DOI: 10.5061/dryad.jm63xsjhs
title: RudLOV—a new optically synchronized cargo transport method reveals unexpected effect of dynasore

description: Live imaging of secretory cargoes is a powerful method for understanding
 the mechanisms of membrane trafficking. Inducing the synchronous release
 of cargoes from an organelle is key for enhancing microscopic observation.
 We developed an optical cargo-releasing method, ‘retention using dark
 state of LOV2’ (RudLOV), which enables exceptional spatial, temporal, and
 quantity control during cargo release. A limited amount of cargo-release
 using RudLOV successfully visualized cargo cisternal-movement and
 cargo-specific exit sites on the Golgi/trans-Golgi network. Moreover, by
 controlling the timing of cargo-release using RudLOV, we revealed
 canonical and non-canonical effects of the well-known dynamin inhibitor
 dynasore, which inhibits early- but not late-Golgi transport and exits
 from the trans-Golgi network where dynamin-2 is active. Accumulation of
 COPI vesicles at the cis-side of the Golgi stacks in dynasore-treated
 cells suggests that dynasore targets COPI-uncoating/tethering/fusion
 machinery in the early-Golgi cisternae or endoplasmic reticulum but not in
 the late-Golgi cisternae. These results provide insight into the cisternal
 maturation of Golgi stacks.
description: The data files included in this datasets are sequence
 files. 
description: #### Information of plasmids used in this work ##### vector name:
 CMV-TfR-2xSca used as marker: TfR::Scarlet * backbone pEGFP-N1 *
 source Clontech * insert 1 human TFRC, Transferrin receptor protein 1 *
 M1..F760 * source mTagBFP2-TFR-20 (Plasmid #55327) * ``` gift from Michael
 Davidson ``` * ``` https://www.addgene.org/55327/ ``` * linker synthetic
 SEFGSTGSTGGGGGSGGGAAAGGRSGGGAPGGGGSGGGGSSA * insert 2 tandem repeat of two
 mScarlet-I (SalI deleted silently) * source Double UP mNeon to mScarlet
 P2A Rac1-wt (Plasmid #125135) * ``` gift from Michael Davidson ``` * ```
 https://www.addgene.org/55327/ ``` * linker of 2FPs GGGSGGGSSA #####
 vector name: CMV-2xRb-GM130 used as marker Ruby::GM130 * backbone pEGFP-N1
 * source Clontech * insert 1 tandem repeat of two mRuby3 * source
 pKanCMV-mClover3-mRuby3 (Plasmid #74252) * gift from Franck Perez *
 [https://www.addgene.org/74252/](https://www.addgene.org/74252/) * linker
 of 2FPs TRGGGSGGT * linker TSGGGGSGGGAAAGGRSGGGAPGGGGSGGGGSTG * insert 2
 GOLGA2 Golgin subfamily A member 2, human * S2..732STOP
 [https://www.uniprot.org/uniprotkb/Q08379/](https://www.uniprot.org/uniprotkb/Q08379/) * source cDNA of HeLa cell ##### vector name: Str-KDEL\_SBP-2NG-VSVG (Str::KDEL\_SBP::NeonGreen::VSVG) used as cargo SBP::NeonGreen::VSVG * backbone Str-KDEL_SBP-EGFP-GPI (Plasmid #65294) * gift from Franck Perez * [https://www.addgene.org/65294/](https://www.addgene.org/65294/) * linker synthetic GSGGSGSGGTT * insert 1 tandem repeat of two mNeonGreen * from CT7-2xNG-COPe in this work * linker synthetic TSGGGGSGGGAAAGGRSGGGAPGGGGSGGGGSS * insert 2 VSV-G * from CMV-SP-Zdk1-2xSca-VSVG ##### **vector name: CMV-TNF-Zdk-2xSca** used as cargo TNFα::Zdk1::Scarlet * backbone CMV-TNF-Zdk-2xCv * insert 1 tandem repeat of two mScarlet-I (SalI deleted silently) * ``` source CMV-TfR-2xSca ``` ##### vector name: CMV-SP-Zdk1-2xSca-VSVG * cargo Zdk1::Scarlet::VSVG * backbone CMV-SP-Zdk1-2xCv-GPI in this paper * insert 1 tandem repeat of two mScarlet-I (SalI deleted silently) * source Double UP mNeon to mScarlet P2A Rac1-wt (Plasmid #125135) * gift from Erik Dent * [https://www.addgene.org/125135/](https://www.addgene.org/125135/) * linker of 2FPs TRGGGSGGT * linker synthetic TSGGGGSGGGAAAGGRSGGGAPGGGGSGGGGSTG ##### **vector