scholarly journals Modulation of Rac Localization and Function by Dynamin

2004 ◽  
Vol 15 (1) ◽  
pp. 256-267 ◽  
Author(s):  
Günther Schlunck ◽  
Hanna Damke ◽  
William B. Kiosses ◽  
Nicole Rusk ◽  
Marc H. Symons ◽  
...  
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cell Spreading ◽  
Small Gtpases ◽  
Dominant Negative ◽  
Membrane Bound ◽  
And Function ◽  
Interfering Rna ◽  
Lamellipodia Formation

The GTPase dynamin controls a variety of endocytic pathways, participates in the formation of phagosomes, podosomal adhesions, and invadopodia, and in regulation of the cytoskeleton and apoptosis. Rac, a member of the Rho family of small GTPases, controls formation of lamellipodia and focal complexes, which are critical in cell migration and phagocytosis. We now show that disruption of dynamin-2 function alters Rac localization and inhibits cell spreading and lamellipodia formation even though Rac is activated. Dominant-negative K44A dynamin-2 inhibited cell spreading and lamellipodia formation on fibronectin without blocking cell adhesion; dynamin-2 depletion by specific small interfering RNA inhibited lamellipodia in a similar manner. Dyn2(K44A) induced Rac mislocalization away from cell edges, into abnormal dorsal ruffles, and led to increased total Rac activity. Fluorescence resonance energy transfer imaging of Rac activity confirmed its predominant localization to aberrant dorsal ruffles in the presence of dominant-negative dyn2(K44A). Dyn2(K44A) induced the accumulation of tubulated structures bearing membrane-bound Rac-GFP. Constitutively active but not wild-type GFP-Rac was found on macropinosomes and Rac-dependent, platelet-derived growth factor-induced macropinocytosis was abolished by Dyn2(K44A) expression. These data suggest an indispensable role of dynamin in Rac trafficking to allow for lamellipodia formation and cell spreading.

scholarly journals Regulatory γ1 subunits defy symmetry in functional modulation of BK channels

2018 ◽  
Vol 115 (40) ◽  
pp. 9923-9928 ◽  
Author(s):  
Vivian Gonzalez-Perez ◽  
Manu Ben Johny ◽  
Xiao-Ming Xia ◽  
Christopher J. Lingle
Keyword(s):  
Single Channel ◽  
Regulatory Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bk Channels ◽  
Bk Channel ◽  
Single Type ◽  
Regulatory Subunits ◽  
And Function

Structural symmetry is a hallmark of homomeric ion channels. Nonobligatory regulatory proteins can also critically define the precise functional role of such channels. For instance, the pore-forming subunit of the large conductance voltage and calcium-activated potassium (BK, Slo1, or KCa1.1) channels encoded by a single KCa1.1 gene assembles in a fourfold symmetric fashion. Functional diversity arises from two families of regulatory subunits, β and γ, which help define the range of voltages over which BK channels in a given cell are activated, thereby defining physiological roles. A BK channel can contain zero to four β subunits per channel, with each β subunit incrementally influencing channel gating behavior, consistent with symmetry expectations. In contrast, a γ1 subunit (or single type of γ1 subunit complex) produces a functionally all-or-none effect, but the underlying stoichiometry of γ1 assembly and function remains unknown. Here we utilize two distinct and independent methods, a Forster resonance energy transfer-based optical approach and a functional reporter in single-channel recordings, to reveal that a BK channel can contain up to four γ1 subunits, but a single γ1 subunit suffices to induce the full gating shift. This requires that the asymmetric association of a single regulatory protein can act in a highly concerted fashion to allosterically influence conformational equilibria in an otherwise symmetric K+channel.

Probing the microscopic structural organization of neat ionic liquids (ILs) and ionic liquid-based gels through resonance energy transfer (RET) studies

2017 ◽  
Vol 19 (34) ◽  
pp. 23194-23203 ◽  
Author(s):  
Debashis Majhi ◽  
Moloy Sarkar
Keyword(s):  
Ionic Liquid ◽  
Energy Transfer ◽  
Ionic Liquids ◽  
Structural Organization ◽  
Rhodamine 6G ◽  
Resonance Energy Transfer ◽  
Resonance Energy

With the aim to understand the role of the ionic constituents of ionic liquids (ILs) in their structural organization, resonance energy transfer (RET) studies between ionic liquids (donor) and rhodamine 6G (acceptor) have been investigated.

scholarly journals Lipid Bilayer Composition Affects Transmembrane Protein Orientation and Function

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Katie D. Hickey ◽  
Mary M. Buhr
Keyword(s):  
Lipid Bilayer ◽  
Lipid Composition ◽  
Protein Function ◽  
Transmembrane Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Outer Leaflet ◽  
Lipid Environment ◽  
Protein Incorporation ◽  
And Function

