scholarly journals Cooperation between Caenorhabditis elegans COMPASS and condensin in germline chromatin organization

2020 ◽  
Author(s):  
M. Herbette ◽  
V. Robert ◽  
A. Bailly ◽  
L. Gely ◽  
R. Feil ◽  
...  
Keyword(s):  
Topoisomerase Ii ◽  
Meiotic Chromosome ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Chromosome Morphology ◽  
Chromatin Organization ◽  
Chromosome Organization ◽  
Fluorescence Lifetime Imaging ◽  
C Elegans

AbstractDeposition of histone H3 lysine 4 (H3K4) methylation at promoters by SET1/COMPASS is associated with context-dependent effects on gene expression and local changes in chromatin organization. Whether SET1/COMPASS also contributes to higher-order chromosome structure has not been investigated. Here, we address this question by quantitative FRET (Förster resonance energy transfer)-based fluorescence lifetime imaging microscopy (FLIM) on C. elegans germ cells expressing histones H2B-eGFP and H2B-mCherry. We find that SET1/COMPASS subunits strongly influence meiotic chromosome organization, with marked effects on the close proximity between nucleosomes. We further show that inactivation of SET-2, the C. elegans homologue of SET1, or CFP-1, the chromatin targeting subunit of COMPASS, strongly enhance chromosome organization defects and loss of fertility resulting from depletion of condensin-II. Defects in chromosome morphology resulting from conditional inactivation of topoisomerase II, another structural component of chromosomes, were also aggravated in the absence of SET-2. Combined, our in vivo findings suggest a model in which the SET1/COMPASS histone methyltransferase complex plays a role in shaping meiotic chromosome in cooperation with the non-histone proteins condensin-II and topoisomerase.

scholarly journals A Role for Caenorhabditis elegans COMPASS in Germline Chromatin Organization

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 2049
Author(s):  
Marion Herbette ◽  
Valérie Robert ◽  
Aymeric Bailly ◽  
Loïc Gely ◽  
Robert Feil ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Topoisomerase Ii ◽  
Meiotic Chromosome ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Chromatin Organization ◽  
Chromosome Organization ◽  
Fluorescence Lifetime Imaging ◽  
C Elegans ◽  
Meiotic Chromosomes

Deposition of histone H3 lysine 4 (H3K4) methylation at promoters is catalyzed by the SET1/COMPASS complex and is associated with context-dependent effects on gene expression and local changes in chromatin organization. The role of SET1/COMPASS in shaping chromosome architecture has not been investigated. Here we used Caenorhabditis elegans to address this question through a live imaging approach and genetic analysis. Using quantitative FRET (Förster resonance energy transfer)-based fluorescence lifetime imaging microscopy (FLIM) on germ cells expressing histones eGFP-H2B and mCherry-H2B, we find that SET1/COMPASS influences meiotic chromosome organization, with marked effects on the close proximity between nucleosomes. We further show that inactivation of set-2, encoding the C. elegans SET1 homologue, or CFP-1, encoding the chromatin targeting subunit of COMPASS, enhances germline chromosome organization defects and sterility of condensin-II depleted animals. set-2 loss also aggravates germline defects resulting from conditional inactivation of topoisomerase II, another structural component of chromosomes. Expression profiling of set-2 mutant germlines revealed only minor transcriptional changes, suggesting that the observed effects are at least partly independent of transcription. Altogether, our results are consistent with a role for SET1/COMPASS in shaping meiotic chromosomes in C. elegans, together with the non-histone proteins condensin-II and topoisomerase. Given the high degree of conservation, our findings expand the range of functions attributed to COMPASS and suggest a broader role in genome organization in different species.

scholarly journals Cooperation between Caenorhabditis elegans COMPASS and condensin in germline chromatin organization

2020 ◽  
Author(s):  
Marion Herbette ◽  
Valérie Robert ◽  
Aymeric Bailly ◽  
Loïc Gely ◽  
Robert Feil ◽  
...  
Keyword(s):  
Gene Expression ◽  
Topoisomerase Ii ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Higher Order ◽  
Chromosome Organization ◽  
Fluorescence Lifetime Imaging ◽  
Chromatin Compaction ◽  
C Elegans ◽  
Meiotic Chromosomes

