Does Golgi Morphology Predict Golgi Trafficking Function?

Presentation Number: SUN 244
Date of Presentation: April 2nd, 2017

Ariel Crocker*1, Matthew D Buschman2, William Fenical3 and Seth Joel Field4
1University of South Florida Morsani College of Medicine, 2University of California, San Diego, 3Scripps Institute of Oceanography, 4University of California, San Diego, San Diego, CA

Abstract

The GOLPH3 pathway is a key component in anterograde trafficking from the Golgi to the plasma membrane. The pathway provides a link from the trans-Golgi membrane to the F-actin cytoskeleton, providing a tensile force that is required for vesicle budding off the Golgi. Previous data from the lab show that inhibition of the GOLPH3 pathway, results in both Golgi compaction and decreased Golgi-to-plasma membrane trafficking. We hypothesized that activation of the GOLPH3 pathway, will result in expansion of Golgi area and an increase in protein trafficking to the plasma membrane. To test this, we used fractionated extracts, a pool of small molecules, that had previously been identified to increase Golgi area, by unknown mechanisms of action. To measure Golgi-to-plasma membrane trafficking, we treated HEK293 cells stabling expressing ts045-VSVG-GFP protein with these fractions. After a 4-hour treatment at the non-permissive temperature (40°C), the cells were placed at the permissive temperature (32°C) for 75 minutes to allow trafficking of the ts045-VSVG-GFP protein to the plasma membrane. Cells were fixed, blocked, and stained with an antibody specific for exofacial VSVG. Trafficking of VSVG to the plasma membrane was measured by summing the exofacial VSVG fluorescence and normalizing to total VSVG-GFP fluorescence. To measure Golgi area we used HeLa cells stably expressing the Golgi marker a-mannosidaseII-GFP, treated with the same fractions for 4 hours. To determine if there is a correlation between Golgi area and Golgi function, we pooled data from several experiments and plotted Golgi area vs. VSVG trafficking for each test fraction. None of the test fractions that cause Golgi expansion show a strong increase in VSVG trafficking to the plasma membrane. However, several strongly block trafficking. Our data shows that there is not a clear correlation between increased Golgi area and Golgi trafficking. We observed a diverse range of expanded Golgi morphologies with various treatment fractions. When treatment fractions were grouped based on similar Golgi morphology and trafficking phenotypes, we observed a trend that cells with an expanded Golgi with a normal ribbon-like morphology were more likely to have minor alterations in trafficking function (normal to low trafficking). While cells displaying a more disrupted punctate and/or diffuse Golgi morphology, were more likely to have major inhibition of trafficking function. Overall, we conclude that disruption of the normal Golgi structure (either compaction or expansion) has significant consequences on protein trafficking.

 

Nothing to Disclose: AC, MDB, WF, SJF