Extracellular Localisation of the C-Terminus of DDX4 Confirmed by Immunocytochemistry and Fluorescence-Activated Cell Sorting

• Published in: Cells (Basel, Switzerland) Switzerland), 2019-06, Vol.8 (6), p.578
• Main Authors: Clarkson, Yvonne L, Weatherall, Emma, Waterfall, Martin, McLaughlin, Marie, Lu, Haojiang, Skehel, Paul A, Anderson, Richard A, Telfer, Evelyn E
• Format: Article
• Language: English
• Subjects: Antibodies - pharmacology; Antibody Specificity; C-Terminus; Cell culture; Cell Membrane Permeability - drug effects; Cell Size - drug effects; Cell surface; DDX4; DEAD-box RNA Helicases - chemistry; DEAD-box RNA Helicases - metabolism; DNA helicase; Epitopes; Epitopes - metabolism; Extracellular Space - metabolism; Female; Females; Flow cytometry; Flow Cytometry - methods; fluorescence-activated cell sorting; Fusion protein; Gene expression; Gonads; HEK293 Cells; human; Humans; Immunocytochemistry; Immunoglobulins; Immunohistochemistry - methods; Laboratories; Localization; Mammals; Myc protein; Oocytes - drug effects; Oocytes - metabolism; Oogenesis; ovarian stem cells; Ovary - metabolism; Polymerase chain reaction; Protein Transport - drug effects; Proteins; Reproducibility of Results; Stem cells
• ISSN: 2073-4409; 2073-4409
• DOI: 10.3390/cells8060578

Putative oogonial stem cells (OSCs) have been isolated by fluorescence-activated cell sorting (FACS) from adult human ovarian tissue using an antibody against DEAD-box helicase 4 (DDX4). DDX4 has been reported to be germ cell specific within the gonads and localised intracellularly. White et al. (2012) hypothesised that the C-terminus of DDX4 is localised on the surface of putative OSCs but is internalised during the process of oogenesis. This hypothesis is controversial since it is assumed that RNA helicases function intracellularly with no extracellular expression. To determine whether the C-terminus of DDX4 could be expressed on the cell surface, we generated a novel expression construct to express full-length DDX4 as a DsRed2 fusion protein with unique C- and N-terminal epitope tags. DDX4 and the C-terminal myc tag were detected at the cell surface by immunocytochemistry and FACS of non-permeabilised human embryonic kidney HEK 293T cells transfected with the DDX4 construct. mRNA expression was detected in the DDX4-positive sorted cells by RT-PCR. This study clearly demonstrates that the C-terminus of DDX4 can be expressed on the cell surface despite its lack of a conventional membrane-targeting or secretory sequence. These results validate the use of antibody-based FACS to isolate DDX4-positive putative OSCs.