A HaloTag® method for assessing the retrograde axonal transport of the p75 neurotrophin receptor and other proteins in compartmented cultures of rat sympathetic neurons.
We have adapted HaloTag® (HT) technology for use in compartmented cultures of rat sympathetic neurons in order to provide a technique that can be broadly applied to studies of the retrograde transport of molecules that play roles in neurotrophin signaling. Transfected neurons expressing HT protein alone, HT protein fused to the p75 neurotrophin receptor (p75NTR) or HT protein fused to tubulin α-1B were maintained in compartmented cultures in which cell bodies and proximal axons of rat sympathetic neurons reside in proximal compartments and their distal axons extend into distal compartments. HT ligand containing a fluorescent tetramethylrhodamine (TMR) label was applied either in the distal compartments or the proximal compartments, and the transport of labeled proteins was assayed by gel fluorescence imaging and TMR immunoblot. HT protein expressed alone displayed little or no retrograde transport. HT protein fused to either the intracellular C-terminus or the extracellular N-terminus of p75NTR was retrogradely transported. The retrograde transport of p75NTR was augmented when the distal axons were provided with nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) or antibodies to BDNF. The anterograde transport of HT protein fused to the N-terminus of tubulin α-1B was also demonstrated. We conclude that retrograde transport of HT fusion proteins provides a powerful and novel approach in studies of axonal transport.
Department of Cell Biology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Sue-Ann
SA
Mok
Karen
K
Lund
Paul
P
Lapointe
Robert B
RB
Campenot
D000595:Amino Acid Sequence; D000818:Animals; D001370:Axonal Transport / Q000502:physiology; D001483:Base Sequence; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods; D002478:Cells, Cultured / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D018274:Electroporation; D005456:Fluorescent Dyes / Q000032:analysis; D005470:Fluorometry; D006867:Hydrolases / Q000032:analysis* / Q000235:genetics; D015151:Immunoblotting; D008024:Ligands; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000378:metabolism*; D009474:Neurons / Q000502:physiology* / Q000648:ultrastructure; D051381:Rats; D017207:Rats, Sprague-Dawley; D017978:Receptors, Growth Factor; D017475:Receptors, Nerve Growth Factor / Q000032:analysis / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D012235:Rhodamines / Q000032:analysis; D017783:Superior Cervical Ganglion / Q000166:cytology; D014162:Transfection; D014404:Tubulin / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
D005456:Fluorescent Dyes; D008024:Ligands; D009419:Nerve Tissue Proteins; D017978:Receptors, Growth Factor; D017475:Receptors, Nerve Growth Factor; D011993:Recombinant Fusion Proteins; D012235:Rhodamines; D014404:Tubulin; C582848:tubulin alpha-1B, rat; C068313:Ngfr protein, rat; C005358:tetramethylrhodamine; D006867:Hydrolases; C046137:haloalkane dehalogenase