Generation of Lentiviral Plasmids for Cell Manipulation

All-in-one doxycycline-inducible lentiviral transfer plasmids (pLV-Dox mCherry-FP4/AP4-MITO) were generated by PCR and subcloning mCherry-FP4-MITO or mCherry-AP4-MITO (a gift from James Bear, University of North Carolina, Chapel Hill, NC, USA; Bear et al., 2000 (link)) in place of Cas9 in pCW-Cas9 (a gift from Eric Lander and David Sabatini (Massachusetts Institute of Technology, Cambridge, MA, USA; Wang et al., 2014 (link); RRID:Addgene_50661). Briefly, Cas9 was cut out and replaced with a multiple cloning site by annealed oligo cloning, and AgeI-BamHI was used to insert FP4/AP4 sequences. TurboRFP-expressing pLKO transfer plasmid was generated by PCR and subcloning TurboRFP in place of the puromycin resistance gene at BamHI-KpnI in pLKO.1 - TRC cloning vector (a gift from David Root, The Broad Institute, Cambridge, MA, USA; Moffat et al., 2006 (link)]; RRID:Addgene_10878). Validated Mus musculus shRNA sequences were obtained from The RNAi Consortium (The Broad Institute via MilliporeSigma; Moffat et al., 2006 (link)) and oligos were ligated between the AgeI-EcoRI sites (replacing the 1.9 kb stuffer) of our pLKO.1 TurboRFP cloning vector using annealed oligo cloning. shRNA sequences and source MilliporeSigma product number are given in Table S1. Lentiviral LifeAct expression vectors were published previously (Padilla-Rodriguez et al., 2018 (link); Parker et al., 2018 (link)): pLenti LifeAct-EGFP BlastR (RRID:Addgene_84383), pLenti-LifeAct-mRuby2 BlastR (RRID:Addgene_84384), pLenti LifeAct-iRFP670 BlastR (RRID:Addgene_84385). Lentiviral cDNA expression vectors were generated by PCR and subcloning cDNA of interest into the transfer plasmids pLenti CMVie-IRES-BlastR or pLenti CMVie-IRES-BlastR alt MCS (pCIB) published previously (Puleo et al., 2019 (link); RRID:Addgene_119863 and RRID:Addgene_120862). To reduce expression of EVL, the CMVie promoter was replaced with the Ef1a short promoter EFS in some constructs. cDNAs were tagged with mEmerald (a gift from Michael Davidson, Florida State University, Tallahassee, FL, USA; RRID:Addgene_53975), mRuby2 (a gift from Michael Davidson; Lam et al., 2012 (link); RRID:Addgene_54768), or piRFP670 (a gift from Vladislav Verkhusha, Albert Einstein College of Medicine, The Bronx, NY, USA; Shcherbakova and Verkhusha, 2013 (link); RRID:Addgene_45457) on the N-terminus of EVL or Arp3, or C-terminus of MTSS1 or MRLC. To generate lentiviral expression vectors for PSD95-FingR-EGFP and -mRuby2, pCAGGs-PSD95-FingR-EGFP was cut with SpeI-AgeI and ligated into pCIB, and mRuby2 was subcloned into SmaI-AgeI sites. For EVL iLID and MIM iLID optogenetic systems, tgRFPt-SspB(R73Q) was PCRed and subcloned from pLL7.0 mTiam1(64–437)-tgRFPt-SSPB R73Q (a gift from Brian Kuhlman, University of North Carolina, Chapel Hill, NC, USA; Guntas et al., 2015 (link); RRID:Addgene_60418) and added to the N-terminus of EVL or C-terminus of MTSS1. Lentiviral expression vectors for MIM-mRuby2, MIM-iRFP670, and MIM iLID were modified to remove the IRES-blasticidin resistance cassette to reduce plasmid size. Additional mammalian expression vectors for co-immunoprecipitation experiments were subcloned into pEF1-MCS-mychis6 B (V92120; Invitrogen) or pCMV 3xFLAG-MCS (Parker et al., 2013 (link)). Mutants of EVL and MIM were generated by PCR or inverse PCR and self-ligation cloning. All plasmids created for this paper will be made available through Addgene where possible. Complete list of plasmids generated and used in this paper can be found in Table S1.