BIBLIOS

Document type
Journal articles

Document subtype
Full paper

Title
A novel microscopy-based assay identifies extended synaptotagmin-1 (ESYT1) as a positive regulator of anoctamin 1 traffic

Participants in the publication
Joana R. Lérias (Author)
BioISI - Biosystems & Integrative Sciences Institute
Madalena C. Pinto (Author)
BioISI
Hugo M. Botelho (Author)
Dep. Química e Bioquímica
Nikhil T. Awatade (Author)
Margarida C. Quaresma (Author)
Iris A.L. Silva (Author)
BioISI
Podchanart Wanitchakool (Author)
Rainer Schreiber (Author)
Rainer Pepperkok (Author)
Karl Kunzelmann (Author)
Margarida D. Amaral (Author)
Dep. Química e Bioquímica
BioISI

Summary
An attractive possibility to treat Cystic Fibrosis (CF), a severe condition caused by dysfunctional CFTR, an epithelial anion channel, is through the activation of alternative (non-CFTR) anion channels. Anoctamin 1 (ANO1) was demonstrated to be a Ca2+-activated chloride channel (CaCC) and thus of high potential to replace CFTR. Despite that ANO1 is expressed in human lung CF tissue, it is present at the cell surface at very low levels. In addition, little is known about regulation of ANO1 traffic, namely which factors promote its plasma membrane (PM) localization. Here, we generated a novel cellular model, expressing an inducible 3HA-ANO1-eGFP construct, and validated its usage as a microscopy tool to monitor for ANO1 traffic. We demonstrate the robustness and specificity of this cell-based assay, by the identification of siRNAs acting both as ANO1 traffic enhancer and inhibitor, targeting respectively COPB1 and ESYT1 (extended synaptotagmin-1), the latter involved in coupling of the endoplasmic reticulum to the PM at specific microdomains. We further show that knockdown of ESYT1 (and family members ESYT2 and ESYT3) significantly decreased ANO1 current density. This ANO1 cell-based assay constitutes an important tool to be further used in high-throughput screens and drug discovery of high relevance for CF and cancer.


Institution
BioISI - Biosystems & Integrative Sciences Institute

Where published
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

Publication Identifiers
ISSN - 0167-4889

Publisher
Elsevier BV



Document Identifiers
URL - http://dx.doi.org/10.1016/j.bbamcr.2017.11.009
DOI - https://doi.org/10.1016/j.bbamcr.2017.11.009

Rankings
SCIMAGO Q1 (2018) - 2.092 - Cell Biology
SCIMAGO Q1 (2018) - 2.092 - Molecular Biology
Web Of Science Q1 (2018) - 4.739 - BIOCHEMISTRY & MOLECULAR BIOLOGY - SCIE

Keywords
Endoplasmic reticulum Intracellular traffic Calcium-activated chloride channels Cystic fibrosis Automated fluorescence microscopy