scholarly journals Live Imaging of Phosphate Levels in Arabidopsis Root Cells Expressing a FRET-Based Phosphate Sensor

Plants ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 1310
Author(s):  
Ana G. L. Assunção ◽  
Sisse K. Gjetting ◽  
Michael Hansen ◽  
Anja T. Fuglsang ◽  
Alexander Schulz
Keyword(s):  
Sustainable Use ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Imaging ◽  
Root Cells ◽  
Cellular Processes ◽  
Mammalian Cell Lines ◽  
Pi Transport ◽  
Seedling Roots ◽  
Use Efficiency

Phosphorous (P) is an essential macronutrient in all organisms serving various fundamental biological processes, and is one of the least available plant nutrients in the soil. The application of inorganic phosphate (Pi) fertilizers is frequent, but it has a high environmental and financial cost. Breeding crops for improved Pi use-efficiency is a promising plant-based solution to pursue a reduction of fertilizer dependency. Availability of tools for monitoring changes of plant cellular Pi concentration in real-time can contribute to advancing knowledge on the molecular basis of Pi transport and homeostasis in plants. Genetically encoded fluorescent sensors have provided new insight on cellular processes. Here, we show that two Pi Fluorescence Resonance Energy Transfer (FRET)-based sensors from the FLIPPi family, the low-affinity FLIPPi-30m and the high-affinity FLIPPi-4µ, can be expressed and analyzed in Arabidopsis thaliana with wild-type background. These FLIPPi sensors had not been tested in plants, but only in mammalian cell lines. We show FRET response and live imaging of Pi levels in seedling roots of Arabidopsis FLIPPi-30m and FLIPPi-4µ lines. Our results reinforce that sensors from the FLIPPi family are valuable tools for studying mechanisms of Pi transport and homeostasis in plants, and for research towards a more sustainable use of Pi fertilization.

scholarly journals Genetically Encoded Fluorescent Sensor for Poly-ADP-Ribose

2020 ◽  
Vol 21 (14) ◽  
pp. 5004
Author(s):  
Ekaterina O. Serebrovskaya ◽  
Nadezda M. Podvalnaya ◽  
Varvara V. Dudenkova ◽  
Anna S. Efremova ◽  
Nadya G. Gurskaya ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Mammalian Cells ◽  
Fluorescent Proteins ◽  
Fluorescent Sensor ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Post Translational Modification ◽  
Cellular Processes ◽  
Hydrogen Peroxide Treatment ◽  
Damage Response

Poly-(ADP-ribosyl)-ation (PARylation) is a reversible post-translational modification of proteins and DNA that plays an important role in various cellular processes such as DNA damage response, replication, transcription, and cell death. Here we designed a fully genetically encoded fluorescent sensor for poly-(ADP-ribose) (PAR) based on Förster resonance energy transfer (FRET). The WWE domain, which recognizes iso-ADP-ribose internal PAR-specific structural unit, was used as a PAR-targeting module. The sensor consisted of cyan Turquoise2 and yellow Venus fluorescent proteins, each in fusion with the WWE domain of RNF146 E3 ubiquitin ligase protein. This bipartite sensor named sPARroW (sensor for PAR relying on WWE) enabled monitoring of PAR accumulation and depletion in live mammalian cells in response to different stimuli, namely hydrogen peroxide treatment, UV irradiation and hyperthermia.

scholarly journals Signal processing for molecular and cellular biological physics: an emerging field

2013 ◽  
Vol 371 (1984) ◽  
pp. 20110546 ◽  
Author(s):  
Max A. Little ◽  
Nick S. Jones
Keyword(s):  
Signal Processing ◽  
Optical Tweezers ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Digital Signal ◽  
Force Microscopy ◽  
Cellular Processes ◽  
Atomic Force ◽  
Non Gaussian ◽  
Autocorrelated Noise

Recent advances in our ability to watch the molecular and cellular processes of life in action—such as atomic force microscopy, optical tweezers and Forster fluorescence resonance energy transfer—raise challenges for digital signal processing (DSP) of the resulting experimental data. This article explores the unique properties of such biophysical time series that set them apart from other signals, such as the prevalence of abrupt jumps and steps, multi-modal distributions and autocorrelated noise. It exposes the problems with classical linear DSP algorithms applied to this kind of data, and describes new nonlinear and non-Gaussian algorithms that are able to extract information that is of direct relevance to biological physicists. It is argued that these new methods applied in this context typify the nascent field of biophysical DSP. Practical experimental examples are supplied.

scholarly journals Bacterial dynamin-like protein DynA mediates lipid and content mixing and shows phospholipid specificity

2018 ◽  
Author(s):  
Lijun Guo ◽  
Marc Bramkamp
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Membrane Stress ◽  
Gtp Hydrolysis ◽  
Cellular Processes ◽  
Organelle Division ◽  
Membrane Tethering ◽  
Hydrolysis Of

