Endocytosis tracking

Single-particle tracking of endocytosis and exocytosis of single-walled carbon nanotubes in NIH-3T3 cells. Nano Letters, 8 (6), 1577-1585. 

Microtubules are used to cover relatively long distances in the cell. Therefore, the short distance between the cell surface and the early endosomes does not require microtubules (94,136). They are involved in the later steps in endocytosis early endosomes accumulate endocytic material for about 10 minutes and then generate transport vesicles (0.5 pm in diameter), which will be taken to the cell center with a relatively high traveling speed of Ipm/sec (with velocities of up to 2.5pm/sec) (95,137). These transport vesicle move on microtubule-tracks to the cell center (and to the ly sosome) (94). When performing live cell imaging studies, it should be kept in mind that microtubules are extremely sensitive to ultraviolet light, which causes their polymerization. 

As gene carriers are internalized by endocytosis, the motion characteristics inside the cell resembles the movement of the endosomal compartments within the cell and the formed vesicles are transported along the microtubule network [38]. Suh et al. [41] quantified the transport of individual internalized polyplexes by multiple-particle tracking and showed that the intracellular transport characteristics of polyplexes depend on spatial location and time posttransfection. Within 30 min, polyplexes accumulated around the nucleus. An average of the transport modes over a 22.5 h period after transfection showed that the largest fraction of polyplexes with active transport was found in the peripheral region of the cells whereas polyplexes close to the nucleus were largely diffusive and subdiffusive. Disruption of the microtubule network by nocodazole completely inhibits active transport and also the perinuclear accumulation of polyplexes [37, 40, 47]. 

This is in contrast to viruses, where the virus particles also show active transport when present in the cytosol after fusion with the plasma membrane or endosomal membrane [60-62], This is due to the ability of specific proteins of the virus particle to bind motor proteins. Single-particle tracking reveals that the quantitative intracellular transport properties of internalized non-viral gene vectors (e.g., polyplexes) are similar to that of viral vectors (e.g., adenovirus) [63]. Suk et al. showed that over 80% of polyplexes and adenoviruses in neurons are subdiffusive and 11-13% are actively transported. However, their trafficking pathways are substantially different. Polyplexes colocalized with endosomal compartments whereas adenovirus particles quickly escaped endosomes after endocytosis. Nevertheless, both exploit the intracellular transport machinery to be actively transported. 

Fig. 8.9 A multifunctional nanoparticle the cRGD sequence binds to a cell surface, the nanoparticle is absorbed by endocytosis and can be tracked by a fluorescent dye before the particle disintegrates to deliver short interfering RNA (siRNA) sequences [23]...

Fluorescent styryl dyes such as FMl-43 have been used to approximate neurotransmitter release by measuring rates of ex-ocytosis (16, 72, 73). These dyes reversibly label endosomal membranes and can be taken up into intracellular synaptic vesicles during endocytosis in systems in which vesicle recycling takes place. Typically, tissue is incubated in the fluorescent dye and then stimulated to promote vesicle cycling and therefore uptake of the dye. The preparation then is washed in fresh buffer to remove dye that remained extracellular. Using fluorescent microscopy, vesicle dynamics can be tracked. Neurotransmitter release is estimated from the rate of destaining (because of exocytosis) usually during stimulation. 

Conversely, several reports have stated evidence of endocytosis or some liposomal-mediated cellular uptake. k49,60 study suggests that uptake of DNArSWCNTs by cells was not only through an endocytotic pathway, but was able to specify that the uptake is dependent on the clatherin-dependent endocytosis pathway. In another study, an endocytotic uptake mechanism for DNArSWCNT conjugates was confirmed by single particle tracking (SPT) within NIH-3T3 cells. ... 

---