scholarly journals Dynamic FRET-FLIM based screening of signal transduction pathways

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Rolf Harkes ◽  
Olga Kukk ◽  
Sravasti Mukherjee ◽  
Jeffrey Klarenbeek ◽  
Bram van den Broek ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Hela Cells ◽  
Source Analysis ◽  
High Temporal Resolution ◽  
Fluorescence Lifetime Imaging ◽  
Decay Kinetics ◽  
Specific Expression ◽  
Breakdown Rates ◽  
Dynamic Screening ◽  
Camp Levels

AbstractFluorescence Lifetime Imaging (FLIM) is an intrinsically quantitative method to screen for protein–protein interactions and is frequently used to record the outcome of signal transduction events. With new highly sensitive and photon efficient FLIM instrumentation, the technique also becomes attractive to screen, with high temporal resolution, for fast changes in Förster Resonance Energy Transfer (FRET), such as those occurring upon activation of cell signaling. The second messenger cyclic adenosine monophosphate (cAMP) is rapidly formed following activation of certain cell surface receptors. cAMP is subsequently degraded by a set of phosphodiesterases (PDEs) which display cell-type specific expression and may also affect baseline levels of the messenger. To study which specific PDEs contribute most to cAMP regulation, we knocked down individual PDEs and recorded breakdown rates of cAMP levels following transient stimulation in HeLa cells stably expressing the FRET/FLIM sensor, Epac-SH189. Many hundreds of cells were recorded at 5 s intervals for each condition. FLIM time traces were calculated for every cell, and decay kinetics were obtained. cAMP clearance was significantly slower when PDE3A and, to a lesser amount, PDE10A were knocked down, identifying these isoforms as dominant in HeLa cells. However, taking advantage of the quantitative FLIM data, we found that knockdown of individual PDEs has a very limited effect on baseline cAMP levels. By combining photon-efficient FLIM instrumentation with optimized sensors, systematic gene knockdown and an automated open-source analysis pipeline, our study demonstrates that dynamic screening of transient cell signals has become feasible. The quantitative platform described here provides detailed kinetic analysis of cellular signals in individual cells with unprecedented throughput.

scholarly journals Dynamic FRET-FLIM based screens of signal transduction pathways: a feasibility study

2021 ◽  
Author(s):  
Rolf Harkes ◽  
Olga Kukk ◽  
Sravasti Mukherjee ◽  
Jeffrey Klarenbeek ◽  
Bram van den Broek ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Protein Interactions ◽  
Hela Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Source Analysis ◽  
High Temporal Resolution ◽  
Fluorescence Lifetime Imaging ◽  
Protein Protein Interactions ◽  
Custom Made

Fluorescence Lifetime Imaging (FLIM) is an intrinsically quantitative method to screen for protein-protein interactions and frequently used to record the outcome of signal transduction events. With new highly sensitive and photon efficient FLIM instrumentation, the technique also becomes attractive to screen, with high temporal resolution, for fast changes in Forster Resonance Energy Transfer (FRET), such as those occurring upon activation of cell signaling. We studied the effects of siRNA-mediated individual knockdown of an extensive set of 22 different phosphodiesterases (PDEs) on baseline levels and agonist-induced changes of the second messenger cAMP. Using HeLa cells stably expressing our FRET-FLIM sensor we imaged many hundreds of cells at 5 second intervals for each condition. Following segmentation of cells by the deep-learning implementation Cellpose, FLIM time traces were calculated and fitted for dynamic analysis with custom-made Python scripts. Taking advantage of the quantitative FLIM data, we found very limited effects of PDE knockdown on baseline and agonist-induced peak levels of cAMP. However, cAMP breakdown in the decay phase was significantly slower when PDE3A and, to a lesser amount, PDE10A were knocked down, identifying these isoforms as dominant in HeLa cells. In conclusion, we present a robust platform that combines photon-efficient FLIM instrumentation with systematic gene knockdown and an automated open-source analysis pipeline. Our quantitative platform provides detailed kinetic analysis of cellular signals in individual cells with unprecedented throughput.

