Dsred2

Transport was assessed on DIV 12 or 13 by adding 6-OHDA to both or either axonal/somal compartment. To label mitochondria, a plasmid containing mitochondrially-targeted DsRed2 was generated by inserting a mitochondrial targeting sequence (MLSLRQSIRFFK, the signal peptide of COX IV) in front of DsRed2 (Clontech, Mountain View, CA). The mitoDsRed2 was then subcloned into a FUGW lentiviral expression vector provided by Dr. Jeffrey Milbrandt (Washington University in St. Louis). The lentivirus was generated in HEK293T cells using procedures previously described [13 (link)]. Cells were transduced with the virus on DIV 2 for 5–6 hours. By limiting viral transduction to obtain 60-70% labeling efficiency, many more singly labeled axons per microchannel were observed. A lentivirus for labeling synaptic vesicles was generated using a plasmid containing synaptophysin fused in frame with cerulean (provided by Dr. Rachel Wong, University of Washington Seattle).

The expression constructs encoding N-terminal GFP-tagged human Trak1 WT (residues 1–953) and Trak1 hyrt (residues 1–824) were generated as previously described (Webber et al., 2008 (link)). The rescue expression constructs encoding shRNA-resistant GFP-tagged Trak1 WT and Trak1 hyrt were generated by site-directed mutagenesis to make two or three silent third-codon substitutions within the shRNA-targeted region of the Trak1 transcript without altering the Trak1 amino acid sequence. The full-length Miro1 and Miro2 expression constructs were provided by Dr. Pontus Aspenstrom (Ludwig Institute for Cancer Research, Uppsala University, Sweden), and full-length Mfn1 and Mfn2 constructs by Dr. David Chan (California Institute of Technology). The DsRed2-Mito plasmid for expressing mitochondrial matrix-targeted red fluorescent protein DsRed2 was obtained from (Clontech), and the mito-Dendra2 construct was a gift from Dr. Michael T. Ryan (La Trobe University, Australia). The shRNA constructs targeting human Trak1 (NM_014965.2-876s1c1 and NM_014965.2-1392s1c1) and a non-targeting shRNA control construct (SHC001) were from Sigma-Aldrich.

For lentiviral experiments, vector eGFP-pWPT (Addgene no. 12255, kind gift from D. Trono) was used to express eGFP, and cDNA encoding human TTL (NP_714923, Origene no. RC207805L2) was cloned in it for TTL expression. PCR amplification and cloning of TTL cDNA were performed with Phusion DNA polymerase (Thermo Scientific) and In-Fusion HD Cloning kit (Clontech), respectively. eGFP cDNA was removed during the cloning process to produce an untagged TTL. For lentiviral shRNA expression, two TTL shRNA sequences, cloned in pLKO.1 vector, were purchased from Sigma-Aldrich: shTTL1 (TRCN0000191515, sequence: 5′-CCG GCA TTC AGA AA GAG TAC TCA ACT CGA GTT GAC TAC TCT TTC TGA ATG CTT TTT TG-3′) and shTTL2 (TRCN0000191227, sequence: 5′-CCG GCT CAA AGA ACT ATG GGA AAT ACT CGA GTA TTT CCC ATA GTT CTT TGA GTT TTT TG-3′).³⁵ The SHC001 pLKO.1-puro empty Vector (Sigma) was used as control (shControl). For the transfection experiments, the plasmid encoding pCMV-EB3-EGFP was a kind gift from Dr Frank Polleux.^{72 (link)} Kind gifts from Dr Erik Dent include the plasmids EB3-tdTomato (Addgene no. 50708) and the plasmid encoding DsRed2 (Clontech), cloned into a pCAX vector. The plasmid pEGFP-N1 with a CMV promoter was also used (Addgene no. 6085-1). All constructs were verified by sequencing (Eurofins and Genewiz). Plasmids were purified with HiPure Plasmid Maxiprep kits (Invitrogen).