Page 189 - ebook

Las1 is involved in the oocyte spindle formation

Juri Yang , Hyemin Kim , Min Kook Kang , Seung Jin Han 1,2,4,5 *
1,2
1,3
1,2

Department of Biological Science, Inje University, Gimhae 50834, Korea
1

2
Institute of Digital Anti-Aging Healthcare, Inje University, Gimhae, 50834, Korea

Department of Research Center, Dong Nam Institute of Radiological and Medical Sciences , Busan 46033, Korea
3

Department of Biotechnology, Inje University, Institute of Basic Science, Inje University , Gimhae 50834, Korea
4 5

Abstract

Las1 was first discovered as a gene which is associated with susceptibility to lung cancer. It is (A)

believed that Las1 binds to tubulin and it is a key factor in the escape from mitotic stress-

induced tumor cell death. However, the exact function and role of Las1 in the cell cycle 120 Negative siRNA 120 Negative siRNA

remains unclear. Las1 RNA and protein are expressed at high levels in reproductive tissues,
100 Las1 siRNA 100 Las1 siRNA

especially in the oocyte. In this study, the function and role of Las1 in oocyte was

investigated. When Las1 was suppressed by microinjection of Las1 siRNA into oocytes, 80 80

abnormal spindle formation and chromosomal alignment were observed in some oocytes.

Las1 is colocalized with spindle assembly checkpoint (SAC) proteins. These results imply that GVBD(%) 60 GVBD(%) 60

Las1 plays a role in spindle formation and cell cycle progression 40 40

20 20
Materials and Methods

0 0

0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7

- Localization of Las1 and spindle. p3xflag-Las1-GFP and mCherry-Tubulin were co-

transfected into HEK293T cells. The cell was fixed with 100% methanol, and then DNA Time(h) Time(h)

was stained with DAPI. The localization of Las1-GFP and mCherry-Tubulin were observed (B)

under the fluorescence microscope. CREST Tubulin DAPI Merge

- Microinjection of Las1 siRNA in to oocyte . Germinal vesicle (GV) oocytes were collected

from C57BL/6 mice and injected with 50pmole control or Las1 siRNA. The cell cycle was

resumed in the α-MEM by removal of milinone. The CREST protein, spindle formation

and DNA position were checked after 16 hours.

- Interaction protein detection using Duolink methood. Cells are treated with Las1 and

one of the SAC proteins (Aurora A, Aurora B or BubRI) antibodies, and treated with the Negative siRNA

secondary antibody bound to the PLA probe. The ligase was added to make DNA

oligoucleotide in a circle shape at 37 °C following adding the polymerase to amplify this

DNA circle at 37 °C. The fluorescent probe was added to confirm the interaction of the

target proteins by amplified DNA.

Results

DAPI 3xflag-Las1-GFP mCherry-Tubulin Merge

Las1 siRNA

Figure 2. Spindle and chromosome alignment after microinjection of Las1 siRNA in mouse

oocytes. (A) The cell cycle progression of Las1 siRNA-microinjected oocytes was monitored by

counting the number of oocytes which undergo GVBD. (B) The meiotic spindle formation and

chromosome alignment were observed after staining kinetochore, spindle, and chromosome with

Fig. 1 Co-localization of Las1 and spindle. 3xFlag-Las1-GFP and mCherry-Tubulin were co-
CREST antibody, tubulin antibody, and DAPI, respectively. Improper spindle formation is indicated

transfected into HEK293T cells. The DNA of cells were stained with DAPI and observed under
with red box and chromosomes which are not located on the equator plate are marked with

the fluorescence microscope. The yellow staining in merged images indicates that Las1 and
arrowhead.
microtubules are in the same location.

Las1 – α-tubulin Las1 – Aurora A las1 – Aurora B Las1 – BubR1

Conclusion

▶ Las1 co-localization with microtubules

#1

▶ Las1 knockdown by siRNA causes abnormal formation of spindle and fragmentation of

chromosome in mouse oocytes.

▶ Las1 interacts with BubRI, a SAC protein.

▶ Las1 may be involved in SAC and spindle formation in the oocyte cell.

#2

References

-Liu, Y., et al., Degradation of lung adenoma susceptibility 1, a major candidate mouse lung

tumor modifier, is required for cell cycle progression. Cancer Res, 2007. 67(21): p. 10207-13.

#3 -Sinnott, R., et al., Mechanisms promoting escape from mitotic stress-induced tumor cell death.

Cancer Res, 2014. 74(14): p. 3857-69.

-Xu, X.L., et al., The microtubule-associated protein ASPM regulates spindle assembly and

meiotic progression in mouse oocytes. PLoS One, 2012. 7(11): p. e49303.

Fig. 3 Interaction protein of Las1 using Duolink. The proteins that interact with Las1 was

identified by Duolink method on the cells. Three different las1 antibody (#1- #3) were used. This research was supported by Basic Science Research Program through the National Research

The red signal indicated the interaction between las1 and checked protein. Las1 interacts Foundation of Korea(NRF) funded by the Ministry of Education(2020R1I1A3070491)

with tubulin and BubR1. DNA was stained with DAPI.