scholarly journals FRCaMP, a Red Fluorescent Genetically Encoded Calcium Indicator Based on Calmodulin from Schizosaccharomyces Pombe Fungus

2020 ◽  
Vol 22 (1) ◽  
pp. 111
Author(s):  
Oksana M. Subach ◽  
Natalia V. Barykina ◽  
Elizaveta S. Chefanova ◽  
Anna V. Vlaskina ◽  
Vladimir P. Sotskov ◽  
...  
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Hela Cells ◽  
Calcium Binding ◽  
Dynamic Range ◽  
Spectral Characteristics ◽  
Binding Domain ◽  
Calcium Transients ◽  
Neuronal Cultures ◽  
Decay Kinetics ◽  
Calcium Binding Domain

Red fluorescent genetically encoded calcium indicators (GECIs) have expanded the available pallet of colors used for the visualization of neuronal calcium activity in vivo. However, their calcium-binding domain is restricted by calmodulin from metazoans. In this study, we developed red GECI, called FRCaMP, using calmodulin (CaM) from Schizosaccharomyces pombe fungus as a calcium binding domain. Compared to the R-GECO1 indicator in vitro, the purified protein FRCaMP had similar spectral characteristics, brightness, and pH stability but a 1.3-fold lower ΔF/F calcium response and 2.6-fold tighter calcium affinity with Kd of 441 nM and 2.4–6.6-fold lower photostability. In the cytosol of cultured HeLa cells, FRCaMP visualized calcium transients with a ΔF/F dynamic range of 5.6, which was similar to that of R-GECO1. FRCaMP robustly visualized the spontaneous activity of neuronal cultures and had a similar ΔF/F dynamic range of 1.7 but 2.1-fold faster decay kinetics vs. NCaMP7. On electrically stimulated cultured neurons, FRCaMP demonstrated 1.8-fold faster decay kinetics and 1.7-fold lower ΔF/F values per one action potential of 0.23 compared to the NCaMP7 indicator. The fungus-originating CaM of the FRCaMP indicator version with a deleted M13-like peptide did not interact with the cytosolic environment of the HeLa cells in contrast to the metazoa-originating CaM of the similarly truncated version of the GCaMP6s indicator with a deleted M13-like peptide. Finally, we generated a split version of the FRCaMP indicator, which allowed the simultaneous detection of calcium transients and the heterodimerization of bJun/bFos interacting proteins in the nuclei of HeLa cells with a ΔF/F dynamic range of 9.4 and a contrast of 2.3–3.5, respectively.

scholarly journals Mutational Activation of a Gαi Causes Uncontrolled Proliferation of Aerial Hyphae and Increased Sensitivity to Heat and Oxidative Stress in Neurospora crassa

Genetics ◽  
1999 ◽  
Vol 151 (1) ◽  
pp. 107-117
Author(s):  
Qi Yang ◽  
Katherine A Borkovich
Keyword(s):  
Signal Transduction ◽  
Neurospora Crassa ◽  
Signal Transduction Pathway ◽  
Sexual Cycle ◽  
Intracellular Camp ◽  
Wild Type ◽  
Independent Functions ◽  
Aerial Hyphae ◽  
Mutational Activation ◽  
Camp Levels

Abstract Heterotrimeric G proteins, consisting of α, β, and γ subunits, transduce environmental signals through coupling to plasma membrane-localized receptors. We previously reported that the filamentous fungus Neurospora crassa possesses a Gα protein, GNA-1, that is a member of the Gαi superfamily. Deletion of gna-1 leads to defects in apical extension, differentiation of asexual spores, sensitivity to hyperosmotic media, and female fertility. In addition, Δgna-1 strains have lower intracellular cAMP levels under conditions that promote morphological abnormalities. To further define the function of GNA-1 in signal transduction in N. crassa, we examined properties of strains with mutationally activated gna-1 alleles (R178C or Q204L) as the only source of GNA-1 protein. These mutations are predicted to inhibit the GTPase activity of GNA-1 and lead to constitutive signaling. In the sexual cycle, gna-1R178C and gna-1Q204L strains are female-fertile, but produce fewer and larger perithecia than wild type. During asexual development, gna-1R178C and gna-1Q204L strains elaborate abundant, long aerial hyphae, produce less conidia, and possess lower levels of carotenoid pigments in comparison to wild-type controls. Furthermore, gna-1R178C and gna-1Q204L strains are more sensitive to heat shock and exposure to hydrogen peroxide than wild-type strains, while Δgna-1 mutants are more resistant. In contrast to Δgna-1 mutants, gna-1R178C and gna-1Q204L strains have higher steady-state levels of cAMP than wild type. The results suggest that GNA-1 possesses several Gβγ-independent functions in N. crassa. We propose that GNA-1 mediates signal transduction pathway(s) that regulate aerial hyphae development and sensitivity to heat and oxidative stresses, possibly through modulation of cAMP levels.

