scholarly journals Aurora B dynamics at centromeres create a diffusion-based phosphorylation gradient

2011 ◽  
Vol 194 (4) ◽  
pp. 539-549 ◽  
Author(s):  
Enxiu Wang ◽  
Edward R. Ballister ◽  
Michael A. Lampson
Keyword(s):  
Cell Division ◽  
Spatial Information ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Aurora B ◽  
Kinase Activation ◽  
Chromosomal Site ◽  
Substrate Phosphorylation ◽  
Dynamic Exchange ◽  
And Diffusion

Aurora B kinase is essential for successful cell division and regulates spindle assembly and kinetochore–microtubule interactions. The kinase localizes to the inner centromere until anaphase, but many of its substrates have distinct localizations, for example on chromosome arms and at kinetochores. Furthermore, substrate phosphorylation depends on distance from the kinase. How the kinase reaches substrates at a distance and how spatial phosphorylation patterns are determined are unknown. In this paper, we show that a phosphorylation gradient is produced by Aurora B concentration and activation at centromeres and release and diffusion to reach substrates at a distance. Kinase concentration, either at centromeres or at another chromosomal site, is necessary for activity globally. By experimentally manipulating dynamic exchange at centromeres, we demonstrate that the kinase reaches its substrates by diffusion. We also directly observe, using a fluorescence resonance energy transfer–based biosensor, phosphorylation spreading from centromeres after kinase activation. We propose that Aurora B dynamics and diffusion from the inner centromere create spatial information to regulate cell division.

scholarly journals Inhibitory and Stimulatory Micropeptides Preferentially Bind to Different Conformations of the Cardiac Calcium Pump

2021 ◽  
Author(s):  
Sean R. Cleary ◽  
Xuan Fang ◽  
Ellen E. Cho ◽  
Marsha P. Pribadi ◽  
Jaroslava Seflova ◽  
...  
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Ion Pump ◽  
Competing Interactions ◽  
Relaxation Cycle ◽  
Reading Frame ◽  
Preferential Binding ◽  
Physical Interactions ◽  
Mechanistic Basis ◽  
Dynamic Exchange

The ATP-dependent ion pump SERCA sequesters Ca2+ in the endoplasmic reticulum to establish a reservoir for cell signaling. Because of its central importance in physiology, this transporter is tightly controlled by physical interactions with tissue-specific regulatory micropeptides that tune SERCA function to match changing physiological conditions. In the heart, phospholamban (PLB) inhibits SERCA, while dwarf open reading frame (DWORF) stimulates SERCA. These competing interactions determine cardiac performance by modulating the amplitude of Ca2+ signals that drive the contraction/relaxation cycle. The distinct functions of these peptides may relate to their reciprocal preferences for SERCA binding. While SERCA binds PLB more avidly at low cytoplasmic Ca2+, it binds DWORF better at high Ca2+. In the present study, we determined that this opposing Ca2+ sensitivity is due to preferential binding of DWORF and PLB to different intermediate conformations that the pump samples during the Ca2+ transport cycle. The results suggest a mechanistic basis for inhibitory and stimulatory micropeptide function. In addition, fluorescence resonance energy transfer (FRET) measurements revealed dynamic shifts in SERCA-micropeptide binding equilibria during cellular Ca2+ elevations. The data suggest Ca2+-dependent dynamic exchange of inhibitory and stimulatory micropeptides from SERCA during the cardiac cycle. Together, these mechanisms provide beat-to-beat modulation of cardiac Ca2+ handling and contribute to the heart's adaptation to the increased physiological demands of exercise.

scholarly journals ZapA tetramerization is required for midcell localization and ZapB interaction in Escherichia coli

2019 ◽  
Author(s):  
Nils Y. Meiresonne ◽  
Tanneke den Blaauwen
Keyword(s):  
Cell Division ◽  
Chromosome Segregation ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bacterial Cell Division ◽  
Cellular Processes ◽  
Interaction Partners ◽  
Associated Proteins

