Visualization of the cancer cell cycle by tissue-clearing technology using the Fucci reporter system.
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ABSTRACT:
SUBMITTER: Takahashi K
PROVIDER: S-EPMC8409402 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:UnlabelledDemonstrating direct interactions between host and virus proteins during infection is a major goal and challenge for the field of virology. Most protein interactions are not binary or easily amenable to structural determination. Using infectious preparations of a polerovirus (Potato leafroll virus [PLRV]) and protein interaction reporter (PIR), a revolutionary technology that couples a mass spectrometric-cleavable chemical cross-linker with high-resolution mass spectrometry, we provide the first report of a host-pathogen protein interaction network that includes data-derived, topological features for every cross-linked site that was identified. We show that PLRV virions have hot spots of protein interaction and multifunctional surface topologies, revealing how these plant viruses maximize their use of binding interfaces. Modeling data, guided by cross-linking constraints, suggest asymmetric packing of the major capsid protein in the virion, which supports previous epitope mapping studies. Protein interaction topologies are conserved with other species in the Luteoviridae and with unrelated viruses in the Herpesviridae and Adenoviridae. Functional analysis of three PLRV-interacting host proteins in planta using a reverse-genetics approach revealed a complex, molecular tug-of-war between host and virus. Structural mimicry and diversifying selection-hallmarks of host-pathogen interactions-were identified within host and viral binding interfaces predicted by our models. These results illuminate the functional diversity of the PLRV-host protein interaction network and demonstrate the usefulness of PIR technology for precision mapping of functional host-pathogen protein interaction topologies.ImportanceThe exterior shape of a plant virus and its interacting host and insect vector proteins determine whether a virus will be transmitted by an insect or infect a specific host. Gaining this information is difficult and requires years of experimentation. We used protein interaction reporter (PIR) technology to illustrate how viruses exploit host proteins during plant infection. PIR technology enabled our team to precisely describe the sites of functional virus-virus, virus-host, and host-host protein interactions using a mass spectrometry analysis that takes just a few hours. Applications of PIR technology in host-pathogen interactions will enable researchers studying recalcitrant pathogens, such as animal pathogens where host proteins are incorporated directly into the infectious agents, to investigate how proteins interact during infection and transmission as well as develop new tools for interdiction and therapy.
| S-EPMC4733995 | biostudies-literature
Project description:Background:Annual killifishes are adapted to surviving and reproducing over alternating dry and wet seasons. During the dry season, all adults die and desiccation-resistant embryos remain encased in dry mud for months or years in a state of diapause where their development is halted in anticipation of the months that have to elapse before their habitats are flooded again. Embryonic development of annual killifishes deviates from canonical teleost development. Epiblast cells disperse during epiboly, and a "dispersed phase" precedes gastrulation. In addition, annual fish have the ability to enter diapause and block embryonic development at the dispersed phase (diapause I), mid-somitogenesis (diapause II) and the final phase of development (diapause III). Developmental transitions associated with diapause entry and exit can be linked with cell cycle events. Here we set to image this transition in living embryos. Results:To visibly explore cell cycle dynamics during killifish development in depth, we created a stable transgenic line in Nothobranchius furzeri that expresses two fluorescent reporters, one for the G1 phase and one for the S/G2 phases of the cell cycle, respectively (Fluorescent Ubiquitination-based Cell Cycle Indicator, FUCCI). Using this tool, we observed that, during epiboly, epiblast cells progressively become quiescent and exit the cell cycle. All embryos transit through a phase where dispersed cells migrate, without showing any mitotic activity, possibly blocked in the G1 phase (diapause I). Thereafter, exit from diapause I is synchronous and cells enter directly into the S phase without transiting through G1. The developmental trajectories of embryos entering diapause and of those that continue to develop are different. In particular, embryos entering diapause have reduced growth along the medio-lateral axis. Finally, exit from diapause II is synchronous for all cells and is characterized by a burst of mitotic activity and growth along the medio-lateral axis such that, by the end of this phase, the morphology of the embryos is identical to that of direct-developing embryos. Conclusions:Our study reveals surprising levels of coordination of cellular dynamics during diapause and provides a reference framework for further developmental analyses of this remarkable developmental quiescent state.
| S-EPMC6842169 | biostudies-literature
Project description:Cellular senescence plays an important role in diverse biological processes such as tumorigenesis and organismal aging. However, lack of methods to specifically identify and isolate live senescent cells hampers the precise understanding of the molecular mechanisms regulating cellular senescence. Here, we report that utilization of fluorescent ubiquitination-based cell cycle indicator (FUCCI) technology allows isolation of live premature senescent cells induced by doxorubicin treatment. Exposure of human foreskin fibroblasts (HFFs) to a low dose of doxorubicin led to cellular senescent phenotypes including formation of γ-H2AX and 53BP1 foci indicative of DNA damage, decreased cell proliferation and increased senescence-associated β-galactosidase (SA-β-gal) activity. Importantly, doxorubicin-induced senescent cells were arrested at S/G2/M phases of cell cycle which can be reported by a construct encoding a fragment of hGeminin fused with monomeric Azami-Green (mAG-hGeminin). Flow cytometric sorting of GFP(+) cells from doxorubicin-treated HFFs carrying mAG-hGeminin reporter enabled isolation and enrichment of live senescent cells in the culture. Our study develops a novel method to identify and isolate live premature senescent cells, thereby providing a new tool to study cellular senescence.
