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Staaf, E., Hedde, P. N., Singh, S. B., Piguet, J., Gratton, E. & Johansson, S. (2018). Educated natural killer cells show dynamic movement of the activating receptor NKp46 and confinement of the inhibitory receptor Ly49A. Science Signaling, 11(517), Article ID eaai9200.
Open this publication in new window or tab >>Educated natural killer cells show dynamic movement of the activating receptor NKp46 and confinement of the inhibitory receptor Ly49A
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2018 (English)In: Science Signaling, ISSN 1945-0877, E-ISSN 1937-9145, Vol. 11, no 517, article id eaai9200Article in journal (Refereed) Published
Abstract [en]

Educated natural killer (NK) cells have inhibitory receptors specific for self major histocompatibility complex (MHC) class I molecules and kill cancer cells more efficiently than do NK cells that do not have such receptors (hyporesponsive NK cells). The mechanism behind this functional empowerment through education has so far not been fully described. In addition, distinctive phenotypic markers of educated NK cells at the single-cell level are lacking. We developed a refined version of the image mean square displacement (iMSD) method (called iMSD carpet analysis) and used it in combination with single-particle tracking to characterize the dynamics of the activating receptor NKp46 and the inhibitory receptor Ly49A on resting educated versus hyporesponsive murine NK cells. Most of the NKp46 and Ly49A molecules were restricted to microdomains; however, individual NKp46 molecules resided in these domains for shorter periods and diffused faster on the surface of educated, compared to hyporesponsive, NK cells. In contrast, the movement of Ly49A was more constrained in educated NK cells compared to hyporesponsive NK cells. Either disrupting the actin cytoskeleton or adding cholesterol to the cells prohibited activating signaling, suggesting that the dynamics of receptor movements within the cell membrane are critical for the proper activation of NK cells. The faster and more dynamic movement of NKp46 in educated NK cells may facilitate a swifter response to interactions with target cells.

Place, publisher, year, edition, pages
AMER ASSOC ADVANCEMENT SCIENCE, 2018
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-223794 (URN)10.1126/scisignal.aai9200 (DOI)000424982900001 ()2-s2.0-85041966098 (Scopus ID)
Funder
Swedish Research CouncilMagnus Bergvall FoundationVINNOVANIH (National Institute of Health), P41-GM103540; P50-GM076516
Note

QC 20180307

Available from: 2018-03-07 Created: 2018-03-07 Last updated: 2018-03-07Bibliographically approved
Staaf, E., Hedde, P. N., Singh, S. B., Piguet, J., Gratton, E. & Johansson, S. (2017). Educated Natural Killer cells exhibit a more dynamic movement of NKp46 and a higher confinement of the Ly49A receptor. Paper presented at 44th Annual Meeting of the Scandinavian-Society-for-Immunology (SSI), OCT 17-20, 2017, Stockholm, SWEDEN. Scandinavian Journal of Immunology, 86(4), 285-285
Open this publication in new window or tab >>Educated Natural Killer cells exhibit a more dynamic movement of NKp46 and a higher confinement of the Ly49A receptor
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2017 (English)In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 86, no 4, p. 285-285Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
WILEY, 2017
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-215791 (URN)000411865200089 ()
Conference
44th Annual Meeting of the Scandinavian-Society-for-Immunology (SSI), OCT 17-20, 2017, Stockholm, SWEDEN
Note

QC 20171018

Available from: 2017-10-18 Created: 2017-10-18 Last updated: 2017-10-18Bibliographically approved
Veya, L., Piguet, J. & Vogel, H. (2015). Single Molecule Imaging Deciphers the Relation between Mobility and Signaling of a Prototypical G Protein-coupled Receptor in Living Cells.. Journal of Biological Chemistry, 290(46), 27723-27735
Open this publication in new window or tab >>Single Molecule Imaging Deciphers the Relation between Mobility and Signaling of a Prototypical G Protein-coupled Receptor in Living Cells.
2015 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 46, p. 27723-27735Article in journal (Refereed) Published
Abstract [en]

