March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Inactivation of JNK Signals Formation Of The Organelle Free Zone (OFZ), A Process That Involves Classical Autophagic Molecules
Author Affiliations & Notes
  • Subhasree Basu
    Pathology Anatomy & Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania
  • Suren Rajakaruna
    Pathology Anatomy & Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania
  • A S. Menko
    Pathology Anatomy & Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania
  • Footnotes
    Commercial Relationships  Subhasree Basu, None; Suren Rajakaruna, None; A. S. Menko, None
  • Footnotes
    Support  EY010577, EY021784, EY014258
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 5591. doi:
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      Subhasree Basu, Suren Rajakaruna, A S. Menko; Inactivation of JNK Signals Formation Of The Organelle Free Zone (OFZ), A Process That Involves Classical Autophagic Molecules. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5591.

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

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Abstract

Purpose: : While an essential element of lens development is the loss of nuclei and organelles during lens fiber cell terminal differentiation there is still much unknown about what signals these events and the mechanism by which the removal of these cellular compartments is executed. We have studied the possibility that blocking JNK signaling, which is responsible for phosphorylation of survival proteins in the Bcl-2 family, could be the inducing signal for loss of nuclei and organelles. In addition, we examined whether an autophagic-like process could be involved in the process of organelle removal.

Methods: : Studies use primary quail lens cultures that mimic differentiation forming lentoid structures or an organ-culture system in which E13 chick embryo lenses are grown ex vivo. Cultures were exposed to the JNK inhibitor (SP600125, 25µM) or vehicle DMSO (24-48hrs). Lenses from organ culture were examined as cryosections. Cells were immunostained for Golgi and ER markers, and/or autophagy markers including Beclin-1 and LC3B, counterstained for F-actin (fluorescent-conjugated phalloidin) and nuclei (TO-PRO-3) and imaged by confocal microscopy. Western blot analysis was used to show expression of the autophagosome form of LC3B, LC3BII.

Results: : Inhibition of JNK in both primary and ex vivo cultures induced loss of nuclei and organelles, suggesting that modulation of this pathway is required for inducing formation of the OFZ. Loss of ER occurred within 24 hrs in ex vivo cultures, and nuclei became pyknotic. By 48 hrs nuclei were lost from central fiber cells, but were retained in controls. Beclin-1-positive vesicles were discovered in the central fiber cells at E15 but were not detected in adjacent cortical lens fiber cells, suggesting a role for autophagy in OFZ formation. Co-localization of the ER marker calreticulin with Beclin-1 and another autophagosome marker, LC3B, provided strong evidence that removal of organelles during fiber cell differentiation involves an autophagic process. Immunoblot analysis confirmed that it is the autophagosome form of LC3B that is expressed in differentiating lens fiber cells.

Conclusions: : JNK inactivation is the inducing signal for organelles loss in the lens fiber cells and autophagy plays a role in the removal of organelles to form the OFZ.

Keywords: differentiation • development • signal transduction 
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