Z-series and time-lapse images of chromosomes and NDC80-deltaSPC-NUF2 microbeads in a monastrol-treated and washed out oocyte
Description
Z-series and time-lapse images of chromosomes and NDC80-deltaSPC-NUF2 microbeads in an oocyte. NDC80-deltaSPC-NUF2 (NDC80-deltaSPC and NUF2 heterodimer) beads were formed in the oocyte as follows; The authors microinjected mRNAs encoding C-terminally green fluorescent protein–tagged NDC80-deltaSPC and NUF2, followed by anti-GFP–conjugated microbeads into the cytoplasm. The mRNA for H2B-mCherry was also injected to visualize the chromosomes. NDC80-deltaSPC is the deletion protein of NDC80 which lacks SPC-25-binding domain at its C-terminal region. The imaging was conducted for 30 minutes in the presence of monastrol, and thereafter, monastrol was washed out. channel1; GFP, channel2; mCherry, channel3;bright field.
Asai K, Zhou Y, Takenouchi O, Kitajima TS. Artificial kinetochore beads establish a biorientation-like state in the spindle. Scince . 2024 Sep 20;385(6715):1366-1375.
Related paper(s)
Kohei Asai, Yuanzhuo Zhou, Osamu Takenouchi, Tomoya S Kitajima (2024) Artificial kinetochore beads establish a biorientation-like state in the spindle., Science (New York, N.Y.), Volume 385, Number 6715, pp. 1366-1375
Published in 2024 Sep 20
(Electronic publication in Sept. 19, 2024, midnight )
(Abstract) Faithful chromosome segregation requires biorientation, where the pair of kinetochores on the chromosome establish bipolar microtubule attachment. The integrity of the kinetochore, a macromolecular complex built on centromeric DNA, is required for biorientation, but components sufficient for biorientation remain unknown. Here, we show that tethering the outer kinetochore heterodimer NDC80-NUF2 to the surface of apolar microbeads establishes their biorientation-like state in mouse cells. NDC80-NUF2 microbeads align at the spindle equator and self-correct alignment errors. The alignment is associated with stable bipolar microtubule attachment and is independent of the outer kinetochore proteins SPC24-SPC25, KNL1, the Mis12 complex, inner kinetochore proteins, and Aurora. Larger microbeads align more rapidly, suggesting a size-dependent biorientation mechanism. This study demonstrates a biohybrid kinetochore design for synthetic biorientation of microscale particles in cells.
Tomoya S Kitajima
, RIKEN
, Center for Biosystems Dynamics Research, Laboratory for Chromosome Segregation
, Center for Biosystems Dynamics Research, Laboratory for Chromosome Segregation