Lateral diffusion enables efficient interactions between membrane proteins, leading to signal transmission across the plasma membrane. An open question is how the spatiotemporal distribution of cell surface receptors influences the transmembrane signaling network. Here we addressed this issue by studying the mobility of a prototypical G protein-coupled receptor, the neurokinin-1 receptor, during its different phases of cellular signaling. Attaching a single quantum dot to individual neurokinin-1 receptors enabled us to follow with high spatial and temporal resolution over long time regimes the fate of individual receptors at the plasma membrane. Single receptor trajectories revealed a very heterogeneous mobility distribution pattern with diffusion constants ranging from 0.0005 to 0.1 μm(2)/s comprising receptors freely diffusing and others confined in 100-600-nm-sized membrane domains as well as immobile receptors. A two-dimensional representation of mobility and confinement resolved two major, broadly distributed receptor populations, one showing high mobility and low lateral restriction and the other showing low mobility and high restriction. We found that about 40% of the receptors in the basal state are already confined in membrane domains and are associated with clathrin. After stimulation with an agonist, an additional 30% of receptors became further confined. Using inhibitors of clathrin-mediated endocytosis, we found that the fraction of confined receptors at the basal state depends on the quantity of membrane-associated clathrin and is correlated to a significant decrease of the canonical pathway activity of the receptors. This shows that the high plasticity of receptor mobility is of central importance for receptor homeostasis and fine regulation of receptor activity.

Place, publisher, year, edition, pages
American Society for Biochemistry and Molecular Biology, 2015
Keywords
Cell signaling
National Category
Biochemistry and Molecular Biology Biophysics
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-178090 (URN)10.1074/jbc.M115.666677 (DOI)000365757500025 ()26363070 (PubMedID)2-s2.0-84946903219 (Scopus ID)
Note

QC 2016-02-11

Available from: 2015-12-07 Created: 2015-12-07 Last updated: 2017-12-01Bibliographically approved
Grasso, L., Wyss, R., Piguet, J., Werner, M., Hassaïne, G., Hovius, R. & Vogel, H. (2013). Downscaling the analysis of complex transmembrane signaling cascades to closed attoliter volumes. PLoS ONE, 8(8)
Open this publication in new window or tab >>Downscaling the analysis of complex transmembrane signaling cascades to closed attoliter volumes
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 8Article in journal (Refereed) Published
Abstract [en]

Cellular signaling is classically investigated by measuring optical or electrical properties of single or populations of living cells. Here we show that ligand binding to cell surface receptors and subsequent activation of signaling cascades can be monitored in single, (sub-)micrometer sized native vesicles with single-molecule sensitivity. The vesicles are derived from live mammalian cells using chemicals or optical tweezers. They comprise parts of a cell's plasma membrane and cytosol and represent the smallest autonomous containers performing cellular signaling reactions thus functioning like minimized cells. Using fluorescence microscopies, we measured in individual vesicles the different steps of G-protein-coupled receptor mediated signaling like ligand binding to receptors, subsequent G-protein activation and finally arrestin translocation indicating receptor deactivation. Observing cellular signaling reactions in individual vesicles opens the door for downscaling bioanalysis of cellular functions to the attoliter range, multiplexing single cell analysis, and investigating receptor mediated signaling in multiarray format.

Place, publisher, year, edition, pages
Public Library of Science, 2013
National Category
Biochemistry and Molecular Biology Biophysics Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-178092 (URN)10.1371/journal.pone.0070929 (DOI)000324465000159 ()23940670 (PubMedID)2-s2.0-84881112332 (Scopus ID)
Note

QC 20151214

Available from: 2015-12-07 Created: 2015-12-07 Last updated: 2017-12-01Bibliographically approved
Piguet, J., Schreiter, C., Segura, J.-M., Vogel, H. & Hovius, R. (2011). Acetylcholine receptor organization in membrane domains in muscle cells: evidence for rapsyn-independent and rapsyn-dependent mechanisms. Journal of Biological Chemistry, 286(1)
Open this publication in new window or tab >>Acetylcholine receptor organization in membrane domains in muscle cells: evidence for rapsyn-independent and rapsyn-dependent mechanisms
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2011 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 1Article in journal (Refereed) Published
Abstract [en]

Nicotinic acetylcholine receptors (nAChR) in muscle fibers are densely packed in the postsynaptic region at the neuromuscular junction. Rapsyn plays a central role in directing and clustering nAChR during cellular differentiation and neuromuscular junction formation; however, it has not been demonstrated whether rapsyn is the only cause of receptor immobilization. Here, we used single-molecule tracking methods to investigate nAChR mobility in plasma membranes of myoblast cells during their differentiation to myotubes in the presence and absence of rapsyn. We found that in myoblasts the majority of nAChR were immobile and that ∼20% of the receptors showed restricted diffusion in small domains of ∼50 nm. In myoblasts devoid of rapsyn, the fraction of mobile nAChR was considerably increased, accompanied by a 3-fold decrease in the immobile population of nAChR with respect to rapsyn-expressing cells. Half of the mobile receptors were confined to domains of ∼120 nm. Measurements performed in heterologously transfected HEK cells confirmed the direct immobilization of nAChR by rapsyn. However, irrespective of the presence of rapsyn, about one-third of nAChR were confined in 300-nm domains. Our results show (i) that rapsyn efficiently immobilizes nAChR independently of other postsynaptic scaffold components; (ii) nAChR is constrained in confined membrane domains independently of rapsyn; and (iii) in the presence of rapsyn, the size of these domains is strongly reduced.

