Abstract
Purpose: :
Synucleins are small highly conserved proteins in vertebrates extensively expressed in the brain. Gamma–synuclein (γ–syn) is genetically linked to Parkinson's diseases. Far less is known about two other members of the synuclein family, alpha–synuclein (α–syn) and beta–synuclein (ß–syn). Previously we have shown that all three members of the synuclein family are abundant in ocular tissues and at least one of them, i.e. γ–syn is implicated in eye diseases. According to recent data, γ–syn is a marker for retinal ganglion cell loss. In spite of high similarity in amino acid sequences, members of the synuclein have different properties. Here we present data on the subcellular localization and properties of γ–syn in several cell lines of ocular origin.
Methods: :
The following cell cultures of ocular origin were used: photoreceptor cell line 661W, retinoblastoma Y79, human astrocytes from the optic nerve head, uveal melanoma cells OM431. Stable clones of Y79 cells expressing α– and γ–syn were isolated by limited dilution method in the presence of G418. The effect of synucleins on neurite outgrowth was measured using Neurite Outgrowth Quantification Assay (Chemicon). Western blotting and immunocytochemical staining were used according to the standard procedures.
Results: :
We found that contrary to the current opinion, γ–syn is not exclusively a cytoplasmic protein, but has a dynamic localization and can associate with subcellular structures. It is present in the perinuclear area in the forms of dots and may be associated to centrosomes. On late steps of mitosis γ–syn is not found in the centrosomes, and redistributes to the midbody in telophase. Under stress conditions (420 C, 60 min) a translocation of γ–syn from the perinuclear area to the nucleus occurs exhibiting nucleocytoplasmic shuttling. γ–Syn may be a component of filopodia–like cell protrusions that also contain vinculin, a known protein component of adherent junctions. γ–Syn overexpression reduces neurite outgrowth in a greater extent than α–syn overexpression.
Conclusions: :
These data support the view that γ–syn may easily change its intracellular localization and associate with subcellular structures in response to intracellular signaling and/or stress. The results are discussed in connection with implication of γ–syn in diseases.
Keywords: immunohistochemistry • cytoskeleton • cell adhesions/cell junctions