HOME> Products> Natural and Chemical compound> Plasma membrane-specific protein translocation platform
SLIPT-PM

For target pathway-specific activation on plasma membrane Plasma membrane-specific protein translocation platform
SLIPT-PM

Date:July 13 2022Web Page No:95015

Funakoshi Co.,Ltd.

SLIPT is a novel chemogenetic platform technology for controlling intracellular localization of proteins by small compounds. The SLIPT-PM reagent can rapidly and stably translocate the protein of interest (POI) fused with designed tag-protein iK6DHFR to plasma membrane (PM). If the POI fused with iK6DHFR -tag can be activated on the PM, SLIPT-PM reagent can induce POI’s downstream signaling pathway without any upstream stimulation. SLIPT-PM is a powerful tool to analyze the target-specific function of signaling molecules.

SLIPT-PM-Abstract

Conventional analysis of ligand-based receptor and its downstream signal activation (Left) and Target specific activation by SLIPT-PM and iK6DHFR-tagged protein (Right).

In the left case, multiple signaling pathways may be simultaneously activated. On the hand, SLIPT-PM can induce translocation of iK6DHFR-tagged protein to the PM and activate it downstream signaling pathway without upstream receptor stimulation. SLIPT-PM/ iK6DHFR platform is versatilely used for various signaling proteins which can be activated on the PM.


What is SLIPT-PM?

About SLIPT

Analysis of signaling pathway is commonly performed by receptor stimulation. However, in almost cases receptor stimulation by specific ligands simultaneously activates various downstream factors and total cellular response could be observed. In some cases, several signaling pathways are crosstalk, analysis of each pathway is more complicated. To solve these problems many research tools are developed but versatile tools are not enough.

About SLIPT-PM

Self-localizing (SL) ligand-induced protein translocation (SLIPT), a novel chemogenetic platform, originally developed by Dr. Shinya Tsukiji (Nagoya Institute of Technology, Japan). SLIPT platform can control localization of protein of interest (POI) fused with genetically engineered tag-proteins by organelle specific accumulation ligand called SL ligands. Previously various types of SL ligands including plasma membrane (PM), endomembrane, Golgi membrane etc, were developed. SLIPT-PM (original compound name, mDcTMP) is a SL ligand for PM coupled with designed tag-protein iK6DHFR. When POI is expressed as a iK6DHFR-fused protein (POI-iK6DHFR), POI-iK6DHFR can be quickly and stably translocated to PM by SLIPT-PM addition. If POI can activate its function on the inner-leaflet of PM, POI-iK6DHFR promotes activation of POI’s downstream signaling factors. Using SLIPT-PM and POI-iK6DHFR, the POI’s downstream signaling pathway can be specifically activated without its original receptor stimulations.
Now, the following signaling pathways are successfully validated by SLIPT-PM/iK6DHFR system.

  • cRaf-MEK-ERK pathway
  • RasGEF-Ras-Raf-MEK-ERK pathway
  • q-PLCβ-PIP2-IP3-Ca2+ pathway
  • s-adenylate cyclase-cAMP pathway
  • PI3K-PIP3-Akt pathway
  • RacGEF-Rac-actin pathway
  • PKCδ-Raf-MEK-ERK pathway

What does we need to establish SLIPT-PM experiments?

SLIPT platform basically requires three components, 1) an organelle specific SL ligand, 2) a ligand-binding tag-protein fused with POI and 3) localization neutralizer. POI fused tag-protein is translocated to a target organelle by a SL ligand and released from the target organelle to the original localization by a neutralizer. Our SLIPT-PM is a specific reagent for PM-targeting and a neutralizer is also included in our kit. Our product does not contain tag-protein-expressing plasmids. Plasmids should be obtained from addgene or constructed by yourself. Please find “How to start SLIPT-PM experiments?”.
SLIPT-PM experiment


Go back to index

Demonstration experiment

PM-translocation of iK6DHFR-EGFP by SLIPT-PM and release from PM to cytosol by Free-TMP

Construction: EGFP-iK6DHFR (Addgene ID:172100)
SLIPT-PM concentration: 10 μM (Addition at 0 min)
Free-TMP concentration: 100 μM (Addition at 30 min)
Cell line: HeLa cells
Reaction medium: serum-free DMEM

Before the addition of SLIPT-PM, EGFP-iK6DHFR is constantly localized in the cytosol in HeLa cells. Immediately after the addition of SLIPT-PM, EGFP-iK6DHFR quickly translocated (t1/2~1.5 min) to the PM and stably tethered in the PM, for at least several hours. By adding Free-TMP (30 min later), EGFP-iK6DHFR gradually released from the PM to cytosol.


