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 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 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.

scholarly journals High-Density Single Particle Tracking on the Plasma Membrane using High-Speed Hyperspectral Imaging

2012 ◽  
Vol 102 (3) ◽  
pp. 581a
Author(s):  
Patrick J. Cutler ◽  
Michael D. Malik ◽  
Sheng Liu ◽  
Jason M. Byars ◽  
Diane S. Lidke ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Hyperspectral Imaging ◽  
Particle Tracking ◽  
Single Particle ◽  
High Speed ◽  
Single Particle Tracking ◽  
High Density

scholarly journals Ca2+-Dependent, Stimulus-Specific Modulation of the Plasma Membrane Ca2+ Pump in Hippocampal Neurons

2009 ◽  
Vol 101 (5) ◽  
pp. 2563-2571 ◽  
Author(s):  
Michael J. Ferragamo ◽  
Jessica L. Reinardy ◽  
Stanley A. Thayer
Keyword(s):  
Plasma Membrane ◽  
Neuronal Activity ◽  
Action Potentials ◽  
Hippocampal Neurons ◽  
Cyclopiazonic Acid ◽  
Synaptic Activity ◽  
Voltage Gated ◽  
Clearance Kinetics ◽  
Transient Elevation ◽  
Kinetics Of

The plasma membrane Ca2+ ATPase (PMCA) plays a major role in restoring Ca2+ to basal levels following transient elevation by neuronal activity. Here we examined the effects of various stimuli that increase [Ca2+]i on PMCA-mediated Ca2+ clearance from hippocampal neurons. We used indo-1-based microfluorimetry in the presence of cyclopiazonic acid to study the rate of PMCA-mediated recovery of Ca2+ elevated by a brief train of action potentials. [Ca2+]i recovery was described by an exponential decay and the time constant provided an index of PMCA-mediated Ca2+ clearance. PMCA function was assessed before and for ≥60 min following a 10-min priming stimulus of either 100 μM N-methyl-d-aspartate (NMDA), 0.1 mM Mg2+ (reduced extracellular Mg2+ induces intense excitatory synaptic activity), 30 mM K+, or control buffer. Recovery kinetics slowed progressively following priming with NMDA or 0.1 mM Mg2+; in contrast, Ca2+ clearance initially accelerated and then slowly returned to initial rates following priming with 30 mM K+-induced depolarization. Treatment with 10 μM calpeptin, an inhibitor of the Ca2+ activated protease calpain, prevented the slowing of kinetics observed following treatment with NMDA but had no affect on the recovery kinetics of control cells. Calpeptin also blocked the rapid acceleration of Ca2+ clearance following depolarization. In calpeptin-treated cells, 0.1 mM Mg2+ induced a graded acceleration of Ca2+ clearance. Thus in spite of producing comparable increases in [Ca2+]i, activation of NMDA receptors, depolarization-induced activation of voltage-gated Ca2+ channels and excitatory synaptic activity each uniquely affected Ca2+ clearance kinetics mediated by the PMCA.

Single particle tracking of cell-surface HLA-DR molecules using R-phycoerythrin labeled monoclonal antibodies and fluorescence digital imaging

1996 ◽  
Vol 109 (8) ◽  
pp. 2101-2109
Author(s):  
K.M. Wilson ◽  
I.E. Morrison ◽  
P.R. Smith ◽  
N. Fernandez ◽  
R.J. Cherry
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Surface ◽  
Particle Tracking ◽  
Single Particle ◽  
Single Particle Tracking ◽  
Lower Intensity ◽  
Directed Motion ◽  
Hla Dr ◽  
Particle Tracks ◽  
Random Diffusion

The mobility of cell surface MHC molecules and their ability to form dynamic associations may be related to the physiological status of the cell and to the potential to bind effector T lymphocytes. To investigate these properties, we have prepared HLA DR specific monoclonal antibodies coupled in a 1:1 mole ratio to the fluorescent phycobiliprotein, R-phycoerythrin (PE). We show that these small particles can be sequentially imaged using a cooled slow-scan charge coupled device camera and hence can be used for single particle tracking experiments. We have applied this technique to investigate the movements of HLA DR molecules on fibroblasts transfected with human DR alpha and DR beta genes. PE-IgG was bound to the transfected fibroblasts and particle tracks were obtained by sequential imaging over a period of typically 30 minutes. Analysis of particle tracks revealed the presence of directed motion and domain-limited diffusion in addition to random diffusion. The contributions of these three types of motion showed cell to cell variability. Velocities of directed motion were of the order of 2 nm second-1 whilst domain diameters were in the range 200–800 nm. Diffusion coefficients for random diffusion were in the range 1 × 10(−13)-5 × 10(−12) cm2 second-1. The higher mobilities were observed for the lower intensity fluorescent spots, which possibly correspond to images of single particles. Much lower mobility was observed with a cell where the spot intensities were approximately double that of the lower intensity spots. These spots could be images of double particles implying the association of at least two HLA DR alpha beta dimers. These data are relevant to the study of MHC class II cell surface redistribution and antigen presentation in specific immunity.

scholarly journals Single-Particle Tracking Palm of Nav1.6 in Hippocampal Neurons Demonstrates Unique Subcellular Diffusion Landscapes

2014 ◽  
Vol 106 (2) ◽  
pp. 36a ◽  
Author(s):  
Elizabeth J. Akin ◽  
Kristen Brown ◽  
Sanaz Sadegh ◽  
Aubrey V. Weigel ◽  
Jean-Baptiste Masson ◽  
...  
Keyword(s):  
Particle Tracking ◽  
Single Particle ◽  
Hippocampal Neurons ◽  
Single Particle Tracking

scholarly journals Single Particle Tracking of α7 Nicotinic AChR in Hippocampal Neurons Reveals Regulated Confinement at Glutamatergic and GABAergic Perisynaptic Sites

PLoS ONE ◽  
2010 ◽  
Vol 5 (7) ◽  
pp. e11507 ◽  
Author(s):  
Thomas Bürli ◽  
Kristin Baer ◽  
Helge Ewers ◽  
Corinne Sidler ◽  
Christian Fuhrer ◽  
...  
Keyword(s):  
Particle Tracking ◽  
Single Particle ◽  
Hippocampal Neurons ◽  
Single Particle Tracking
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