Abstract Background Histone-modifying activities play important roles in gene expression and influence higher-order genome organization. SET1/COMPASS (Complex Proteins Associated with Set1) deposits h istone H3 lysine 4 (H3K4) methylation at promoter regions and is associated with context-dependent effects on gene expression. Whether it also contributes to higher-order chromosome organization has not been explored. Results Using a quantitative FRET (Förster resonance energy transfer)-based fluorescence lifetime imaging microscopy (FLIM) approach to assay nanometer scale chromatin compaction in live animals, we reveal a novel role for SET1/COMPASS in structuring meiotic chromosomes in the C. elegans germline . Inactivation of SET-2, the C. elegans homologue of SET1, strongly enhanced chromosome organization defects and loss of fertility resulting from depletion of condensin-II, and aggravated defects in chromosome morphology resulting from inactivation of topoisomerase II, another major structural component of chromosomes. Loss of CFP-1, the chromatin targeting subunit of COMPASS, similarly affected germline chromatin compaction measured by FLIM-FRET and enhanced condensin-II knock-down phenotypes. Conclusions The data presented here are consistent with a role of SET1/ COMPASS in shaping meiotic chromosomes in the C. elegans germline. This new insight has important implications for how c hromatin-modifying complexes and histone modifications may cooperate with non histone-proteins to achieve proper chromosome organization, not only in meiosis, but also in mitosis.

scholarly journals Discs large 1 controls daughter-cell polarity after cytokinesis in vertebrate morphogenesis

2018 ◽  
Vol 115 (46) ◽  
pp. E10859-E10868 ◽  
Author(s):  
Yuwei Li ◽  
Jason A. Junge ◽  
Cosimo Arnesano ◽  
Garrett G. Gross ◽  
Jeffrey H. Miner ◽  
...  
Keyword(s):  
Cell Polarity ◽  
Protein Interactions ◽  
Protein Function ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Explant Culture ◽  
Scaffolding Protein ◽  
Fluorescence Lifetime Imaging ◽  
Discs Large

Vertebrate embryogenesis and organogenesis are driven by cell biological processes, ranging from mitosis and migration to changes in cell size and polarity, but their control and causal relationships are not fully defined. Here, we use the developing limb skeleton to better define the relationships between mitosis and cell polarity. We combine protein-tagging and -perturbation reagents with advanced in vivo imaging to assess the role of Discs large 1 (Dlg1), a membrane-associated scaffolding protein, in mediating the spatiotemporal relationship between cytokinesis and cell polarity. Our results reveal that Dlg1 is enriched at the midbody during cytokinesis and that its multimerization is essential for the normal polarity of daughter cells. Defects in this process alter tissue dimensions without impacting other cellular processes. Our results extend the conventional view that division orientation is established at metaphase and anaphase and suggest that multiple mechanisms act at distinct phases of the cell cycle to transmit cell polarity. The approach employed can be used in other systems, as it offers a robust means to follow and to eliminate protein function and extends the Phasor approach for studying in vivo protein interactions by frequency-domain fluorescence lifetime imaging microscopy of Förster resonance energy transfer (FLIM-FRET) to organotypic explant culture.

scholarly journals The in vivo mechanics of the magnetotactic backbone as revealed by correlative FLIM-FRET and STED microscopy

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Erika Günther ◽  
André Klauß ◽  
Mauricio Toro-Nahuelpan ◽  
Dirk Schüler ◽  
Carsten Hille ◽  
...  
Keyword(s):  
Magnetic Particles ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Stimulated Emission ◽  
Fluorescence Lifetime Imaging ◽  
Sted Microscopy

AbstractProtein interaction and protein imaging strongly benefit from the advancements in time-resolved and superresolution fluorescence microscopic techniques. However, the techniques were typically applied separately and ex vivo because of technical challenges and the absence of suitable fluorescent protein pairs. Here, we show correlative in vivo fluorescence lifetime imaging microscopy Förster resonance energy transfer (FLIM-FRET) and stimulated emission depletion (STED) microscopy to unravel protein mechanics and structure in living cells. We use magnetotactic bacteria as a model system where two proteins, MamJ and MamK, are used to assemble magnetic particles called magnetosomes. The filament polymerizes out of MamK and the magnetosomes are connected via the linker MamJ. Our system reveals that bacterial filamentous structures are more fragile than the connection of biomineralized particles to this filament. More importantly, we anticipate the technique to find wide applicability for the study and quantification of biological processes in living cells and at high resolution.

scholarly journals Intravital quantification reveals dynamic calcium concentration changes across B cell differentiation stages

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Carolin Ulbricht ◽  
Ruth Leben ◽  
Asylkhan Rakhymzhan ◽  
Frank Kirchhoff ◽  
Lars Nitschke ◽  
...  
Keyword(s):  
B Cells ◽  
Stress Responses ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Affinity Maturation ◽  
Fluorescence Lifetime Imaging ◽  
B Cell Differentiation ◽  
Concentration Changes ◽  
Activated B Cells

