Engineering Orthogonal, Plasma Membrane-Specific SLIPT Systems for Multiplexed Chemical Control of Signaling Pathways in Living Single Cells

2020 ◽  
Vol 15 (4) ◽  
pp. 1004-1015 ◽  
Author(s):  
Akinobu Nakamura ◽  
Choji Oki ◽  
Kenya Kato ◽  
Satoko Fujinuma ◽  
Gembu Maryu ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Signaling Pathways ◽  
Chemical Control ◽  
Single Cells ◽  
Living Single

Multiplexed Chemical Control of Signaling Pathways by Orthogonal, Plasma Membrane-Specific SLIPT Systems

2019 ◽  
Author(s):  
Akinobu Nakamura ◽  
Choji Oki ◽  
Kenya Kato ◽  
Satoko Fujinuma ◽  
Gembu Maryu ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Signaling Pathways ◽  
Mammalian Cells ◽  
Protein Translocation ◽  
Single Cells ◽  
Cellular Systems ◽  
Cell Manipulation ◽  
Signal Control ◽  
Synthetic Cell ◽  
Multiple Signaling

Most cell behaviors are the outcome of processing information from multiple signals generated upon cell stimulation. A systematic understanding of cellular systems requires methods that activate multiple signaling molecules or pathways in single cells. However, the construction of tools for such multiplexed signal control is challenging. Here we present orthogonal chemogenetic systems that allow control of multiple signaling pathways in living mammalian cells based on self-localizing ligand-induced protein translocation (SLIPT). Two orthogonal SLIPT systems were constructed to enable chemically inducible, individual translocation of two proteins from the cytoplasm to the inner-leaflet of the plasma membrane in the same cell. The SLIPT systems combined with fluorescent reporters achieved simultaneous multiplexed activation and monitoring of endogenous Ras/ERK and PI3K/Akt pathways in single cells. Thus, orthogonal SLIPT systems provide a powerful platform for multiplexed chemical signal control in single cells, offering new opportunities for dissecting cell signaling networks and synthetic cell manipulation.<br>

Multiplexed Chemical Control of Signaling Pathways by Orthogonal, Plasma Membrane-Specific SLIPT Systems

2019 ◽  
Author(s):  
Akinobu Nakamura ◽  
Choji Oki ◽  
Kenya Kato ◽  
Satoko Fujinuma ◽  
Gembu Maryu ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Signaling Pathways ◽  
Mammalian Cells ◽  
Protein Translocation ◽  
Single Cells ◽  
Cellular Systems ◽  
Cell Manipulation ◽  
Signal Control ◽  
Synthetic Cell ◽  
Multiple Signaling

Most cell behaviors are the outcome of processing information from multiple signals generated upon cell stimulation. A systematic understanding of cellular systems requires methods that activate multiple signaling molecules or pathways in single cells. However, the construction of tools for such multiplexed signal control is challenging. Here we present orthogonal chemogenetic systems that allow control of multiple signaling pathways in living mammalian cells based on self-localizing ligand-induced protein translocation (SLIPT). Two orthogonal SLIPT systems were constructed to enable chemically inducible, individual translocation of two proteins from the cytoplasm to the inner-leaflet of the plasma membrane in the same cell. The SLIPT systems combined with fluorescent reporters achieved simultaneous multiplexed activation and monitoring of endogenous Ras/ERK and PI3K/Akt pathways in single cells. Thus, orthogonal SLIPT systems provide a powerful platform for multiplexed chemical signal control in single cells, offering new opportunities for dissecting cell signaling networks and synthetic cell manipulation.<br>

Multimodal Atomic Force Imaging of Open Hemichannelinduced Modulation of Cell Volume and Viscoelastic Properties

