scholarly journals Activity of Rho-family GTPases during cell division as visualized with FRET-based probes

2003 ◽  
Vol 162 (2) ◽  
pp. 223-232 ◽  
Author(s):  
Hisayoshi Yoshizaki ◽  
Yusuke Ohba ◽  
Kazuo Kurokawa ◽  
Reina E. Itoh ◽  
Takeshi Nakamura ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Division ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Nucleotide Exchange ◽  
Temporal Regulation ◽  
Rhoa Activity ◽  
Rho Family Gtpases ◽  
Rho Family ◽  
Spatio Temporal

Rho-family GTPases regulate many cellular functions. To visualize the activity of Rho-family GTPases in living cells, we developed fluorescence resonance energy transfer (FRET)–based probes for Rac1 and Cdc42 previously (Itoh, R.E., K. Kurokawa, Y. Ohba, H. Yoshizaki, N. Mochizuki, and M. Matsuda. 2002. Mol. Cell. Biol. 22:6582–6591). Here, we added two types of probes for RhoA. One is to monitor the activity balance between guanine nucleotide exchange factors and GTPase-activating proteins, and another is to monitor the level of GTP-RhoA. Using these FRET probes, we imaged the activities of Rho-family GTPases during the cell division of HeLa cells. The activities of RhoA, Rac1, and Cdc42 were high at the plasma membrane in interphase, and decreased rapidly on entry into M phase. From after anaphase, the RhoA activity increased at the plasma membrane including cleavage furrow. Rac1 activity was suppressed at the spindle midzone and increased at the plasma membrane of polar sides after telophase. Cdc42 activity was suppressed at the plasma membrane and was high at the intracellular membrane compartments during cytokinesis. In conclusion, we could use the FRET-based probes to visualize the complex spatio-temporal regulation of Rho-family GTPases during cell division.

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.

Regulation of RalA GTPase by phosphatidylinositol 3-kinase as visualized by FRET probes

2006 ◽  
Vol 34 (5) ◽  
pp. 851-854 ◽  
Author(s):  
H. Yoshizaki ◽  
K. Aoki ◽  
T. Nakamura ◽  
M. Matsuda
Keyword(s):  
Plasma Membrane ◽  
Time Course ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Small Gtpases ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Subsequent Activation ◽  
Phosphoinositide 3 Kinase

Small GTPases, which are binary switches regulating various signal transduction cascades, function not only to relay signals but also to integrate them from multiple signalling branches. For example, RalA activity is regulated by at least three signalling cascades involving Ras, Rac or PI3K (phosphoinositide 3-kinase). To untangle such complicated regulatory mechanisms, we have been developing probes for GTPases, kinases and phosphatidylinositols based on the principle of FRET (fluorescence resonance energy transfer). We demonstrated previously that, upon EGF (epidermal growth factor) stimulation, Ras activity increases diffusely in the plasma membrane, whereas RalA activity increases predominantly in lamellipodial protrusions. Here, we show that the level of PtdIns(3,4,5)P3 is increased diffusely in the plasma membrane, whereas, in the central region, the level of PtdIns(3,4)P2 is increased more in the nascent lamellipodia than in the plasma membrane. The distribution and time course of Akt activation are similar to those of increased PtdIns(3,4)P2 levels. These observations suggest that the increase in PtdIns(3,4)P2 and the subsequent activation of Akt may be responsible for the localized activation of RalA. Thus the signals from Ras and PI3K converge at the level of Ral GEFs (guanine nucleotide-exchange factors), and this convergence restricts the area of RalA activation.

scholarly journals Direct Spatial Control of Epac1 by Cyclic AMP

2009 ◽  
Vol 29 (10) ◽  
pp. 2521-2531 ◽  
Author(s):  
Bas Ponsioen ◽  
Martijn Gloerich ◽  
Laila Ritsma ◽  
Holger Rehmann ◽  
Johannes L. Bos ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Adhesion ◽  
Cyclic Amp ◽  
Conformational Change ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cell Junction ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

ABSTRACT Epac1 is a guanine nucleotide exchange factor (GEF) for the small G protein Rap and is directly activated by cyclic AMP (cAMP). Upon cAMP binding, Epac1 undergoes a conformational change that allows the interaction of its GEF domain with Rap, resulting in Rap activation and subsequent downstream effects, including integrin-mediated cell adhesion and cell-cell junction formation. Here, we report that cAMP also induces the translocation of Epac1 toward the plasma membrane. Combining high-resolution confocal fluorescence microscopy with total internal reflection fluorescence and fluorescent resonance energy transfer assays, we observed that Epac1 translocation is a rapid and reversible process. This dynamic redistribution of Epac1 requires both the cAMP-induced conformational change as well as the DEP domain. In line with its translocation, Epac1 activation induces Rap activation predominantly at the plasma membrane. We further show that the translocation of Epac1 enhances its ability to induce Rap-mediated cell adhesion. Thus, the regulation of Epac1-Rap signaling by cAMP includes both the release of Epac1 from autoinhibition and its recruitment to the plasma membrane.

