RudLOV—a new optically synchronized cargo transport method reveals unexpected effect of dynasore

Satoh, Akiko 1 ; Satoh, Takunori1 ; Tago, Tatsuya1

Published Nov 08, 2024 on Dryad. https://doi.org/10.5061/dryad.jm63xsjhs

Abstract

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.

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/
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: 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/
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/
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/
- linker GLEGRAHGAPRP
- source Str-KDEL_TNF-SBP-EGFP (Plasmid #65278)
- gift from Franck Perez
- 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/
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/
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/
- source Str-Ii_VSVG-SBP-EGFP (Plasmid #65300)
- gift from Franck Perez
- 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/
linker synthetic NSGSGSGS
insert 2 Zdk1 MVDNKFNKEKTRAGAEIHSLPNLNVEQKFAFIVSLFDDPSQSANLLAEAKKLNDAQAPK
- source pTriEx-mVenus-Zdk1-Rac1 Q61L (Plasmid #81137)
- gift from Klaus Hahn
- 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/
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/
- source Str-KDEL_SBP-EGFP-GPI (Plasmid #65294)
- gift from Franck Perez
- 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/
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/
- 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/
- 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-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/
- 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)

RudLOV—a new optically synchronized cargo transport method reveals unexpected effect of dynasore

Data files

Abstract

README

Information of plasmids used in this work

vector name: CMV-TfR-2xSca

vector name: CMV-2xRb-GM130

vector name: Str-KDEL_SBP-2NG-VSVG (Str::KDEL_SBP::NeonGreen::VSVG)

vector name: CMV-TNF-Zdk-2xSca

vector name: CMV-SP-Zdk1-2xSca-VSVG

vector name: CMV-TNF-Zdk-2xCv

vector name: CMV-TNF-Zdk-2xSca

vector name: CMV-SP-Zdk-2xCv-VSVG

vector name: CMV-SP-Zdk-2xCv-GPI

vector name: pEBP-Sec61b-mTagBFP2-LL-LOV2

vector name: CT7-2xNG-COPe

vector name CT7-2xSca-Rab6a

vector name pEGFP VAMP3

vector name: CMV-TfR-2xSca

vector name:CMV-2xCv-GM130

vector name: CMV-ManII-EGFP

vector name: CMV-2xCv-GM130

vector name: CMV-ManII-EGFP

Methods

Works referencing this dataset