Related Experiment Videos
The human autoantigen MCP1 is required during early stages of DNA replication
E Bronze-da-Rocha1, A Nóvoa, C Cunha
1Laboratório de Genética Molecular da Mitose, Instituto de Biologia Molecular e Celular da Universidade do Porto, Portugal. erocha@ibmc.up.pt
Abstract:
Metaphase chromosome protein 1 (MCP1) is a nuclear autoantigen that is associated with condensed chromosomes throughout mitosis. During interphase, this antigen shows a speckle distribution in the nucleus, excluding the nucleolus. Additionally, MCP1 binds tightly to the scaffold/matrix component of nuclei and isolated chromosomes. In order to determine the in-vivo localization of the antigen, we have expressed MCP1 fused to EGFP in tissue culture cells. The results demonstrate that MCP1 is located in the nucleus during interphase and during mitosis associates tightly to condensed chromosomes. Furthermore, microinjection of specific antibody confirms these results. We have used a specific monoclonal antibody (mAb 402) against MCP1 to assess the function of this antigen during cell cycle progression. HeLa and Ptk-2 cells that were microinjected into the nucleus and/or cytoplasm at G1/S and very early S phase were not able to progress and complete DNA replication. However, injection of mAb 402 at mid or late S phase does not prevent completion of DNA replication and subsequent progression into mitosis. Microinjection of mAb 402 in Ptk-2 cells synchronized in mitosis did not interfere with progression of mitosis and cells divided. Our results suggest that MCP1 is required at the G1/S transition and during early S phase.
Insights
Metaphase chromosome protein 1 (MCP1) is crucial for DNA replication. Blocking MCP1 function at the G1/S transition prevents DNA synthesis, while later inhibition has no effect.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Metaphase chromosome protein 1 (MCP1) is a nuclear autoantigen.
- MCP1 localizes to condensed chromosomes during mitosis and has a speckled nuclear distribution during interphase.
- MCP1 binds to nuclear scaffolds and isolated chromosomes.
Purpose of the Study:
- To determine the in-vivo localization of MCP1.
- To investigate the function of MCP1 during cell cycle progression.
Main Methods:
- Expressing MCP1 fused to EGFP in tissue culture cells to observe localization.
- Microinjection of a specific monoclonal antibody (mAb 402) against MCP1 into cells at different cell cycle phases.
Main Results:
- MCP1 localizes to the nucleus during interphase and associates with condensed chromosomes during mitosis.
- Microinjection of mAb 402 at G1/S and early S phase inhibited DNA replication.
- Microinjection of mAb 402 at mid/late S phase or during mitosis did not affect DNA replication or cell division.
Conclusions:
- MCP1 is essential for DNA replication at the G1/S transition and early S phase.
- MCP1's function is critical during specific stages of the cell cycle.