Demonstration


Go back to index

References

  1. Ishida, et al., J. Am. Chem. Soc., 135, 12684~12689 (2013). "Synthetic self-localizing ligands that control the spatial location of proteins in living cells."
  2. Nakamura, et al., ACS Chem. Biol., 15, 837~843 (2020). "Designer palmitoylation motif-based self-localizing ligand for sustained control of protein localization in living cells and Caenorhabditis elegans."
  3. Suzuki, et al., Cell Chem. Biol., in press. DOI: 10.1016/j.chembiol.2022.06.005 "A chemogenetic platform for controlling plasma membrane signaling and synthetic signal oscillation."

Go back to index

Product components

SLIPT-PM (catalog No. #FDV-0045) contains following two components.
  • SLIPT-PM(0.2 mg/vial)
  • SLIPT supplement (Free-TMP) 5 mg/vial *Optionally used for PM release

This product does not include iK6DHFR-expression plasmids. iK6DHFR-expressing plasmids should be got from addgene or constructed from eDHFRWT-expressing plasmids by yourself. Detail plasmid information are shown in “SLIPT-PM Experimental Guide Book”.

Go back to index

How to start SLIPT-PM experiments?

To establish SLIPT-PM experiments, you need not only our products but also iK6DHFR-expression plasmids. Before order of our SLIPT-PM product, please read following processes.

How to get iK6DHFR-expression plasmids?

iK6DHFR-expression plasmids containing a multi-cloning site and EGFP gene are distributed by plasmid bank addgene (Table 1). You can also construct plasmids from eDHFRWT gene by yourself, according to the reference paper (Ref. 3). Plasmids for control experiments mentioned in the “SLIPT-PM Experimental Guide Book” are also distributed by addgene (Table 2)

Table 1
Purpose Plasmid name addgene ID
To fuse your POI to C-terminal of iK6DHFR
(EGFP-iK6DHFR-POI)
pCMV-EGFP-eDHFR(69K6)-MCS 172100
To fuse your POI to N-terminal of iK6DHFR
(POI- iK6DHFR-EGFP)
pCMV-MCS-eDHFR(69K6)-EGFP 172101

Table 2 : List of control plasmids for pathway specific expriments
Pathway Construct name in the guidebook addgene plasmid name addgene ID
cRaf-MEK-ERK pathway EGFP-iK6DHFR-cRaf pPBpuro-EGFP-eDHFR(69K6)-cRaf 178849
cRaf-mNeonGreen(mNG)-iK6DHFR pPBpuro-cRaf-mNG-eDHFR(69K6) 172107
qpathway mNeonGreen(mNG)-iK6DHFR-Gαq pCAGGS-mNG-eDHFR(69K6)-G-alpha-q 172106
mNeonGreen(mNG)-iK6DHFR-Gαq (L254A) pCAGGS-mNG-eDHFR(69K6)-G-alpha-q(L254A) 178851
s pathway miRFP670-iK6DHFR-Gαs pPBpuro-miRFP670-eDHFR(69K6)-G-alpha-s 172105
miRFP670-iK6DHFR pCSIIpuro-miRFP670-eDHFR(69K6) 178852
PI3K-Akt pathway mNeonGreen(mNG)-iK6DHFR-p85iSH2 pPBpuro-mNG-eDHFR(69K6)-p85iSH2 172103
mNeonGreen(mNG)-iK6DHFR pCSIIpuro-mNG-eDHFR(69K6) 178853
RacGEF-Rac pathway mNeonGreen(mNG)-iK6DHFR-Tiam1DH-PH pPBpuro-mNG-eDHFR(69K6)-Tiam1 172102
mNeonGreen(mNG)-iK6DHFR pCSIIpuro-mNG-eDHFR(69K6) 178853
Grb2/SOS1-Ras-Raf-MEK-ERK pathway Grb2mimic-iRFP713 pCAGGS-chGrb2/eDHFR(69K6)-iRFP713 178854
K6DHFR-iRFP713 pCAGGS-eDHFR(69K6)-iRFP713 178855

Please find each application note in the “SLIPT-PM Experimental Guide Book”.

Detail information of SLIPT platform and SLIPT-PM

You can see the technology background, principle and guides for experimental setting in our “SLIPT-PM Experimental Guide Book” below URL. If you are interested in SLIPT-PM experiments more detail, please find it prefer to order of our product. The other application notes are also described in the “SLIPT-PM Experimental Guide Book”.
Download “SLIPT-PM Experimental Guide Book” here

