Send to

Choose Destination
See comment in PubMed Commons below
Am J Physiol Cell Physiol. 2013 Feb 15;304(4):C312-23. doi: 10.1152/ajpcell.00311.2012. Epub 2012 Nov 14.

Definitive evidence using enucleated cytoplasts for a nongenomic basis for the cystic change in endoplasmic reticulum structure caused by STAT5a/b siRNAs.

Author information

Department of Cell Biology and Anatomy, New York Medical College, Valhalla, NY 10595, USA.


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.

[Indexed for MEDLINE]
Free PMC Article
PubMed Commons home

PubMed Commons

How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for HighWire Icon for PubMed Central
    Loading ...
    Support Center