Place, publisher, year, edition, pages
American Society for Biochemistry and Molecular Biology, 2011
National Category
Biophysics Cell Biology
Identifiers
urn:nbn:se:kth:diva-178097 (URN)10.1074/jbc.M110.139782 (DOI)000285782800039 ()20978122 (PubMedID)2-s2.0-78650959800 (Scopus ID)
Note

QC 20151211

Available from: 2015-12-07 Created: 2015-12-07 Last updated: 2017-12-01Bibliographically approved
Roizard, S., Danelon, C., Hassaïne, G., Piguet, J., Schulze, K., Hovius, R., . . . Vogel, H. (2011). Activation of G-protein-coupled receptors in cell-derived plasma membranes supported on porous beads. Journal of the American Chemical Society, 133(42)
Open this publication in new window or tab >>Activation of G-protein-coupled receptors in cell-derived plasma membranes supported on porous beads
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2011 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 133, no 42Article in journal (Refereed) Published
Abstract [en]

G-protein-coupled receptors (GPCRs) are ubiquitous mediators of signal transduction across cell membranes and constitute a very important class of therapeutic targets. In order to study the complex biochemical signaling network coupling to the intracellular side of GPCRs, it is necessary to engineer and control the downstream signaling components, which is difficult to realize in living cells. We have developed a bioanalytical platform enabling the study of GPCRs in their native membrane transferred inside-out from live cells to lectin-coated beads, with both membrane sides of the receptor being accessible for molecular interactions. Using heterologously expressed adenosine A(2A) receptor carrying a yellow fluorescent protein, we showed that the tethered membranes comprised fully functional receptors in terms of ligand and G protein binding. The interactions between the different signaling partners during the formation and subsequent dissociation of the ternary signaling complex on single beads could be observed in real time using multicolor fluorescence microscopy. This approach of tethering inside-out native membranes accessible from both sides is straightforward and readily applied to other transmembrane proteins. It represents a generic platform suitable for ensemble as well as single-molecule measurements to investigate signaling processes at plasma membranes.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2011
National Category
Biophysics Biochemistry and Molecular Biology Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-178093 (URN)10.1021/ja205302g (DOI)000296678200034 ()21910424 (PubMedID)2-s2.0-80055011129 (Scopus ID)
Note

QC 20160125

Available from: 2015-12-07 Created: 2015-12-07 Last updated: 2017-12-01Bibliographically approved
Werner, M., Merenda, F., Piguet, J., Salathé, R.-P. & Vogel, H. (2011). Microfluidic array cytometer based on refractive optical tweezers for parallel trapping, imaging and sorting of individual cells.. Lab on a Chip, 11(14), 2432-2439
Open this publication in new window or tab >>Microfluidic array cytometer based on refractive optical tweezers for parallel trapping, imaging and sorting of individual cells.
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2011 (English)In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 11, no 14, p. 2432-2439Article in journal (Refereed) Published
Abstract [en]

Analysis of genetic and functional variability in populations of living cells requires experimental techniques capable of monitoring cellular processes such as cell signaling of many single cells in parallel while offering the possibility to sort interesting cell phenotypes for further investigations. Although flow cytometry is able to sequentially probe and sort thousands of cells per second, dynamic processes cannot be experimentally accessed on single cells due to the sub-second sampling time. Cellular dynamics can be measured by image cytometry of surface-immobilized cells, however, cell sorting is complicated under these conditions due to cell attachment. We here developed a cytometric tool based on refractive multiple optical tweezers combined with microfluidics and optical microscopy. We demonstrate contact-free immobilization of more than 200 yeast cells into a high-density array of optical traps in a microfluidic chip. The cell array could be moved to specific locations of the chip enabling us to expose in a controlled manner the cells to reagents and to analyze the responses of individual cells in a highly parallel format using fluorescence microscopy. We further established a method to sort single cells within the microfluidic device using an additional steerable optical trap. Ratiometric fluorescence imaging of intracellular pH of trapped yeast cells allowed us on the one hand to measure the effect of the trapping laser on the cells' viability and on the other hand to probe the dynamic response of the cells upon glucose sensing.