Mechanism and role of localized activation of Rho-family GTPases in growth factor-stimulated fibroblasts and neuronal cells

2005 ◽  
Vol 33 (4) ◽  
pp. 631-634 ◽  
Author(s):  
K. Kurokawa ◽  
T. Nakamura ◽  
K. Aoki ◽  
M. Matsuda
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cell Function ◽  
Leading Edge ◽  
Dependent Manner ◽  
Rhoa Activity ◽  
Rho Family Gtpases ◽  
Epidermal Growth ◽  
Rho Family

Rho-family GTPases regulate various aspects of cell function by controlling cytoskeletal changes; however, their spatial regulation within the cells remains largely unknown. To understand this regulation, we have studied the spatiotemporal activity of Rho-family GTPases in migrating cells and growth factor-stimulated cells by using probes based on the principle of fluorescence resonance energy transfer. In migrating fibroblasts and epithelial cells, the level of RhoA activity is high both at the contractile tail and at the leading edge, whereas Rac1 and Cdc42 activities are high only at the leading edge. In cells stimulated with epidermal growth factor or nerve growth factor, activities of Rac1 and Cdc42 were transiently elevated in a broad area of the plasma membrane, followed by a localized activation at nascent lamellipodia. In contrast, on epidermal growth factor stimulation, RhoA activity decreased diffusely at the plasma membrane. Notably, RhoA activity persisted at the tip of growth factor-induced membrane ruffles and, in agreement with this finding, RhoA is required for membrane ruffling. These observations suggest that the activities of Rho-family GTPases are elaborately regulated in a time- and space-dependent manner to control cytoskeletal changes and that the basic mechanism of controlling cell shape via Rho-family GTPases is common to various cell types.

Single-molecule FRET for Ultrasensitive Detection of Biomolecules

2014 ◽  
Vol 1 (1) ◽  
Author(s):  
Kun Yang ◽  
Yong Yang ◽  
Chun-yang Zhang
Keyword(s):  
Single Molecule ◽  
New Technologies ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Molecular Probes ◽  
Ultrasensitive Detection ◽  
Single Molecule Fret ◽  
Biological Reaction ◽  
Spatio Temporal ◽  
Detection Of Biomolecules

AbstractSingle-molecule Förster resonance energy transfer (sm- FRET) has been widely employed to detect biomarkers and to probe the structure and dynamics of biomolecules. By monitoring the biological reaction in a spatio-temporal manner, smFRET can reveal the transient intermediates of biological processes that cannot be obtained by conventional ensemble measurements. This review provides an overview of singlemolecule FRET and its applications in ultrasensitive detection of biomolecules, including the major techniques and the molecular probes used for smFRET as well as the biomedical applications of smFRET. Especially, the combination of sm- FRET with new technologies might expand its applications in clinical diagnosis and biomedical research

scholarly journals Comparative analysis of the coordinated motion of Hsp70s from different organelles observed by single-molecule three-color FRET

2021 ◽  
Vol 118 (33) ◽  
pp. e2025578118
Author(s):  
Lena Voith von Voithenberg ◽  
Anders Barth ◽  
Vanessa Trauschke ◽  
Benjamin Demarco ◽  
Swati Tyagi ◽  
...  
Keyword(s):  
Single Molecule ◽  
Structural Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Conformational Space ◽  
Distribution Analysis ◽  
Nucleotide Exchange ◽  
Nucleotide Analogs ◽  
Recognition Mechanism ◽  
Substrate Peptide

Cellular function depends on the correct folding of proteins inside the cell. Heat-shock proteins 70 (Hsp70s), being among the first molecular chaperones binding to nascently translated proteins, aid in protein folding and transport. They undergo large, coordinated intra- and interdomain structural rearrangements mediated by allosteric interactions. Here, we applied a three-color single-molecule Förster resonance energy transfer (FRET) combined with three-color photon distribution analysis to compare the conformational cycle of the Hsp70 chaperones DnaK, Ssc1, and BiP. By capturing three distances simultaneously, we can identify coordinated structural changes during the functional cycle. Besides the known conformations of the Hsp70s with docked domains and open lid and undocked domains with closed lid, we observed additional intermediate conformations and distance broadening, suggesting flexibility of the Hsp70s in adopting the states in a coordinated fashion. Interestingly, the difference of this distance broadening varied between DnaK, Ssc1, and BiP. Study of their conformational cycle in the presence of substrate peptide and nucleotide exchange factors strengthened the observation of additional conformational intermediates, with BiP showing coordinated changes more clearly compared to DnaK and Ssc1. Additionally, DnaK and BiP were found to differ in their selectivity for nucleotide analogs, suggesting variability in the recognition mechanism of their nucleotide-binding domains for the different nucleotides. By using three-color FRET, we overcome the limitations of the usual single-distance approach in single-molecule FRET, allowing us to characterize the conformational space of proteins in higher detail.

scholarly journals Apical polarity proteins recruit the RhoGEF Cysts to promote junctional myosin assembly

