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Injection of anticentromere antibodies in interphase disrupts events required for chromosome movement at mitosis
R L Bernat1, G G Borisy, N F Rothfield
1Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Abstract:
We have used autoantibodies to probe the function of three human centromere proteins in mitosis. These antibodies recognize three human polypeptides in immunoblots: CENP-A (17 kD), CENP-B (80 kD), and CENP-C (140 kD). Purified anticentromere antibodies (ACA-IgG) disrupt mitosis when introduced into tissue culture cells during interphase. We have identified two execution points for antibody inhibition. Antibodies injected into the nucleus greater than or equal to 3 h before mitosis prevent the chromosomes from undergoing normal prometaphase movements in the subsequent mitosis. Antibodies injected in the nucleus during late G2 cause cells to arrest in metaphase. Surprisingly, antibodies introduced subsequent to the beginning of prophase do not block mitosis. These results suggest that the CENP antigens are involved in two essential interphase events that are required for centromere action in mitosis. These may include centromere assembly coordinate with the replication of alpha-satellite DNA at the end of S phase and the structural maturation of the kinetochore that begins at prophase.
Insights
Autoantibodies targeting centromere proteins CENP-A, CENP-B, and CENP-C disrupt mitosis by interfering with essential interphase events. This impacts chromosome movement and cell cycle progression, revealing key roles for these proteins in mitosis.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Centromere proteins are crucial for accurate chromosome segregation during mitosis.
- Autoantibodies provide a tool to investigate the function of specific proteins in cellular processes.
Purpose of the Study:
- To investigate the function of human centromere proteins CENP-A, CENP-B, and CENP-C in mitosis using autoantibodies.
- To identify critical time points during the cell cycle when these proteins are essential for mitotic progression.
Main Methods:
- Utilized autoantibodies against human centromere proteins (CENP-A, CENP-B, CENP-C).
- Introduced purified anticentromere antibodies (ACA-IgG) into tissue culture cells at different interphase stages.
- Observed effects on chromosome movement and cell cycle progression using microscopy and immunoblotting.
Main Results:
- Antibodies injected into the nucleus >= 3 hours before mitosis prevented normal prometaphase chromosome movements.
- Antibodies injected during late G2 caused cells to arrest in metaphase.
- Antibodies introduced after prophase initiation did not block mitosis, indicating specific interphase requirements.
Conclusions:
- CENP proteins are involved in essential interphase events required for centromere function during mitosis.
- These events likely include centromere assembly coordinated with DNA replication and kinetochore maturation.
- The timing of CENP protein function is critical for successful mitotic progression.