The Gi/o protein family transduces signals from a diverse group of G protein-coupled receptors (GPCRs). The observed specificity of Gi/o-GPCR coupling and the high rate of Gi/o signal transduction have been hypothesized to be enabled by the existence of stable associates between Gi/o proteins and their cognate GPCRs in the inactive state (Gi/o-GPCR preassembly). To test this hypothesis, we applied the recently developed technique of two-photon polarization microscopy (2PPM) to Gαi1 subunits labeled with fluorescent proteins and four GPCRs: the α2A-adrenergic receptor, GABAB, cannabinoid receptor type 1 (CB1R), and dopamine receptor type 2. Our experiments with non-dissociating mutants of fluorescently labeled Gαi1 subunits (exhibiting impaired dissociation from activated GPCRs) showed that 2PPM is capable of detecting GPCR-G protein interactions. 2PPM experiments with non-mutated fluorescently labeled Gαi1 subunits and α2A-adrenergic receptor, GABAB, or dopamine receptor type 2 receptors did not reveal any interaction between the Gi1 protein and the non-stimulated GPCRs. In contrast, non-stimulated CB1R exhibited an interaction with the Gi1 protein. Further experiments revealed that this interaction is caused solely by CB1R basal activity; no preassembly between CB1R and the Gi1 protein could be observed. Our results demonstrate that four diverse GPCRs do not preassemble with non-active Gi1 However, we also show that basal GPCR activity allows interactions between non-stimulated GPCRs and Gi1 (basal coupling). These findings suggest that Gi1 interacts only with active GPCRs and that the well known high speed of GPCR signal transduction does not require preassembly between G proteins and GPCRs.

• MeSH
• HEK293 buňky MeSH
• lidé MeSH
• mutace MeSH
• proteiny vázající GTP - alfa-podjednotky Gi-Go genetika   metabolismus   MeSH
• receptory spřažené s G-proteiny genetika   metabolismus   MeSH
• signální transdukce fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

• MeSH
• buňky * MeSH
• molekulární biologie MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• molekulární biologie, molekulární medicína
• NLK Publikační typ
• učebnice vysokých škol

ArcLight, a genetically encoded fluorescent protein voltage probe with a large ΔF/ΔV, is a fusion between the voltage sensing domain of the Ciona instestinalis voltage sensitive phosphatase and super ecliptic pHluorin carrying a single mutation (A227D in the fluorescent protein). Without this mutation the probe produces only a very small change in fluorescence in response to voltage deflections (∼ 1%). The large signal afforded by this mutation allows optical detection of action potentials and sub-threshold electrical events in single-trials in vitro and in vivo. However, it is unclear how this single mutation produces a probe with such a large modulation of its fluorescence output with changes in membrane potential. In this study, we identified which residues in super ecliptic pHluorin (vs eGFP) are critical for the ArcLight response, as a similarly constructed probe based on eGFP also exhibits large response amplitude if it carries these critical residues. We found that D147 is responsible for determining the pH sensitivity of the fluorescent protein used in these probes but by itself does not result in a voltage probe with a large signal. We also provide evidence that the voltage dependent signal of ArcLight is not simply sensing environmental pH changes. A two-photon polarization microscopy study showed that ArcLight's response to changes in membrane potential includes a reorientation of the super ecliptic pHluorin. We also explored different changes including modification of linker length, deletion of non-essential amino acids in the super ecliptic pHluorin, adding a farnesylation site, using tandem fluorescent proteins and other pH sensitive fluorescent proteins.

• MeSH
• akční potenciály * MeSH
• aminokyseliny chemie   genetika   metabolismus   MeSH
• fluorescence MeSH
• fluorescenční barviva chemie   metabolismus   MeSH
• fluorescenční spektrometrie MeSH
• HEK293 buňky MeSH
• kinetika MeSH
• koncentrace vodíkových iontů MeSH
• konfokální mikroskopie MeSH
• krysa rodu rattus MeSH
• kultivované buňky MeSH
• lidé MeSH
• luminescentní proteiny chemie   genetika   metabolismus   MeSH
• membránové potenciály MeSH
• metoda terčíkového zámku MeSH
• missense mutace MeSH
• neurony metabolismus   fyziologie   MeSH
• prenylace MeSH
• rekombinantní fúzní proteiny chemie   genetika   metabolismus   MeSH
• zelené fluorescenční proteiny chemie   genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, American Recovery and Reinvestment Act MeSH
• Research Support, N.I.H., Extramural MeSH

