scholarly journals Live cell micropatterning reveals the dynamics of signaling complexes at the plasma membrane

2014 ◽  
Vol 207 (3) ◽  
pp. 407-418 ◽  
Author(s):  
Sara Löchte ◽  
Sharon Waichman ◽  
Oliver Beutel ◽  
Changjiang You ◽  
Jacob Piehler
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Spatial Organization ◽  
Polymer Brush ◽  
Effector Proteins ◽  
Live Cells ◽  
Type I ◽  
Rgd Peptides ◽  
Single Molecule Level ◽  
Cell Micropatterning

Interactions of proteins in the plasma membrane are notoriously challenging to study under physiological conditions. We report in this paper a generic approach for spatial organization of plasma membrane proteins into micropatterns as a tool for visualizing and quantifying interactions with extracellular, intracellular, and transmembrane proteins in live cells. Based on a protein-repellent poly(ethylene glycol) polymer brush, micropatterned surface functionalization with the HaloTag ligand for capturing HaloTag fusion proteins and RGD peptides promoting cell adhesion was devised. Efficient micropatterning of the type I interferon (IFN) receptor subunit IFNAR2 fused to the HaloTag was achieved, and highly specific IFN binding to the receptor was detected. The dynamics of this interaction could be quantified on the single molecule level, and IFN-induced receptor dimerization in micropatterns could be monitored. Assembly of active signaling complexes was confirmed by immunostaining of phosphorylated Janus family kinases, and the interaction dynamics of cytosolic effector proteins recruited to the receptor complex were unambiguously quantified by fluorescence recovery after photobleaching.

scholarly journals Directed manipulation of membrane proteins by fluorescent magnetic nanoparticles

2020 ◽  
Author(s):  
Jia Hui Li ◽  
Paula Santos-Otte ◽  
Braedyn Au ◽  
Jakob Rentsch ◽  
Stephan Block ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Magnetic Nanoparticles ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Single Molecule ◽  
Super Resolution ◽  
Single Particle Tracking ◽  
Live Cells ◽  
Single Molecule Level ◽  
Membrane Components

AbstractThe plasma membrane is the interface through which cells interact with their environment. Membrane proteins are embedded in the lipid bilayer of the plasma membrane and their function in this context is often linked to their specific location and dynamics within the membrane. However, few methods are available for nanoscale manipulation of membrane protein location at the single molecule level. Here, we report the use of fluorescent magnetic nanoparticles (FMNPs) to track membrane molecules and to manipulate their movement. FMNPs allow single-particle tracking (SPT) at 10 nm spatial and 5 ms temporal resolution, and using a magnetic needle, we pull membrane components laterally through the membrane with femtonewton-range forces. In this way, we successfully dragged lipid-anchored and transmembrane proteins over the surface of living cells. Doing so, we detected submembrane barriers and in combination with super-resolution microscopy could localize these barriers to the actin cytoskeleton. We present here a versatile approach to probe membrane processes in live cells via the magnetic control of membrane protein motion.

scholarly journals A method for imaging single molecules at the plasma membrane of live cells within tissue slices

2020 ◽  
Vol 153 (1) ◽  
Author(s):  
Gregory I. Mashanov ◽  
Tatiana A. Nenasheva ◽  
Tatiana Mashanova ◽  
Catherine Maclachlan ◽  
Nigel J.M. Birdsall ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Lines ◽  
Single Molecule ◽  
Cardiac Tissue ◽  
Single Molecules ◽  
Live Cells ◽  
Biological Macromolecules ◽  
Tissue Slices ◽  
Tissue Samples ◽  
Mammalian Cell Lines

Recent advances in light microscopy allow individual biological macromolecules to be visualized in the plasma membrane and cytosol of live cells with nanometer precision and ∼10-ms time resolution. This allows new discoveries to be made because the location and kinetics of molecular interactions can be directly observed in situ without the inherent averaging of bulk measurements. To date, the majority of single-molecule imaging studies have been performed in either unicellular organisms or cultured, and often chemically fixed, mammalian cell lines. However, primary cell cultures and cell lines derived from multi-cellular organisms might exhibit different properties from cells in their native tissue environment, in particular regarding the structure and organization of the plasma membrane. Here, we describe a simple approach to image, localize, and track single fluorescently tagged membrane proteins in freshly prepared live tissue slices and demonstrate how this method can give information about the movement and localization of a G protein–coupled receptor in cardiac tissue slices. In principle, this experimental approach can be used to image the dynamics of single molecules at the plasma membrane of many different soft tissue samples and may be combined with other experimental techniques.

