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First published online 16 August 2005
doi: 10.1242/jcs.02517

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Contribution of sequence variation in Drosophila actins to their incorporation into actin-based structures in vivo

Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Anatomy, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK

View larger version (80K): [in a new window]   Fig. 1. Sequence, phylogenetic analysis and timing of expression of all actin proteins in Drosophila. (A) Alignment of the six Drosophila and two human actin proteins. Nomenclature of the fly actins indicates the chromosome location of the gene, e.g. actin5C is located at 5C on the X. Hs ß-actin: human cytoplasmic ß-actin; Hs 2-actin: human enteric 2-actin. Colours indicate residues conserved between groups of actins in flies or human (see also B): blue indicates cytoplasmic actin-specific residues; red indicates residues specific to fly cytoplasmic actins; yellow, human cytoplasmic actins (including in Hs 1-actin, which is not shown); green, fly muscle-actin-specific residues; pink and purple, residues specific for actin79B and 88F, respectively; orange, human muscle-specific residues (conserved within the other human muscle actins not shown); grey, all other non-conserved residues. The asterisk indicates the single amino acid exchange in the mutant GFPactin79BR291H (see Fig. 7); the arrowhead, the point of domain swapping for the chimeric actins (see Fig. 8). (B) The phylogenetic relationship between all fly and human actins. Note that all fly actins are more closely related to vertebrate cytoplasmic actins than to vertebrate muscle actins. Coloured bars correspond to the highlighting of conserved residues in A. Hs ß-actin: human cytoplasmic ß-actin; Hs 1-actin: human cytoplasmic 1-actin; Hs 2-actin: human enteric 2-actin; Hs 1-actin: human skeletal 1-actin; Hs 2-actin: human smooth muscle 2-actin; Hs -cardiac-actin: human cardiac -actin. (C) Diagram of the timing of expression of actins during development of Drosophila as defined by Fyrberg et al. using northern blot analysis (Fyrberg et al., 1983). White equals no expression, and the darker the boxes are shaded, the higher the expression level.

View larger version (80K): [in a new window]   Fig. 2. Actin isoform incorporation into filopodia and lamellipodia. Using the ptc-Gal4 driver all six actins were expressed in the embryonic epidermis and amnioserosa, an extraembryonic membrane covering the dorsal region of the embryo, which is displaced by the embryonic epidermis moving dorsally (Jacinto et al., 2002). (A) Diagram illustrating the contact between embryonic epidermis and amnioserosa during stage 14 of embryogenesis. The box indicates the approximate area of scanning shown in the panels below. The row of epidermal cells contacting the amnioserosa (the leading edge cells) display numerous filopodia and some lamellipodia (highlighted by the staining with phalloidin in B'-G'). The amioserosa cells also have many lamellipodia and filopodia contacting neighbouring cells. (B-G) Most actins strongly label the leading edge filopodia and F-actin-rich cellular protrusions within the amnioserosa. Only actin79B (E) did not appear to be strongly incorporated into these but rather filled the cytoplasm more uniformly.

View larger version (136K): [in a new window]   Fig. 3. Actin isoform incorporation into cortical actin and stress-fibre-like assemblies. Using the CY2-Gal4 driver all six actins were ectopically expressed in the follicular epithelium during oogenesis. (A) Diagram illustrating the level of the confocal sections in B-G' and H-N'. (B-G') Confocal sections through the follicle epithelium; the apical surface of the epithelium facing the oocyte is up, basal surface is down, as depicted in A. In the colour panels, GFPactins are green, labelling with phalloidin is red (B-G), the individual GFPactin channels are also shown as black and white images (B'-G'). The arrows in B,C,F,G indicate phalloidin-labelling of the muscle sheet surrounding the egg chambers, the arrowhead in B indicates the strong phalloidin labelling within the oocyte. Both structures were not labelled with the GFPactins as the CY2-Gal4 driver is only expressed in the follicle cells. Different actins varied in the level of their incorporation into the apical terminal web and microvilli [e.g. strong incorporation for actin5C (B') and actin42A (C')] and incorporation into the cortical actin cytoskeleton lining the lateral sides of follicle cells [e.g. strong labelling for actin87E (F')]. (H-N') Confocal scans of a basal face-on view of the follicle cells (with the muscle sheath removed), showing the basal actin stress fibres as depicted in A. In the colour panels, GFPactins are green, labelling with phalloidin is red (H-N) and the individual GFPactin channels are shown as black and white images (H'-N'). Levels of incorporation into the bundles vary, with actin42A being highly concentrated (I') and actin79B being only weakly incorporated (L') into the fibres.

