scholarly journals High-throughput, single-particle tracking reveals nested membrane domains that dictate KRasG12D diffusion and trafficking

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Yerim Lee ◽  
Carey Phelps ◽  
Tao Huang ◽  
Barmak Mostofian ◽  
Lei Wu ◽  
...  
Keyword(s):  
Particle Tracking ◽  
Single Particle ◽  
Single Particle Tracking ◽  
Ras Signaling ◽  
Fast Diffusion ◽  
Membrane Domains ◽  
Membrane Diffusion ◽  
Spatial Regulation ◽  
Insight Into ◽  
Distinct Membrane

Membrane nanodomains have been implicated in Ras signaling, but what these domains are and how they interact with Ras remain obscure. Here, using single particle tracking with photoactivated localization microscopy (spt-PALM) and detailed trajectory analysis, we show that distinct membrane domains dictate KRasG12D (an active KRas mutant) diffusion and trafficking in U2OS cells. KRasG12D exhibits an immobile state in ~70 nm domains, each embedded in a larger domain (~200 nm) that confers intermediate mobility, while the rest of the membrane supports fast diffusion. Moreover, KRasG12D is continuously removed from the membrane via the immobile state and replenished to the fast state, reminiscent of Ras internalization and recycling. Importantly, both the diffusion and trafficking properties of KRasG12D remain invariant over a broad range of protein expression levels. Our results reveal how membrane organization dictates membrane diffusion and trafficking of Ras and offer new insight into the spatial regulation of Ras signaling.

scholarly journals High-throughput single-particle tracking reveals nested membrane nanodomain organization that dictates Ras diffusion and trafficking

2019 ◽  
Author(s):  
Yerim Lee ◽  
Carey Phelps ◽  
Tao Huang ◽  
Barmak Mostofian ◽  
Lei Wu ◽  
...  
Keyword(s):  
High Throughput ◽  
Particle Tracking ◽  
Single Particle ◽  
Single Particle Tracking ◽  
Ras Signaling ◽  
Fast Diffusion ◽  
Membrane Domains ◽  
Spatial Mechanisms ◽  
Insight Into ◽  
Distinct Membrane

AbstractMembrane nanodomains have been implicated in Ras signaling, but what these domains are and how they interact with Ras remain obscure. Using high throughput single particle tracking with photoactivated localization microscopy and detailed trajectory analysis, here we show that distinct membrane domains dictate KRas diffusion and trafficking in U2OS cells. KRas exhibits an immobile state in domains ∼70 nm in size, each embedded in a larger domain (∼200 nm) that confers intermediate mobility, while the rest of the membrane supports fast diffusion. Moreover, KRas is continuously removed from the membrane via the immobile state and replenished to the fast state, likely coupled to internalization and recycling. Importantly, both the diffusion and trafficking properties of KRas remain invariant over a broad range of protein expression levels. Our results reveal how membrane organization dictates KRas diffusion and trafficking and offer insight into how Ras signaling may be regulated through spatial mechanisms.

scholarly journals Organization of the axon initial segment: Actin like a fence

2016 ◽  
Vol 215 (1) ◽  
pp. 9-11 ◽  
Author(s):  
Yu-Mei Huang ◽  
Matthew N. Rasband
Keyword(s):  
Particle Tracking ◽  
Single Particle ◽  
High Speed ◽  
Initial Segment ◽  
Single Particle Tracking ◽  
Axon Initial Segment ◽  
Membrane Domains ◽  
Superresolution Microscopy ◽  
Cell Biol ◽  
Actin Rings

What prevents the movement of membrane molecules between axonal and somatodendritic domains is unclear. In this issue, Albrecht et. al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201603108) demonstrate via high-speed single-particle tracking and superresolution microscopy that lipid-anchored molecules in the axon initial segment are confined to membrane domains separated by periodically spaced actin rings.

scholarly journals Rapid diffusion of large TatA complexes detected using single particle tracking microscopy

2020 ◽  
Author(s):  
Aravindan Varadarajan ◽  
Felix Oswald ◽  
Holger Lill ◽  
Erwin J.G. Peterman ◽  
Yves J. M. Bollen
Keyword(s):  
Particle Tracking ◽  
Single Particle ◽  
Transmembrane Protein ◽  
Single Particle Tracking ◽  
Fast Diffusion ◽  
Translocation Event ◽  
Twin Arginine Translocation ◽  
Rhomboid Proteases ◽  
Folded Proteins ◽  
Membrane Spanning

AbstractThe twin-arginine translocation (Tat) system transports folded proteins across the cytoplasmic membrane of most bacteria and archaea. TatA, which contains a single membrane-spanning helix, is believed to be responsible for the actual translocation. According to the prevalent model, multiple TatA subunits form a transient protein-conducting pore, which disassembles after each translocation event. An alternative model exists, in which TatA proteins locally weaken the lipid bilayer to translocate folded proteins. Here, we imaged eGFP-fused TatA expressed from the genome in live E. coli cells. Images showed TatA occuring both in highly mobile monomers or small oligomers and in large, stable complexes that do not dissociate. Single-particle tracking revealed that large TatA complexes switch between fast and slow diffusion. The fast diffusion is too fast for a transmembrane protein complex consisting of multiple TatA monomers. In line with recent data on rhomboid proteases, we propose that TatA complexes switch between a slowly diffusing transmembrane conformation and a rapidly diffusing membrane-disrupting state that enables folded proteins to cross the membrane, in accordance with the membrane-weakening model.

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.

Nanoparticle diffusion during gelation of tetra poly(ethylene glycol) provides insight into nanoscale structural evolution

Soft Matter ◽  
2020 ◽  
Vol 16 (9) ◽  
pp. 2256-2265 ◽  
Author(s):  
Emmabeth Parrish ◽  
Katie A. Rose ◽  
Matteo Cargnello ◽  
Christopher B. Murray ◽  
Daeyeon Lee ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Particle Tracking ◽  
Single Particle ◽  
Structural Evolution ◽  
Single Particle Tracking ◽  
Poly Ethylene Glycol ◽  
Nanoparticle Diffusion ◽  
Poly Ethylene ◽  
Grafted Nanoparticles ◽  
Insight Into

Single particle tracking (SPT) of PEG grafted nanoparticles (NPs) was used to examine the gelation of tetra poly(ethylene glycol) (TPEG) succinimidyl glutarate (TPEG-SG) and amine (TPEG-A) terminated 4-armed stars.

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