scholarly journals Continuous imaging of plasmon rulers in live cells reveals early-stage caspase-3 activation at the single-molecule level

2009 ◽  
Vol 106 (42) ◽  
pp. 17735-17740 ◽  
Author(s):  
Y.-w. Jun ◽  
S. Sheikholeslami ◽  
D. R. Hostetter ◽  
C. Tajon ◽  
C. S. Craik ◽  
...  
Keyword(s):  
Single Molecule ◽  
Caspase 3 ◽  
Early Stage ◽  
Live Cells ◽  
Single Molecule Level ◽  
Plasmon Rulers

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 Quantifying transcription factor binding dynamics at the single-molecule level in live cells

Methods ◽  
2017 ◽  
Vol 123 ◽  
pp. 76-88 ◽  
Author(s):  
Diego M. Presman ◽  
David A. Ball ◽  
Ville Paakinaho ◽  
Jonathan B. Grimm ◽  
Luke D. Lavis ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Single Molecule ◽  
Transcription Factor Binding ◽  
Live Cells ◽  
Single Molecule Level ◽  
Factor Binding

Protein clustering and spatial organization in T-cells

2015 ◽  
Vol 43 (3) ◽  
pp. 315-321 ◽  
Author(s):  
Michael J. Shannon ◽  
Garth Burn ◽  
Andrew Cope ◽  
Georgina Cornish ◽  
Dylan M. Owen
Keyword(s):  
T Cell ◽  
Single Molecule ◽  
Spatial Organization ◽  
Super Resolution ◽  
Cell Protein ◽  
Single Molecule Level ◽  
Composition And Structure ◽  
Resolution Analysis ◽  
And Function ◽  
Super Resolution Microscopy

T-cell protein microclusters have until recently been investigable only as microscale entities with their composition and structure being discerned by biochemistry or diffraction-limited light microscopy. With the advent of super resolution microscopy comes the ability to interrogate the structure and function of these clusters at the single molecule level by producing highly accurate pointillist maps of single molecule locations at ~20nm resolution. Analysis tools have also been developed to provide rich descriptors of the pointillist data, allowing us to pose questions about the nanoscale organization which governs the local and cell wide responses required of a migratory T-cell.

Probing plasma membrane dynamics at the single-molecule level

2013 ◽  
Vol 18 (11) ◽  
pp. 617-624 ◽  
Author(s):  
Xiaojuan Li ◽  
Doan-Trung Luu ◽  
Christophe Maurel ◽  
Jinxing Lin
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Membrane Dynamics ◽  
Single Molecule Level

scholarly journals BRCA2 diffuses as oligomeric clusters with RAD51 and changes mobility after DNA damage in live cells

2014 ◽  
Vol 207 (5) ◽  
pp. 599-613 ◽  
Author(s):  
Marcel Reuter ◽  
Alex Zelensky ◽  
Ihor Smal ◽  
Erik Meijering ◽  
Wiggert A. van Cappellen ◽  
...  
Keyword(s):  
Dna Damage ◽  
Single Molecule ◽  
Fluorescent Proteins ◽  
Single Particle Tracking ◽  
Correlation Spectroscopy ◽  
Live Cells ◽  
Physical Interaction ◽  
Single Molecule Level ◽  
Before And After ◽  
Endogenous Loci

Genome maintenance by homologous recombination depends on coordinating many proteins in time and space to assemble at DNA break sites. To understand this process, we followed the mobility of BRCA2, a critical recombination mediator, in live cells at the single-molecule level using both single-particle tracking and fluorescence correlation spectroscopy. BRCA2-GFP and -YFP were compared to distinguish diffusion from fluorophore behavior. Diffusive behavior of fluorescent RAD51 and RAD54 was determined for comparison. All fluorescent proteins were expressed from endogenous loci. We found that nuclear BRCA2 existed in oligomeric clusters, and exhibited heterogeneous mobility. DNA damage increased BRCA2 transient binding, presumably including binding to damaged sites. Despite its very different size, RAD51 displayed mobility similar to BRCA2, which indicates physical interaction between these proteins both before and after induction of DNA damage. We propose that BRCA2-mediated sequestration of nuclear RAD51 serves to prevent inappropriate DNA interactions and that all RAD51 is delivered to DNA damage sites in association with BRCA2.