| S-EPMC4977570 | biostudies-literature
Project description:ObjectivesDental implants are major treatment options for restoring teeth loss. Biological processes at the implant-tissue interface are critical for implant osseointegration. Superior mechanical properties of the implant constitute a major challenge for traditional histological techniques. It is imperative to develop new technique to investigate the implant-tissue interface.Materials and methodsOur laboratory developed the polyethylene glycol (PEG)-associated solvent system (PEGASOS) tissue clearing method. By immersing samples into various chemical substances, bones and teeth could be turned to transparent with intact internal structures and endogenous fluorescence being preserved. We combined the PEGASOS tissue clearing method with transgenic mouse line and other labelling technique to investigate the angiogenesis and osteogenesis processes occurring at the implant-bone interface.ResultsClearing treatment turned tissue highly transparent and implant could be directly visualized without sectioning. Implant, soft/hard tissues and fluorescent labels were simultaneously imaged in decalcified or non-decalcified mouse mandible samples without disturbing their interfaces. Multi-channel 3-dimensional image stacks at high resolution were acquired and quantified. The processes of angiogenesis and osteogenesis surrounding titanium or stainless steel implants were investigated.ConclusionsBoth titanium and stainless steel implants support angiogenesis at comparable levels. Successful osseointegration and calcium precipitation occurred only surrounding titanium, but not stainless steel implants. PEGASOS tissue clearing method provides a novel approach for investigating the interface between implants and hard tissue.
| S-EPMC6536405 | biostudies-literature
Project description:During pancreas organogenesis, Neurog3HI endocrine-committing cells are generated from a population of Sox9+ mitotic progenitors with only a low level of Neurog3 transcriptional activity (Neurog3TA.LO ). Low-level Neurog3 protein, in Neurog3TA.LO cells, is required to maintain their mitotic endocrine-lineage-primed status. Herein, we describe a Neurog3-driven FUCCI cell-cycle reporter (Neurog3P2A.FUCCI ) derived from a Neurog3 BAC transgenic reporter that functions as a loxed cassette acceptor (LCA). In cycling Sox9+ Neurog3TA.LO progenitors, the majority of cells in S-G2 -M phases have undetectable levels of Neurog3 with increased expression of endocrine progenitor markers, while those in G1 have low Neurog3 levels with increased expression of endocrine differentiation markers. These findings support a model in which variations in Neurog3 protein levels are coordinated with cell-cycle phase progression in Neurog3TA.LO progenitors with entrance into G1 triggering a concerted effort, beyond increasing Neurog3 levels, to maintain an endocrine-lineage-primed state by initiating expression of the downstream endocrine differentiation program prior to endocrine-commitment.
| S-EPMC5750046 | biostudies-literature
Project description:RATIONALE:Understanding and manipulating the cardiomyocyte cell cycle has been the focus of decades of research, however the ultimate goal of activating mitotic activity in adult mammalian cardiomyocytes remains elusive and controversial. The relentless pursuit of controlling cardiomyocyte mitosis has been complicated and obfuscated by a multitude of indices used as evidence of cardiomyocyte cell cycle activity that lack clear identification of cardiomyocyte "proliferation" versus cell cycle progression, endoreplication, endomitosis, and even DNA damage. Unambiguous appreciation of the complexity of cardiomyocyte replication that avoids oversimplification and misinterpretation is desperately needed. OBJECTIVE:Track cardiomyocyte cell cycle activity and authenticate fidelity of proliferation markers as indicators of de novo cardiomyogenesis in post-mitotic cardiomyocytes. METHODS AND RESULTS:Cardiomyocytes expressing the FUCCI construct driven by the α-myosin heavy chain promoter were readily and uniformly detected through the myocardium of transgenic mice. Cardiomyocyte cell cycle activity peaks at postnatal day 2 and rapidly declines thereafter with almost all cardiomyocytes arrested at the G1/S cell cycle transition. Myocardial infarction injury in adult hearts prompts transient small increases in myocytes progressing through cell cycle without concurrent mitotic activity, indicating lack of cardiomyogenesis. In comparison, cardiomyogenic activity during early postnatal development correlated with coincidence of FUCCI and cKit+ cells that were undetectable in the adult myocardium. CONCLUSIONS:Cardiomyocyte-specific expression of Fluorescence Ubiquitination-based Cell Cycle Indicators (FUCCI) reveals previously unappreciated aspects of cardiomyocyte cell cycle arrest and biological activity in postnatal development and in response to pathologic damage. Compared to many other methods and model systems, the FUCCI transgenic (FUCCI-Tg) mouse represents a valuable tool to unambiguously track cell cycle and proliferation of the entire cardiomyocyte population in the adult murine heart. FUCCI-Tg provides a desperately needed novel approach in the armamentarium of tools to validate cardiomyocyte proliferative activity that will reveal cell cycle progression, discriminate between cycle progression, DNA replication, and proliferation, and provide important insight for enhancing cardiomyocyte proliferation in the context of adult myocardial tissue.
