Tdtomato c1

Plasmids used for tdTomato-Tau overexpression in HEK293T cells and the tdTomato-C1 control plasmid were a gift from Michael Davidson: tdTomato-MAPTau-C-10 (RRID: Addgene_58112), tdTomato-MAPTau-N-10 (RRID:Addgene_58113) and tdTomato-C1 (RRID:Addgene_54653). Control tdTomato-N1 plasmid was a gift from Michael Davidson, Nathan Shaner and Roger Tsien (RRID:Addgene_54642) [129 (link)]. For Tau overexpression in SH-SY5Y cells, the cDNA sequence of either the 441 amino acids-long human 2N4R Tau isoform or that of the 758 amino acids-long human Big Tau isoform, respectively, was cloned into the pSMPUW-IRES-Bsd vector backbone (Cell Biolabs, cat. no. VPK-219). The empty vector was used as a control. Lentiviral particles generated using these plasmids were used to create SH-SY5Y cell lines that stably overexpress Tau, as well as corresponding control cell lines.

WPRE was amplified by PCR using the pCSII-EF-mRFP1-RfA plasmid (RIKEN BioResource Center) as a template, and inserted and fused into EcoRV and SacI sites in pCAG-LSL-ZsGreen (Addgene #51269)⁴⁸ using NEBuilder HiFi DNA Assembly Master Mix to exchange the ZsGreen and WPRE sequences. Then, oligoDNA (5′-TCGACCCGCCACCAATCATTTAAATAGGTCCCTCGACCTGCA-3′) was ligated into PstI and SalI sites in pCAG-LSL-WPRE-pA to eliminate the LSL sequence. The tdTomato coding sequence was amplified by PCR using tdTomato-C1 (Addgene #54653) as a template, and inserted and fused into EcoRI and KpnI sites in pCAG-WPRE-pA using NEBuilder HiFi DNA Assembly Master Mix. The mouse genomic 500-bp upstream and downstream DNA sequences of the crRNA target site of the Hipp11 locus were amplified by PCR using the C57BL/6JJcl mouse genomic DNA as a template, and were used as left and right homology arms, respectively. The CAG-tdTomato-WPRE-pA fragment was purified from the SpeI-digested pCAG-tdTomato-WPRE-pA. Then, the three fragments were fused and inserted into KpnI and BamHI sites of pCriMGET_9-12a using NEBuilder HiFi DNA Assembly Master Mix.

Human myosin 1e and truncated constructs tagged with EGFP have been previously described^{78 (link)}. Human myosin 1f (NM_012335.3) cDNA from the Mammalian Gene Consortium was amplified and cloned into pEGFP-C1 (Clontech). Both myosins were also cloned into mEmerald-C1 and tdTomato-C1 (gifts from Michael Davidson; Addgene plasmids #53975 and 54653) and mScarlet-C1. The following constructs were purchased from Addgene: EGFP-PLCδ-PH, EGFP-AKT-PH, and EGFP-PKCδ-C1 (gifts from Tobias Meyer, plasmids #211789, 21218, and 21216), mScarlet-PM (gift from Dorus Gadella, #98821), EGFP-VAMP3 (a gift from Thierry Galli, #42310), Ruby-Lifeact, mEGFP-Lifeact, mEmerald-Lifeact and EGFP-actin (gifts from Michael Davidson, #54674, #54610, #54148, and #56421). EGFP-FcγRIIA was a gift from Sergio Grinstein (Hospital for Sick Kids, Toronto, ON), and EGFP-TAPP1 was a gift from Aaron Marshall (University of Manitoba, Winnipeg, MB). YFP-myo1c and EGFP-myo1g were gifts from Matt Tyska (Vanderbilt University, Nashville, TN) that were cloned into mEmerald-C1. All primers used for construct generation are listed in Supplementary Table 1.