View larger version (95K): [in a new window]   Fig. 4. Actin isoform incorporation into ring canals and actin cages. Using the nanosVP16-Gal4 driver all six actins were expressed in the germline during oogenesis. (A-F') Confocal sections through germaria of GFPactin-expressing ovarioles showing actin incorporation into ring canals. In the colour panels, GFPactins are green, labelling with phalloidin is red (A-F) and the individual GFPactin channels are shown as black and white images (A'-F'). Arrows in A'-F' indicate individual ring canals. (G,H) High levels of actin overexpression can disrupt cyst formation during oogenesis. Overexpression of either actin5C or actin57B in the germline resulted in a fraction of ovarioles failing to form cysts containing 16 cells. The actins localized to cell cortices and ring canals, but these ring canals appeared to contain excess actin and cystocytes were multinucleate (G; GFPactin is shown in green, phalloidin in red and DNA labelled with TOTO-3 is blue; the arrow indicates a ring canal). Despite the reduced cell number, several stage 10-11 egg chambers showed a morphologically distinct oocyte (H; GFPactin is in green, phalloidin in red; the numbers indicate the three nurse cells of this egg chamber, the asterisk denotes the oocyte). (I-O') Confocal sections through the nurse cell part of a stage 10B egg chamber. Egg chambers at this stage, prior to dumping of their cytoplasmic contents into the oocyte, develop a specialized array of actin filaments, `cages', around each nurse cell nucleus. GFPactins are incorporated to varying degrees into these actin cages, e.g. actin87E being incorporated very strongly (N,N') and actin79B only being incorporated at a very low level (M,M'). In the colour panels, GFPactins are green, labelling with phalloidin is red (I-O) and the individual GFPactin channels are shown as black and white images (I'-O').

View larger version (125K): [in a new window]   Fig. 5. Actin isoform incorporation into sarcomeric assemblies in larval muscles. Using the mef2-Gal4 driver all six actins were expressed in the visceral and body wall musculature of third larval instar. (A) The incorporation patterns for each actin into the visceral muscles. The coloured panels are the merged images of the GFP channel (green and left column) and phalloidin (red and middle column). (B) Lateral views of the individual GFPactins in a single longitudinal third instar body wall muscle, with a diagram indicating the sarcomeric localization on the right (Z indicates Z-lines). Note that GFPactin88F is excluded from Z-lines. (C) The muscle ends that interdigitate with the tendon cells (not visible) within the epidermis to anchor the muscle. This end of the sarcomeric structure of a muscle is a modified and enlarged Z-line (Reedy and Beall, 1993b).

View larger version (78K): [in a new window]   Fig. 6. Actin isoform incorporation into sarcomeric assemblies in adult indirect flight muscles. (A) Using the mef2-Gal4 driver all six actins were expressed in the adult indirect flight muscles. A single muscle myofibril of an adult indirect flight muscle is shown for each actin. GFP is shown in the left column and in green in the colour images, and phalloidin is shown in the middle column and in red in the colour images. Note that, apart from the Z-lines, GFPactins appear to be more strongly incorporated into a `core' structure of the indirect flight muscle. (B) The `core' incorporation pattern of GFPactins is preserved when actins are expressed with other Gal4-drivers that have different levels and timing of expression; shown are GFPactin5C and GFPactin42A using CY2-Gal4, and GFPactin88F using 24B-Gal4 (colour panels show GFP in green and phalloidin in red and the black and white panels show GFP alone). (C) Schematic showing the continuous assembly of indirect flight muscle myofibrils during pupal stages (Reedy and Beall, 1993a). A section through thin filaments of a sarcomere is shown and a cross section of an individual sarcomere between two Z-lines. The darker the colour, the earlier the corresponding thin filaments have been assembled. (D) Disorganisation of the indirect flight muscles caused by GFPactin overexpression. Overview of a field of indirect flight muscle fibres expressing actin5C. Note that some fibres appear to split and fuse irregularly (arrows), a feature not observed in wild-type fibres.

View larger version (121K): [in a new window]   Fig. 7. A point-mutant form of GFPactin79B, actin79BR291H, is specifically incorporated only into Z-lines. (A) In the process of cloning actin79B we also recovered and expressed a point-mutant version of the protein that leads to an R-H amino acid exchange in position 291 of the protein. (B-F) This single amino acid change had striking consequences on the localization of actin79BR291H in muscles: the protein is nearly exclusively incorporated into Z-lines (most dramatically seen in the indirect flight muscles; E,F). (B-F) Incorporation into (B) larval visceral muscles, (C,D) third instar larval body wall muscles (D shows the modified Z-line at the tendon ends of the muscles) and (E-F) indirect flight muscles (F shows the modified Z-line at the muscle end). Analysis in epithelial tissues showed that actin79BR291H is not enriched in the filopodia of the leading edge or the amnioserosa in embryos (G shows GFPactin; G', phalloidin). (H-I') Within the follicle epithelium, actin79BR291H was only weakly found in the cell cortex (H,H'), but was incorporated into apical microvilli and the basal actin bundles (I,I'). In all colour panels GFPactins is shown in green, labelling with phalloidin is in red. All black and white panels show either GFPactin or phalloidin single channels as indicated on each panel.

View larger version (97K): [in a new window]   Fig. 8. Chimera analysis of actin79BR291H and GFPactin88F, two proteins specifically incorporated into or excluded from Z-lines. To determine, whether the single amino acid change in actin79BR291H determined its preference for Z-lines, and to determine which residues in GFPactin88F prevent its incorporation into Z-lines, we exchanged the N-terminal 149 amino acid between the two proteins. (A-D) The C-terminal half of GFPactin88F excludes the chimeric protein from Z-lines (A,C), whereas the C terminus of actin79BR291H brings the N-terminal half of GFPactin88F into Z-lines (B,D). (A,B) GFPactin in third instar larval body wall muscles, (C,C',D,D') adult indirect flight muscles (green in C and D); the phalloidin channel is shown in C and D (red) and in C''and D''.