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First published online 14 January 2004
doi: 10.1242/dev.00976

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Moesin contributes an essential structural role in Drosophila photoreceptor morphogenesis

Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA

View larger version (173K): [in a new window]   Fig. 1. Moesin and Crumbs define complementary subdomains of the photoreceptor apical surface. (A) Distinct apical membrane domains are evident in a transmission electron micrograph cross-section of an adult wild-type ommatidium: rhabdomeres, closely-packed photosensitive microvilli and the stalk (S), collaring the rhabdomere and connecting to the adherens junctions (AJ). Both stalk and rhabdomere face the inter-rhabdomeral space (IRS). Stalks of photoreceptors R1-R6 are typically folded, often with coated pits in the pocket (inset). (B) Confocal micrograph shows activated Moesin (p-Moesin, green) localized to the rhabdomere base, and Crumbs (blue) localized to the stalk. Rhodamine-phalloidin (red) stains axial actin filaments of rhabdomere microvilli and the rhabdomere terminal web (RTW). (C) Rhabdomere and stalk primordial domains differentiate by 50% pupal development (pd). Rhabdomere primordia are covered with short finger-like microvilli, and the smooth membrane of the future stalk displays a cytoplasmic density typical of membranes heavily invested with cytoskeleton. (D) 50% pd, p-Moesin (green) has resolved to the rhabdomere base and Crumbs (blue) to the stalk. Crumbs localization parallels stalk morphology. Crumbs staining is not present in R1, R3 and R6 at this stage. Anterior is to the right in all figures. Scale bars: 1 µm in A,C; 10 µm in B,D.

View larger version (163K): [in a new window]   Fig. 2. Rescue of MoePL54 lethality. (A) Confocal image of 50% pd hs>Moesin Myc retina shows F-actin staining (red) and full-length wild-type Moesin (anti-Myc, green). Myc-tagged Moesin concentrates at the rhabdomere base and is distributed throughout the cell cytoplasm (asterisks). Gal4/UAS transgenes show variable levels of protein expression in pupal eyes. (B) Confocal micrograph of a live 65% pd hs>Moesin GFP retina. Strong GFP signal is seen in rhabdomeres and pigment cell cytoplasm; lesser signal is also evident in photoreceptor cytoplasm. (C) Act5C>Moe-GFP rescues MoePL54 lethality. Confocal image of 70% pd retina shows F-actin staining (red) and Moesin-GFP (green) in the MoePL54 hemizygous background. Photoreceptors with the most severe defects show the lowest detectable levels of Moesin-GFP (arrowheads). Moesin-GFP concentrates at the rhabdomere base in cells expressing lower protein amounts (arrow). (D) Electron micrograph cross-section of a 1-day post-eclosion MoePL54/Y; Act5C>Moe-GFP adult ommatidium. Photoreceptors of Act5C>Moesin-GFP rescued MoePL54 contain imperfect rhabdomeres, frequently exhibiting defects in size and microvillar organization. Scale bars: 10 µm in A,B,C; 1 µm in D.

View larger version (124K): [in a new window]   Fig. 3. Moesin RNAi disorganizes F-actin and apical microvilli. (A, C) Confocal micrographs show F-actin staining (red) and Moesin immunolocalization (green). (A) In a 57% pd hs>IR-Moesin retina, photoreceptors lacking Moesin (arrowheads) are reduced in size and exhibit abnormal F-actin distribution. The asterisked ommatidium (enlarged, right) shows lack of Moesin in R1, R3 and R7. (B) An electron micrograph of 57% pd hs>IR-Moesin ommatidium shows loosely organized rhabdomere microvilli (compare with Fig. 5A); the rhabdomere of R5 is reduced. (C) GMR>IR-Moesin photoreceptor apical membrane organization is devastated by Moesin loss. A single photoreceptor (arrowhead) retains strong Moesin signal and an intact rhabdomere. (D) Electron micrograph of 70% pd GMR>IR-Moesin ommatidium shows severe rhabdomere defects. The asterisked photoreceptor lacks a rhabdomere. Adherens junctions appear normal (B,D; arrows). Scale bars: 10 µm in A,C; 1 µm in B,D.

View larger version (160K): [in a new window]   Fig. 4. Genetic Moesin loss-of-function disrupts rhabdomere morphogenesis. Confocal micrographs show F-actin staining (red) and Moesin immunolocalization (green). (A) Photoreceptors lacking Moesin exhibit extreme F-actin disorganization at the apical surface (arrowheads) in homozygous null MoePL54 65% pd eye clones. (B) Photoreceptors are missing (asterisks) in 65% pd eye clones of a second allele, MoeX5. (C,D) Retinas of 1-day post-eclosion MoeG0323/MoeG0323; Rho172R/CyO flies show wild-type Moesin immunolocalization at the rhabdomere base in cross-section (C) and side view (D, arrowheads). In side view, Moesin is prominent on the apical surfaces of cone cells lying above the rhabdomeres. Scale bars: 10 µm.

View larger version (122K): [in a new window]   Fig. 5. Dominant-active T559D Moesin expression prevents photoreceptor apical regionalization. (A,B) Electron micrographs of 65% pd wild type (A) and hs>T559D Moesin Myc (B) ommatidium. The apical membrane, spanning between adherens junctions (A,B; arrows), is severely disorganized by T559D Moesin expression (B). In this ommatidium (B), R5 is less affected, showing clear rhabdomere and stalk domains, presumably due to lower levels of T559D Moesin expression. (C,D) The apical actin cytoskeleton is severely disrupted in dominant-active Moesin-expressing cells, and Crumbs (C) does not properly relocalize to the stalk. (D) The field in C, showing Crumbs staining (blue) merged with staining for F-actin (red) and Myc (green). Red rhabdomeres of `escaper' cells, not expressing T559D Moesin Myc, appear normal. (E) The ommatidium marked with an asterisk in D is shown at a higher magnification. T559D Moesin was not expressed equally in all cells in this ommatidium, as reflected by the lack of Myc staining in the asterisked R7. F-actin in the rhabdomere of R7 is orderly, and Crumbs was properly cleared from the rhabdomere membrane and localizes to the stalk. Compare with neighboring photoreceptors in D. Scale bars: 1 µm in A,B; 10 µm in D.

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