2019 ◽  
Vol 218 (10) ◽  
pp. 3397-3414 ◽  
Author(s):  
Jordan T. Silver ◽  
Frederik Wirtz-Peitz ◽  
Sérgio Simões ◽  
Milena Pellikka ◽  
Dong Yan ◽  
...  
Keyword(s):  
Rho Gtpases ◽  
Rho Gtpase ◽  
Adherens Junctions ◽  
Nucleotide Exchange ◽  
Temporal Regulation ◽  
Apical Polarity ◽  
Polarity Proteins ◽  
Spatio Temporal ◽  
Crumbs Complex

The spatio-temporal regulation of small Rho GTPases is crucial for the dynamic stability of epithelial tissues. However, how RhoGTPase activity is controlled during development remains largely unknown. To explore the regulation of Rho GTPases in vivo, we analyzed the Rho GTPase guanine nucleotide exchange factor (RhoGEF) Cysts, the Drosophila orthologue of mammalian p114RhoGEF, GEF-H1, p190RhoGEF, and AKAP-13. Loss of Cysts causes a phenotype that closely resembles the mutant phenotype of the apical polarity regulator Crumbs. This phenotype can be suppressed by the loss of basolateral polarity proteins, suggesting that Cysts is an integral component of the apical polarity protein network. We demonstrate that Cysts is recruited to the apico-lateral membrane through interactions with the Crumbs complex and Bazooka/Par3. Cysts activates Rho1 at adherens junctions and stabilizes junctional myosin. Junctional myosin depletion is similar in Cysts- and Crumbs-compromised embryos. Together, our findings indicate that Cysts is a downstream effector of the Crumbs complex and links apical polarity proteins to Rho1 and myosin activation at adherens junctions, supporting junctional integrity and epithelial polarity.

Dynamic receptor superstructures at the plasma membrane

1997 ◽  
Vol 30 (1) ◽  
pp. 67-106 ◽  
Author(s):  
S. DAMJANOVICH ◽  
R. GÁSPÁR, Jr. ◽  
C. PIERI
Keyword(s):  
Plasma Membrane ◽  
Energy Transfer ◽  
Single Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Scanning Force Microscopy ◽  
Physical Parameters ◽  
Conformational States ◽  
Lipid Domain ◽  
Receptor Clusters

1. INTRODUCTION 681.1 Receptor patterns in the plasma membrane 681.2 Different types of receptor patterns 712. METHODS TO INVESTIGATE NON-RANDOM RECEPTOR CLUSTERING 732.1 Fluorescence resonance energy transfer 732.2 Flow cytometric energy transfer measurement 782.3 Fluorescence anisotropy and energy transfer 792.4 Photobleaching energy transfer on single cells 812.5 Two-dimensional mapping of receptor superstructures 822.6 Detecting single receptor molecules 852.7 Chemical identification of receptor clusters 862.8 Electron microscopy 872.9 Scanning force microscopy 883. CONFORMATIONAL STATES OF RECEPTORS 903.1 Multi-subunit receptor structures 903.2 Physical parameters influencing conformational states 913.3 Chemical interactions and receptor conformations 924. ON THE ORIGIN OF NATURALLY OCCURRING RECEPTOR CLUSTERS 934.1 Synthesis of receptors and their localization in the plasma membrane 934.2 Lipid domain structure of the plasma membrane 944.3 The validity of the Singer–Nicolson model 945. CONCLUSIONS 966. ACKNOWLEDGEMENTS 967. REFERENCES 97

scholarly journals Spatiotemporal Analysis of Differential Akt Regulation in Plasma Membrane Microdomains

2008 ◽  
Vol 19 (10) ◽  
pp. 4366-4373 ◽  
Author(s):  
Xinxin Gao ◽  
Jin Zhang
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Lipid Rafts ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Spatiotemporal Analysis ◽  
Membrane Microdomains ◽  
Cholesterol Depletion ◽  
Growth Factor Signaling

As a central kinase in the phosphatidylinositol 3-kinase pathway, Akt has been the subject of extensive research; yet, spatiotemporal regulation of Akt in different membrane microdomains remains largely unknown. To examine dynamic Akt activity in membrane microdomains in living cells, we developed a specific and sensitive fluorescence resonance energy transfer-based Akt activity reporter, AktAR, through systematic testing of different substrates and fluorescent proteins. Targeted AktAR reported higher Akt activity with faster activation kinetics within lipid rafts compared with nonraft regions of plasma membrane. Disruption of rafts attenuated platelet-derived growth factor (PDGF)-stimulated Akt activity in rafts without affecting that in nonraft regions. However, in insulin-like growth factor-1 (IGF)-1 stimulation, Akt signaling in nonraft regions is dependent on that in raft regions. As a result, cholesterol depletion diminishes Akt activity in both regions. Thus, Akt activities are differentially regulated in different membrane microdomains, and the overall activity of this oncogenic pathway is dependent on raft function. Given the increased abundance of lipid rafts in some cancer cells, the distinct Akt-activating characteristics of PDGF and IGF-1, in terms of both effectiveness and raft dependence, demonstrate the capabilities of different growth factor signaling pathways to transduce differential oncogenic signals across plasma membrane.

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