Compositional analysis of non-caveolar Caveolin-1-containing domains in Müller glia

Abstract

Purpose : Caveolae, flask-shaped plasma membrane containing caveolin-1 (Cav1) and Cavin1/PTRF, are thought to play important roles in several ocular diseases. Cav1 and Cavin1/PTRF are co-expressed in many tissues, and deletion of one significantly downregulates expression of the other, resulting in loss of caveolae. Cavin1/PTRF is required to sequester Cav1 into caveolae and is the only protein reported to have a stabilizing effect on Cav1. However, our data suggest that in Müller glia, Cav1 protein is stable without Cavin1/PTRF and exists preferentially outside caveolae (non-caveolar Cav1). Our goal is to identify the mechanism of Cav1 stabilization and define the protein components of non-caveolar Cav1 domains in Müller glia. Herein, we present proteomic data on Cav1-containing domains in the presence and absence of Cavin1/PTRF

Methods : We used quantitative mass spectrometry (MS), to assess the protein composition of non-caveolar Cav1 immunoprecipitates (IPs) from immortalized Müller glia (MIO-M1) cells and compared this composition to Cav1 IPs from cells transduced with adenovirus driving expression of Cavin1/PTRF. The Core Laboratory for Molecular and Cytometry Research at the University of Oklahoma Health Sciences Center performed MS analysis using the Thermo Lumos Fusion tribrid Orbitrap mass spectrometer. We used STRING database to predict protein interaction networks and molecular pathways.

Results : We have previously identified that Cav1 protein is stable in MIO-M1 cells without Cavin1/PTRF and are devoid of morphologically-identifiable caveolae. Expression of recombinant Cavin1/PTRF in MIO-M1 cells resulted in dramatic increases in caveolae as determined by transmission electron microscopy. Mass spectrometric analysis of Cav1 immunoprecipitates from Cavin1/PTRF-transduced and control cells revealed 175 proteins differentially-associated with Cav1 when Cavin1/PTRF is expressed (40 with increased association, including the expressed Cavin1/PTRF; 135 with decreased association). Proteins with significantly increased association included actin cytoskeletal components. Intriguingly, a number of ribosomal proteins showed significantly decreased association with Cav1 when Cavin1/PTRF was expressed

Conclusions : Cavin1/PTRF expression in MIO-M1 significantly increases actin cytoskeleton interaction with caveolae and reduces ribosomal association with non-caveolar domains

This is a 2021 ARVO Annual Meeting abstract.