June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Role of Caspases in the Process of Nuclear Removal
Author Affiliations & Notes
  • Rifah Gheyas
    Pathology and Genomic Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania, United States
  • A Sue Menko
    Pathology and Genomic Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania, United States
  • Footnotes
    Commercial Relationships   Rifah Gheyas None; A Sue Menko None
  • Footnotes
    Support  NEI Grant RO1 EY02478
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 523. doi:
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      Rifah Gheyas, A Sue Menko; Role of Caspases in the Process of Nuclear Removal. Invest. Ophthalmol. Vis. Sci. 2023;64(8):523.

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

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Abstract

Purpose : Members of the caspase family of proteases are involved in the targeted degradation of nuclear proteins, contributing to nuclear breakdown during apoptosis. Among the direct substrates of apoptosis-linked caspases are Intermediate Filament Proteins and the Inhibitor of Caspase-Activated DNA (ICAD), whose cleavage releases and activates its binding partner Caspase-Activated DNase (CAD). As caspases have long been suspected to play a role in Organelle Free Zone (OFZ) formation, we have investigated the role of caspases in the nuclear removal process during lens development required to form the OFZ.

Methods : Confocal image analysis was performed throughout development to examine the timing of removal of the nuclear membrane (LAP2), removal the nuclear lamina (lamin B), nuclear condensation (DAPI), and DNA cleavage (pH2AX, TUNEL) in the process of nuclear removal. To examine the role of caspases in inducing nuclear elimination to form the OFZ, chick embryo lenses were placed in organ culture at E10, a developmental time prior to induction of nuclear condensation and cultured for 48 hrs in the presence of the pan-caspase inhibitor Z-VAD-FMK, the caspase 3/7 inhibitor Z-DEVD-FMK, or their vehicle control DMSO. Immunoblot analysis was performed following microdissection of the lenses into differentiation state-specific regions to examine impact on degradation of the inner nuclear membrane protein LAP2 and the nuclear lamina protein lamin B. Confocal image analysis was performed on cryosections co-labeled for various combinations of LAP2, lamin B, ICAD, CAD, the DNA cleavage marker pH2AX, TUNEL, and DAPI.

Results : Our studies showed that the nuclear membrane disassembles prior to the nuclear lamina and that ICAD and ICAD localize to the nucleus prior to removal of its envelope. Nuclear ICAD is degraded, freeing CAD just prior to nuclear condensation and DNA cleavage. CAD localization is not altered following caspase inhibition. Removal of the nuclear membrane, fragmentation of lamin B, DNA cleavage and nuclear condensation in the central fiber cells are suppressed when caspase activity is blocked.

Conclusions : Caspase 3/7 plays a role in nuclear elimination to form the OFZ, most likely through its involvement in removal of the nuclear lamina and activation of the nuclease CAD.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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