scholarly journals Developmental control of plant Rho GTPase nano-organization by the lipid phosphatidylserine

Science ◽  
2019 ◽  
Vol 364 (6435) ◽  
pp. 57-62 ◽  
Author(s):  
Matthieu Pierre Platre ◽  
Vincent Bayle ◽  
Laia Armengot ◽  
Joseph Bareille ◽  
Maria del Mar Marquès-Bueno ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Root Development ◽  
Rho Gtpase ◽  
Plant Root ◽  
Small Gtpase ◽  
Auxin Signaling ◽  
Master Regulators ◽  
Downstream Signaling ◽  
Cellular Context

Rho guanosine triphosphatases (GTPases) are master regulators of cell signaling, but how they are regulated depending on the cellular context is unclear. We found that the phospholipid phosphatidylserine acts as a developmentally controlled lipid rheostat that tunes Rho GTPase signaling in Arabidopsis. Live superresolution single-molecule imaging revealed that the protein Rho of Plants 6 (ROP6) is stabilized by phosphatidylserine into plasma membrane nanodomains, which are required for auxin signaling. Our experiments also revealed that the plasma membrane phosphatidylserine content varies during plant root development and that the level of phosphatidylserine modulates the quantity of ROP6 nanoclusters induced by auxin and hence downstream signaling, including regulation of endocytosis and gravitropism. Our work shows that variations in phosphatidylserine levels are a physiological process that may be leveraged to regulate small GTPase signaling during development.

scholarly journals Title: In vivo phosphatidylserine variations steer Rho GTPase signaling in a cell-context dependent manner

2018 ◽  
Author(s):  
Matthieu Pierre Platre ◽  
Vincent Bayle ◽  
Laia Armengot ◽  
Joseph Bareille ◽  
Maria Mar Marques-Bueno ◽  
...  
Keyword(s):  
Single Molecule ◽  
Rho Gtpases ◽  
Rho Gtpase ◽  
Plant Root ◽  
Super Resolution ◽  
Small Gtpase ◽  
Auxin Signaling ◽  
Dependent Manner ◽  
Downstream Signaling

AbstractRho GTPases are master regulators of cell signaling, but how they are regulated depending on the cellular context is unclear. Here, we show that the phospholipid phosphatidylserine acts as a developmentally-controlled lipid rheostat that tunes Rho GTPase signaling in Arabidopsis. Live super-resolution single molecule imaging revealed that RHO-OF-PLANT6 (ROP6) is stabilized by phosphatidylserine into plasma membrane (PM) nanodomains, which is required for auxin signaling. Furthermore, we uncovered that the PM phosphatidylserine content varies during plant root development and that the level of phosphatidylserine modulates the quantity of ROP6 nanoclusters induced by auxin and hence downstream signaling, including regulation of endocytosis and gravitropism. Our work reveals that variations in phosphatidylserine levels are a physiological process that may be leveraged to regulate small GTPase signaling during development.One Sentence SummaryPhosphatidylserine acts as a developmentally-controlled lipid rheostat that regulates cellular auxin sensitivity and plant development.

Cell biochemistry studied by single-molecule imaging

2006 ◽  
Vol 34 (5) ◽  
pp. 983-988 ◽  
Author(s):  
G.I. Mashanov ◽  
T.A. Nenasheva ◽  
M. Peckham ◽  
J.E. Molloy
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Molecular Complexes ◽  
Live Cells ◽  
Diffusion Constants ◽  
Powerful Analytical Tool ◽  
Cellular Context ◽  
Molecular Information ◽  
Temporal And Spatial ◽  
Spatial Trajectories

Over the last decade, there have been remarkable developments in live-cell imaging. We can now readily observe individual protein molecules within living cells and this should contribute to a systems level understanding of biological pathways. Direct observation of single fluorophores enables several types of molecular information to be gathered. Temporal and spatial trajectories enable diffusion constants and binding kinetics to be deduced, while analyses of fluorescence lifetime, intensity, polarization or spectra give chemical and conformational information about molecules in their cellular context. By recording the spatial trajectories of pairs of interacting molecules, formation of larger molecular complexes can be studied. In the future, multicolour and multiparameter imaging of single molecules in live cells will be a powerful analytical tool for systems biology. Here, we discuss measurements of single-molecule mobility and residency at the plasma membrane of live cells. Analysis of diffusional paths at the plasma membrane gives information about its physical properties and measurement of temporal trajectories enables rates of binding and dissociation to be derived. Meanwhile, close scrutiny of individual fluorophore trajectories enables ideas about molecular dimerization and oligomerization related to function to be tested directly.

scholarly journals Nanodomain-mediated lateral sorting drives polarization of the small GTPase ROP2 in the plasma membrane of root hair cells