AbstractBacterial cell division is guided by FtsZ treadmilling precisely at midcell. FtsZ itself is regulated by FtsZ associated proteins (Zaps) that couple it to different cellular processes. ZapA is known to enhance FtsZ bundling but also forms the synchronizing link with chromosome segregation through ZapB and matS bound MatP. ZapA exists as dimers and tetramers in the cell. Using the ZapAI83E mutant that only forms dimers, this paper investigates the effects of ZapA multimerization state on its interaction partners and cell division. By employing (fluorescence) microscopy and Förster Resonance Energy Transfer in vivo it is shown that; dimeric ZapA is unable to complement a zapA deletion strain and localizes diffusely through the cell but still interacts with FtsZ that is not part of the cell division machinery. Dimeric ZapA is unable to recruit ZapB, which localizes in its presence unipolarly in the cell. Interestingly, the localization profiles of the chromosome and unipolar ZapB anticorrelate. The work presented here confirms previously reported in vitro effects of ZapA multimerization in vivo and further places it in a broader context by revealing the strong implications for ZapB localization and ter linkage.

scholarly journals Investigating molecular crowding during cell division in budding yeast with FRET

2021 ◽  
Author(s):  
Sarah Lecinski ◽  
Jack Shepherd ◽  
Lewis Frame ◽  
Immy Hayton ◽  
Chris MacDonald ◽  
...  
Keyword(s):  
Cell Division ◽  
Single Molecule ◽  
Budding Yeast ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Molecular Crowding ◽  
Yeast Saccharomyces Cerevisiae ◽  
Recent Advances ◽  
Spatial Reorganization ◽  
Mother Cells

Cell division, aging, and stress recovery triggers the spatial reorganization of cellular components in the cytoplasm, but also of membrane bound organelles, with molecular changes in their compositions and structures. However, it is not clear how these events are coordinated and how they integrate with regulation of molecular crowding. We use the budding yeast Saccharomyces cerevisiae as a model eukaryotic unicellular organism to study these questions using recent progress in optical fluorescence microscopy and crowding sensing probe technology. We used a F&oumlrster Resonance Energy Transfer (FRET) based sensor, illuminated by confocal microscopy for high throughput analyses and Slimfield microscopy for single-molecule resolution, to quantify molecular crowding. We determine crowding in response to growth and osmotic stress, and its dependence on mother and daughter cells during division, and find unexpectedly the presence of hot spots of crowding across the bud neck in the burgeoning daughter cell, which might be rationalized by the packing of inherited material, like the vacuole, from mother cells. We discuss recent advances in understanding the role of crowding in cellular regulation and key current challenges, and conclude by presenting our recent advances in optimizing FRET-based measurements of crowding whilst simultaneously imaging a third colour, which can be used as an organelle marker and to readout membrane morphology. Our approaches can be combined with synchronised cell populations to increase experimental throughput and correlate molecular crowding information with different stages in the cell cycle.

scholarly journals Two Orders of Magnitude Variation of Diffusion-Enhanced Förster Resonance Energy Transfer in Polypeptide Chains

Polymers ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 1079 ◽  
Author(s):  
Maik Jacob ◽  
Indrajit Ghosh ◽  
Roy D’Souza ◽  
Werner Nau
Keyword(s):  
Energy Transfer ◽  
Dynamic Properties ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Forster Resonance Energy Transfer ◽  
Förster Resonance Energy Transfer ◽  
Donor And Acceptor ◽  
The Impact ◽  
And Diffusion ◽  
Förster Resonance