Single-molecule live-cell imaging of bacterial DNA repair and damage tolerance

2017 ◽  
Vol 46 (1) ◽  
pp. 23-35 ◽  
Author(s):  
Harshad Ghodke ◽  
Han Ho ◽  
Antoine M. van Oijen
Keyword(s):  
Dna Repair ◽  
Single Molecule ◽  
Live Cell Imaging ◽  
Spatial Organization ◽  
Cell Imaging ◽  
Live Cell ◽  
Live Cells ◽  
Temporal Regulation ◽  
Repair Processes ◽  
Wide Range

Genomic DNA is constantly under threat from intracellular and environmental factors that damage its chemical structure. Uncorrected DNA damage may impede cellular propagation or even result in cell death, making it critical to restore genomic integrity. Decades of research have revealed a wide range of mechanisms through which repair factors recognize damage and co-ordinate repair processes. In recent years, single-molecule live-cell imaging methods have further enriched our understanding of how repair factors operate in the crowded intracellular environment. The ability to follow individual biochemical events, as they occur in live cells, makes single-molecule techniques tremendously powerful to uncover the spatial organization and temporal regulation of repair factors during DNA–repair reactions. In this review, we will cover practical aspects of single-molecule live-cell imaging and highlight recent advances accomplished by the application of these experimental approaches to the study of DNA–repair processes in prokaryotes.

scholarly journals The Type Iα Inositol Polyphosphate 4-Phosphatase Generates and Terminates Phosphoinositide 3-Kinase Signals on Endosomes and the Plasma Membrane

2005 ◽  
Vol 16 (5) ◽  
pp. 2218-2233 ◽  
Author(s):  
Ivan Ivetac ◽  
Adam D. Munday ◽  
Marina V. Kisseleva ◽  
Xiang-Ming Zhang ◽  
Susan Luff ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Effector Proteins ◽  
Type I ◽  
Endosomal Trafficking ◽  
Inositol Polyphosphate ◽  
Ph Domain ◽  
Membrane Recruitment ◽  
Early Endosomes ◽  
Phosphoinositide 3 Kinase

Endosomal trafficking is regulated by the recruitment of effector proteins to phosphatidylinositol 3-phosphate [PtdIns(3)P] on early endosomes. At the plasma membrane, phosphatidylinositol-(3,4)-bisphosphate [PtdIns(3,4)P2] binds the pleckstrin homology (PH) domain-containing proteins Akt and TAPP1. Type Iα inositol polyphosphate 4-phosphatase (4-phosphatase) dephosphorylates PtdIns(3,4)P2, forming PtdIns(3)P, but its subcellular localization is unknown. We report here in quiescent cells, the 4-phosphatase colocalized with early and recycling endosomes. On growth factor stimulation, 4-phosphatase endosomal localization persisted, but in addition the 4-phosphatase localized at the plasma membrane. Overexpression of the 4-phosphatase in serum-stimulated cells increased cellular PtdIns(3)P levels and prevented wortmannin-induced endosomal dilatation. Furthermore, mouse embryonic fibroblasts from homozygous Weeble mice, which have a mutation in the type I 4-phosphatase, exhibited dilated early endosomes. 4-Phosphatase translocation to the plasma membrane upon growth factor stimulation inhibited the recruitment of the TAPP1 PH domain. The 4-phosphatase contains C2 domains, which bound PtdIns(3,4)P2, and C2-domain-deletion mutants lost PtdIns(3,4)P2 4-phosphatase activity, did not localize to endosomes or inhibit TAPP1 PH domain membrane recruitment. The 4-phosphatase therefore both generates and terminates phosphoinositide 3-kinase signals at distinct subcellular locations.

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