1999 ◽  
Vol 5 (S2) ◽  
pp. 998-999
Author(s):  
Seung K. Rhee ◽  
Arjan P. Quist ◽  
Hai Lin ◽  
Nils Almqvist ◽  
Ratneshx Lai
Keyword(s):  
Plasma Membrane ◽  
Cell Volume ◽  
Lucifer Yellow ◽  
Single Cells ◽  
Dye Uptake ◽  
Aortic Endothelial Cells ◽  
Atomic Force ◽  
Cell Plasma ◽  
Uptake Assay ◽  
Gap Junctional

Hemichannels from two single cells can join upon contact between these cells to form gap junctions - an intercellular pathway for the direct exchange of ions and small metabolites. Using techniques of fluorescent dye-uptake assay, laser confocal fluorescence imaging and atomic force microscopy (AFM), we have examined the role of hemichannels, present in the non-junctional regions of single cell plasma membrane, in the modulation of cell volume.Antibodies against a gap junctional protein connexin43, were immunolocalized to nonjunctional plasma membrane regions of single BICR-MlRk k (breast tumor epithelial) cells, KOM-1 (bovine aortic endothelial) cells, and GM04260 (AD-free human) fibroblast cells. In the absence of extracellular calcium, cytoplasmic uptake of Lucifer yellow (LY) but not of dextran-conjugated LY was observed in single cells. Dye uptake was prevented by gap junctional inhibitors, ẞ-glycyrrhetinic acid (ẞGCA) and oleamide.

scholarly journals Ykt6 membrane-to-cytosol cycling regulates exosomal Wnt secretion

2018 ◽  
Author(s):  
Karen Linnemannstöns ◽  
Pradhipa Karuna M ◽  
Leonie Witte ◽  
Jeanette Clarissa Kittel ◽  
Adi Danieli ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Wnt Signaling ◽  
Signaling Pathways ◽  
Long Range ◽  
Protein Trafficking ◽  
Secretory Pathway ◽  
Multivesicular Bodies ◽  
Phosphorylation Sites ◽  
Wnt Proteins ◽  
C Terminus

Protein trafficking in the secretory pathway, for example the secretion of Wnt proteins, requires tight regulation. These ligands activate Wnt signaling pathways and are crucially involved in development and disease. Wnt is transported to the plasma membrane by its cargo receptor Evi, where Wnt/Evi complexes are endocytosed and sorted onto exosomes for long-range secretion. However, the trafficking steps within the endosomal compartment are not fully understood. The promiscuous SNARE Ykt6 folds into an auto-inhibiting conformation in the cytosol, but a portion associates with membranes by its farnesylated and palmitoylated C-terminus. Here, we demonstrate that membrane detachment of Ykt6 is essential for exosomal Wnt secretion. We identified conserved phosphorylation sites within the SNARE domain of Ykt6, which block Ykt6 cycling from the membrane to the cytosol. In Drosophila, Ykt6-RNAi mediated block of Wg secretion is rescued by wildtype but not phosphomimicking Ykt6. The latter accumulates at membranes, while wildtype Ykt6 regulates Wnt trafficking between the plasma membrane and multivesicular bodies. Taken together, we show that a regulatory switch in Ykt6 fine-tunes sorting of Wnts in endosomes.

scholarly journals Cell wound repair in Drosophila occurs through three distinct phases of membrane and cytoskeletal remodeling

2011 ◽  
Vol 193 (3) ◽  
pp. 455-464 ◽  
Author(s):  
Maria Teresa Abreu-Blanco ◽  
Jeffrey M. Verboon ◽  
Susan M. Parkhurst
Keyword(s):  
Cell Death ◽  
Plasma Membrane ◽  
Single Cell ◽  
Wound Repair ◽  
Single Cells ◽  
Repair Process ◽  
Wound Edge ◽  
Cytoskeletal Remodeling ◽  
Tissue Integrity ◽  
E Cadherin