scholarly journals Transcriptome changes reveal the genetic mechanisms of the reproductive plasticity of workers in lower termites

BMC Genomics ◽  
2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Chenxu Ye ◽  
Humaira Rasheed ◽  
Yuehua Ran ◽  
Xiaojuan Yang ◽  
Lianxi Xing ◽  
...  
Keyword(s):  
Gene Expression ◽  
Signal Transduction ◽  
Molecular Mechanisms ◽  
Environmental Changes ◽  
Mapk Pathway ◽  
Specific Expression ◽  
Expression Of Genes ◽  
Genetic Mechanisms ◽  
Ecological Success ◽  
Reproductive Plasticity

Abstract Background The reproductive plasticity of termite workers provides colonies with tremendous flexibility to respond to environmental changes, which is the basis for evolutionary and ecological success. Although it is known that all colony members share the same genetic background and that differences in castes are caused by differences in gene expression, the pattern of the specific expression of genes involved in the differentiation of workers into reproductives remains unclear. In this study, the isolated workers of Reticulitermes labralis developed into reproductives, and then comparative transcriptomes were used for the first time to reveal the molecular mechanisms underlying the reproductive plasticity of workers. Results We identified 38,070 differentially expressed genes and found a pattern of gene expression involved in the differentiation of the workers into reproductives. 12, 543 genes were specifically upregulated in the isolated workers. Twenty-five signal transduction pathways classified into environmental information processing were related to the differentiation of workers into reproductives. Ras functions as a signalling switch regulates the reproductive plasticity of workers. The catalase gene which is related to longevity was up-regulated in reproductives. Conclusion We demonstrate that workers leaving the natal colony can induce the expression of stage-specific genes in the workers, which leads to the differentiation of workers into reproductives and suggests that the signal transduction along the Ras-MAPK pathway crucially controls the reproductive plasticity of the workers. This study also provides an important model for revealing the molecular mechanism of longevity changes.

scholarly journals Stimulation of tissue plasminogen activator production by retinoic acid: synergistic effect on protein kinase C-mediated activation

Blood ◽  
1992 ◽  
Vol 80 (4) ◽  
pp. 981-987 ◽  
Author(s):  
RD Medh ◽  
L Santell ◽  
EG Levin
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase C ◽  
Retinoic Acid ◽  
Protein Kinase ◽  
Hela Cells ◽  
Additive Effect ◽  
Kinase C ◽  
Tissue Plasminogen ◽  
Camp Levels ◽  
Stimulation Of

