Molecular Mechanisms of Golgi Structure Alterations During Stress

Abstract

In mammals, the Golgi apparatus has a unique stacked structure that is important for its function; yet, the Golgi structure is dynamic and its structure and function changes in response to different stress conditions applied to the cell. Since Golgi functions are abnormal in stress, it implies that Golgi structure may play a critical role in human diseases.

This research project studied how the Golgi apparatus responds to stress. The study process followed in a stepwise progression: First, it was necessary to determine how the Golgi responded to endoplasmic reticulum (ER) stress; findings indicated that the Golgi is fragmented in response to thapsigargin (TG) but not to other ER stress inducers such as tunicamycin (Tm) and dithiothreitol (DTT). Since TG treatment is known to cause an increase in cytosolic calcium, it was useful to determine whether a calcium-sensitive kinase could act as the trigger of Golgi morphological change. This led to identification of protein kinase C-α (PKCα) and its substrate GRASP55 in calcium stress-induced Golgi fragmentation, and abnormal Golgi functions. More generally, cytosolic calcium is a logical trigger in the Golgi structure alteration process during stress, as it has been shown to be regulated during several cellular processes. Subsequently, this study confirmed that activating or inflammatory agents induce Golgi fragmentation and cytosolic calcium increase via a similar mechanism.

These findings are of great interest to cell biologists who are working on Golgi dynamics, membrane trafficking, cellular responses to stresses, and histamine biology.

Next, several novel findings are described where, reactive oxidative species (ROS) induce the degradation of Golgi structural proteins in the trans-Golgi, including Arl1, Golgin-97, and Golgin-245, and thereby impair membrane trafficking.

This work is believed to be the first systematic study of how ROS affects Golgi structure and function. It revealed the trans-Golgi and trafficking at the trans-Golgi as novel targets of ROS in cells, which may help understand the toxicity of ROS in human diseases. It also found that the degradation of Arl1, Golgin-97 and Golgin-245 is not mediated by proteasomes nor lysosomes, but rather by cytosolic proteases. This finding underscores the importance of cytosolic proteases whose importance has been often underestimated in recent studies.

During this study, some molecular tools were designed and constructed to detect Golgi calcium signals in cells using GCaMP-based sensors. The main advantage of this sensor is that the GCaMP calcium probe is covalently linked to GRASP55 on the Golgi and can directly sense and detect fluctuations of local cytoplasmic calcium in the vicinity of the Golgi. This tool is essentially a de facto Golgi stress sensor, which can be used by others who wish to quickly screen for compounds that reduce calcium-related Golgi fragmentation during stress.

Besides calcium imaging was an opportunity to develop another set of tools for imaging specific proteins of interest at the ultrastructural level. These sensors utilize a recombinant ascorbate peroxidase (apex) enzyme to catalytically precipitate an electron dense product in the vicinity of a cellular protein of interest. In the lab the apex gene was linked to each GRASP55 and GRASP65 with the hopes that future researchers will utilize this fusion protein to identify the precise intracellular localizations of these proteins with high resolution.