title: GFP-moesin illuminates actin cytoskeleton dynamics in living tissue and demonstrates cell shape changes during morphogenesis in Drosophila.
abstract: Moesin, ezrin, and radixin (MER) are components of the cortical actin cytoskeleton and membrane processes such as filopodia and microvilli. Their C-terminal tails contain an extended region that is predicted to be helical, an actin binding domain, and a region(s) that participates in self-association. We engineered an in vivo fluorescent actin binding protein (GFP-moe) by joining sequences that encode the jellyfish green fluorescent protein (GFP) to sequences that encode the C-terminal end of the sole Drosophila MER homolog, moesin [Moesin-like gene product, referred to previously as the D17 MER-like protein; Edwards et al., 1994, Proc. Natl. Acad. Sci. USA 91, 4589], and Dmoesin [McCartney and Fehon, 1996, J. Cell Biol. 133, 843]. Transgenic flies expressing this fusion protein under control of the hsp70 promoter were generated and used for analysis of cell shape changes during morphogenesis of various developmental stages and tissues. Following heat shock, high levels of stable fusion protein are produced by all somatic tissues. GFP-moe localizes to the cortical actin cytoskeleton, providing a strong in vivo marker for cell shape and pattern during epithelial morphogenesis. The protein also becomes highly enriched in pseudopods, microvilli, axons, denticles, the border cell process, and other membrane projections, potentially by binding to endogenous moesin as well as actin. We show that GFP-moe can be used to examine the development and behavior of these dynamic structures in live specimens. We observe a bright green fluorescent, presumably actin-rich, polar cell proboscis that inserts itself into the forming micropyle and appears to maintain an opening for sperm passage around which the chorion is formed. We also confirm the existence of an actin-rich purse string at the leading edge of the lateral epidermis and provide a dynamic analysis of its behavior as it migrates during dorsal closure. Observations of embryos, larvae, and pupae show that GFP-moe is also useful for labeling the developing nervous system and will be a good general marker of dynamic cell behavior during morphogenesis in live tissues and demonstrate that fusion of a subcellular localization signal to GFP greatly increases its utility as a cell marker.
authors.affiliation: Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: K A
authors.initials: KA
authors.lastname: Edwards
authors.forename: M
authors.initials: M
authors.lastname: Demsky
authors.forename: R A
authors.initials: RA
authors.lastname: Montague
authors.forename: N
authors.initials: N
authors.lastname: Weymouth
authors.forename: D P
authors.initials: DP
authors.lastname: Kiehart
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D004330:Drosophila / Q000196:embryology*; D004625:Embryo, Nonmammalian / Q000166:cytology / Q000502:physiology*; D005260:Female; D018507:Gene Expression Regulation, Developmental; D049452:Green Fluorescent Proteins; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics; D008164:Luminescent Proteins / Q000096:biosynthesis; D008840:Microfilament Proteins; D009024:Morphogenesis; D009420:Nervous System / Q000166:cytology / Q000196:embryology; D009474:Neurons / Q000166:cytology / Q000502:physiology; D010053:Ovary / Q000502:physiology; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry; D011679:Pupa; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D037401:Scyphozoa
chemical_list: D018840:HSP70 Heat-Shock Proteins; D008164:Luminescent Proteins; D008840:Microfilament Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; C071058:moesin; D049452:Green Fluorescent Proteins