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M Joseph Costello, Ashik Mohamed, Kurt Gilliland, Sönke Johnsen, Kevin Schey; High resolution confocal microscopy of potential newly described nuclear excisosomes in primate lenses. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1213.
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© ARVO (1962-2015); The Authors (2016-present)
The final stage of the formation of the organelle-free zone (OFZ) in vertebrate lenses involves the degradation of fiber cell nuclei. Recent reports (PLoS One 2016) of a specific organelle, the nuclear excisosome, proposed to degrade the nuclear envelope near the OFZ in chick embryo lenses suggests that this may be a general phenomenon that may also occur in primate lenses. Preliminary transmission electron microscopy (TEM) data (ARVO2016 E-Abstract 3076) showed a large membrane complex associated with monkey lens nuclei near the OFZ. Additional confocal microscopy evidence was sought to substantiate the proposal that primate lenses also employed the nuclear excisosome.
Transparent human donor lenses (India, n=3, ages 55, 67, 70) and macaque monkey lenses (Vanderbilt, n=3, ages 8, 9, 13) were initially fixed in formalin then paraformaldehyde and stored in buffer until Vibratome sectioned and processed for TEM or confocal microscopy as reported previously. The confocal images were obtained with a Zeiss LSM880 with Airyscan on sections stained with DiI for membranes and DAPI for nuclei.
Sections midway from the anterior pole to the equator displayed numerous nuclei near the OFZ in a narrow band about 100 µm from the surface. These nuclei were in the remodeling zone where irregular cell shapes and interdigitations were prominent. Nuclei in this region had irregular shapes and displayed large spherical objects often about 3 µm in diameter, associated mainly with the tapered ends of nuclei and intensely labeled with DiI, consistent with the TEM data that showed large membrane complexes containing multilamellar lipid aggregates. These nuclei were distinct from nuclei in the epithelium or the bow region that had smooth contours and no such associated objects. Within about 10 fiber cell layers deeper, no intact nuclei were visible. In this region, small DAPI stained nuclear degradation remnants, about 200 nm in diameter, were visible.
Nuclear degradation in primate lenses occurred in the remodeling zone near the OFZ over a very narrow band of about 10 cell layers. Intact nuclei were often observed in direct contact with large spherical objects that contained multilamellar lipid and intense DiI fluorescence suggesting that these were nuclear excisosomes that rapidly degraded the nuclear envelope leading to loss of nuclei leaving only small nuclear fragments.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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