title: Using in vivo imaging to measure RNA mobility in Xenopus laevis oocytes.
abstract: RNA localization in the Xenopus oocyte is responsible for the establishment of polarity during oogenesis as well as the specification of germ layers during embryogenesis. However, the inability to monitor mRNA localization in live vertebrate oocytes has posed a major barrier to understanding the mechanisms driving directional transport. Here we describe a method for imaging MS2 tagged RNA in live Xenopus oocytes to study the dynamics of RNA localization. We also focus on methods for implementing and analyzing FRAP data. This protocol is optimized for imaging of the RNAs in stage II oocytes but it can be adapted to study dynamics of other molecules during oogenesis. Using this approach, mobility can be measured in different regions of the oocyte, enabling the direct observation of molecular dynamics throughout the oocyte.
authors.affiliation: Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI 02912, USA.
authors.forename: Erin A
authors.initials: EA
authors.lastname: Powrie
authors.affiliation: Department of Applied Mathematics, Brown University, Providence, RI 02912, USA.
authors.forename: Veronica
authors.initials: V
authors.lastname: Ciocanel
authors.affiliation: Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI 02912, USA.
authors.forename: Jill A
authors.initials: JA
authors.lastname: Kreiling
authors.affiliation: Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
authors.forename: James A
authors.initials: JA
authors.lastname: Gagnon
authors.affiliation: Department of Applied Mathematics, Brown University, Providence, RI 02912, USA.
authors.forename: Bjӧrn
authors.initials: B
authors.lastname: Sandstede
authors.affiliation: Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI 02912, USA. Electronic address: kimberly_mowry@brown.edu.
authors.forename: Kimberly L
authors.initials: KL
authors.lastname: Mowry
mesh_terms: D000818:Animals; D005260:Female; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D018507:Gene Expression Regulation, Developmental; D009865:Oocytes / Q000378:metabolism / Q000648:ultrastructure*; D009866:Oogenesis / Q000235:genetics; D034443:RNA Transport; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000072760:Single Molecule Imaging / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods; D016707:Tissue Fixation / Q000379:methods; D029867:Xenopus Proteins / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D055544:beta-Globins / Q000235:genetics / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D012333:RNA, Messenger; D029867:Xenopus Proteins; D055544:beta-Globins
keywords: Confocal microscopy; Fluorescence recovery after photobleaching; Live cell imaging; Oogenesis; RNA localization; Xenopus