Controllable by light! Chromosome Movement Regulation Reagent in Mitosis PCEI-HU | Funakoshi

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

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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.

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.
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.

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.

Mafy, N. N., Matsuo, K., Hiruma, S., Uehara, R., and Tamaoki, N., J. Am. Chem. Soc., 142(3), 1763～1767 (2020). [PMID: 31927956]
"Photoswitchable CENP-E Inhibitor Enabling the Dynamic Control of Chromosome Movement and Mitotic Progression."

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