Studying the different roles and behaviors of Syntaxin 5 conformers in trafficking using advanced bioimaging

  • Maria CARDOSO DA ROCHA

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

Abstract

Retro-2 was recently discovered as the first pharmacological inhibitor of the ER exit site (ERES). This compound protects cells against Shiga-like toxins and Ricin by inhibiting their retrograde trafficking from the Endoplasmic Reticulum (ER) to the Golgi. Retro-2 induces the partial relocalization of Syntaxin5 (Stx5) from the Golgi to the ER, blocking its interaction with GPP130, which is required for retrograde transport. Interestingly, the interaction of Stx5 with canonical SNARE proteins is maintained, suggesting Retro-2 has differential effects on these two sets of interactions, but the reason behind it is still unknown. In this project, we hypothesise that the two conformers of Stx5 could be responsible for the different effects of Retro-2 on Stx5 interactions. Therefore, the aims were to determine if Retro-2 causes I) redistribution of one conformation over the other, II) determine whether the two conformations are responsible for the differential effects of Retro-2 on Stx5’s interaction with canonical SNAREs versus GPP130 and III) identify if specific stages of trafficking are dependent on certain conformations. The first steps to address this question were the optimisation of immunofluorescence and transfection conditions and the identification of tools to study the conformations of Stx5 and their interaction with GPP130. I established a panel of antibodies that were suitable for use in human or rat cell lines. Using advanced imaging approaches, I used these tools to determine that Stx5 colocalizes with GPP130 at the cis-Golgi. To address the hypothesis that the closed conformation may be primarily targeted by Retro2 treatment, using an antibody specific to open Stx5, I showed that there was a higher redistribution of open Stx5 than closed Stx5 from Golgi to ER in response to Retro-2 treatment. Finally, I started to optimize the tools to study the interaction between Stx5 and GPP130. This work forms the foundation to compare the interaction and localization of the two Stx5 conformations with GPP130 and the canonical SNARE partners, which will be continued in Dr Forrester’s group, thus elucidating a specific role for Syn5 conformers in retrograde trafficking.
Date of Award16 Jan 2024
Original languageEnglish
Awarding Institution
  • University of Namur
SupervisorAlison Forrester (Supervisor)

Keywords

  • Syntaxin 5; SNAREs; Retro-2; Trafficking; Bioimaging

Cite this

'