QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 28 November 2007
doi: 10.1242/dev.011437

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Development Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Liu, R. Articles by Parkhurst, S. M. Search for Related Content PubMed PubMed Citation Articles by Liu, R. Articles by Parkhurst, S. M.

Sisyphus, the Drosophila myosin XV homolog, traffics within filopodia transporting key sensory and adhesion cargos

Raymond Liu¹, Sarah Woolner^1,2,*, James E. Johndrow^1,*, David Metzger^1,*, Adriana Flores^1,* and Susan M. Parkhurst^1,

¹ Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
² MRC-LMCB, University College London, London WC1E 6BT, UK.

View larger version (84K): [in this window] [in a new window]   Fig. 1. Distribution of Syph in Drosophila embryos and S2R+ cells. (A-D) Dorsal view of successively older wild-type stage 14 Drosophila embryos stained with an anti-Syph antibody. Boxed regions are shown at higher magnification in A'-D'. Syph is predominantly cytoplasmic at this stage. Note Syph accumulation in the leading edge (LE) cells (arrows in A',B') before the onset of dorsal closure (DC). This accumulation resolves to the dorsal-most edge of LE cells (arrows in C') and seam edge (arrowhead) during DC, and then subsides once closure is complete (arrow in D'). (E-G) S2R+ cells stained with an anti-Syph antibody (E,E'; green in G,G') and phalloidin to visualize actin (F,F'; red in G,G'). The boxed region in F is shown at higher magnification in E'-G'. Syph is found along filopodial extensions and at a subset of their tips (arrows in G'), but not all tips (arrowhead in E' and G'). Scale bars: 20 µm in A-D'; 2 µm in E-G'.

View larger version (80K): [in this window] [in a new window]   Fig. 2. Syph is dynamically expressed in LE cells and filopodia during DC. (A-E) Dorsal view of an embryo expressing Syph-RFP in an En (striped) pattern during DC. Boxed region in A and equivalent regions of B-E are shown at higher magnification in A'-E'. Note that Syph accumulates in LE cells as the epithelial sheets meet (arrows in A'), and decreases after midline fusion. (F-F''') Syph accumulates in puncta at the apex of LE cells expressing GFP-Syph (arrow in F). These puncta accumulate in filopodia as the epithelial sheets meet (arrowhead in F,F') and disperse after cell fusion (brackets in F''-F'''). See Movie 1 in the supplementary material. (G-J) Syph localization in filopodia. Boxed region in G and equivalent regions of H-J are shown at higher magnification in G'-J'. Levels of Syph are dynamic within a growing filopodia: yellow lines in G'-J' follow the movement of a Syph puncta within a single filopodia. See Movie 2 in the supplementary material. (K-L'') Filopodia in LE cells in an embryo expressing both Syph-GFP (K,L; green in K'',L'') and mRFP-actin (K',L'; red in K'',L'') in an En pattern. As epithelial sheets approach each other, both Syph and actin accumulate in the LE cells and in their filopodia (K-K''), before resolving to a thin band of expression at the dorsal-most edge after fusion (L-L''). Scale bars: 20 µm in A-E'; 5 µm in F-L''.

View larger version (38K): [in this window] [in a new window]   Fig. 3. Syph interacts with DE-cadherin and several MT-associated proteins through its FERM domain. (A) 35S-labelled in vitro translated (IVT)-Syph C-terminal tail (input) binds to GST-cadICD, -Katanin, -aPKC, -Milt and -EB1. (B) DE-cadherin was immunoprecipitated from embryo lysate with an anti-Syph antibody, but not when primary antibody was omitted. (C) Diagram of the Syph protein fragments and substitution point mutations used to map protein-protein interactions. (D,E) GST-cadICD binds 35S-labelled IVT-Syph tail, -Syph t3, and the -L1 and -L3 regions of the Syph FERM domain. GST-cadICD binding maps to the L1a3 fragment within the L1 region and the L3b fragment within the L3 region. (F,G) Substitution point mutations in the L1 or L3 regions do not selectively inhibit cadICD binding to Syph. GST-cadICD and -Milt bind to 35S-labelled IVT-L1, -L3, -LGVE*, -QEF* and -HWS*, but show significantly reduced binding to IVT-IVQG*, -DAFT*, -STR* and -DMK*. GST-Katanin binds to IVT-L1, -L3, and all of the mutant fragments except IVT-DMK*. GST-aPKC binds to IVT-L1, -L3, and all the mutant proteins tested. EB1 binding does not map to the L1 or L3 regions and serves as a negative control. (H) Diagram of DE-cadherin protein and the protein fragments used to map its interaction with Syph. JMD: juxtamembrane domain, CBD: catenin binding domain; TMD: transmembrane domain. (I) 35S-labelled Syph-tail and -t3 proteins bind to GST-cadICD and -cad-D. (J-J'') Computer model of the Merlin FERM domain crystal structure. The regions corresponding to L1 and L3 in Syph are shown in green and blue, respectively, and lie on the same surface of the protein as shown in side (J) and topdown (J') views. (J'') The position of the point mutations in L1 and L3 that disrupt Syph binding to cadICD are shown in purple and pink, respectively.