Place, publisher, year, edition, pages
Royal Society of Chemistry, 2011
Keywords
yeast saccharomyces-cerevisiae, single-cell, intracellular PH, flow-cytometry, gene-expression, biological cells, mammalian-cells, manipulation, growth, noise
National Category
Biophysics Analytical Chemistry Atom and Molecular Physics and Optics
Identifiers
urn:nbn:se:kth:diva-178096 (URN)10.1039/c1lc20181f (DOI)000292168500018 ()21655617 (PubMedID)2-s2.0-80051757580 (Scopus ID)
Note

QC 2016-01-18

Available from: 2015-12-07 Created: 2015-12-07 Last updated: 2017-12-01Bibliographically approved
Brun, M. A., Griss, R., Reymond, L., Tan, K.-T., Piguet, J., Peters, R. J., . . . Johnsson, K. (2011). Semisynthesis of fluorescent metabolite sensors on cell surfaces.. Journal of the American Chemical Society, 133(40)
Open this publication in new window or tab >>Semisynthesis of fluorescent metabolite sensors on cell surfaces.
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2011 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 133, no 40Article in journal (Refereed) Published
Abstract [en]

Progress in understanding signal transduction and metabolic pathways is hampered by a shortage of suitable sensors for tracking metabolites, second messengers, and neurotransmitters in living cells. Here we introduce a class of rationally designed semisynthetic fluorescent sensor proteins, called Snifits, for measuring metabolite concentrations on the cell surface of mammalian cells. Functional Snifits are assembled on living cells through two selective chemical labeling reactions of a genetically encoded protein scaffold. Our best Snifit displayed fluorescence intensity ratio changes on living cells significantly higher than any previously reported cell-surface-targeted fluorescent sensor protein. This work establishes a generally applicable and rational strategy for the generation of cell-surface-targeted fluorescent sensor proteins for metabolites of interest.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2011
National Category
Biochemistry and Molecular Biology Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-178095 (URN)10.1021/ja206915m (DOI)000296036700069 ()21879732 (PubMedID)2-s2.0-80053525802 (Scopus ID)
Note

QC 20151215

Available from: 2015-12-07 Created: 2015-12-07 Last updated: 2017-12-01Bibliographically approved
Piguet, J. (2010). Advanced Fluorescence Microscopy to Study Plasma Membrane Protein Dynamics. (Doctoral dissertation). Lausanne: EPFL
Open this publication in new window or tab >>Advanced Fluorescence Microscopy to Study Plasma Membrane Protein Dynamics
2010 (English)Doctoral thesis, monograph (Other academic)
Abstract [en]

Membrane protein dynamics is of great importance for living organisms. The precise localization of proteins composing a synapse on the membrane facing a nerve terminus is essential for proper functioning of the nervous system. In muscle fibers, the nicotinic acetylcholine is densely packed under the motor nerve termini. A receptor associated protein, rapsyn, acts as a linker between the receptor and the other components of the synaptic suramolecular assembly. Advances in fluorescence microscopy have allowed to measure the behavior of a single receptor in the cell membrane. In this work single-molecule microscopy was used to track the motion of ionotropic acetylcholine (nAChR) and serotonin (5HT3R) receptors in the plasma membrane of cells. We present methods for measuring single-molecule diffusion and their analysis. Single molecule tracking has shown a high dependence of acetylcholine receptors diffusion on its associated protein rapsyn. Comparing muscle cells that either express rapsyn or are devoid of it, we found that rapsyn plays an important role on receptor immobilization. A three-fold increase of receptor mobility was observed in muscle cells devoid of rapsyn. However, in these cells, a certain fraction of immobilized receptors was also found immobile. Furthermore, nAChR were strongly confined in membrane domains of few tens of nanometers. This showed that membrane composition and membrane associated proteins influence on receptor localization. During muscle cell differentiation, the fraction of immobile nAChR diminished along with the decreasing nAChR and stable rapsyn expression levels. The importance of rapsyn in nAChR immobilization has been further confirmed by measurements in HEK 293 cells, where co-expression of rapsyn increased immobilization of the receptor. nAChR is a ligand-gated ion-channel of the Cys-loop family. In mammals, members of this receptor family share general structural and functional features. They are homo- or hetero-pentamers and form a membrane-spanning ion channel. Subunits have three major regions, an extracellular ligand binding domain, a transmembrane channel and a large intracellular loop. 5HT3R was used as a model to study the effect of this loop on receptor mobility. Single-molecule tracking experiments on receptors with progressively larger deletions in the intracellular loop did not show a dependence of the size of the loop on the diffusion coefficient of mobile receptors. However, two regions were identified to play a role in receptor mobility by changing the fractions of immobile and directed receptors. Interestingly, a prokaryotic homologue of cys-loop receptors, ELIC, devoid of a large cytoplasmic loop was found to be immobile or to show directed diffusion similar as the wild-type 5HT3R. The scaffolding protein rapsyn stabilizes nAChR clusters in a concentration dependent manner. We have measured the density and self-interactions of rapsyn using FRET microscopy. Point-mutations of rapsyn, known to provoke myopathies, destabilized rapsyn self-interactions. Rapsyn-N88K, and R91L were found at high concentration in the cytoplasm suggesting that this modification disturbs membrane association of rapsyn. A25V was found to accumulate in the endoplasmic reticulum. Fluorescent tools to measure intracellular concentration of calcium ions are of great value to study the function of neurons. Rapsyn is highly abundant at the neuromuscular junction and thus is a genuine synaptic marker. A fusion protein of rapsyn with a genetically encoded ratiometric calcium sensor has been made to probe synapse activity. This thesis has shown that the combined use of biologically relevant system and modern fluorescence microscopy techniques deliver important information on pLGIC behaviour in the cell membrane.