2021 ◽  
Author(s):  
Vanessa Aphaia Fiona Fuchs ◽  
Philipp Denninger ◽  
Milan Župunski ◽  
Yvon Jaillais ◽  
Ulrike Engel ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Root Hair ◽  
Root Hairs ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Cellular Growth ◽  
Epifluorescence Microscopy ◽  
Anionic Phospholipids ◽  
Downstream Effectors

Formation of root hairs involves the targeted recruitment of the cellular growth machinery to the root hair initiation domain (RHID), a specialized site at the plasma membrane (PM) of trichoblast cells. Early determinants in RHID establishment are small GTPases of the Rho-of-plants (ROP) protein family, which are required for polarization of downstream effectors, membrane modification and targeted secretion during tip growth. It remains, however, not fully understood how ROP GTPases themselves are polarized. To investigate the mechanism underlying ROP2 recruitment, we employed Variable Angle Epifluorescence Microscopy (VAEM) and exploited mCitrine fluorophore blinking for single molecule localization, particle tracking and super-resolved imaging of the trichoblast plasma membrane. We observed the association of mCit-ROP2 within distinct membrane nanodomains, whose polar occurrence at the RHID was dependent on the presence of the RopGEF GEF3, and found a gradual, localized decrease of mCit-ROP2 protein mobility that preceded polarization. We provide evidence for a step-wise model of ROP2 polarization that involves (i) an initial non-polar recruitment to the plasma membrane via interactions with anionic phospholipids, (ii) ROP2 assembly into membrane nanodomains independent of nucleotide-binding state and, sub-sequently, (iii) lateral sorting into the RHID, driven by GEF3-mediated localized reduction of ROP2 mobility.

scholarly journals Amphetamine activates Rho GTPase signaling to mediate dopamine transporter internalization and acute behavioral effects of amphetamine

2015 ◽  
Vol 112 (51) ◽  
pp. E7138-E7147 ◽  
Author(s):  
David S. Wheeler ◽  
Suzanne M. Underhill ◽  
Donna B. Stolz ◽  
Geoffrey H. Murdoch ◽  
Edda Thiels ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Dopamine Transporter ◽  
Rho Gtpase ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Dopamine Neurons ◽  
Rhoa Activity ◽  
Dependent Inactivation ◽  
Independent Process ◽  
Dopamine Efflux

Acute amphetamine (AMPH) exposure elevates extracellular dopamine through a variety of mechanisms that include inhibition of dopamine reuptake, depletion of vesicular stores, and facilitation of dopamine efflux across the plasma membrane. Recent work has shown that the DAT substrate AMPH, unlike cocaine and other nontransported blockers, can also stimulate endocytosis of the plasma membrane dopamine transporter (DAT). Here, we show that when AMPH enters the cytoplasm it rapidly stimulates DAT internalization through a dynamin-dependent, clathrin-independent process. This effect, which can be observed in transfected cells, cultured dopamine neurons, and midbrain slices, is mediated by activation of the small GTPase RhoA. Inhibition of RhoA activity with C3 exotoxin or a dominant-negative RhoA blocks AMPH-induced DAT internalization. These actions depend on AMPH entry into the cell and are blocked by the DAT inhibitor cocaine. AMPH also stimulates cAMP accumulation and PKA-dependent inactivation of RhoA, thus providing a mechanism whereby PKA- and RhoA-dependent signaling pathways can interact to regulate the timing and robustness of AMPH’s effects on DAT internalization. Consistent with this model, the activation of D1/D5 receptors that couple to PKA in dopamine neurons antagonizes RhoA activation, DAT internalization, and hyperlocomotion observed in mice after AMPH treatment. These observations support the existence of an unanticipated intracellular target that mediates the effects of AMPH on RhoA and cAMP signaling and suggest new pathways to target to disrupt AMPH action.

scholarly journals Rap1 Spatially Controls ArhGAP29 To Inhibit Rho Signaling during Endothelial Barrier Regulation

2015 ◽  
Vol 35 (14) ◽  
pp. 2495-2502 ◽  
Author(s):  
A. Post ◽  
W. J. Pannekoek ◽  
B. Ponsioen ◽  
M. J. Vliem ◽  
J. L. Bos
Keyword(s):  
Plasma Membrane ◽  
Barrier Function ◽  
Rho Gtpase ◽  
Small Gtpase ◽  
Endothelial Barrier ◽  
Nucleotide Exchange ◽  
Endothelial Barrier Function ◽  
Nucleotide Exchange Factor ◽  
Barrier Regulation ◽  
Spatiotemporal Control

