Trak1 and Mitochondrial Dynamics Regulation

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.