View larger version (63K): [in this window] [in a new window]   Fig. 4. syph-deficient embryos exhibit delayed and mis-matched epithelial alignment during DC. (A-E'''') Confocal micrographs showing dorsal view of an embryo expressing actin-GFP (A-C'''') or DE-cadherin-GFP (D-E'''') under the control of the En-Gal4 driver (striped pattern) that were injected with buffer alone (A-A'''',D-D'''), with Syph dsRNA#1 (B-B'''',E-E'''), or with Syph dsRNA#2 (C-C'''') undergoing DC. Syph RNAi-injected embryos expressing the actin-GFP reporter complete DC but show mismatching of stripes, whereas those expressing the cadherin-GFP reporter display mismatched, puckered stripes and delayed or incomplete hole closure (see oval in E''''). Note: the intense actin-GFP labeled filopodia on the upper side of the embryo in B'-B''' are observed as a result of the orientation of the embryo, not as a result of the Syph RNAi. (F-G) Confocal micrograph of a single optical plane from two time points (F, F', and G, respectively) of Syph RNAi-injected embryo expressing En-Gal4 driven actin-GFP showing a single cell from one epithelial sheet (dashed line) contacting across eight to ten cells from the opposite epithelial sheet. (H-J') BG2 cell co-stained for Syph (H) and DE-cadherin (I) show co-localization of the two proteins in filopodia (J). (H'-J') Higher magnification view of the area boxed in J for H-J, respectively. (K-M) High magnification dorsal view of embryos expressing DE-cadherin-GFP under the control of the En-Gal4 driver (striped pattern) that were injected with buffer alone (K) or with Syph dsRNA#1 (L,M) undergoing DC. Note accumulation of GFP-cadherin at the dorsalmost side of the LE cells and the long filopodial extensions in control (arrowhead, K) embryos. Syph-deficient embryos that exhibit epithelial mismatching do not accumulate GFP-cadherin on the dorsal-most side of their LE cells or appreciable filopodia (M), whereas Syph-deficient embryos that can still match their epithelial sheets display an intermediate phenotype with some dorsal edge GFP-cadherin accumulation and lamellipodia-like projections from their LE cells (arrowheads, L). See Movie 3 in the supplementary material. Scale bars: 20 µm in A-E''''; 10 µm in F-M.

View larger version (115K): [in this window] [in a new window]   Fig. 5. syph-deficient embryos exhibit disrupted MT networks. (A-D''''') Confocal micrographs showing dorsal view of embryos expressing nuclear GFP (A-B''''') or -tubulin-GFP (C-D''''') under the control of the En-Gal4 driver, which were injected with buffer alone (A-A'''', C-C'''') or with Syph dsRNA (B-B''''', D-D''''') undergoing DC (time elapsed intervals given in minutes). Syph RNAi-injected embryos exhibit misaligned stripes and delayed or incomplete dorsal hole closure. Scale bars: 20 µm.

View larger version (67K): [in this window] [in a new window]   Fig. 6. Aberrant localization of Syph cargos in syph-deficient embryos. (A-C) High-magnification dorsal view of embryos expressing EB1-EYFP under the control of the En-Gal4 driver (striped pattern) that were injected with buffer alone (A) or with Syph dsRNA (B,C) undergoing DC. Note trafficking of EB1-EYFP LE cell filopodia (brackets) in control embryos (A) that is mostly absent from Syph-deficient embryos (bracket, B; C). Syph-deficient embryos exhibiting the strongest phenotypes also show disrupted EB1-EYFP trafficking within the LE and more ventrally located cells (C). See Movie 4 in the supplementary material. (D-G'') Actin expression (as visualized by phalloidin staining; D-E'') and -tubulin expression (F-G'') in buffer-injected (D-D''; F-F'') and Syph dsRNA-treated (E-E''; G-G'') nuclear-GFP-expressing stage 15 embryos. (H-I') -tubulin expression in wild-type (H,H') and Syph dsRNA-treated (I,I') S2R+ cells. Note reduction of cellular extensions and disruption of MT lattice networks in the Syph RNAi-treated cells. Scale bars: 10 µm in A-C; 20 µm in D-I'.