The small GTPase Rap1 controls the actin cytoskeleton by regulating Rho GTPase signaling. We recently established that the Rap1 effectors Radil and Rasip1, together with the Rho GTPase activating protein ArhGAP29, mediate Rap1-induced inhibition of Rho signaling in the processes of epithelial cell spreading and endothelial barrier function. Here, we show that Rap1 induces the independent translocations of Rasip1 and a Radil-ArhGAP29 complex to the plasma membrane. This results in the formation of a multimeric protein complex required for Rap1-induced inhibition of Rho signaling and increased endothelial barrier function. Together with the previously reported spatiotemporal control of the Rap guanine nucleotide exchange factor Epac1, these findings elucidate a signaling pathway for spatiotemporal control of Rho signaling that operates by successive protein translocations to and complex formation at the plasma membrane.

scholarly journals Biosensors Monitor Ligand-Selective Effects at Kappa Opioid Receptors

2021 ◽  
Author(s):  
Lucie Oberhauser ◽  
Miriam Stoeber
Keyword(s):  
Subcellular Location ◽  
Receptor Activation ◽  
Cellular Level ◽  
Two Dimensions ◽  
Kappa Opioid Receptor ◽  
Kappa Opioid ◽  
Downstream Signaling ◽  
Regulator Protein ◽  
Cellular Context ◽  
Selective Effects

AbstractThe kappa opioid receptor (KOR) has emerged as a promising therapeutic target for pain and itch treatment. There is growing interest in biased agonists that preferentially activate select signaling pathways downstream of KOR activation on the cellular level due to their therapeutic promise in retaining the analgesic and antipruritic effects and eliminating the sedative and dysphoric effects of KOR signaling on the physiological level. The concept of ligand-selective signaling includes that biased ligands promote KOR to selectively recruit one transducer or regulator protein over another, introducing bias into the signaling cascade at the very receptor-proximal level. Measuring agonist effects directly at the receptor has remained challenging and previous studies have focused on inferring agonist-selective KOR engagement with G protein relative to β-arrestin based on downstream signaling readouts. Here we discuss novel strategies to directly assess ligand-selective effects on receptor activation using KOR-interacting biosensors. The conformation-specific cytoplasmic biosensors are disconnected from the endogenous signaling machinery and provide a direct receptor-proxy readout of ligand effects in living cells. Receptor–biosensor interaction is ligand concentration dependent and can be used to determine relative ligand potency and efficacy. In addition, the biosensors reveal the existence of two dimensions of agonist bias in the cellular context: Firstly, agonists can selectively produce discrete protein-engaged KOR states and secondly, agonists can differ in the precise subcellular location at which they activate KOR. We discuss the value and the limitations of using orthogonal receptor-interacting biosensors in the quest to understand functional selectivity amongst KOR agonists in the cellular context.

scholarly journals Post-Translational Modification and Subcellular Compartmentalization: Emerging Concepts on the Regulation and Physiopathological Relevance of RhoGTPases

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1990
Author(s):  
Inmaculada Navarro-Lérida ◽  
Miguel Sánchez-Álvarez ◽  
Miguel Ángel del Pozo
Keyword(s):  
Rho Gtpases ◽  
Rho Gtpase ◽  
Protein Complexes ◽  
Specific Protein ◽  
Cell Polarization ◽  
Small Gtpase ◽  
Post Translational Modification ◽  
Functional Regulation ◽  
Subcellular Compartmentalization ◽  
Cellular Compartments

Cells and tissues are continuously exposed to both chemical and physical stimuli and dynamically adapt and respond to this variety of external cues to ensure cellular homeostasis, regulated development and tissue-specific differentiation. Alterations of these pathways promote disease progression—a prominent example being cancer. Rho GTPases are key regulators of the remodeling of cytoskeleton and cell membranes and their coordination and integration with different biological processes, including cell polarization and motility, as well as other signaling networks such as growth signaling and proliferation. Apart from the control of GTP–GDP cycling, Rho GTPase activity is spatially and temporally regulated by post-translation modifications (PTMs) and their assembly onto specific protein complexes, which determine their controlled activity at distinct cellular compartments. Although Rho GTPases were traditionally conceived as targeted from the cytosol to the plasma membrane to exert their activity, recent research demonstrates that active pools of different Rho GTPases also localize to endomembranes and the nucleus. In this review, we discuss how PTM-driven modulation of Rho GTPases provides a versatile mechanism for their compartmentalization and functional regulation. Understanding how the subcellular sorting of active small GTPase pools occurs and what its functional significance is could reveal novel therapeutic opportunities.

Four-Color Single-Molecule Imaging with Engineered Tags Resolves the Molecular Architecture of Signaling Complexes in the Plasma Membrane

2021 ◽  
Author(s):  
Junel Sotolongo Bellón ◽  
Oliver Birkholz ◽  
Christian Paolo Richter ◽  
Florian Eull ◽  
Hella Kenneweg ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Single Molecule Imaging ◽  
Molecular Architecture
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