Identifying the localization of Sec16 and interacting partners during steady state protein secretion and perturbation

  • Randy-Jo MPIA MASSA

Student thesis: Master typesMaster in Biomedecine, professional focus in preclinical research

Abstract

The Endoplasmic Reticulum (ER) is a key part of the early secretory pathway. Proteins are folded in the ER, then transported to the Golgi and then secreted or distributed through the cell. To leave the ER, proteins are packaged at the ER exit sites (ERES) which are areas of the ER made up of different proteins that interact dynamically to form COPII coated carriers that recruit cargo and transport them to the Golgi. Retro-2 has been shown to inhibit specific anterograde trafficking through targeting ERES protein Sec16A. It has previously been shown that Retro-2 treatment decreases interactions between Sec16A and ERES proteins. Therefore, I hypothesize that loss of these interactions may change the available pools of ERES proteins in the cell, allowing them to play increased roles at non-ERES processes. Using confocal microscopy, I have optimized immunofluorescence and transfection protocols to analyze the colocalization of ERES proteins at ERES at steady state or treated with Retro-2. Retro-2 causes a change in recruitment of specific ERES components to the ERES. Interestingly, the disruption of inner – outer coat recruitment is potentially responsible for the decreased ERES function induced by Retro-2. I present an image analysis workflow that I use to quantify fluorescence intensity of proteins colocalizing at the ERES. Using this protocol, I examine the free pools of ERES proteins, and the possibility that they play increased roles non-ERES processes within the cells as a result of Retro-2 treatment. Although preliminary, the data here suggest that Retro-2 may have a regulatory role on the mTOR pathway. This project explores which cellular processes are linked to the acute inhibition of ERES by Retro-2 and shows that Retro-2 treatment may modulate the mTOR pathway. It also provides a new hypothesis that needs to be studied in more depth, that Retro-2 disrupts inner and outer COPII coat recruitment at a low level, a mechanism that could account for the partial decrease in ERES function previously reported. This project is one of the first performed in Dr Forrester’s group, and therefore has established a number of protocols and produced results that will be build upon in years to come.
Date of Award16 Jan 2024
Original languageEnglish
Awarding Institution
  • University of Namur
SupervisorAlison Forrester (Supervisor)

Keywords

  • ERES
  • Sec16A
  • Retro-2
  • trafficking
  • microscopy

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