Place, publisher, year, edition, pages
Lausanne: EPFL, 2010
Keywords
fluorescence, microscopy, plasma membrane, membrane proteins, membrane proteins diffusion, single-molecule imaging, single-molecule tracking, pentameric ligand-gated ion channels, nicotinic acetylcholine receptor, serotonin receptor, synapse, neuromuscular junction, post-synaptic scaffold, rapsyn, myopathies, fluorescent proteins, Förster resonance energy transfer, FRET imaging, protein-protein interactions, protein trafficking, photo-activatable proteins
National Category
Biophysics Biochemistry and Molecular Biology Atom and Molecular Physics and Optics
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-178147 (URN)10.5075/epfl-thesis-4869 (DOI)
Public defence
2010-11-05, EPFL, Lausanne, 15:49 (French)
Opponent
Supervisors
Note

QC 20151217

Available from: 2015-12-17 Created: 2015-12-07 Last updated: 2015-12-17Bibliographically approved
Perez, J.-B., Segura, J.-M., Abankwa, D., Piguet, J., Martinez, K. L. & Vogel, H. (2006). Monitoring the diffusion of single heterotrimeric G proteins in supported cell-membrane sheets reveals their partitioning into microdomains.. Journal of Molecular Biology, 363(5)
Open this publication in new window or tab >>Monitoring the diffusion of single heterotrimeric G proteins in supported cell-membrane sheets reveals their partitioning into microdomains.
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2006 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 363, no 5Article in journal (Refereed) Published
Abstract [en]

Supported cell-membrane sheets are promising in vitro systems to investigate the properties of membranes with native protein/lipid composition, in particular their sub-compartmentalization and the differential localization of proteins associated to them. While such studies are usually performed using static microscopy techniques, we demonstrate here the potential offered by dynamic diffusion measurements. Whereas the overall fluidity of the lipid bilayer was preserved, the preparation of the membrane sheets led to the selective immobilization of extracellular and transmembrane (TM) glycosylated proteins and the anchored proteins/lipids associated with them. Taking advantage of this, we investigated the association of the G protein Gq with TM proteins, in particular G-protein coupled receptors (GPCRs), by monitoring the changes in diffusion occurring after preparation of the supported membranes. Two fluorescently tagged Galphaq proteins were constructed, which remained either mostly monomeric in the plasma membrane or associated with Gbetagamma in heterotrimers. While both constructs diffused similarly in living cells, the preparation of the supported membranes led to the selective immobilization of the heterotrimers with minimal changes of the diffusion of the monomeric Galphaq. The diverse mobility of monomeric and heterotrimeric Galphaq was a result of their different lipid anchors as demonstrated by monitoring the diffusion of the corresponding anchors alone. We propose that the immobilization of the heterotrimer was caused by its partitioning inside membrane microdomains surrounding GPCRs.

Place, publisher, year, edition, pages
Elsevier, 2006
National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-178098 (URN)10.1016/j.jmb.2006.08.036 (DOI)000241757100005 ()16996083 (PubMedID)2-s2.0-33750009848 (Scopus ID)
Note

QC 20151211

Available from: 2015-12-07 Created: 2015-12-07 Last updated: 2017-12-01Bibliographically approved
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-4762-4887

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