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Photoswitchable CENP-E Inhibitor PCEI-HU | Funakoshi

Date:July 16 2020Web Page No:81511

Funakoshi Co.,Ltd.

Controllable by light! Chromosome movement regulation reagent in mitosis
Photoswitchable CENP-E Inhibitor : PCEI-HU

PCEI-HU (Photoswitchable CENP-E inhibitor) is the world's first reagent which can control movements of Centromere protein E (CENP-E), a kinesin-like motor protein in metapahse of mitosis.
Visible light stops the movement of chromosome, and UV light resumes the movement of chromosome.
This movement is controllable until all chromosomes are aligned on the metaphase plate in metaphase.

PCEI-HU is a powerful tool for the fundamental investigation of prometaphase/metaphase and spindle assembly checkpoint (SAC).

This product has been commercialized under the license from Hokkaido University.

Product Background

Product Background (click here)

Mitosis is a fundamental process of cell duplication for the growth and maintenance of tissues in multicellular organisms. During mitosis, many kinds of proteins play crucial roles in accurate distribution of the replicated genome into two daughter cells. The replicated genomes are condensed into chromosomes in the prophase and subsequently, chromosomes move to the center of the mitotic cells, frequently called metaphase plate, along spindle microtubules in prometaphase.
Chromosomes bind to spindle microtubules via kinetochore which is a unique protein complex in centromere.
Only when all chromosomes are aligned to the metaphase plate in metaphase, separation of sister chromosome begins (anaphase).
SAC monitors accurate alignment of chromosomes in metaphase plate in mitosis to exact separation of genomic information into daughter cells. Accurate segregation of all chromosomes is the most important matter to satisfy the SAC criteria. Centromere protein E (CENP-E) is a key component for chromosome congression along microtubule spindles to the metaphase plate. CENP-E is a dimeric protein which has three domains, N-terminal motor domain, C-terminal kinetochore binding domain and coiled-coiled linker region. Recent studies suggest inhibition of ATPase activity of the CENP-E motor domain suppresses chromosome congression to the metaphase plate, subsequently SAC detects the misalignment of chromosomes and as a result, anaphase does not start.
Controlling CENP-E activity has a large potential to control mitosis progression.
Although some potent CENP-E inhibitors have been developed so far, these inhibitors are not suitable for reversibly controlling CENP-E activity.

Fig.1 Overview of CENP-E in mitosis

Fig.1 Overview of CENP-E in mitosis

Dr. Tamaoki, Dr. Uehara and co-workers, Hokkaido University, succeeded in obtaining the world’s first photoswitchable CENP-E inhibitor (PCEI-HU) which contains an azobenzene-based photo-isomerization unit.
PCEI-HU can regulate CENP-E activity ON and OFF repeatedly by UV and visible light (Vis) irradiation.

PCEI-HU in water without light shows potent CENP-E inhibitory activity, but once UV light irradiation, PCEI-HU lacks its inhibitory activity. Under the condition, CENP-E keeps its motor activity for chromosome congression to the metaphase plate. This UV-induced loss of inhibitor action is recovered by Vis light.
After Vis irradiation, PCEI-HU blocks CENP-E activity again and chromatin congression is clearly suppressed.
PCEI-HU is a powerful tool for the fundamental investigation of prometaphase/metaphase and SAC.

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Features

  • PCEI-HU works as CENP-E blocking form after visible light irradiation.
  • PCEI-HU works as CENP-E non-bloking form after UVlight irradiation.
  • Until the all chromosomes are aligned to the metaphase plate in metaphase, movement can be regulated by visible / UV light.
 Fig.2 Principle and overview of PCEI-HU animation

Fig.2 Principle and overview of PCEI-HU

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Data

Absorption spectrum

Fig.3 Absorption spectrum
Left: Absorption spectrum of PCEI-HU. Without light irradiation (w/o light; gray), after 365 nm light irradiation
(UV irradiation; red) and after 510 nm light irradiation (Vis irradiation; blue).
Right : Absorbance changes at 338 nm under alternating 365/510 nm illumination.

Live cell imaging of mitotic chromosomes in PCEI-HU treated LLC-PK 1 cells

Fig.4 Live cell imaging of mitotic chromosomes in PCEI-HU treated LLC-PK 1 cells LLC-PK1 cells were treated with near-infrared DNA staining dye (SiR-DNA, 1 μM) and subsequently 1 μM PCEI-HU and 20 μM MG132 for 2 hours in darkroom. Cells were irradiated with UV light (365 nm) and Vis (510 nm) repeatedly and monitored in live-cell (Upper figure).
Movement of a specific chromosome (yellow arrow) was analyzed in kymograph (Lower figure).
While after UV irradiation, chromosomes moved to metaphase plate, but after Vis irradiation chromosomes left from metaphase plate. The chromosome was repeatedly regulated by UV/Vis cycles until chromosomes reaching to the metaphase plate.

