Journal: eLife
Article Title: Rab10 regulates the sorting of internalised TrkB for retrograde axonal transport
doi: 10.7554/eLife.81532
Figure Lengend Snippet: ( a ) Schematic of two-compartment microfluidic chambers highlighting compartmentalisation of somata (left) and axon terminals (right). Micro-flow from somatic to axonal compartments provides fluidic isolation of the somatic compartment to probes added to the other chamber. ( b ) Representative images from the cell bodies of neurons incubated for 2.5 hr with anti-TrkB in the axonal compartment, with (+) or without (-) brain-derived neurotrophic factor (BDNF). Pink arrowheads indicate examples of retrogradely transported TrkB-positive organelles. Scale bar: 10 µm. ( c ) Neurons treated with the shRNA targeting Rab10 were compared to control transduced neurons. Immunofluorescence revealed similar neuronal density (see ßIII-tubulin in orange and nuclear staining in green, top panel), but a decrease in both, expression of Rab10 (grey, middle panel) and retrograde accumulation of TrkB after 2.5 hr (colour intensity scale, bottom panel). Scale bar: 50 µm. ( d ) Quantification of retrograde TrkB accumulation in three independent experiments show statistically significant differences (unpaired Student’s t -test, t(140), p<0.0001). ( e ) Correlation between expression level of Rab10 and retrograde TrkB accumulation in control and Rab10-knockdown neurons show a significant linear correlation (goodness-of-fit R 2 =0.61; Pearson r, XY pairs = 131, p<0.0001). ( f ) Axonal stimulation with BDNF for 2.5 hr leads to robust appearance of phosphorylated CREB in the nucleus of control neurons (left panel). This response was impaired in neurons depleted of Rab10 (middle panel), and rescued by the co-expression of a shRNA-resistant mutant Rab10 (right panel). Immunofluorescence for Rab10 is shown in grey, with the nuclei indicated with a pink mask, and nuclear phosphorylated CREB is shown in a colour intensity scale. Scale bar: 50 µm. ( g ) Quantification from three independent experiments showing the statistically significant effect of manipulating Rab10 expression on the levels of phosphorylated CREB in the nucleus (one-way ANOVA, F(2,280), p<0.0001; p values for the Bonferroni multiple comparison tests, t(280), are indicated in the plot). Source data of the plots have been included in . Figure 2—source data 1. Data tables for each plot presented in are given as individual CSV files.
Article Snippet: Antibody , Chicken polyclonal anti-tubulin ßIII , Synaptic Systems , Cat#302 306; RRID: AB_2620048 , 1:300.
Techniques: Isolation, Incubation, Derivative Assay, shRNA, Control, Immunofluorescence, Staining, Expressing, Knockdown, Mutagenesis, Comparison
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