Sperm membranes change in structure and composition upon ejaculation to undergo capacitation, a molecular transformation which enables spermatozoa to undergo the acrosome reaction and be capable of fertilization. Changes to the membrane environment including lipid composition, specifically lipid microdomains, may be responsible for enabling capacitation. To study the effect of lipid environment on proteins, liposomes were created using lipids extracted from bull sperm membranes, with or without a protein (Na+K+-ATPase or -amylase). Protein incorporation, function, and orientation were determined. Fluorescence resonance energy transfer (FRET) confirmed protein inclusion in the lipid bilayer, and protein function was confirmed using a colourometric assay of phosphate production from ATP cleavage. In the native lipid liposomes, ATPase was oriented with the subunit facing the outer leaflet, while changing the lipid composition to 50% native lipids and 50% exogenous lipids significantly altered this orientation of Na+K+-ATPase within the membranes.

scholarly journals Visualization of growth signal transduction cascades in living cells with genetically encoded probes based on Förster resonance energy transfer

2008 ◽  
Vol 363 (1500) ◽  
pp. 2143-2151 ◽  
Author(s):  
Kazuhiro Aoki ◽  
Etsuko Kiyokawa ◽  
Takeshi Nakamura ◽  
Michiyuki Matsuda
Keyword(s):  
Signal Transduction ◽  
Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Small Gtpases ◽  
Forster Resonance Energy Transfer ◽  
Fluorescence Probes ◽  
Förster Resonance Energy Transfer ◽  
Förster Resonance

Fluorescence probes based on the principle of Förster resonance energy transfer (FRET) have shed new light on our understanding of signal transduction cascades. Among them, unimolecular FRET probes containing fluorescence proteins are rapidly increasing in number because these genetically encoded probes can be easily loaded into living cells and allow simple acquisition of FRET images. We have developed probes for small GTPases, tyrosine kinases, serine–threonine kinases and phosphoinositides. Images obtained with these probes have revealed that membrane protrusions such as nascent lamellipodia or neurites provide an active signalling platform in the growth factor-stimulated cells.

scholarly journals Focal adhesions are sites of integrin extension

2010 ◽  
Vol 188 (6) ◽  
pp. 891-903 ◽  
Author(s):  
Janet A. Askari ◽  
Christopher J. Tynan ◽  
Stephen E.D. Webb ◽  
Marisa L. Martin-Fernandez ◽  
Christoph Ballestrem ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Focal Adhesions ◽  
Resonance Energy ◽  
Cell Spreading ◽  
Integrin Subunit ◽  
Adherent Cells ◽  
Extended Form ◽  
Integrin Α5β1 ◽  
Mediate Cell

Integrins undergo global conformational changes that specify their activation state. Current models portray the inactive receptor in a bent conformation that upon activation converts to a fully extended form in which the integrin subunit leg regions are separated to enable ligand binding and subsequent signaling. To test the applicability of this model in adherent cells, we used a fluorescent resonance energy transfer (FRET)–based approach, in combination with engineered integrin mutants and monoclonal antibody reporters, to image integrin α5β1 conformation. We find that restricting leg separation causes the integrin to adopt a bent conformation that is unable to respond to agonists and mediate cell spreading. By measuring FRET between labeled α5β1 and the cell membrane, we find extended receptors are enriched in focal adhesions compared with adjacent regions of the plasma membrane. These results demonstrate definitely that major quaternary rearrangements of β1-integrin subunits occur in adherent cells and that conversion from a bent to extended form takes place at focal adhesions.

scholarly journals Recruitment of Eph receptors into signaling clusters does not require ephrin contact

2004 ◽  
Vol 164 (5) ◽  
pp. 661-666 ◽  
Author(s):  
Sabine H. Wimmer-Kleikamp ◽  
Peter W. Janes ◽  
Anthony Squire ◽  
Philippe I.H. Bastiaens ◽  
Martin Lackmann
Keyword(s):  
Binding Capacity ◽  
Cluster Formation ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Lapse ◽  
Eph Receptors ◽  
Biological Responses ◽  
Ephrin Ligands ◽  
Membrane Bound ◽  
Large Clusters

Eph receptors and their cell membrane–bound ephrin ligands regulate cell positioning and thereby establish or stabilize patterns of cellular organization. Although it is recognized that ephrin clustering is essential for Eph function, mechanisms that relay information of ephrin density into cell biological responses are poorly understood. We demonstrate by confocal time-lapse and fluorescence resonance energy transfer microscopy that within minutes of binding ephrin-A5–coated beads, EphA3 receptors assemble into large clusters. While remaining positioned around the site of ephrin contact, Eph clusters exceed the size of the interacting ephrin surface severalfold. EphA3 mutants with compromised ephrin-binding capacity, which alone are incapable of cluster formation or phosphorylation, are recruited effectively and become phosphorylated when coexpressed with a functional receptor. Our findings reveal consecutive initiation of ephrin-facilitated Eph clustering and cluster propagation, the latter of which is independent of ephrin contacts and cytosolic Eph signaling functions but involves direct Eph–Eph interactions.