Calcium is a universal second messenger present in all eukaryotic cells. The mobilization and storage of Ca2+ ions drives a number of signaling-related processes, stress–responses, or metabolic changes, all of which are relevant for the development of immune cells and their adaption to pathogens. Here, we introduce the Förster resonance energy transfer (FRET)-reporter mouse YellowCaB expressing the genetically encoded calcium indicator TN-XXL in B lymphocytes. Calcium-induced conformation change of TN-XXL results in FRET-donor quenching measurable by two-photon fluorescence lifetime imaging. For the first time, using our novel numerical analysis, we extract absolute cytoplasmic calcium concentrations in activated B cells during affinity maturation in vivo. We show that calcium in activated B cells is highly dynamic and that activation introduces a persistent calcium heterogeneity to the lineage. A characterization of absolute calcium concentrations present at any time within the cytosol is therefore of great value for the understanding of long-lived beneficial immune responses and detrimental autoimmunity.

scholarly journals Podoplanin Associates with CD44 to Promote Directional Cell Migration

2010 ◽  
Vol 21 (24) ◽  
pp. 4387-4399 ◽  
Author(s):  
Ester Martín-Villar ◽  
Beatriz Fernández-Muñoz ◽  
Maddy Parsons ◽  
Maria M. Yurrita ◽  
Diego Megías ◽  
...  
Keyword(s):  
Cell Migration ◽  
Epithelial Mesenchymal Transition ◽  
Resonance Energy Transfer ◽  
Mouse Skin ◽  
Resonance Energy ◽  
Fluorescence Lifetime Imaging ◽  
Mesenchymal Transition ◽  
Tumor Cell Migration ◽  
Podoplanin Expression

Podoplanin is a transmembrane glycoprotein up-regulated in different human tumors, especially those derived from squamous stratified epithelia (SCCs). Its expression in tumor cells is linked to increased cell migration and invasiveness; however, the mechanisms underlying this process remain poorly understood. Here we report that CD44, the major hyaluronan (HA) receptor, is a novel partner for podoplanin. Expression of the CD44 standard isoform (CD44s) is coordinately up-regulated together with that of podoplanin during progression to highly aggressive SCCs in a mouse skin model of carcinogenesis, and during epithelial-mesenchymal transition (EMT). In carcinoma cells, CD44 and podoplanin colocalize at cell surface protrusions. Moreover, CD44 recruitment promoted by HA-coated beads or cross-linking with a specific CD44 antibody induced corecruitment of podoplanin. Podoplanin–CD44s interaction was demonstrated both by coimmunoprecipitation experiments and, in vivo, by fluorescence resonance energy transfer/fluorescence lifetime imaging microscopy (FRET/FLIM), the later confirming its association on the plasma membrane of cells with a migratory phenotype. Importantly, we also show that podoplanin promotes directional persistence of motility in epithelial cells, a feature that requires CD44, and that both molecules cooperate to promote directional migration in SCC cells. Our results support a role for CD44-podoplanin interaction in driving tumor cell migration during malignancy.

scholarly journals Measuring NDC80 binding reveals the molecular basis of tension-dependent kinetochore-microtubule attachments

2018 ◽  
Author(s):  
Tae Yeon Yoo ◽  
Jeong-Mo Choi ◽  
William Conway ◽  
Che-Hang Yu ◽  
Rohit V. Pappu ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Molecular Basis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Aurora B ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Ndc80 Complex

AbstractProper kinetochore-microtubule attachments, mediated by the NDC80 complex, are required for error-free chromosome segregation. Erroneous attachments are corrected by the tension dependence of kinetochore-microtubule interactions. Here, we present a method, based on fluorescence lifetime imaging microscopy and Förster resonance energy transfer, to quantitatively measure the fraction of NDC80 complexes bound to microtubules at individual kinetochores in living human cells. We found that NDC80 binding is modulated in a chromosome autonomous fashion over prometaphase and metaphase, and is predominantly regulated by centromere tension. We show that this tension dependency requires phosphorylation of the N-terminal tail of Hec1, a component of the NDC80 complex, and the proper localization of Aurora B kinase, which modulates NDC80 binding. Our results lead to a mathematical model of the molecular basis of tension-dependent NDC80 binding to kinetochore microtubules in vivo.

scholarly journals Imaging molecular interactions in cells by dynamic and static fluorescence anisotropy (rFLIM and emFRET)

2003 ◽  
Vol 31 (5) ◽  
pp. 1020-1027 ◽  
Author(s):  
D.S. Lidke ◽  
P. Nagy ◽  
B.G. Barisas ◽  
R. Heintzmann ◽  
J.N. Post ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Laser Scanning ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Wide Field ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging

We report the implementation and exploitation of fluorescence polarization measurements, in the form of anisotropy fluorescence lifetime imaging microscopy (rFLIM) and energy migration Förster resonance energy transfer (emFRET) modalities, for wide-field, confocal laser-scanning microscopy and flow cytometry of cells. These methods permit the assessment of rotational motion, association and proximity of cellular proteins in vivo. They are particularly applicable to probes generated by fusions of visible fluorescence proteins, as exemplified by studies of the erbB receptor tyrosine kinases involved in growth-factor-mediated signal transduction.

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