The Journal of Neuroscience, November 23, 2005, ():
The Small GTPase Rab7 Controls the Endosomal Trafficking and Neuritogenic Signaling of the Nerve Growth Factor Receptor TrkA
J. Neurosci. Saxena et al.
25: 10930
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplementary Figure 1
Expression of DN-Rab7/GFP in PC12 cells does not affect the recycling pathway nor the secretory pathway
(A) Unaltered subcellular localization of Rab5 and Rab11 in cells expressing DN-Rab7/GFP. PC12 cells were transiently transfected with DN-Rab7/GFP and then processed for indirect immunofluorescence with antibodies against Rab5 (upper panel) or Rab11 (lower panel), followed by confocal microscopy. Unaltered subcellular localization of these two key Rab family members in DN-Rab7/GFP expressing cells (green) compared to neighbouring control cells. Scale bar = 10 µm
(B) Unaltered trafficking of internalized Transferrin (Tf) in cells expressing DN-Rab7/GFP. PC12 cells were transiently transfected with DN-Rab7/GFP. Subsequently, cells were washed with DMEM/BSA and red-fluorescently labelled Tf (AlexaFluor546) was added for 5 min. Subsequently, the DMEM/BSA containing Tf was removed and substituted by prewarmed DMEM/BSA for 10 min to allow the internalized Tf to proceed through the recycling pathway. Then, cells were cooled on ice, surface stripped with high salt/low pH buffer, washed and fixed, followed by confocal microscopy. Unaltered subcellular localization of Tf (red) in DN-Rab7/GFP expressing cells (green) compared to neighbouring control cells. Scale Bar=10 µm
(C) Unaltered regulated and constitutive secretion of BDNF in cells expressing DN-Rab7/GFP. PC12 cells were transiently co-transfected with plasmids coding for DN-Rab7/GFP+BDNF or with GFP+BDNF. The next day, cells were washed 3 times with “physiological buffer” containing 125 mM NaCl and 5 mM KCl (Kruttgen et al., 1998), followed by either incubation in “physiological buffer” to measure constitutive secretion or by incubation in “high potassium buffer” containing 75 mM NaCl and 55 mM KCl to trigger regulated secretion from neuronal cells. After 30 min incubation at 37°C, supernatants were taken, cleared of debris by centrifugation and subjected in triplicates to a BDNF specific ELISA (Promega). We found unaltered regulated secretion (released BDNF concentration by “high potassium buffer” – constitutively released BDNF concentration by “physiological buffer”) of BDNF from cells expressing DN-Rab7/GFP.
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Supplementary Figure 2
Association of phosphorylated TrkA with Rab7.
PC12 cells were transfected with WT-Rab7/GFP and stimulated with NGF for different timepoints. Equal amounts of lysates were immunoprecipitated with antibodies against GFP to pull down Rab7/GFP, followed by immunoblotting with antibodies directed against TrkA phosphorylated at Y490. A representative experiment is shown.
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Supplementary Figure 3
Schematic on the regulation of TrkA signalling endosome motility
Previous work showed that retrograde transport of TrkA signalling endosomes depends on dynein (Heerssen et al., 2004) and that the juxtamembrane domain of TrkA binds directly to dynein light chain (Yano et al., 2001), thereby linking the signalling endosome to the classical minus-end transport apparatus (Vale, 2003). We now show that Rab7, a small GTPase attached to the cytoplasmic surface of endosomes via lipid anchors, is found in a complex with TrkA and regulates the trafficking and signalling of this RTK. It remains to be clarified how Rab7 controls the motility of TrkA endosomes, and, on the other hand, how Rab7 activity is being regulated. These studies appear relevant to our understanding of neurodegenerative conditions recently associated with mutations in Rab7 (Verhoeven et al., 2003; Houlden et al., 2004).