ABSTRACTThe dynamins family of GTPases is involved in key cellular processes in eukaryotes, including vesicle trafficking and organelle division. The GTP hydrolysis cycle of dynamin translates to a conformational change in the protein structure, which forces the underlying lipid layer into an energetically unstable conformation that promotes membrane rearrangements. Many bacterial genomes encode dynamin-like proteins, but the biological function of these proteins has remained largely enigmatic. In recent years, our group has reported that the dynamin-like protein DynA from Bacillus subtilis mediates nucleotide-independent membrane tethering in vitro and contributes to the innate immunity of bacteria against membrane stress and phage infection. However, so far the mechanism of membrane stress response and the role of GTP hydrolysis remain unclear. Here, we employed content mixing and lipid mixing assays in reconstituted systems to study if the dynamin-like protein DynA from B. subtilis induces membrane full fusion, and further test the possibility that GTP hydrolysis of DynA may act on the fusion-through-hemifusion pathway. Our results based on fluorescence resonance energy transfer (FRET) indicated that DynA could induce aqueous content mixing even in absence of GTP. Moreover, DynA-induced membrane fusion in vitro is a thermo-promoted slow response. Surprisingly, digestion of protein mediated an instantl rise of content exchange, supporting the assumption that disassembly of DynA is the fundamental power for fusion-through-hemifusion.

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 Signaling diversity enabled by Rap1-regulated plasma membrane ERK with distinct temporal dynamics

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Jeremiah Keyes ◽  
Ambhighainath Ganesan ◽  
Olivia Molinar-Inglis ◽  
Archer Hamidzadeh ◽  
Jinfan Zhang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Temporal Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Signaling Specificity ◽  
Temporal Regulation ◽  
Cellular Processes ◽  
Extracellular Signal ◽  
Transient Activity ◽  
Erk Activity

A variety of different signals induce specific responses through a common, extracellular-signal regulated kinase (ERK)-dependent cascade. It has been suggested that signaling specificity can be achieved through precise temporal regulation of ERK activity. Given the wide distrubtion of ERK susbtrates across different subcellular compartments, it is important to understand how ERK activity is temporally regulated at specific subcellular locations. To address this question, we have expanded the toolbox of Förster Resonance Energy Transfer (FRET)-based ERK biosensors by creating a series of improved biosensors targeted to various subcellular regions via sequence specific motifs to measure spatiotemporal changes in ERK activity. Using these sensors, we showed that EGF induces sustained ERK activity near the plasma membrane in sharp contrast to the transient activity observed in the cytoplasm and nucleus. Furthermore, EGF-induced plasma membrane ERK activity involves Rap1, a noncanonical activator, and controls cell morphology and EGF-induced membrane protrusion dynamics. Our work strongly supports that spatial and temporal regulation of ERK activity is integrated to control signaling specificity from a single extracellular signal to multiple cellular processes.

scholarly journals BacMam System for FRET-Based cAMP Sensor Expression in Studies of Melanocortin MC1 Receptor Activation

2012 ◽  
Vol 17 (8) ◽  
pp. 1096-1101 ◽  
Author(s):  
Olga Mazina ◽  
Reet Reinart-Okugbeni ◽  
Sergei Kopanchuk ◽  
Ago Rinken
Keyword(s):  
High Throughput Screening ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cyclic Adenosine Monophosphate ◽  
Second Messenger ◽  
Adenosine Monophosphate ◽  
Receptor Activation ◽  
Dependent Manner ◽  
Endogenous Expression ◽  
Mammalian Cell Lines

Cyclic adenosine monophosphate (cAMP) is a second messenger of many G-protein-coupled receptors (GPCRs) and a useful readout molecule to estimate the biological activity of various GPCR-specific agents. Here we report the development and use of a Förster resonance energy transfer (FRET) biosensor for cAMP (Epac2-camps) combined with a baculovirus-based BacMam transduction system. The constructed BacMam-Epac2-camps viral transduction system is a simple and robust tool for ligand screening at the second-messenger level in a variety of mammalian cell lines. The level of biosensor protein expression can easily be adjusted in a dose-dependent manner depending on the multiplicity of viral infection. For setting up the assay, we used a B16F10 murine melanoma cell line with endogenous expression of melanocortin-1 receptor (MC1R). The receptor activation was characterized by a set of MC1R full and partial agonists. Bivalent ions Ca2+ as well as Mg2+ modulated ligand potencies, whereas the effect was ligand and ion specific. Results obtained for MC1R indicate that the BacMam-Epac2-camps system may also be applicable for studying the activation of other GPCRs and may be implemented in routine analysis as well as in high-throughput screening.

