photoreceptor Fate Determination in the Vertebrate Retina
摘要： Photoreceptors are highly specialized primary sensory neurons that sense light and initiate vision. This critical role is well demonstrated by the fact that visual impairment accompanies photoreceptor loss or dysfunction in many human diseases. With the remarkable advances in stem cell research, one therapeutic approach is to use stem cells to generate photoreceptors and then engraft them into diseased eyes. Knowledge of the molecular mechanisms that control photoreceptor genesis during normal development can greatly aid in the production of photoreceptor cells for this approach. This article will discuss advances in our understanding of the molecular mechanisms that regulate photoreceptor fate determination during development. Recent lineage studies have shown that there are distinct retinal progenitor cells (RPCs) that produce specific combinations of daughter cell types, including photoreceptors and other types of retinal cells. Gene regulatory networks, in which transcription factors interact via cis-regulatory DNA elements, have been discovered that operate within distinct RPCs, and/or newly postmitotic cells, to direct the choice of photoreceptor fate.
关键词： retinal development,photoreceptor fate determination,transcription factors,gene regulatory network,cell lineages
更新于2025-09-19 17:15:36
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Transcription-coupled nucleotide excision repair is coordinated by ubiquitin and SUMO in response to ultraviolet irradiation
摘要： Cockayne Syndrome (CS) is a severe neurodegenerative and premature aging autosomal-recessive disease, caused by inherited defects in the CSA and CSB genes, leading to defects in transcription-coupled nucleotide excision repair (TC-NER) and consequently hypersensitivity to ultraviolet (UV) irradiation. TC-NER is initiated by lesion-stalled RNA polymerase II, which stabilizes the interaction with the SNF2/SWI2 ATPase CSB to facilitate recruitment of the CSA E3 Cullin ubiquitin ligase complex. However, the precise biochemical connections between CSA and CSB are unknown. The small ubiquitin-like modifier SUMO is important in the DNA damage response. We found that CSB, among an extensive set of other target proteins, is the most dynamically SUMOylated substrate in response to UV irradiation. Inhibiting SUMOylation reduced the accumulation of CSB at local sites of UV irradiation and reduced recovery of RNA synthesis. Interestingly, CSA is required for the efficient clearance of SUMOylated CSB. However, subsequent proteomic analysis of CSA-dependent ubiquitinated substrates revealed that CSA does not ubiquitinate CSB in a UV-dependent manner. Surprisingly, we found that CSA is required for the ubiquitination of the largest subunit of RNA polymerase II, RPB1. Combined, our results indicate that the CSA, CSB, RNA polymerase II triad is coordinated by ubiquitin and SUMO in response to UV irradiation. Furthermore, our work provides a resource of SUMO targets regulated in response to UV or ionizing radiation.
关键词： SUMOylation,DNA damage response,transcription-coupled nucleotide excision repair,Cockayne Syndrome,UV irradiation,ubiquitination
更新于2025-09-12 10:27:22
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Arabidopsis E2Fc is required for the DNA damage response under UV-B radiation epistatically over the microRNA396 and independently of E2Fe
摘要： UV-B radiation inhibits plant growth, and this inhibition is, to a certain extent, regulated by miR396-mediated repression of Growth Regulating transcription Factors (GRFs). Moreover, E2Fe transcription factor also modulates Arabidopsis leaf growth. Here, we provide evidence that, at UV-B intensities that induce DNA damage, E2Fc participates in the inhibition of cell proliferation. We demonstrate that E2Fc deficient plants show a lower inhibition of leaf size under UV-B conditions that damage DNA, decreased cell death after exposure and altered SOG1 and ATR expression. Interestingly, the previously reported participation of E2Fe in UV-B responses, which is a transcriptional target of E2Fc, is independent and different of that described for E2Fc. On the other hand, we here demonstrate that E2Fc has an epistatic role over the miR396 pathway under UV-B conditions. Finally, we show that inhibition of cell proliferation by UV-B is independent of the regulation of class II TCP transcription factors. Together, our results demonstrate that E2Fc is required for miR396 activity on cell proliferation under UV-B, and that its role is independent of E2Fe, probably modulating DNA damage responses through the regulation of SOG1 and ATR transcript levels.
