A joint project of the Graduate School, Peabody College, and the Jean & Alexander Heard Library

Title page for ETD etd-07262010-135944


Type of Document Dissertation
Author Rickmyre, Jamie Lyn
URN etd-07262010-135944
Title Developmental requirement and regulation of the Drosophila homolog of MCPH1, a human microcephaly gene
Degree PhD
Department Cell and Developmental Biology
Advisory Committee
Advisor Name Title
Kathleen Gould Committee Chair
David Cortez Committee Member
Ethan Lee Committee Member
Laura Lee Committee Member
Keywords
  • cell cycle
  • MCPH1
  • microcephaly
  • Drosophila
  • Anaphase Promoting Complex
Date of Defense 2010-05-04
Availability unrestricted
Abstract
Mutations in human microcephalin (MCPH1) result in a form of autosomal recessive primary microcephaly, a disorder of fetal brain growth characterized by a severely reduced cerebral cortex and head size with mental retardation. Both human and Drosophila MCPH1 contain BRCA1 C-terminal domains (BRCT domains), which are found in many proteins that function in DNA repair and cell-cycle control. Maternal-effect lethal mutations in Drosophila mcph1 result in cell-cycle arrest due to triggering of the centrosome inactivation pathway, a Checkpoint kinase 2 (Chk2)-mediated response following DNA damage or incomplete DNA replication in early Drosophila embryos (Rickmyre et al. 2007). mcph1 embryos exhibit genomic instability as evidenced by frequent chromatin bridging in anaphase. Furthermore, in contrast to studies of human MCPH1, the ATR/Chk1-mediated DNA checkpoint appears to be intact in Drosophila mcph1 mutants. In order to further understand how MCPH1 functions, I used tandem affinity purification (TAP)-mass spectrometry to find interactors. This approach revealed several regulators of chromatin structure, RNAi machinery components, and proteins involved in DNA replication in complex with Drosophila MCPH1.

In collaboration with Dr. Marc Kirschner’s laboratory, we identified Drosophila MCPH1 in a genome-scale biochemical screen for substrates of the Anaphase-Promoting Complex (APC). We have identified the destruction box of MCPH1 required for APC-mediated degradation in vitro. Our in vivo data indicate that MCPH1 protein levels are elevated in embryos from females carrying a maternal-effect allele of APC2, which encodes the functional ligase of the APC. We have also demonstrated that human MCPH1 is an in vitro substrate of the APC and have shown that its levels oscillate in a cell-cycle dependent manner in cultured human cells with lower protein levels when APC is activated during late mitosis and G1. Finally, injection of 2- or 4-cell staged Xenopus embryos with human or Drosophila mcph1 RNA results in cell-cycle arrest. Injected cells undergo a few rounds of normal cleavage before exhibiting failed cytokinesis while the uninjected cells continue to divide unperturbed.

Files
  Filename       Size       Approximate Download Time (Hours:Minutes:Seconds) 
 
 28.8 Modem   56K Modem   ISDN (64 Kb)   ISDN (128 Kb)   Higher-speed Access 
  Rickmyre_dissertation.pdf 12.78 Mb 00:59:09 00:30:25 00:26:37 00:13:18 00:01:08

Browse All Available ETDs by ( Author | Department )

If you have more questions or technical problems, please Contact LITS.