Identification of Retina-Specific Dynamin-1 Protein Complexes

Abstract

Purpose: : Dynamin-1 is a highly-expressed neuronal-specific GTPase implicated in endocytosis and vesicle movement at presynaptic terminals of the brain; however, its role in the retina is unknown. We previously identified a retinal-specific isoform of Dynamin-1 that localizes to photoreceptor synaptic terminals and binds Tulp1, a photoreceptor-specific protein thought to be involved in vesicle movement. To further investigate this protein complex, we sought to identify additional Dynamin-1-binding partners in retinal tissue.

Methods: : Immunoprecipitation and Western blot experiments were performed with bovine and mouse retinal homogenates using a Dynamin-1 antibody raised against the epitope exclusive to the retinal-specific isoform. Immunoprecipitation products were separated by SDS-PAGE, protein bands were excised and in-gel digested with trypsin and identified by LC MS/MS. Identified proteins were localized in mouse retinal sections by immunohistochemistry.

Results: : We identified several canonical endocytic proteins by immunoprecipitation, including alpha-tubulin and clathrin. Most notably, we identified Dynamin-3, a protein thought to mediate vesicle cycling, in both bovine and murine retinal homogenates. Western blot analysis indicates that Dynamin-3 is expressed in the retina, and immunohistochemistry shows that it co-localizes with Dynamin-1 and Tulp1 in photoreceptor synaptic terminals. Reciprocal immunoprecipitation, immunohistochemistry and proximity ligand analyses are in progress to verify and further evaluate these interactions.

Conclusions: : Our data indicate that Dynamin-1 is likely involved in the endocytic pathway of the retina. Dynamin-3 is expressed in photoreceptor cells and may interact with the Dynamin-1/Tulp1 complex in the movement of vesicles in synaptic terminals. Further investigation into the nature of the interactions of these proteins will be carried out.

Keywords: photoreceptors • synapse • protein structure/function