Fig.5 Cellular localization of chromosomes, CENP-E and tubulin under photo-dependent PCEI-HU HeLa cells were treated with 1 μM PCEI-HU and 20 μM MG132*1 under following three conditions.

Fig.5 Cellular localization of chromosomes, CENP-E and tubulin under photo-dependent PCEI-HU HeLa cells were treated with 1 μM PCEI-HU and 20 μM MG132*1 under following three conditions.
After these conditions, cells were fixed with PFA and visualized by specific antibodies for CENP-E and α-tubulin and DAPI staining for chromosomes.
1) Add PCEI-HU without light for 2 hours → fixation = “w/o light”
2) Add PCEI-HU without light for 2 hours → after UV irradiation, culture for 30 min = “UV irradiation”
3) Add PCEI-HU without light for 2 hours → after UV irradiation, culture for 5 min
→ after Vis irradiation, culture for 30 min = “Vis irradiation”
Under the condition 1), PCEI-HU w/o light works as the “CENP-E block form” and induced misalignment of chromosomes.
CENP-E was highly accumulated in the centrosome. Under the condition 2), UV irradiation converted PCEI-HU to “CENP-E non-block form” and all chromosomes reached to the metaphase plate. CENP-E was distributed near metaphase plate.
Under condition 3), Vis irradiation recovered CENP-E inhibitory activity of PCEI-HU and shows a similar result with 1) w/o light.
NOTE *1: MG132 is used as an anaphase inhibitor. Under MG132 treatment, mitosis did not enter anaphase even when all chromosomes were aligned in the metaphase plate.

Functional assay to monitor a movement of Mad2, a SAC-regulatory protein

Fig.6 Functional assay to monitor a movement of Mad2, a SAC-regulatory protein HeLa cells co-expressing Mad2-GFP and histone H2B-mCherry were treated with PCEI-HU and MG132 under the following two conditions.
1) PCEI-HU w/o light for 2 hours →after UV irradiation, culture for 5 min
2) PCEI-HU w/o light for 2 hours →after UV irradiation, culture for 30 min
→after Vis irradiation, culture for 30 min →fixed and observed In both conditions, PCEI-HU w/o light-induced misalignment of chromosomes and aggregation of Mad2 in misaligned chromosomes.
In condition 1), short-term culture after UV irradiation Mad2 remained aggregated in the misaligned chromosome.
On the other hand, condition 2) showed long-term culture after UV irradiation induces correct alignment of chromosomes and Mad2 was clearly diffused in the cytosol.
These results indicate Mad2 specifically accumulated in misaligned chromosomes and after diffused in the cell by a complete alignment of chromosome in the metaphase plate.

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Reference

Mafy, N. N. et al., J. Am. Chem. Soc., 142, 1763-1767 (2020)

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Product Information

[Date : July 02 2022 00:30]

Detail Product Name Product Code Supplier Size Price
PCEI-HU, Photoswitchable CENP-E Inhibitor
DatasheetThis may not be the latest data sheet.
FDV-0037 FNAFunakoshi Co.,Ltd. 50 µg $400

Description PCEI-HU (Photoswitchable CENP-E inhibitor) is the world's first reagent which can control movements of Centromere protein E (CENP-E), a kinesin-like motor protein in metapahse of mitosis. Visible light stops the movement of chromosome, and UV light resumes the movement of chromosome. This movement is controllable until all chromosomes are aligned on the metaphase plate in metaphase. PCEI-HU is a powerful tool for the fundamental investigation of prometaphase/metaphase and spindle assembly checkpoint (SAC).
Storage -20°C CAS
Link

Gatastatin G2 | Funakoshi

[Date : July 02 2022 00:30]

PCEI-HU, Photoswitchable CENP-E Inhibitor


  • Product Code: FDV-0037
  • Supplier: FNA
  • Size: 50µg
  • Price: $400

Description PCEI-HU (Photoswitchable CENP-E inhibitor) is the world's first reagent which can control movements of Centromere protein E (CENP-E), a kinesin-like motor protein in metapahse of mitosis. Visible light stops the movement of chromosome, and UV light resumes the movement of chromosome. This movement is controllable until all chromosomes are aligned on the metaphase plate in metaphase. PCEI-HU is a powerful tool for the fundamental investigation of prometaphase/metaphase and spindle assembly checkpoint (SAC).
Storage -20°C CAS
Link

Gatastatin G2 | Funakoshi



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