When single cells or tissues are injured, the wound must be repaired quickly in order to prevent cell death, loss of tissue integrity, and invasion by microorganisms. We describe Drosophila as a genetically tractable model to dissect the mechanisms of single-cell wound repair. By analyzing the expression and the effects of perturbations of actin, myosin, microtubules, E-cadherin, and the plasma membrane, we define three distinct phases in the repair process—expansion, contraction, and closure—and identify specific components required during each phase. Specifically, plasma membrane mobilization and assembly of a contractile actomyosin ring are required for this process. In addition, E-cadherin accumulates at the wound edge, and wound expansion is excessive in E-cadherin mutants, suggesting a role for E-cadherin in anchoring the actomyosin ring to the plasma membrane. Our results show that single-cell wound repair requires specific spatial and temporal cytoskeleton responses with distinct components and mechanisms required at different stages of the process.

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 β-Arrestin 2 and ERK1/2 Are Important Mediators Engaged in Close Cooperation between TRPV1 and µ-Opioid Receptors in the Plasma Membrane

2020 ◽  
Vol 21 (13) ◽  
pp. 4626
Author(s):  
Barbora Melkes ◽  
Vendula Markova ◽  
Lucie Hejnova ◽  
Jiri Novotny
Keyword(s):  
Plasma Membrane ◽  
Signaling Pathways ◽  
Opioid Receptors ◽  
The Other ◽  
Hek293 Cells ◽  
Lateral Mobility ◽  
Pain Pathways ◽  
Close Cooperation ◽  
Signaling Interactions

The interactions between TRPV1 and µ-opioid receptors (MOR) have recently attracted much attention because these two receptors play important roles in pain pathways and can apparently modulate each other’s functioning. However, the knowledge about signaling interactions and crosstalk between these two receptors is still limited. In this study, we investigated the mutual interactions between MOR and TRPV1 shortly after their activation in HEK293 cells expressing these two receptors. After activation of one receptor we observed significant changes in the other receptor’s lateral mobility and vice versa. However, the changes in receptor movement within the plasma membrane were not connected with activation of the other receptor. We also observed that plasma membrane β-arrestin 2 levels were altered after treatment with agonists of both these receptors. Knockdown of β-arrestin 2 blocked all changes in the lateral mobility of both receptors. Furthermore, we found that β-arrestin 2 can play an important role in modulating the effectiveness of ERK1/2 phosphorylation after activation of MOR in the presence of TRPV1. These data suggest that β-arrestin 2 and ERK1/2 are important mediators between these two receptors and their signaling pathways. Collectively, MOR and TRPV1 can mutually affect each other’s behavior and β-arrestin 2 apparently plays a key role in the bidirectional crosstalk between these two receptors in the plasma membrane.

scholarly journals Ligands and receptors mediating signal transduction in sea urchin spermatozoa

Reproduction ◽  
2004 ◽  
Vol 127 (2) ◽  
pp. 141-149 ◽  
Author(s):  
Anna T Neill ◽  
Victor D Vacquier
Keyword(s):  
Signal Transduction ◽  
Plasma Membrane ◽  
Signaling Pathways ◽  
Sea Urchin ◽  
Acrosome Reaction ◽  
Sea Urchins ◽  
Model System ◽  
Surrounding Seawater ◽  
Sea Urchin Sperm

Sea urchins have long been a model system for the study of fertilization. Much has been learned about how sea urchin sperm locate and fertilize the egg. Sperm and eggs are spawned simultaneously into the surrounding seawater. Sperm signaling pathways lead to downstream events that ensure fertilization. Upon spawning, sperm must acquire motility and then they must swim towards or respond to the egg in some way. Finally, they must undergo a terminal exocytotic event known as the acrosome reaction that allows the sperm to bind to the vitelline layer of the egg and then to fuse with the egg plasma membrane. Motility is stimulated by exposure to seawater, while later events are orchestrated by factors from the egg. The sperm signaling pathways are exquisitely tuned to bring the sperm to the egg, bind, and fuse the two cells as quickly as possible.

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