name: CMV-TNF-Zdk-2xCv** * used as cargo TNFα::Zdk1::Clover * backbone pEGFP-N1 * source Clontech * insert 1 human TNF, Tumor necrosis factor * M1.. [https://www.uniprot.org/uniprotkb/P01375/](https://www.uniprot.org/uniprotkb/P01375/) * linker GLEGRAHGAPRP * source Str-KDEL_TNF-SBP-EGFP (Plasmid #65278) * gift from Franck Perez * [https://www.addgene.org/65278/](https://www.addgene.org/65278/) * linker synthetic TSGGGGSGGGAAAGGRSGGGAPGGGGSGGGGGSGS * insert 2 Zdk1 MVDNKFNKEKTRAGAEIHSLPNLNVEQKFAFIVSLFDDPSQSANLLAEAKKLNDAQAPK * source pTriEx-mVenus-Zdk1-Rac1 Q61L (Plasmid #81137) * gift from Klaus Hahn * [https://www.addgene.org/81137/](https://www.addgene.org/81137/) * linker synthetic GSGGSGSGSGGSSA * insert 3 tandem repeat of two mClover3 (KpnI deleted silently) * source pKanCMV-mClover3-mRuby3 (Plasmid #74252) * gift from Franck Perez * [https://www.addgene.org/74252/](https://www.addgene.org/74252/) * linker of 2FPsGGGSGGGSSA ##### **vector name: CMV-TNF-Zdk-2xSca** used as cargo TNFα::Zdk1::Scarlet * backbone CMV-TNF-Zdk-2xCv * insert 1 tandem repeat of two mScarlet-I (SalI deleted silently) * source CMV-TfR-2xSca * this work ##### **vector name: CMV-SP-Zdk-2xCv-VSVG** used as cargo Zdk1::Clover::VSVG * backbone CMV-SP-Zdk-2xCv-GPI * source this paper * insert 1 VSV-G, vesicular stomatitis Indiana virus G protein * F18..I500 [https://www.uniprot.org/uniprotkb/B7UCZ5/](https://www.uniprot.org/uniprotkb/B7UCZ5/) * source Str-Ii_VSVG-SBP-EGFP (Plasmid #65300) * gift from Franck Perez * [https://www.addgene.org/65300/](https://www.addgene.org/65300/) ##### vector name: CMV-SP-Zdk-2xCv-GPI  used as cargo Zdk1::Clover::GPI * backbone pEGFP-N1 * source Clontech * insert 1 Signal peptide of Interleukin-2 * M1..T18 MYRMQLLSCIALSLALVT * source Str-KDEL_SBP-EGFP-GPI (Plasmid #65294) * gift from Franck Perez * [https://www.addgene.org/65294/](https://www.addgene.org/65294/) * linker synthetic NSGSGSGS * insert 2 Zdk1 MVDNKFNKEKTRAGAEIHSLPNLNVEQKFAFIVSLFDDPSQSANLLAEAKKLNDAQAPK * source pTriEx-mVenus-Zdk1-Rac1 Q61L (Plasmid #81137) * gift from Klaus Hahn * [https://www.addgene.org/81137/](https://www.addgene.org/81137/) * Linker ggatccggtggttctggttctggtGGTACC * insert 3 tandem repeat of two mClover3 (KpnI deleted silently) * source pKanCMV-mClover3-mRuby3 (Plasmid #74252) * gift from Franck Perez * [https://www.addgene.org/74252/](https://www.addgene.org/74252/) * linker of 2FPs TRGGGSGGT * linker synthetic TSGGGGSGGGAAAGGRSGGGAPGGGGSGGGGSS * insert 4 GPI-anker signal of human CD59 glycoprotein * L100..STOP129 LENGGTSLSEKTVLLLVTPFLAAAWSLHP* * [https://www.uniprot.org/uniprotkb/P13987/](https://www.uniprot.org/uniprotkb/P13987/) * source Str-KDEL_SBP-EGFP-GPI (Plasmid #65294) * gift from Franck Perez * [https://www.addgene.org/65294/](https://www.addgene.org/65294/) ##### vector name: pEBP-Sec61b-mTagBFP2-LL-LOV2 used to express hook * backbone pEB-multi-TGT-puro * ``` source FujiFilm ``` * insert 1 kan-r pEGFP-N1 * ``` source Clontech ``` * replacing puro-r * insert 2 Sec61b Protein transport protein Sec61 subunit beta * ``` M1..S96 https://www.uniprot.org/uniprotkb/P60468/ ``` * ``` source cDNA of HeLa cell ``` * linker synthetic AAAT * insert 3 mTagBFP2 * ``` source "mTagBFP2-TFR-20 (Plasmid #55327) " ``` * ``` gift from Michael Davidson ``` * ``` https://www.addgene.org/55327/ ``` * linker synthetic GAPGGGGSGGGAGSGSGSGGGGSGGGGSGGGSGGGSGGGSGGGSGGGGSGGGGSGGGSGGGTGTGTGTAAAGGRSGGSGSGDAPGGGGSGGGGSS * insert 4 LOV2 * ``` source pTriEx-NTOM20-LOV2 (Plasmid #81009) ``` * ``` gift from Klaus Hahn ``` * ``` https://www.addgene.org/81009/ ``` ##### vector name: CT7-2xNG-COPe used as marker NeonGreen::εCOP * backbone pEGFP-N1 * ``` source Clontech ``` * insert 