SSBD:database v3

Dataset Fig4G_Mut9A_10

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Project

Project
422-Asai-KinetochoreBeads
Title
Dynamics of chromosomes and artificial kinetochore beads in mouse oocytes and embryos
Description
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. The authors 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.
License
CC BY 4.0
Funding
RIKEN intramural grants; RIKEN Pioneering Project “Long-timescale Molecular Chronobiology” (T.S.K.); JSPS KAKENHI 23H04948, 21H02407, and 18H05549 (T.S.K.); Mitsubishi Foundation (T.S.K.).

Dataset

Title
Z-series and time-lapse images of chromosomes and NDC80-deltaSPC-9A-NUF2 microbeads in a monastrol-treated and washed out oocyte
Description
Z-series and time-lapse images of chromosomes and NDC80-deltaSPC-9A-NUF2 microbeads in an oocyte. NDC80-deltaSPC-9A-NUF2 (NDC80-9A-deltaSPC and NUF2 heterodimer) beads were formed in the oocyte as follows; The authors microinjected mRNAs encoding C-terminally green fluorescent protein–tagged NDC80-9A-deltaSPC and NUF2, followed by anti-GFP–conjugated microbeads into the cytoplasm. The mRNA for H2B-mCherry was also injected to visualize the chromosomes. NDC80-9A-deltaSPC is phosphodeficient form of NDC80-deltaSPC. 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.
License
CC BY 4.0
Submitted at
None
Released at
None
Updated at

Biosamples

Description
Mouse oocyte which was microinjected mRNAs encoding NDC80-deltaSPC-9A-NUF2 followed by anti-GFP–conjugated microbeads (1.7 to 2.6 mm in diameter). The H2B-mCherry mRNA was also injexcted.
Organisms
Mus musculus (NCBI:txid10090)
Strains
B6D2F1
Cells
oocyte (CL_0000023)
Cell lines
Intrinsic variables
Extrinsic variables
Mouse oocyte was microinjected mRNAs encoding NDC80-deltaSPC-9A-NUF2 followed by anti-GFP–conjugated microbeads (1.7 to 2.6 mm in diameter). The H2B-mCherry mRNA was also injexcted. The oocyte was pretreated with monastrol and washedout during imaging.

Ontologies

UBERON
oocyte (CL_0000023)
Anatomical entities
MeSH: Oocytes
Gene ontology: Biological processes
female meiosis chromosome segregation (GO:0016321) kinetochore assembly (GO:0051382)
Gene ontology: Cellular components
kinetochore (GO:0000776) chromosome (GO:0005694)
Gene ontology: Molecular functions

Genetics

Gene names
Protein names
Kinetochore protein NDC80 homolog (Q9D0F1) Kinetochore protein Nuf2 (Q99P69) Histone H2B type 1-J (P06899)
Genetic methods
injection
Protein tags
mCherryFP (FBbi:00000525)
Probes
Oligo Primer
Genotype

Treatments

Reagent or Compound
Concentrations
100 micromolar (UO_0000064)
FoldDilution

Imaging Methods

Dimensions
512x512x25x3x120
X scales
0.063 micrometer
Y scales
0.063 micrometer
Z scales
1.25 micrometer
T scales
5 minutes
Channels
3 channel
Microscopy types
confocal microscopy (FBbi:00000251) time lapse microscopy (FBbi:00000249)
Detection methods
Visualization methods
Green fluorescent protein from Aequorea mCherry
Illumination methods
Sources of contrast
Contrast enhancing methods
Resolution enhancing methods
Airyscan
Image parameters
Sample preparation methods
living tissue

Instruments

Body
Zeiss LSM880
Model
Light source
Detector
Objective
Zeiss 40×C-Apochromat 1.2NA
Filter set
Dichroic