title: Cytokines, polarity proteins, and endosomal protein trafficking and signaling-the sertoli cell blood-testis barrier system in vitro as a study model.
abstract: Endosomal signaling is emerging as one of the most important cellular events that regulate signaling function in mammalian cells or an epithelium in response to changes in environment such as the presence of stimuli mediated by cytokines, toxicants, heat, ions during growth and development, and other cellular processes such as cytokinesis and spermatogenesis. Recent studies have shown that protein endocytosis-the initial step of endosomal signaling-involves the participation of polarity proteins, such as partitioning defective protein 6 (Par6), Cdc42 and 14-3-3 (also known as Par5), which in turn is regulated by cytokines (e.g., TGF-β2, TGF-β3) and testosterone at the Sertoli cell blood-testis barrier (BTB) in the mammalian testis. In this short method paper, we provide a detailed protocol of assessing protein endocytosis, the initial and also the most critical step of endosomal signaling at the Sertoli cell BTB. This biochemical endocytosis assay summarizes our experience for the last decade, which should likely be performed in conjunction with the dual-labeled immunofluorescence analysis to assess protein endocytosis. While we are using a Sertoli cell in vitro system that mimics the BTB in vivo, this approach should be applicable to virtually all mammalian cells.
authors.affiliation: The Mary M. Wohlford Laboratory for Male Contraceptive Research, Center for Biomedical Research, Population Council, New York, USA; Department of Reproductive Physiology, Zhejiang Academy of Medical Sciences, Hangzhou, Zhejiang, China.
authors.forename: Xiang
authors.initials: X
authors.lastname: Xiao
authors.affiliation: The Mary M. Wohlford Laboratory for Male Contraceptive Research, Center for Biomedical Research, Population Council, New York, USA.
authors.forename: Elissa W P
authors.initials: EW
authors.lastname: Wong
authors.affiliation: The Mary M. Wohlford Laboratory for Male Contraceptive Research, Center for Biomedical Research, Population Council, New York, USA.
authors.forename: Pearl P Y
authors.initials: PP
authors.lastname: Lie
authors.affiliation: The Mary M. Wohlford Laboratory for Male Contraceptive Research, Center for Biomedical Research, Population Council, New York, USA.
authors.forename: Dolores D
authors.initials: DD
authors.lastname: Mruk
authors.affiliation: Department of Biology, Hong Kong Baptist University, Hong Kong, China.
authors.forename: Chris K C
authors.initials: CK
authors.lastname: Wong
authors.affiliation: The Mary M. Wohlford Laboratory for Male Contraceptive Research, Center for Biomedical Research, Population Council, New York, USA. Electronic address: Y-Cheng@popcbr.rockefeller.edu.
authors.forename: C Yan
authors.initials: CY
authors.lastname: Cheng
mesh_terms: D000818:Animals; D001360:Avidin / Q000737:chemistry; D001681:Biological Assay; D001710:Biotin / Q000737:chemistry; D001814:Blood-Testis Barrier / Q000378:metabolism*; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis / Q000235:genetics*; D011992:Endosomes / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D008297:Male; D021381:Protein Transport; D051381:Rats; D017207:Rats, Sprague-Dawley; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D012708:Sertoli Cells / Q000166:cytology / Q000378:metabolism*; D015398:Signal Transduction; D013091:Spermatogenesis / Q000235:genetics*; D013739:Testosterone / Q000378:metabolism; D053781:Transforming Growth Factor beta2 / Q000235:genetics / Q000378:metabolism; D053782:Transforming Growth Factor beta3 / Q000235:genetics / Q000378:metabolism
chemical_list: C568773:Clmp protein, rat; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D011956:Receptors, Cell Surface; D053781:Transforming Growth Factor beta2; D053782:Transforming Growth Factor beta3; D001360:Avidin; D013739:Testosterone; D001710:Biotin
keywords: Blood–testis barrier; Endocytosis; Endosome; Protein endocytosis; Recycling; Sertoli cell; Spermatogenesis; Testis; Transcytosis