scholarly journals Single-molecule diffusion measurements of H-Ras at the plasma membrane of live cells reveal microdomain localization upon activation

2005 ◽  
Vol 118 (9) ◽  
pp. 1799-1809 ◽  
Author(s):  
P. H. M. Lommerse
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Live Cells

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 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.

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 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.

The fence and picket structure of the plasma membrane of live cells as revealed by single molecule techniques (Review)

2003 ◽  
Vol 20 (1) ◽  
pp. 13-18 ◽  
Author(s):  
Ken Ritchie ◽  
Ryota Iino ◽  
Takahiro Fujiwara ◽  
Kotono Murase ◽  
Akihiro Kusumi
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Live Cells

scholarly journals Motional dynamics of single Patched1 molecules in cilia are controlled by Hedgehog and cholesterol

2019 ◽  
Vol 116 (12) ◽  
pp. 5550-5557 ◽  
Author(s):  
Lucien E. Weiss ◽  
Ljiljana Milenkovic ◽  
Joshua Yoon ◽  
Tim Stearns ◽  
W. E. Moerner
Keyword(s):  
Single Molecule ◽  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Transmembrane Protein ◽  
Cellular Level ◽  
Live Cells ◽  
Cholesterol Depletion ◽  
Motional Dynamics ◽  
Directional Transport ◽  
Bulk Behavior

The Hedgehog-signaling pathway is an important target in cancer research and regenerative medicine; yet, on the cellular level, many steps are still poorly understood. Extensive studies of the bulk behavior of the key proteins in the pathway established that during signal transduction they dynamically localize in primary cilia, antenna-like solitary organelles present on most cells. The secreted Hedgehog ligand Sonic Hedgehog (SHH) binds to its receptor Patched1 (PTCH1) in primary cilia, causing its inactivation and delocalization from cilia. At the same time, the transmembrane protein Smoothened (SMO) is released of its inhibition by PTCH1 and accumulates in cilia. We used advanced, single molecule-based microscopy to investigate these processes in live cells. As previously observed for SMO, PTCH1 molecules in cilia predominantly move by diffusion and less frequently by directional transport, and spend a fraction of time confined. After treatment with SHH we observed two major changes in the motional dynamics of PTCH1 in cilia. First, PTCH1 molecules spend more time as confined, and less time freely diffusing. This result could be mimicked by a depletion of cholesterol from cells. Second, after treatment with SHH, but not after cholesterol depletion, the molecules that remain in the diffusive state showed a significant increase in the diffusion coefficient. Therefore, PTCH1 inactivation by SHH changes the diffusive motion of PTCH1, possibly by modifying the membrane microenvironment in which PTCH1 resides.

scholarly journals A Photoactivatable Push−Pull Fluorophore for Single-Molecule Imaging in Live Cells

2008 ◽  
Vol 130 (29) ◽  
pp. 9204-9205 ◽  
Author(s):  
Samuel J. Lord ◽  
Nicholas R. Conley ◽  
Hsiao-lu D. Lee ◽  
Reichel Samuel ◽  
Na Liu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Live Cells ◽  
Single Molecule Imaging

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
Sign in / Sign up

Export Citation Format

Share Document