Abstract Trans retinoic acid (t-RA) stimulated the production of tissue plasminogen activator (tPA) in HeLa-S3 and human umbilical vein endothelial cells (huvecs) in a dose-dependent manner with maximal release (four to five times control) at 40 nmol/L and 40 mumol/L, respectively. In endothelial cells, the stimulation of tPA production by phorbol 12-myristate 13-acetate (PMA) was potentiated 1.9-fold by 10 mumol/L t-RA, or 1.8 times the additive effect. In HeLa cells, total tPA secretion with 10 nmol/L PMA was increased from 43 ng/mL to 96 ng/mL by 40 nmol/L t-RA, which was two times the additive effect. Higher concentrations of t-RA (400 nmol/L) depressed tPA secretion by itself and also suppressed PMA-induced tPA production by 50%. Histamine and thrombin also synergized with t-RA. t-RA (40 nmol/L) and 10 micrograms/mL histamine or 10 U/mL thrombin combined to induce tPA production 3.4 and 1.3 times the additive effect in HeLa cells. Cyclic adenosine monophosphate (cAMP) levels were not significantly affected by 10 nmol/L to 10 mumol/L t-RA. Nor did 10 nmol/L PMA and 40 nmol/L t- RA together affect cAMP levels, suggesting that t-RA-mediated potentiation of PMA-induced tPA production occurred via a mechanism that was independent of cAMP levels. Downregulation of protein kinase C (PKC) by pretreatment of huvecs with 100 nmol/L PMA completely blocked a secondary response to PMA, but did not have a significant effect on t- RA induction. Pretreatment with 10 mumol/L t-RA, on the other hand, did not significantly affect a secondary stimulus by 100 nmol/L PMA, but completely suppressed a secondary stimulation by 10 mumol/L t-RA alone. These studies suggest that the mechanism mediating t-RA stimulation of tPA production interacts with the PKC pathway, resulting in synergism.

scholarly journals Adenosine 3':5'-monophosphate content and actions in the division cycle of synchronized HeLa cells.

1976 ◽  
Vol 71 (2) ◽  
pp. 515-534 ◽  
Author(s):  
C E Zeilig ◽  
R A Johnson ◽  
E W Sutherland ◽  
D L Friedman
Keyword(s):  
Cell Cycle ◽  
Cyclic Amp ◽  
Hela Cells ◽  
S Phase ◽  
Intracellular Camp ◽  
Specific Effects ◽  
Low Concentrations ◽  
High Concentrations ◽  
Camp Levels ◽  
Stimulation Of

The involvement of adenosine 3':5'-monophosphate (cAMP) in the regulation of the cell cycle was studied by determining intracellular fluctuations in cAMP levels in synchronized HeLa cells and by testing the effects of experimentally altered levels on cell cycle traverse. Cyclic AMP levels were lowest during mitosis and were highest during late G-1 or early S phase. These findings were supported by results obtained when cells were accumulated at these points with Colcemid or high levels of thymidine. Additional fluctuations in cAMP levels were observed during S phase. Two specific effects of cAMP on cell cycle traverse were found. Elevation of cAMP levels in S phase or G-2 caused arrest of cells in G-2 for as long as 10 h and lengthened M. However, once cells reached metaphase, elevation of cAMP accelerated the completion of mitosis. Stimulation of mitosis was also observed after addition of CaCl2. The specificity of the effects of cAMP was verified by demonstrating that: (a) intracellular cAMP was increased after exposure to methylisobutylxanthine (MIX) before any observed effects on cycle traverse; (b) submaximal concentrations of MIX potentiated the effects of isoproterenol; and (c) effects of MIX and isoproterenol were mimicked by 8-Br-cAMP. MIX at high concentrations inhibited G-1 traverse, but this effect did not appear to be mediated by cAMP. Isoproterenol slightly stimulated G-1 traverse and partially prevented the MIX-induced delay. Moreover, low concentrations of 8-Br-cAMP (0.10-100 muM) stimulated G-1 traverse, whereas high concentrations (1 mM) inhibited. Both of these effects were also observed with the control, Br-5'-AMP, at 10-fold lower concentrations.

scholarly journals Intracellular elevations of free calcium induced by activation of histamine H1 receptors in interphase and mitotic HeLa cells: hormone signal transduction is altered during mitosis.

