Graduation Year

2024

Graduation Month

May

Document Type

Thesis

Degree Name

Bachelor of Science

School or Department

Biological Sciences, Division of

Major

Neuroscience – Cellular and Molecular

Faculty Mentor Department

Biological Sciences, Division of

Faculty Mentor

Jesse C. Hay

Keywords

COPII, Protein Transport, ALG-2, Peflin, Neuroblastoma, Glioblastoma

Subject Categories

Cell Biology | Molecular and Cellular Neuroscience | Molecular Biology

Abstract

Regulatory mechanisms of protein trafficking from the endoplasmic reticulum (ER) are critical to understand since neurodegenerative diseases involve defects in this process leading to chronic ER stress and cell death. This study aimed to better understand the calcium regulatory mechanisms of ER-to-Golgi trafficking in hybrid neuroglioblastoma cells (NG108). Specifically, we asked whether proteasomal degradation of transport machinery was involved in the previously demonstrated upregulation of ER-to-Golgi transport evoked by calcium signaling. Based upon previous literature, we believe that the calcium induced increase in transport is due to the activation of the calcium sensitive regulatory protein apoptosis linked gene 2 (ALG-2) and its interactions with the inhibitory regulatory protein peflin. A potential mechanism is that after calcium induced ALG-2 dissociation from peflin, peflin is degraded by the proteasome and the removal of its inhibitory effects cause the increase in transport. This hypothesis was tested by NG108 transfection with a fluorescent cargo protein engineered to allow inducible, synchronous trafficking from the ER. Cargo transport was completed for 15 minutes and the cells were subsequently fixed and immunolabeled for detection of the Golgi complex. Cells were imaged on a widefield microscope and the images were analyzed to quantitate the transport index, a measure of the rate of cargo transport. Prior to transport cells were treated with the calcium agonists histamine or ATP and/or the proteasome inhibitor MG132. Results indicated that calcium signaling caused an increase in cargo transport, as expected. Importantly, inhibition of the proteasome caused a decrease in cargo transport. However, treatment with both a calcium agonist and the proteasome inhibitor caused an increase compared to treatment with just the proteasome inhibitor. Our new results show that proteasomal activity is required for optimum transport but is not involved in the calcium stimulation of transport per se. This suggests that proteasomal activity and calcium fluctuations function to regulate transport rates independently.

Honors College Research Project

1

GLI Capstone Project

no

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© Copyright 2024 Samuel A. Galindo