PES 2013 Loader With Hamachi.33
the findings above indicate that ampars are recycled back to the plasma membrane in neurons. to further validate this hypothesis, we determined the recycling kinetics of ampars in neurons using a biochemical approach, in which cell-surface ampars were first labeled using a flow cytometer, and subsequently analyzed by flow cytometry. as shown in supplementary figure 7, the labeled cell-surface fraction on the cell surface decreased in parallel with an increase in incubation time. these results clearly demonstrate that cell-surface ampars are recycled in neurons.
finally, we also analyzed the differential distribution of ampars in dendrites and spines. to verify this hypothesis, primary hippocampal neurons prepared from wild-type mice were incubated with cam2(tco) and subsequently labeled with different fluorophores. we first obtained a population of dendritic spines by sparse cell-labeling with 2μm cellmask orange, and subsequently performed live imaging and biochemical analyses on cam2(tco)-labeled spines and dendritic shafts using confocal live imaging and a biochemical approach. as shown in fig. 4a -b, labeled spines were clearly observed with high resolution after 2h of incubation at 37c in culture medium. fluorescence signal intensities of spine head regions were stronger than those of spine neck regions (fig. 4a ). in addition, ampars in spine heads were much more internalized than those in spine necks (fig. 4b ). moreover, we investigated the recovery of cell-surface ampars in both dendritic spines and shafts. in dendrites, cell-surface ampars were almost fully recovered within a 2h incubation (fig. 4c ), while recovery in spines was quite slow. this suggests that ampars are recycled back to the plasma membrane only in spines. collectively, our findings demonstrated that a labeling method based on affinity-based biotinylation and bioorthogonal click chemistry can selectively and rapidly label cell-surface iglurs in live cells under physiological temperatures in culture medium. our method is expected to be used as a powerful tool to characterize the recycling kinetics of iglurs in vivo.
to examine the effects of ampars, hek293t cells were transfected with glua2flip(q) (ampar(+)), and control glua2(q) (ampar()) were treated with 2m cam2(tco) for 4 h followed by the addition of 1m tz(fl) for 5 min, or treated with 2m cam2(fl) for 4 h in the presence or absence of 50m nbqx in culture medium at 37c. cells were fixed with 4% pfa and immunostained with anti-psd95 or anti-map2 antibody. the cells were observed using confocal microscopy. scale bars, 10m. to examine the effects of ampars, hek293t cells were transfected with glua2flip(q) (ampar(+)), and control glua2(q) (ampar()) were treated with 2m cam2(tco) for 4 h followed by the addition of 1m tz(fl) for 5 min, or treated with 2m cam2(fl) for 4 h in the presence or absence of 50m nbqx in culture medium at 37c. a western blotting analyses of hek293t cells after the two-step labeling. hek293t cells transfected with glua2flfl(q), which carries ampar(+), or glua2(q) were treated with 2m cam2(fl) for 4 h followed by the addition of 1m tz(fl) for 5min, or treated with 2m cam2(tco) for 4 h in the presence or absence of 50m nbqx at 37c. next, we examined whether two-step chemical labeling could be applied to living animals. to address this question, we conducted an in vivo chemical labeling experiment using mice. as shown in fig. 7a, neurons in the primary somatosensory cortex were clearly labeled with cam2(tco) at 0.1m, but not with cam2(fl). then, 1m tz(fl) was added to the labeled brain slices; subsequent live imaging revealed the internalization of ampars within 5min (fig. 7b, c). the chemical labeling of ampars was blocked by the addition of 0.1mm nbqx (supplementary fig. 24). the chemical labeling of nmdars was also conducted, and the results were similar to those for ampars. in addition, cam2(tco) labeled the surface of synapses as visualized by two-photon imaging of fluorescently labeled ampa receptors (fig. 7d, e). 5ec8ef588b