Although most heterotrimeric G proteins are thought to dissociate into Gα and Gβγ subunits upon activation, the evidence in the Gi/o family has long been inconsistent and contradictory. The Gi/o protein family mediates inhibition of cAMP production and regulates the activity of ion channels. On the basis of experimental evidence, both heterotrimer dissociation and rearrangement have been postulated as crucial steps of Gi/o protein activation and signal transduction. We have now investigated the process of Gi/o activation in living cells directly by two-photon polarization microscopy and indirectly by observations of G protein-coupled receptor kinase-derived polypeptides. Our observations of existing fluorescently labeled and non-modified Gαi/o constructs indicate that the molecular mechanism of Gαi/o activation is affected by the presence and localization of the fluorescent label. All investigated non-labeled, non-modified Gi/o complexes dissociate extensively upon activation. The dissociated subunits can activate downstream effectors and are thus likely to be the major activated Gi/o form. Constructs of Gαi/o subunits fluorescently labeled at the N terminus (GAP43-CFP-Gαi/o) seem to faithfully reproduce the behavior of the non-modified Gαi/o subunits. Gαi constructs labeled within the helical domain (Gαi-L91-YFP) largely do not dissociate upon activation, yet still activate downstream effectors, suggesting that the dissociation seen in non-modified Gαi/o proteins is not required for downstream signaling. Our results appear to reconcile disparate published data and settle a long running dispute.

• MeSH
• aktivace enzymů fyziologie   MeSH
• HEK293 buňky MeSH
• lidé MeSH
• protein GAP-43 genetika   metabolismus   MeSH
• proteiny vázající GTP - alfa-podjednotky Gi-Go genetika   metabolismus   MeSH
• proteiny vázající GTP - beta-podjednotky genetika   metabolismus   MeSH
• proteiny vázající GTP - gama-podjednotky genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Multifunctional two-photon laser scanning microscopy provides attractive advantages over conventional two-photon laser scanning microscopy. For the first time, simultaneous measurement of the second harmonic generation (SHG) signals in the forward and backward directions and two photon excitation fluorescence were achieved from the deep shade plant Selaginella erythropus. RESULTS: These measurements show that the S. erythropus leaves produce high SHG signals in both directions and the SHG signals strongly depend on the laser's status of polarization and the orientation of the dipole moment in the molecules that interact with the laser light. The novelty of this work is (1) uncovering the unusual structure of S. erythropus leaves, including diverse chloroplasts, various cell types and micromophology, which are consistent with observations from general electron microscopy; and (2) using the multifunctional two-photon laser scanning microscopy by combining three platforms of laser scanning microscopy, fluorescence microscopy, harmonic generation microscopy and polarizing microscopy for detecting the SHG signals in the forward and backward directions, as well as two photon excitation fluorescence. CONCLUSIONS: With the multifunctional two-photon laser scanning microscopy, one can use noninvasive SHG imaging to reveal the true architecture of the sample, without photodamage or photobleaching, by utilizing the fact that the SHG is known to leave no energy deposition on the interacting matter because of the SHG virtual energy conservation characteristic.

• MeSH
• chloroplasty chemie   ultrastruktura   MeSH
• konfokální mikroskopie metody   MeSH
• listy rostlin chemie   ultrastruktura   MeSH
• mikroskopie fluorescenční multifotonová metody   MeSH
• počítačové zpracování obrazu metody   MeSH
• Selaginellaceae chemie   ultrastruktura   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Testate amoebae (TA) are a group of free-living protozoa, important in ecology and paleoecology. Testate amoebae taxonomy is mainly based on the morphological features of the shell, as examined by means of light microscopy or (environmental) scanning electron microscopy (SEM/ESEM). We explored the potential applications of confocal laser scanning microscopy (CLSM), two photon excitation microscopy (TPEM), phase contrast, differential interference contrast (DIC Nomarski), and polarization microscopy to visualize TA shells and inner structures of living cells, which is not possible by SEM or environmental SEM. Images captured by CLSM and TPEM were utilized to create three-dimensional (3D) visualizations and to evaluate biovolume inside the shell by stereological methods, to assess the function of TA in ecosystems. This approach broadens the understanding of TA cell and shell morphology, and inner structures including organelles and endosymbionts, with potential implications in taxonomy and ecophysiology.

• MeSH
• Amoebozoa klasifikace   ultrastruktura   MeSH
• mikroskopie metody   MeSH
• zobrazování trojrozměrné MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• biologie buňky MeSH
• molekulární biologie MeSH
• Publikační typ
• monografie MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• biologie
• cytologie, klinická cytologie