A flexible peptide chain displays structural and dynamic properties that correspond to its folding and biological activity. These properties are mirrored in intrachain site-to-site distances and diffusion coefficients of mutual site-to-site motion. Both distance distribution and diffusion determine the extent of Förster resonance energy transfer (FRET) between two sites labeled with a FRET donor and acceptor. The relatively large Förster radii of traditional FRET methods (R0 > 20 Å) lead to a fairly low contribution of diffusion. We introduced short-distance FRET (sdFRET) where Dbo, an asparagine residue conjugated to 2,3-diazabicyclo[2.2.2]octane, acts as acceptor paired with donors, such as naphtylalanine (NAla), tryptophan, 5-l-fluorotryptophan, or tyrosine. The Förster radii are always close to 10 Å, which makes sdFRET highly sensitive to diffusional motion. We recently found indications that the FRET enhancement caused by diffusion depends symmetrically on the product of the radiative fluorescence lifetime of the donor and the diffusion coefficient. In this study, we varied this product by two orders of magnitude, using both donors of different lifetime, NAla and FTrp, as well as a varying viscogen concentration, to corroborate this statement. We demonstrate the consequences of this relationship in evaluating the impact of viscogenic coadditives on peptide dimensions.

scholarly journals A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C

2003 ◽  
Vol 161 (5) ◽  
pp. 899-909 ◽  
Author(s):  
Jonathan D. Violin ◽  
Jin Zhang ◽  
Roger Y. Tsien ◽  
Alexandra C. Newton
Keyword(s):  
Hela Cells ◽  
Mammalian Cells ◽  
Mdck Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Calcium Oscillation ◽  
Fluorescent Reporter ◽  
Temporal Regulation ◽  
Fluorescent Reporters ◽  
Substrate Phosphorylation

Signals transduced by kinases depend on the extent and duration of substrate phosphorylation. We generated genetically encoded fluorescent reporters for PKC activity that reversibly respond to stimuli activating PKC. Specifically, phosphorylation of the reporter expressed in mammalian cells causes changes in fluorescence resonance energy transfer (FRET), allowing real time imaging of phosphorylation resulting from PKC activation. Targeting of the reporter to the plasma membrane, where PKC is activated, reveals oscillatory phosphorylation in HeLa cells in response to histamine. Each oscillation in substrate phosphorylation follows a calcium oscillation with a lag of ∼10 s. Novel FRET-based reporters for PKC translocation, phosphoinositide bisphosphate conversion to IP3, and diacylglycerol show that in HeLa cells the oscillatory phosphorylations correlate with Ca2+-controlled translocation of conventional PKC to the membrane without oscillations of PLC activity or diacylglycerol. However, in MDCK cells stimulated with ATP, PLC and diacylglycerol fluctuate together with Ca2+ and phosphorylation. Thus, specificity of PKC signaling depends on the local second messenger-controlled equilibrium between kinase and phosphatase activities to result in strict calcium-controlled temporal regulation of substrate phosphorylation.

scholarly journals Disconnect between signalling potency and in vivo efficacy of pharmacokinetically optimised biased glucagon-like peptide-1 receptor agonists

2019 ◽  
Author(s):  
Maria Lucey ◽  
Philip Pickford ◽  
James Minnion ◽  
Jan Ungewiss ◽  
Katja Schoeneberg ◽  
...  
Keyword(s):  
Therapeutic Potential ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Trafficking ◽  
Appetite Suppression ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
And Diffusion

AbstractObjectiveTo determine how pharmacokinetically advantageous acylation impacts on glucagon-like peptide-1 receptor (GLP-1R) signal bias, trafficking, anti-hyperglycaemic efficacy and appetite suppression.MethodsIn vitro signalling responses were measured using biochemical and biosensor assays. GLP-1 receptor trafficking was determined by confocal microscopy and diffusion-enhanced resonance energy transfer. Pharmacokinetics, glucoregulatory effects and appetite suppression were measured in acute, sub-chronic and chronic settings in mice.ResultsA C-terminally acylated ligand, exendin-phe1-C16, was identified with undetectable β-arrestin recruitment and GLP-1R internalisation. Depending on the cellular system used, this molecule was up to 1000-fold less potent than the comparator exendin-asp-3-C16 for cyclic AMP signalling, yet was considerably more effective in vivo, particularly for glucose regulation.ConclusionsC-terminal acylation of biased GLP-1R agonists increases their degree of signal bias in favour of cAMP production and improves their therapeutic potential.

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