scholarly journals Revealing compartmentalised membrane diffusion in living cells with interferometric scattering microscopy

2016 ◽  
Author(s):  
G. de Wit ◽  
D. Albrecht ◽  
H. Ewers ◽  
P. Kukura
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Particle Tracking ◽  
Single Particle ◽  
Hippocampal Neurons ◽  
Living Cells ◽  
Single Particle Tracking ◽  
Unique Combination ◽  
Membrane Diffusion ◽  
Mammalian Cell Lines

AbstractSingle-particle tracking is a powerful tool for studying single molecule behaviour involving plasma membrane-associated events in cells. Here, we show that interferometric scattering microscopy (iSCAT) combined with gold nanoparticle labeling can be used to follow the motion of membrane proteins in the plasma membrane of live cultured mammalian cell lines and hippocampal neurons. The unique combination of microsecond temporal resolution and nanometer spatial precision reveals signatures of a compartmentalised plasma membrane in neurons.

scholarly journals Neuronal receptors display cytoskeleton-independent directed motion on the plasma membrane

2018 ◽  
Author(s):  
R. D. Taylor ◽  
M. Heine ◽  
N. J. Emptage ◽  
L. C. Andreae
Keyword(s):  
Plasma Membrane ◽  
Neuronal Activity ◽  
Particle Tracking ◽  
Hippocampal Neurons ◽  
Intracellular Transport ◽  
Transmembrane Proteins ◽  
Single Particle Tracking ◽  
Directed Motion ◽  
Surface Movement ◽  
Transport Vesicles

AbstractDirected transport of transmembrane proteins is generally believed to occur via intracellular transport vesicles. However, using single particle tracking in rat hippocampal neurons with a pH-sensitive quantum dot probe which specifically reports surface movement of receptors, we have identified a subpopulation of neuronal EphB2 receptors that exhibit directed motion between synapses within the plasma membrane itself. This receptor movement occurs independently of the cytoskeleton but is dependent on cholesterol and is regulated by neuronal activity.

scholarly journals Single-molecule diffusometry reveals no catalysis-induced diffusion enhancement of alkaline phosphatase as proposed by FCS experiments

2020 ◽  
Vol 117 (35) ◽  
pp. 21328-21335
Author(s):  
Zhijie Chen ◽  
Alan Shaw ◽  
Hugh Wilson ◽  
Maxime Woringer ◽  
Xavier Darzacq ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Single Molecule ◽  
Particle Tracking ◽  
Single Particle ◽  
Theoretical Models ◽  
Single Particle Tracking ◽  
Correlation Spectroscopy ◽  
Single Molecule Level ◽  
Diffusion Phenomena ◽  
Diffusion Enhancement

Theoretical and experimental observations that catalysis enhances the diffusion of enzymes have generated exciting implications about nanoscale energy flow, molecular chemotaxis, and self-powered nanomachines. However, contradictory claims on the origin, magnitude, and consequence of this phenomenon continue to arise. To date, experimental observations of catalysis-enhanced enzyme diffusion have relied almost exclusively on fluorescence correlation spectroscopy (FCS), a technique that provides only indirect, ensemble-averaged measurements of diffusion behavior. Here, using an anti-Brownian electrokinetic (ABEL) trap and in-solution single-particle tracking, we show that catalysis does not increase the diffusion of alkaline phosphatase (ALP) at the single-molecule level, in sharp contrast to the ∼20% enhancement seen in parallel FCS experiments usingp-nitrophenyl phosphate (pNPP) as substrate. Combining comprehensive FCS controls, ABEL trap, surface-based single-molecule fluorescence, and Monte Carlo simulations, we establish thatpNPP-induced dye blinking at the ∼10-ms timescale is responsible for the apparent diffusion enhancement seen in FCS. Our observations urge a crucial revisit of various experimental findings and theoretical models––including those of our own––in the field, and indicate that in-solution single-particle tracking and ABEL trap are more reliable means to investigate diffusion phenomena at the nanoscale.