Go back to index

Application note; Activation of Raf-ERK pathway

(1) Stable activation model

■Target signaling pathway: cRaf → MEK → ERK
■Constructs in the experiment:
- iK6DHFR-fused protein: EGFP-iK6DHFR-cRaf*1 (addgene ID:172107)
*1In this experiment, cRaf indicates the full-length cRaf protein
- Reporter protein: Nucleus ERK activity sensor (ERKKTR*2-mKusabiraOrange (mKO))
*2 ERKKTR is kinase translocation reporter for ERK
■SLIPT-PM concentration: SLIPT-PM 5 μM
■Cells: HeLa cells
■Reaction medium: serum-free DMEM
■Experimental design:
cRaf, a downstream factor of Ras, can be activated by Ras on the PM and subsequently activate MEK-ERK pathway. Phosphorylated ERK is subsequently translocated into the nucleus and activates further downstream transcription factors. In this model experiment, iK6DHFR-fused cRaf protein (EGFP-iK6DHFR-cRaf) is translocated to the PM by SLIPT-PM and promotes activation of MEK-ERK pathway on the PM without Ras activity. ERK activity is monitored by a nucleus ERK activity sensor (ERKKTR-mKO).

SLIPT-PM

Before the addition of SLIPT-PM, EGFP-iK6DHFR-cRaf and ERKKTR-mKO were constantly localized in cytosol and mainly nucleus, respectively. By the addition of SLIPT-PM, at first, EGFP-iK6DHFR-cRaf rapidly translocated to the PM (t1/2~1 min) and subsequent elimination of nuclear ERKKTR-mKO to cytosol was observed. In the presence of MEK inhibitor, although EGFP-iK6DHFR-cRaf translocated to the PM, ERKKTR-mKO localization was not changed. This result indicates SLIPT-PM promotes membrane-binding of EGFP-iK6DHFR-cRaf and EGFP-iK6DHFR-cRaf activates the MEK-ERK signaling pathway.

SLIPT-PM



(2) Reversible activation model

■Target signaling pathway: cRaf → MEK → ERK
■Constructs in the experiment:
- iK6-DHFR-fused protein: cRaf*1-mNeonGreen (mNG) -iK6DHFR(addgene ID:172107)
*1 In this experiment, cRaf indicates the full-length cRaf protein
- Reporter protein: mCherry-ERK
■SLIPT-PM concentration: 5 μM
■Free-TMP concentration: 50 μM
■Cells: HeLa cells
■Reaction medium: serum-free DMEM
■Experimental design:
cRaf, a downstream factor of Ras, can be activated by Ras on the PM and subsequently activate MEK-ERK pathway. Phosphorylated ERK is translocated into the nucleus and activates further downstream transcription factors. In this model experiment, iK6DHFR-fused cRaf protein (cRaf-mNG-iK6DHFR) is translocated to the PM by SLIPT-PM and promotes activation of MEK-ERK pathway. Nuclear translocation of mCherry-ERK is monitored as a sensor of MEK-ERK pathway. After then, by addition of Free-TMP induced release of cRaf-mNG-iK6DHFR from the PM to cytosol. Furthermore, wash out of Free-TMP reproduces PM-recruitment of cRaf-mNG-iK6DHFR by remaining SLIPT-PM on the PM again and reactivates MEK-ERK pathway.

SLIPT-PM

Before the addition of SLIPT-PM, both cRaf-mNG-iK6DHFR and mCherry-ERK constantly localized in cytosol. By the addition of SLIPT-PM, firstly, cRaf-mNG-iK6DHFR translocated to the PM (t1/2~7 min) and subsequently, nuclear translocation of mCherry-ERK was observed. The addition of Free-TMP at 20 min clearly canceled the PM-tethering of cRaf-mNG-iK6DHFR and nuclear localization of mCherry-ERK was decreased. The cell medium containing Free-TMP was continuously exchanged using a peristaltic pump from 40 min to 60 min recovered PM-translocation of cRaf-mNG-iK6DHFR and nuclear localization of mCherry-ERK again without the addition of SLIPT-PM. In this experiment, reversible regulation of cRaf-mNG-iK6DHFR and mCherry-ERK was successfully observed at least two cycles of Free-TMP addition and medium change. This result indicates SLIPT-PM is still tethering on the PM after Free-TMP addition and medium change.

SLIPT-PM

Scale bar 10 μm




Go back to index

Product information

[Date : June 15 2024 00:05]

Detail Product Name Product Code Supplier Size Price
SLIPT-PM
DatasheetThis may not be the latest data sheet.
FDV-0045 FNAFunakoshi Co.,Ltd. 1 kit $400

Description
Storage -20°C CAS
Link

[Date : June 15 2024 00:05]

SLIPT-PM


  • Product Code: FDV-0045
  • Supplier: FNA
  • Size: 1kit
  • Price: $400

Description
Storage -20°C CAS
Link


Go back to index

CONTACT

export@funakoshi.co.jp

  • Prices are for USA / Canada customers. Prices do not include shipping and handling charges, VAT, import tariffs and service charge etc.
  • Please note that Product Information or Price may change without notice.