关键词： E2F transcription factor,DNA damage response,UV-B,cell proliferation,miR396
更新于2025-09-10 09:29:36
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Changes in Fluorescence Recovery After Photobleaching (FRAP) as an indicator of SOX9 transcription factor activity
摘要： Cells respond to their environment via an intricate cellular signaling network, directing cell fate. Changes in cell fate are characterized by changes in gene transcription, dictated by (master) transcription factor activity. SOX9 is the master transcription factor for chondrocyte development. Its impaired function is implicated in osteoarthritis and growth disorders, such as dwarfism. However, the factors regulating SOX9 transcriptional activity are not yet fully mapped. Current methods to study transcription factor activity are indirect and largely limited to quantification of SOX9 target gene and protein expression levels after several hours or days of stimulation, leading to poor temporal resolution. We used Fluorescence Recovery After Photobleaching (FRAP) to study the mobility of SOX9 and correlated the changes in mobility to changes in its transcriptional activity by cross-validating with chromatin immunoprecipitation and qPCR. We show that using FRAP, we can quantify the changes in SOX9 mobility on short time scales as an indication of transcriptional activity, which correlated to changes of SOX9 DNA-binding and long-term target gene expression.
关键词： FRAP,transcription factor dynamics,transcriptional activity,osteoarthritis,cartilage,SOX9
更新于2025-09-10 09:29:36
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Bringing Light to Transcription: The Optogenetics Repertoire
摘要： The ability to manipulate expression of exogenous genes in particular regions of living organisms has profoundly transformed the way we study biomolecular processes involved in both normal development and disease. Unfortunately, most of the classical inducible systems lack fine spatial and temporal accuracy, thereby limiting the study of molecular events that strongly depend on time, duration of activation, or cellular localization. By exploiting genetically engineered photo sensing proteins that respond to specific wavelengths, we can now provide acute control of numerous molecular activities with unprecedented precision. In this review, we present a comprehensive breakdown of all of the current optogenetic systems adapted to regulate gene expression in both unicellular and multicellular organisms. We focus on the advantages and disadvantages of these different tools and discuss current and future challenges in the successful translation to more complex organisms.
关键词： transcription,optogenetics,cryptochrome,phytochrome B,LOV,gene expression,UVR8,light
更新于2025-09-09 09:28:46
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Fabrication of the Fresnel lens with liquid silicone rubber using rapid injection mold
摘要： Liquid silicone rubber (LSR) parts are used in aerospace application due to their stable properties. In order to compress time to market for a new product, it is necessary to develop an injection mold for LSR injection mold swiftly and effectively. Rapid tooling (RT) technology can reduce time to market compared to the conventional machining methods. The aluminum-filled epoxy resin mold is capable of replacing the conventional steel mold for small batch production using injection molding. In this study, an injection mold with heating element was developed using RT technology for LSR injection molding. It was found that the replication rate for the average microgroove depth and width of the Al-filled epoxy resin mold is about 90.5% and 98.9%, respectively. The transcription rate for the average microgroove depth and width of the LSR molded parts is about 91.5% and 99.2%, respectively. The variations in dimension of microgroove depth and width of the LSR molded parts can be controlled within ± 1 μm. The average surface roughness of the Al-filled epoxy resin mold was only increased by about 12.5 nm after 200 test runs of LSR injection molding.
关键词： Liquid silicone rubber injection molding,Aluminum-filled epoxy resin,Fresnel lens,Replication rate,Transcription rate
更新于2025-09-09 09:28:46
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Shining light on plant hormones with genetically encoded biosensors
摘要： Signalling molecules are produced, degraded, modified and transported throughout the development of higher organisms. Understanding their mode of action implies understanding these dynamics in vivo and in real time. Genetically encoded biosensors are being more and more used as tools to ‘follow’ signalling molecules and their responses inside an organism. This is the case for plants, where important progresses have been made in the development of such biosensors. Here, we summarize the main genetically encoded biosensors built for plant hormones, constructed using diverse components and steps of their signalling pathways.
关键词： fluorescent protein,transcription,signalling,sensor,phytohormone
更新于2025-09-09 09:28:46