LMO7, AN ACTIN-ASSOCIATED PROTEIN, AND THE MITOTIC SPINDLE CHECKPOINT

• Main Authors: Wei-Chun Hsich, 謝偉鈞
• Other Authors: Yue-Li Juang
• Format: Others
• Language: en_US
• Online Access: http://ndltd.ncl.edu.tw/handle/27688117525806534667

碩士 === 慈濟大學 === 醫學研究所 === 90 === Abstract Mitotic spindle checkpoint can ensure precise chromosome separation during mitosis progression. Mad1 (mitotic arrest defective) is conserved between human and yeast and plays a key role in spindle assembly checkpoint. To clone and characterize the genes associated with mitotic spindle checkpoint, we have used Human MAD1 (HsMAD1) as a bait to perform yeast two-hybrid screening from human liver cDNA library. We have cloned LMO7, a gene containing a tissue-specific altermatively spliced LIM domain. By in vitro binding assays, bacterially expressed GST-HsMAD1 could interact with bacterially expressed LMO7 C298 and coprecipitate with LMO7 from HeLa cell extracts. We have constructed several deletion mutants of both HsMAD1 and LMO7 to perform yeast two-hybrid analysis and found that the region from amino acids 320 to 480 in HsMAD1 was required to interact with LMO7 and LMO7 C-terminal 298 amino acids to interact with HsMAD1. Immunofluorescence staining showed that LMO7 localized to nucleus as dots and was associated with actin filaments but not with microtubules in cytoplasm. LMO7 was not localized to centromere and telomere in nucleus. Overexpression of dominant-negative mutant LMO7 disrupted checkpoint arrest in human cells during microtubule depolymerization. We conclude that LMO7 was the first discovered actin-associated protein required for the spindle checkpoint.