Mito-GFP construct (pABCb10aa1-35-GFP; Addgene) was used as a mitochondrial marker (Graf et al., 2004 (link)). pMito-KR (Evrogen) was used to ablate mitochondria in growth cones and in some cases as a mitochondrial marker. Transfection with pTagBFP (Evrogen) was used to visualize axons and growth cones. The Myc-tagged Miro1 (WT) and Myc-Miro1E208K/E328K (Miro-KK) expression constructs were purchased from Addgene (plasmid 47888 and 47894, respectively). Quikchange site-directed mutagenesis kit (Agilent; 200521) was used to introduce point mutations into the Myc-Miro1 WT plasmid to generate the nonacetylatable (K to A) and acetyl-mimetic (K to Q) constructs. mCherry-tagged α-tubulin expression plasmids (WT, nonacetylatable K40A, acetyl-mimetic K40Q) were previously described (Lee et al., 2015 (link)).
DRG cultures were transfected using the Amaxa Nucleofector with basic neuron SCN Nucleofector Kit (Lonza) and analyzed by live-cell imaging 48–72 h later. SiRNAs for HDAC6 have been described previously (Rivieccio et al., 2009 (link)); these were transfected with DarmaFect3 (Dharmacon), and cells were analyzed in live-cell imaging as above. siRNAs used for Miro1 knockdown were: #1, 5′-CAACAAACAUUCUAUUGAUAAGGTA-3′, and #2, 5′-CCUGCAUGAAGUCAAGCAAGAACAC-3′ (Integrated DNA Technologies).
DRG cultures were transfected using the Amaxa Nucleofector with basic neuron SCN Nucleofector Kit (Lonza) and analyzed by live-cell imaging 48–72 h later. SiRNAs for HDAC6 have been described previously (Rivieccio et al., 2009 (link)); these were transfected with DarmaFect3 (Dharmacon), and cells were analyzed in live-cell imaging as above. siRNAs used for Miro1 knockdown were: #1, 5′-CAACAAACAUUCUAUUGAUAAGGTA-3′, and #2, 5′-CCUGCAUGAAGUCAAGCAAGAACAC-3′ (Integrated DNA Technologies).
