SINGLE-PARTICLE TRACKING:Applications to Membrane Dynamics

Perillo

et al. 2016

Biophysical Journal

Whereas important discoveries made by single-particle tracking have changed our view of the plasma membrane organization and motor protein dynamics in the past three decades, experimental studies of intracellular processes using singleparticle tracking are rather scarce because of the lack of three-dimensional (3D) tracking capacity. In this study we use a newly developed 3D single-particle tracking method termed TSUNAMI (Tracking of Single particles Using Nonlinear And Multiplexed Illumination) to investigate epidermal growth factor receptor (EGFR) trafficking dynamics in live cells at 16/43 nm (xy/z) spatial resolution, with track duration ranging from 2 to 10 min and vertical tracking depth up to tens of microns. To analyze the long 3D trajectories generated by the TSUNAMI microscope, we developed a trajectory analysis algorithm, which reaches 81% segment classification accuracy in control experiments (termed simulated movement experiments). When analyzing 95 EGF-stimulated EGFR trajectories acquired in live skin cancer cells, we find that these trajectories can be separated into three groups-immobilization (24.2%), membrane diffusion only (51.6%), and transport from membrane to cytoplasm (24.2%). When EGFRs are membrane-bound, they show an interchange of Brownian diffusion and confined diffusion. When EGFRs are internalized, transitions from confined diffusion to directed diffusion and from directed diffusion back to confined diffusion are clearly seen. This observation agrees well with the model of clathrin-mediated endocytosis.

Section: Revealing Membrane Dynamics Of Egfrsmentioning

confidence: 90%

Section: Introductionmentioning

confidence: 99%

Segmentation of 3D Trajectories Acquired by TSUNAMI Microscope: An Application to EGFR Trafficking

Perillo

et al. 2016

Biophysical Journal

Journal of Controlled Release

“…From the trajectories, the diffusion coefficient and mode of motion (free diffusion, anomalous diffusion or directed motion) can be derived on a particle by particle basis. [129] SPT was applied to study the diffusion of polystyrene nanoparticles with different surface modifications in living, hydrated Burkholderia multivorans and P. aeruginosa biofilms. [130] These two pathogens are able to cause chronic pulmonary infections in CF patients.…”

Section: Studying the Interaction And Transport Of Nanoparticles In Bmentioning

confidence: 99%

Lipid and polymer nanoparticles for drug delivery to bacterial biofilms

Forier

Raemdonck

Smedt

et al. 2014

370

320

Biofilms are matrix-enclosed communities of bacteria that show increased antibiotic resistance and the capability to evade the immune system. They can cause recalcitrant infections which cannot be cured with classical antibiotic therapy. Drug delivery by lipid or polymer nanoparticles is considered a promising strategy for overcoming biofilm resistance. These particles are able to improve the delivery of antibiotics to the bacterial cells, thereby increasing the efficacy of the treatment. In this review we give an overview of the types of polymer and lipid nanoparticles that have been developed for this purpose. The antimicrobial activity of nanoparticle encapsulated antibiotics compared to the activity of the free antibiotic is discussed in detail. In addition, targeting and triggered drug release strategies to further improve the antimicrobial activity are reviewed. Finally, ample attention is given to advanced microscopy methods that shed light on the behavior of nanoparticles inside biofilms, allowing further optimization of the nanoformulations. Lipid and polymer nanoparticles were found to increase the antimicrobial efficacy in many cases. Strategies such as the use of fusogenic liposomes, targeting of the nanoparticles and triggered release of the antimicrobial agent ensured the delivery of the antimicrobial agent in close proximity of the bacterial cells, maximizing the exposure of the biofilm to the antimicrobial agent. The majority of the discussed papers still present data on the in vitro anti-biofilm activity of nanoformulations, indicating that there is an urgent need for more in vivo studies in this field.

“…Leaving one frame out will double the lag time, leaving two frames out will triple it. Linearity over such time scales indicates diffusion, and divergence from linearity can be related to corralled movement or active transport (Saxton and Jacobson, 1997).…”

Section: Eq (3)mentioning

confidence: 99%

Chapter 27 Cell Biology of mRNA Decay

Grünwald

Singer

Czaplinski

2008

Methods in Enzymology

Studying single mRNA molecules has added new dimensions to our understanding of gene expression and the life cycle of mRNA in cells. Advances in microscopes and detection technology have opened access to single molecule research to most researchers interested in molecular biology. Here we provide an overview technique for single molecule studies of RNA in either fixed samples or in living cells. As part of a volume on mRNA turnover, it is increasingly relevant, because many of the recent advances in studies of mRNA turnover have suggested that there is non-homogeneous distribution of turnover factors in the cell. For this reason, understanding of spatial relationships between mRNA and mRNA turnover factors should enrich our understanding of this process.