scholarly journals Real-Time 3D Single Particle Tracking: Towards Active Feedback Single Molecule Spectroscopy in Live Cells

Molecules ◽  
2019 ◽  
Vol 24 (15) ◽  
pp. 2826 ◽  
Author(s):  
Shangguo Hou ◽  
Courtney Johnson ◽  
Kevin Welsher
Keyword(s):  
Fluorescence Spectroscopy ◽  
Real Time ◽  
Temporal Resolution ◽  
Single Molecule ◽  
Particle Tracking ◽  
Single Particle ◽  
Single Particle Tracking ◽  
Single Molecule Fluorescence ◽  
Single Molecule Fluorescence Spectroscopy ◽  
Active Feedback

Single molecule fluorescence spectroscopy has been largely implemented using methods which require tethering of molecules to a substrate in order to make high temporal resolution measurements. However, the act of tethering a molecule requires that the molecule be removed from its environment. This is especially perturbative when measuring biomolecules such as enzymes, which may rely on the non-equilibrium and crowded cellular environment for normal function. A method which may be able to un-tether single molecule fluorescence spectroscopy is real-time 3D single particle tracking (RT-3D-SPT). RT-3D-SPT uses active feedback to effectively lock-on to freely diffusing particles so they can be measured continuously with up to photon-limited temporal resolution over large axial ranges. This review gives an overview of the various active feedback 3D single particle tracking methods, highlighting specialized detection and excitation schemes which enable high-speed real-time tracking. Furthermore, the combination of these active feedback methods with simultaneous live-cell imaging is discussed. Finally, the successes in real-time 3D single molecule tracking (RT-3D-SMT) thus far and the roadmap going forward for this promising family of techniques are discussed.

scholarly journals A global sampler of single particle tracking solutions for single molecule microscopy

PLoS ONE ◽  
2019 ◽  
Vol 14 (10) ◽  
pp. e0221865
Author(s):  
Michael Hirsch ◽  
Richard Wareham ◽  
Ji W. Yoon ◽  
Daniel J. Rolfe ◽  
Laura C. Zanetti-Domingues ◽  
...  
Keyword(s):  
Single Molecule ◽  
Particle Tracking ◽  
Single Particle ◽  
Single Particle Tracking ◽  
Single Molecule Microscopy

scholarly journals The cell wall regulates dynamics and size of plasma-membrane nanodomains inArabidopsis

2019 ◽  
Vol 116 (26) ◽  
pp. 12857-12862 ◽  
Author(s):  
J. F. McKenna ◽  
D. J. Rolfe ◽  
S. E. D. Webb ◽  
A. F. Tolmie ◽  
S. W. Botchway ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Confocal Microscopy ◽  
Particle Tracking ◽  
Single Particle ◽  
Cluster Size ◽  
Particle Dynamics ◽  
Single Particle Tracking ◽  
Signaling Cascade ◽  
Plant Plasma Membrane

Plant plasma-membrane (PM) proteins are involved in several vital processes, such as detection of pathogens, solute transport, and cellular signaling. For these proteins to function effectively there needs to be structure within the PM allowing, for example, proteins in the same signaling cascade to be spatially organized. Here we demonstrate that several proteins with divergent functions are located in clusters of differing size in the membrane using subdiffraction-limited Airyscan confocal microscopy. Single particle tracking reveals that these proteins move at different rates within the membrane. Actin and microtubule cytoskeletons appear to significantly regulate the mobility of one of these proteins (the pathogen receptor FLS2) and we further demonstrate that the cell wall is critical for the regulation of cluster size by quantifying single particle dynamics of proteins with key roles in morphogenesis (PIN3) and pathogen perception (FLS2). We propose a model in which the cell wall and cytoskeleton are pivotal for regulation of protein cluster size and dynamics, thereby contributing to the formation and functionality of membrane nanodomains.

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