scholarly journals Dynamic remodeling of scaffold interactions in dendritic spines controls synaptic excitability

2012 ◽  
Vol 198 (2) ◽  
pp. 251-263 ◽  
Author(s):  
Enora Moutin ◽  
Fabrice Raynaud ◽  
Jonathan Roger ◽  
Emilie Pellegrino ◽  
Vincent Homburger ◽  
...  
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Membrane Receptors ◽  
Physical Interaction ◽  
Scaffolding Proteins ◽  
Cellular Functions ◽  
Electrophysiological Recordings ◽  
Living Neurons ◽  
Activity Dependent

Scaffolding proteins interact with membrane receptors to control signaling pathways and cellular functions. However, the dynamics and specific roles of interactions between different components of scaffold complexes are poorly understood because of the dearth of methods available to monitor binding interactions. Using a unique combination of single-cell bioluminescence resonance energy transfer imaging in living neurons and electrophysiological recordings, in this paper, we depict the role of glutamate receptor scaffold complex remodeling in space and time to control synaptic transmission. Despite a broad colocalization of the proteins in neurons, we show that spine-confined assembly/disassembly of this scaffold complex, physiologically triggered by sustained activation of synaptic NMDA (N-methyl-d-aspartate) receptors, induces physical association between ionotropic (NMDA) and metabotropic (mGlu5a) synaptic glutamate receptors. This physical interaction results in an mGlu5a receptor–mediated inhibition of NMDA currents, providing an activity-dependent negative feedback loop on NMDA receptor activity. Such protein scaffold remodeling represents a form of homeostatic control of synaptic excitability.

scholarly journals Selection and Characterization of a Nanobody Biosensor of GTP-Bound RHO Activities

Antibodies ◽  
2019 ◽  
Vol 8 (1) ◽  
pp. 8 ◽  
Author(s):  
Laura Keller ◽  
Nicolas Bery ◽  
Claudine Tardy ◽  
Laetitia Ligat ◽  
Gilles Favre ◽  
...  
Keyword(s):  
Dynamic Range ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bound State ◽  
Phage Display Library ◽  
Small Gtpases ◽  
Intracellular Expression ◽  
Co Precipitation ◽  
Cytoskeleton Dynamics

RHO (Ras HOmologous) GTPases are molecular switches that activate, in their state bound to Guanosine triphosphate (GTP), key signaling pathways, which involve actin cytoskeleton dynamics. Previously, we selected the nanobody RH12, from a synthetic phage display library, which binds the GTP-bound active conformation of RHOA (Ras Homologous family member A). However, when expressed as an intracellular antibody, its blocking effect on RHO signaling led to a loss of actin fibers, which in turn affected cell shape and cell survival. Here, in order to engineer an intracellular biosensor of RHOA-GTP activation, we screened the same phage nanobody library and identified another RHO-GTP selective intracellular nanobody, but with no apparent toxicity. The recombinant RH57 nanobody displays high affinity towards GTP-bound RHOA/B/C subgroup of small GTPases in vitro. Intracellular expression of the RH57 allowed selective co-precipitation with the GTP-bound state of the endogenous RHOA subfamily. When expressed as a fluorescent fusion protein, the chromobody GFP-RH57 was localized to the inner plasma membrane upon stimulation of the activation of endogenous RHO. Finally, the RH57 nanobody was used to establish a BRET-based biosensor (Bioluminescence Resonance Energy Transfer) of RHO activation. The dynamic range of the BRET signal could potentially offer new opportunities to develop cell-based screening of RHOA subfamily activation modulators.

Electrogenerated chemiluminescence resonance energy transfer between ZnGa2O4/g-C3N4 and gold nanoparticles/graphene and its application in the detection of thrombin

The Analyst ◽  
2020 ◽  
Vol 145 (22) ◽  
pp. 7412-7420
Author(s):  
Hui Liu ◽  
Hao Yin ◽  
TingTing Yang ◽  
HouCheng Ding ◽  
YongPing Dong
Keyword(s):  
Gold Nanoparticles ◽  
Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Electrogenerated Chemiluminescence ◽  
Label Free ◽  
Spinel Type ◽  
Semiconductor Oxides ◽  
Type Semiconductor

A label-free ECL sensor for thrombin was fabricated based on ECL resonance energy transfer occurred between ZnGa2O4/g-C3N4 and AuNP/GR nanocomposites, which revealed a new role of spinel-type semiconductor oxides in the fabrication of ECL sensors.

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