1 tandem repeat of two mNeonGreen * ``` source Double UP mNeon to mScarlet P2A Rac1-wt (Plasmid #125135) ``` * ``` gift from Erik Dent ``` * ``` https://www.addgene.org/125135/ ``` * linker of 2FPs TRGGGSGGT * linker synthetic TSGGGGSGGGAAAGGRSGGGAPGGGGSGGGGSS * insert 2 human COPE, Coatomer subunit epsilon * ``` A2..STOP309 https://www.uniprot.org/uniprotkb/O14579/ ``` * ``` source cDNA of HeLa cell ``` ##### vector name CT7-2xSca-Rab6a used as marker Scarlet::Rab6 * backbone pEGFP-N1 * source Clontech - insert 1 tandem repeat of two mScarlet-I (SalI deleted silently) * source Double UP mNeon to mScarlet P2A Rac1-wt (Plasmid #125135) * gift from Erik Dent * [https://www.addgene.org/125135/](https://www.addgene.org/125135/) - linker of 2FPs TRGGGSGGT - linker synthetic TSGGGGSGGGAAAGGRSGGGAPGGGGSGGGGSTG - insert 2 human Rab6a, Ras-related protein Rab-6A ,  isoform P20340-1 * M1..STOP209 [https://www.uniprot.org/uniprotkb/P20340/](https://www.uniprot.org/uniprotkb/P20340/) * source cDNA of HeLa cell ##### vector name pEGFP VAMP3 used as marker GFP::Vamp3 * ``` gift from Thierry Galli ``` * ``` https://www.addgene.org/42310/ ``` ##### vector name: CMV-TfR-2xSca used as marker TfR::Scarlet * backbone pEGFP-N1 * source Clontech * insert 1 human TFRC, Transferrin receptor protein 1 * M1..F760 * source mTagBFP2-TFR-20 (Plasmid #55327) * gift from Michael Davidson * [https://www.addgene.org/55327/](https://www.addgene.org/55327/) * linker synthetic SEFGSTGSTGGGGGSGGGAAAGGRSGGGAPGGGGSGGGGSSA * insert 2 tandem repeat of two mScarlet-I (SalI deleted silently) * source Double UP mNeon to mScarlet P2A Rac1-wt (Plasmid #125135) * gift from Michael Davidson * [https://www.addgene.org/55327/](https://www.addgene.org/55327/) * linker of 2FPs GGGSGGGSSA ##### vector name:CMV-2xCv-GM130 used as marker Clover::GM130 * backbone pEGFP-N1 * source Clontech - insert 1 tandem repeat of two mClover3 (KpnI deleted silently) * ``` source pKanCMV-mClover3-mRuby3 (Plasmid #74252) ``` * ``` gift from Franck Perez ``` * ``` https://www.addgene.org/74252/ ``` - linker of 2FPs TRGGGSGGT - linker TSGGGGSGGGAAAGGRSGGGAPGGGGSGGGGSTG - insert 2 GOLGA2 Golgin subfamily A member 2, human * ``` S2..732STOP https://www.uniprot.org/uniprotkb/Q08379/ ``` * ``` source cDNA of HeLa cell ``` ##### vector name: CMV-ManII-EGFP used as marker ManII::EGFP * backbone: pEGFP-N1 * ``` source Clontech ``` * insert 1 MAN2A1 Alpha-mannosidase 2, mouse * ``` M1..L116 https://www.uniprot.org/uniprotkb/P27046/ ``` * ``` source Str-KDEL_ManII-SBP-tagBFP (Plasmid #65254) ``` * ``` gift from Franck Perez ``` * ``` https://www.addgene.org/65254/ ``` - Linker synthetic DPPVAT - insert 2 EGFP * ``` source pEGFP-N1 (Clontech) ``` ##### vector name: CMV-2xCv-GM130 used as marker Clover::GM130 * backbone pEGFP-N1 * ``` source Clontech ``` * insert 1 tandem repeat of two mClover3 (KpnI deleted silently) * ``` source pKanCMV-mClover3-mRuby3 (Plasmid #74252) ``` * ``` gift from Franck Perez ``` * ``` https://www.addgene.org/74252/ ``` * linker of 2FPs TRGGGSGGT * linker TSGGGGSGGGAAAGGRSGGGAPGGGGSGGGGSTG * insert 2 GOLGA2 Golgin subfamily A member 2, human * ``` S2..732STOP https://www.uniprot.org/uniprotkb/Q08379/ ``` * ``` source cDNA of HeLa cell ``` ##### vector name: CMV-ManII-EGFP used as marker ManII::EGFP * backbone: pEGFP-N1 * source Clontech * insert 1 MAN2A1 Alpha-mannosidase 2, mouse  * M1..L116 [https://www.uniprot.org/uniprotkb/P27046/](https://www.uniprot.org/uniprotkb/P27046/) * source Str-KDEL_ManII-SBP-tagBFP (Plasmid #65254) * gift from Franck Perez * [https://www.addgene.org/65254/](https://www.addgene.org/65254/) - Linker synthetic DPPVAT - insert 2 EGFP * source pEGFP-N1 (Clontech)