Characterization of Small Objects in Homogenates of the Squid Optic Lobe

Abstract

The squid (Loligo pealei) optic lobe is unique among sources of CNS synaptosomes via differential centrifugation, both in its ease and speed of dissection and preparation, and in the purity of the final result (Pekkurnaz et al. Biol. Bull. 2011). Despite the high purity of ∼1 µm-diameter synaptosomes in the synaptosome fraction, another class of 2-10 μm diameter objects were detected by one us (GP) in DIC microscopy of thin layers of this fraction. Recently, a publication assumed that these inclusions were large synaptosomes (Cefaliello et al. Molec. Neurobio. 2019). To test that assumption, and to further characterize these inclusions, we carried out a series of fluorescent labeling and dye exclusion experiments by direct imaging for size classification. Two major classes of objects were found, a larger class (L) 5-10 μm in diameter and a smaller class (S) that appeared more refractile, perfectly spherical, and averaged 2 μm in diameter. To identify some of the macromolecular content of these objects, we labeled them with fluorescent dyes for DNA, lipid, and protein aggregates. ‘L’ stained positive for the membrane dye BODIPY-sphingomyelin and the DNA marker Hoechst. The exclusion of rhodamine-dextran (10kDa) further corroborated the membrane-enclosed nature of the ‘L’ object. Electron microscopy showed a large ∼10 μm spheroid with a double membrane and dark material draping the internal membrane, suggesting that the ‘L’ inclusions were nuclei. The majority of the inclusions in the ‘S’ class did not exclude dyes, did not stain with the DNA dye Hoechst, and were positive for a rotamer fluorescent marker for intracellular aggregated misfolded proteins. Currently, efforts are underway to identify the protein composition of the ‘S’ droplets.