April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Knockdown of RanGAP1 leads to apoptosis by inducing mitotic defects in lens epithelial cells
Author Affiliations & Notes
  • Weike Ji
    ophthalmology and visual sciences, university of Nebraska medical center, omaha, NE
    Biochemistry & Molecular Biol, Univ of Nebraska Med Center, Omaha, NE
  • Footnotes
    Commercial Relationships Weike Ji, None
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Investigative Ophthalmology & Visual Science April 2014, Vol.55, 3566. doi:
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      Weike Ji; Knockdown of RanGAP1 leads to apoptosis by inducing mitotic defects in lens epithelial cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3566.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: We have previously shown that RanGAP1 (Ran GTPase activating protein) is a major lens protein. Previous studies showed that the RanGAP1 knockout in mice is lethal during embryonic development, suggesting RanGAP1 played vital roles. However, regarding its functions, it remains largely unknown. Our present study reports that RanGAP1 is indispensible for cell viability and loss of RanGAP1 leads to apoptosis by inducing mitotic defects in lens epithelial cells.

Methods: Confocal microscope was used to record the cell cycle progress. Immunocytochemistry was used to detect the expression and localization of RanGAP1 and related protein components. SiRNA-mediated knockdown of RanGAP1 and pEGFP-RanGAP1-mediated over expression were used to change the expression levels of RanGAP1. Cell flow cytometry and detection of caspase activity were used to analyze apoptosis.

Results: RanGAP1 expression was knocked down in mouse lens epithelial cells (αTN4-1) and human lens epithelial cells (HLE) via siRNA or shRNA respectively, and consequently both two types of lens cells were shown significantly higher levels of nuclear condensation, cleavage of lamin B1, and activation of caspase 2 and 3, classic markers for apoptosis. Annexin V staining also confirmed the above results. Mechanistically, we demonstrated that the enhanced level of apoptosis was due to the fact that RanGap1 knockdown resulted in mitotic spindle defects, leading to chromosome misalignment with abnormal cytokinesis and multinuclear cells.

Conclusions: RanGAP1 is a major lens protein that executes important functions in cell viability by regulating normal cell division. (Supported by NEI, Zhongshan Ophthalmic Center and HNU)

Keywords: 426 apoptosis/cell death • 657 protein modifications-post translational • 445 cataract  
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