| S-EPMC6588545 | biostudies-literature
Project description:Spatial information of cells in their tissue microenvironment is necessary to understand the complexity of pathophysiological processes. Volumetric imaging of cleared organs provides this information; however, current protocols are often elaborate, expensive, and organ specific. We developed a simplified, cost-effective, non-hazardous approach for efficient tissue clearing and multi-organ volumetric imaging (EMOVI). EMOVI enabled multiplexed antibody-based immunolabeling, provided adequate tissue transparency, maintained cellular morphology and preserved fluorochromes. Exemplarily, EMOVI allowed the detection and quantification of scarce cell populations during pneumonitis. EMOVI also permitted histo-cytometric analysis of MHC-II expressing cells, revealing distinct populations surrounding or infiltrating glomeruli of nephritic kidneys. Using EMOVI, we found widefield microscopy with real-time computational clearing as a valuable option for rapid image acquisition and detection of rare cellular events in cleared organs. EMOVI has the potential to make tissue clearing and volumetric imaging of immune cells applicable for a broad audience by facilitating flexibility in organ, fluorochrome and microscopy usage.
| S-EPMC7869862 | biostudies-literature
Project description:Blood vessels are three-dimensional (3D) in structure and precisely connected. Conventional histological methods are unsuitable for their analysis because of the destruction of functionally important topological 3D vascular structures. Tissue optical clearing techniques enable extensive volume imaging and data analysis without destroying tissue. This study therefore applied a tissue clearing technique to acquire high-resolution 3D images of rat brain vasculature using light-sheet and confocal microscopies. Rats underwent middle cerebral artery occlusion for 45 min followed by 24 h reperfusion with lectin injected directly into the heart for vascular staining. For acquiring 3D images of rat brain vasculature, 3-mm-thick brain slices were reconstructed using tissue clearing and light-sheet microscopy. Subsequently, after 3D rendering, the fitting of blood vessels to a filament model was used for analysis. The results revealed a significant reduction in vessel diameter and density in the ischemic region compared to those in contralesional non-ischemic regions. Immunostaining of 0.5-mm-thick brain slices revealed considerable neuronal loss and increased astrocyte fluorescence intensity in the ipsilateral region. Thus, these methods can provide more accurate data by broadening the scope of the analyzed regions of interest for examining the 3D cerebrovascular system and neuronal changes occurring in various brain disorders.
| S-EPMC9508267 | biostudies-literature
Project description:Cell cycle phase transitions are tightly orchestrated to ensure efficient cell cycle progression and genome stability. Interrogating these transitions is important for understanding both normal and pathological cell proliferation. By quantifying the dynamics of the popular FUCCI reporters relative to the transitions into and out of S phase, we found that their dynamics are substantially and variably offset from true S phase boundaries. To enhance detection of phase transitions, we generated a new reporter whose oscillations are directly coupled to DNA replication and combined it with the FUCCI APC/C reporter to create "PIP-FUCCI". The PIP degron fusion protein precisely marks the G1/S and S/G2 transitions; shows a rapid decrease in signal in response to large doses of DNA damage only during G1; and distinguishes cell type-specific and DNA damage source-dependent arrest phenotypes. We provide guidance to investigators in selecting appropriate fluorescent cell cycle reporters and new analysis strategies for delineating cell cycle transitions.
| S-EPMC6342071 | biostudies-literature
Project description:Several pathologic conditions of the heart lead to cardiac structural remodelling. Given the high density and the opaque nature of the myocardium, deep three dimensional (3D) imaging is difficult to achieve and structural analysis of pathological myocardial structure is often limited to two dimensional images and of thin myocardial sections. Efficient methods to obtain optical clearing of the tissue for 3D visualisation are therefore needed. Here we describe a rapid, simple and versatile Free-of-Acrylamide SDS-based Tissue Clearing (FASTClear) protocol specifically designed for cardiac tissue. With this method 3D information regarding collagen content, collagen localization and distribution could be easily obtained across a whole 300 µm-thick myocardial slice. FASTClear does not induce structural or microstructural distortion and it can be combined with immunostaining to identify the micro- and macrovascular networks. In summary, we have obtained decolorized myocardial tissue suitable for high resolution 3D imaging, with implications for the study of complex cardiac tissue structure and its changes during pathology.
| S-EPMC5507863 | biostudies-other