1988 ◽  
Vol 107 (6) ◽  
pp. 2533-2539 ◽  
Author(s):  
M Volpi ◽  
R D Berlin
Keyword(s):  
Signal Transduction ◽  
Intracellular Calcium ◽  
Hela Cells ◽  
Calcium Influx ◽  
Tumor Promoter ◽  
Free Calcium ◽  
Interphase Cells ◽  
Underlying Mechanisms ◽  
Histamine H1 Receptors ◽  
Transient Elevation

A broad range of membrane functions, including endocytosis and exocytosis, are strongly inhibited during mitosis. The underlying mechanisms are unclear, however, but will probably be important in relation to the mitotic cycle and the regulation of surface phenomena generally. A major unanswered question is whether membrane signal transduction is altered during mitosis; suppression of an intracellular calcium [( Ca2+]i) transient could inhibit exocytosis; [Ca2+]i elevation could disassemble the mitotic spindle. Activation of the histamine H1 receptor interphase in HeLa cells is shown here by Indo-1 fluorescence to produce a transient elevation of [Ca2+]i. The [Ca2+]i transient consists of an initial sharp rise that is at least partially dependent on intracellular calcium followed by an elevated plateau that is absolutely dependent on extracellular calcium. The [Ca2+]i transient is completely suppressed by preincubation with the tumor promoter, phorbol myristate acetate, but is unaffected by preincubation with pertussis toxin (islet-activating protein). In mitotic (metaphase-arrested) HeLa cells, the [Ca2+]i transient is largely limited to the initial peak. Measurement of 45Ca2+ uptake shows that it is stimulated by histamine in interphase cells, but not in mitotics. We conclude that the histamine-stimulated generation of the second messenger, [Ca2+]i, in mitotic cells is limited by failure to activate a sustained calcium influx. The initial phase of calcium mobilization from intracellular stores is comparable to that in interphase cells. Hormone signal transduction thus appears to be altered during mitosis.

scholarly journals Neuro-Current Response Functions: A Unified Approach to MEG Source Analysis under the Continuous Stimuli Paradigm

2019 ◽  
Author(s):  
Proloy Das ◽  
Christian Brodbeck ◽  
Jonathan Z. Simon ◽  
Behtash Babadi
Keyword(s):  
Auditory Processing ◽  
Source Analysis ◽  
High Temporal Resolution ◽  
Linear Filter ◽  
Current Response ◽  
Response Functions ◽  
Continuous Speech ◽  
Spatiotemporal Resolution ◽  
Linear Filters ◽  
Neural Processes

AbstractCharacterizing the neural dynamics underlying sensory processing is one of the central areas of investigation in systems and cognitive neuroscience. Neuroimaging techniques such as magnetoencephalography (MEG) and Electroencephalography (EEG) have provided significant insights into the neural processing of continuous stimuli, such as speech, thanks to their high temporal resolution. Existing work in the context of auditory processing suggests that certain features of speech, such as the acoustic envelope, can be used as reliable linear predictors of the neural response manifested in M/EEG. The corresponding linear filters are referred to as temporal response functions (TRFs). While the functional roles of specific components of the TRF are well-studied and linked to behavioral attributes such as attention, the cortical origins of the underlying neural processes are not as well understood. In this work, we address this issue by estimating a linear filter representation of cortical sources directly from neuroimaging data in the context of continuous speech processing. To this end, we introduce Neuro-Current Response Functions (NCRFs), a set of linear filters, spatially distributed throughout the cortex, that predict the cortical currents giving rise to the observed ongoing MEG (or EEG) data in response to continuous speech. NCRF estimation is cast within a Bayesian framework, which allows unification of the TRF and source estimation problems, and also facilitates the incorporation of prior information on the structural properties of the NCRFs. To generalize this analysis to M/EEG recordings which lack individual structural magnetic resonance (MR) scans, NCRFs are extended to free-orientation dipoles and a novel regularizing scheme is put forward to lessen reliance on fine-tuned coordinate co-registration. We present a fast estimation algorithm, which we refer to as the Champ-Lasso algorithm, by leveraging recent advances in optimization, and demonstrate its utility through application to simulated and experimentally recorded MEG data under auditory experiments. Our simulation studies reveal significant improvements over existing methods that typically operate in a two-stage fashion, in terms of spatial resolution, response function reconstruction, and recovering dipole orientations. The analysis of experimentally-recorded MEG data without MR scans corroborates existing findings, but also delineates the distinct cortical distribution of the underlying neural processes at high spatiotemporal resolution. In summary, we provide a principled modeling and estimation paradigm for MEG source analysis tailored to extracting the cortical origin of electrophysiological responses to continuous stimuli.

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