scholarly journals Twisting and subunit rotation in single F O F 1 -ATP synthase

2013 ◽  
Vol 368 (1611) ◽  
pp. 20120024 ◽  
Author(s):  
Hendrik Sielaff ◽  
Michael Börsch
Keyword(s):  
Energy Transfer ◽  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Step Size ◽  
Storage Mechanism ◽  
Cellular Processes ◽  
Recent Developments ◽  
Subunit Rotation ◽  
Atp Synthases

F O F 1 -ATP synthases are ubiquitous proton- or ion-powered membrane enzymes providing ATP for all kinds of cellular processes. The mechanochemistry of catalysis is driven by two rotary nanomotors coupled within the enzyme. Their different step sizes have been observed by single-molecule microscopy including videomicroscopy of fluctuating nanobeads attached to single enzymes and single-molecule Förster resonance energy transfer. Here we review recent developments of approaches to monitor the step size of subunit rotation and the transient elastic energy storage mechanism in single F O F 1 -ATP synthases.

scholarly journals A practical method for monitoring FRET-based biosensors in living animals using two-photon microscopy

2015 ◽  
Vol 309 (11) ◽  
pp. C724-C735 ◽  
Author(s):  
Wen Tao ◽  
Michael Rubart ◽  
Jennifer Ryan ◽  
Xiao Xiao ◽  
Chunping Qiao ◽  
...  
Keyword(s):  
Cell Biology ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Practical Method ◽  
Two Photon Microscopy ◽  
Cellular Processes ◽  
Two Photon ◽  
Photon Excitation ◽  
Wide Range

The commercial availability of multiphoton microscope systems has nurtured the growth of intravital microscopy as a powerful technique for evaluating cell biology in the relevant context of living animals. In parallel, new fluorescent protein (FP) biosensors have become available that enable studies of the function of a wide range of proteins in living cells. Biosensor probes that exploit Förster resonance energy transfer (FRET) are among the most sensitive indicators of an array of cellular processes. However, differences between one-photon and two-photon excitation (2PE) microscopy are such that measuring FRET by 2PE in the intravital setting remains challenging. Here, we describe an approach that simplifies the use of FRET-based biosensors in intravital 2PE microscopy. Based on a systematic comparison of many different FPs, we identified the monomeric (m) FPs mTurquoise and mVenus as particularly well suited for intravital 2PE FRET studies, enabling the ratiometric measurements from linked FRET probes using a pair of experimental images collected simultaneously. The behavior of the FPs is validated by fluorescence lifetime and sensitized emission measurements of a set of FRET standards. The approach is demonstrated using a modified version of the AKAR protein kinase A biosensor, first in cells in culture, and then in hepatocytes in the liver of living mice. The approach is compatible with the most common 2PE microscope configurations and should be applicable to a variety of different FRET probes.

scholarly journals Involvement of Sphingosine-1-Phosphate in Glutamate Secretion in Hippocampal Neurons

2007 ◽  
Vol 27 (9) ◽  
pp. 3429-3440 ◽  
Author(s):  
Taketoshi Kajimoto ◽  
Taro Okada ◽  
Huan Yu ◽  
Sravan K. Goparaju ◽  
Saleem Jahangeer ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Lipid Mediator ◽  
Cellular Processes ◽  
Sphingosine 1 Phosphate ◽  
Key Enzyme ◽  
The Central Nervous System ◽  
Interfering Rna

ABSTRACT Neuronal activity greatly influences the formation and stabilization of synapses. Although receptors for sphingosine-1-phosphate (S1P), a lipid mediator regulating diverse cellular processes, are abundant in the central nervous system, neuron-specific functions of S1P remain largely undefined. Here, we report two novel actions of S1P using primary hippocampal neurons as a model system: (i) as a secretagogue where S1P triggers glutamate secretion and (ii) as an enhancer where S1P potentiates depolarization-evoked glutamate secretion. Sphingosine kinase 1 (SK1), a key enzyme for S1P production, was enriched in functional puncta of hippocampal neurons. Silencing SK1 expression by small interfering RNA as well as SK1 inhibition by dimethylsphingosine resulted in a strong inhibition of depolarization-evoked glutamate secretion. Fluorescence recovery after photobleaching analysis showed translocation of SK1 from cytosol to membranes at the puncta during depolarization, which resulted in subsequent accumulation of S1P within cells. Fluorescent resonance energy transfer analysis demonstrated that the S1P1 receptor at the puncta was activated during depolarization and that depolarization-induced S1P1 receptor activation was inhibited in SK1-knock-down cells. Importantly, exogenously added S1P at a nanomolar concentration by itself elicited glutamate secretion from hippocampal cells even when the Na+-channel was blocked by tetrodotoxin, suggesting that S1P acts on presynaptic membranes. Furthermore, exogenous S1P at a picomolar level potentiated depolarization-evoked secretion in the neurons. These findings indicate that S1P, through its autocrine action, facilitates glutamate secretion in hippocampal neurons both by secretagogue and enhancer actions and may be involved in mechanisms underlying regulation of synaptic transmission.

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