NYMC Faculty Publications

Definitive Evidence Using Enucleated Cytoplasts for a Nongenomic Basis for the Cystic Change in Endoplasmic Reticulum Structure Caused by STAT5a/b siRNAs

Author Type(s)

Faculty

DOI

10.1152/ajpcell.00311.2012

Journal Title

American Journal of Physiology. Cell Physiology

First Page

312

Last Page

323

Document Type

Article

Publication Date

2-15-2013

Department

Cell Biology and Anatomy

Keywords

Cells, Cultured, Cycloheximide, Dichlororibofuranosylbenzimidazole, Endoplasmic Reticulum, Endothelial Cells, Gene Knockdown Techniques, Humans, Membrane Proteins, Nucleic Acid Synthesis Inhibitors, Protein Synthesis Inhibitors, Pulmonary Artery, RNA, Small Interfering, STAT5 Transcription Factor, Single-Cell Analysis, Tumor Suppressor Proteins

Disciplines

Medical Cell Biology | Medical Physiology | Medicine and Health Sciences

Abstract

STAT5a/b species are well known as transcription factors that regulate nuclear gene expression. In a novel line of research in human pulmonary arterial endothelial cells (HPAECs), we previously observed that STAT5a associated with the Golgi apparatus and that siRNA-mediated knockdown of STAT5a/b led to the rapid development of a dramatic cystic change in the endoplasmic reticulum (ER) characterized by deposition along cyst membranes and tubule-to-cyst boundaries of the proteins reticulon-4 (RTN4; also called Nogo-B) and the ER-resident GTPase atlastin-3 (ATL3) and Golgi fragmentation. We now report that STAT5a can be observed in ER sheets in digitonin-permeabilized HPAECs and that anti-STAT5a cross- immunopanned ATL3 but not RTN4. Moreover, there was marked accumulation of the 63-kDa cytoskeleton-linking membrane protein and ER-spacer CLIMP63 (also called cytoskeleton-associated protein 4, CKAP4) and KDEL-mCherry within the cysts. That the STAT5a/b-siRNA-induced cystic ER phenotype developed in the presence of the transcription inhibitor 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) had suggested that the mechanism was independent of the transcription factor functions of STAT5a/b, i.e., was "nongenomic." We have now definitively tested the requirement for the nucleus in eliciting the STAT5a/b-siRNA-induced cystic ER phenotype. Enucleated HPAEC cytoplasts were prepared using adherent 35-mm cultures using the cytochalasin B-centrifugation method (typically yielding 65-75% enucleation). STAT5a/b siRNAs readily elicited the cystic ER phenotype including the marked luminal accumulation of CLIMP63 and Golgi fragmentation in the recovered HPAEC cytoplasts demonstrably lacking a nucleus. These studies provide unequivocal evidence using enucleated cytoplasts for a nongenomic mechanism(s) underlying the cystic change in ER structure elicited by STAT5a/b knockdown.

Link to Full Text

Share

COinS