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Actin filament turnover removes bundles from Drosophila bristle cells

Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018, USA

View larger version (26K): [in a new window]   Fig. 1. Extensive actin filament loss from bundle modules is seen in maturing Drosophila bristles. Wild-type bristles of increasing age were stained with rhodamine-labeled phalloidin and imaged by confocal microscopy. Images of bristles at the indicated age (32-56 hours after puparium formation) were examined for smooth bundles with no gaps (a), bundles with overlapping modules (c), bundles with gaps (e), apically tapering modules (g) and for bundles with extensive filament ghosts remaining (i). The percentage of bristles with bundles of each type was plotted as a function of developmental time. Note that individual bristles could contain more than one type of bundle. For example, 43-hour bristles often contain bundles with overlapping modules and bundles with gaps (see text for details). A total of 195 thoracic bristles were examined. Portions of bristles illustrating smooth bundles (b), bundles with overlapping modules (indicated with arrowheads) (d), and bundles with gaps (indicated with arrowheads) (f) are shown to the right. A portion of a bristle illustrating tapering modules is shown in (h). Arrowheads indicate alignment of modules. A portion of a bristle illustrating extensive filament ghosts is shown in (j). Arrowheads indicate two bundles with ghost connections. Bars, 2 µm.

View larger version (180K): [in a new window]   Fig. 2. Longitudinal thin sections through actin bundles in bristles of 54-hour pupae. These sections were selected because they show junctions between adjacent modules. If this plate is rotated 90° so the viewer is looking across the bundles, the 12-nm period owing to the fascin crosslinks can be easily recognized. This period is indicated by the bars in (b) and (c), but it is especially visible near the base of each micrograph. If the bristle is bent before fixation, the overlap between adjacent modules separates at the point of overlap (a). As the module shortens, the number of filaments found in the overlap region decreases (b). Shortly thereafter a distinct gap is seen between adjacent modules (c).

View larger version (73K): [in a new window]   Fig. 3. Time-lapse confocal microscopy of living bristle cells shows barbed-end loss from actin filaments. (a) Actin bundles decorated with GFP-moesin from a single bristle-cell extension were visualized over a 90-minute period. Images were taken at 30-minute intervals (0, 30, 60 and 90 minutes) and are displayed from left to right. These images were aligned using a constellation of fluorescent irregularities on the bundles. The position of one of these bright spots is adjacent to the asterisk (*). Bristle orientation is indicated in the rightmost panel. The top region of these images is characterized by gaps in eight bundles that do not appear to widen during this time course. The upper and lower boundaries of one of these gaps are indicated by the closely spaced arrows for each time point. The bottom region of these images — near the bristle base — is characterized by widening gaps in eight bundles. The upper and lower boundaries of two of these gaps are indicated by the pair of arrows (left side of image) and the pair of double-headed arrows (right side of image). Note that the lower boundaries of these gaps, defined by the barbed ends of the modules below, are moving away from the fixed upper boundaries. (b and c) The rate of barbed-end module shortening was determined as described in the text by measuring the length of five well defined gaps in the upper region of the bristle (b) and in the lower region of the bristle (c). A least-squares fit for each lengthening gap generates a line with a slope representing the rate of module shortening. All modules in the five non-widening gaps (b) showed negligible shortening: average rate of 0.08±0.05 (SEM) µm/hour. All modules in the five widening gaps (c) showed similar shortening rates: average rate of 1.10±0.04 (SEM) µm/hour.

View larger version (89K): [in a new window]   Fig. 4. Jasplakinolide prevents module shortening. Thoraces from 48-hour animals were cultured for 5 hours in the presence or in the absence of jasplakinolide (3 µM), stained with rhodamine-labeled phalloidin and imaged by confocal microscopy. (a) Bundles in control bristles develop the expected gaps owing to actin depolymerization. (b) In contrast, bundles in jasplakinolide-treated bristles failed to undergo normal barbed-end depolymerization and remained smooth.

View larger version (14K): [in a new window]   Fig. 5. Cycloheximide does not affect bristle growth but does promote premature bundle breakdown. (a) A group of same-age thoraces (dissected from a single cohort of pupae 36 hours after puparium formation) were divided into two groups and cultured in the presence or in the absence (control) of 1 µM cycloheximide for 5 hours. Bristle lengths were measured from confocal images of phalloidin-stained microchaetes. Bristle length was unaffected when cultured in cycloheximide. (b) A group of same-age thoraces (dissected from a single cohort of pupae 42 hours after puparium formation) were divided into two groups and cultured in the presence or in the absence (control) of 1 µM cycloheximide for two hours. Actin bundles were imaged by confocal microscopy and evaluated by the criteria illustrated in Fig. 1. A total of 56 bristles were examined and the percentages of bristles exhibiting tapering modules or bundle ghosts characteristic of depolymerizing bundles are presented. Of interest here is that cycloheximide promotes the breakdown of modules usually seen in older bristles (e.g., Fig. 1).

View larger version (200K): [in a new window]   Fig. 6. Transverse thin section through a macrochaete from a thorax of a 36-hour-old pupa that was cultured in the presence of cycloheximide for 5 hours before fixation. Of interest is the actin bundles indicated by the arrows as they are small yet the filaments in the bundles are hexagonally packed (see insert) and maximally crosslinked by fascin.

View larger version (175K): [in a new window]   Fig. 7. Solid modules separate longitudinally into a series of subbundles. All of these figures come from 54- and 55-hour old pupae. Panels (a and b) show thin transverse sections through a bristle. The chitinous exoskeleton of the bristle is being deposited outside the plasma membrane surrounding the bristle. Located at the top of panel (a) are three bundles (arrows) illustrated at higher magnification in (b). Each bundle has separated into two subbundles. The dots in the bundles are the actin filaments cut in cross section. Panel (c) shows a section through a module with a flat basal end and a pointed apical end that is splitting into two subbundles. The 12-nm period attributable to the cross-bridge fascin is indicated on this micrograph by the three bars. This period is best seen by looking across the bundle from its side. Panel (d) shows a deeper split of a bundle into two subbundles. Panel (e) shows two tandem modules splitting into a series of subbundles. At least one subbundle remains associated with the plasma membrane.

View larger version (135K): [in a new window]   Fig. 8. Transverse sections through a microchaete from a 54-hour pupa. This section illustrates the first step in subbundle formation where holes or areas lacking in filaments appear within the actin bundle (e.g., arrow in bundle at 7 o'clock).

View larger version (76K): [in a new window]   Fig. 9. Cytochalasin promotes the premature breakdown of modules into subbundles. Macrochaetes from wild-type thoraces cultured in the presence of cytochalasin (35-42 hours) were fixed, stained with rhodamine-labeled phalloidin and imaged by confocal microscopy. Panel (b) shows an increased number of bundles in the bristle shaft (arrowhead). The modules seem to dissolve into subbundles at the base of the shaft (arrow) and accumulate in the cell body. Panel (a) shows a higher magnification of the extra bundles present in a different bristle. Panel (c) shows another bristle and what appear to be subbundles pouring off the end of the bristle shaft (arrow) and into the cell body. Bars, 5 µm.

View larger version (161K): [in a new window]   Fig. 10. A thin section through the base of the bristle cell from an isolated thorax that had been treated for two hours with cytochalasin D before fixation. The thorax was dissected from a 41-hour pupa. By 43 hours, bundle disassembly in the controls had not yet occurred. In the cytoplasm surrounding the nucleus (N) are numerous small bundles (a) each containing about 50 filaments. One of the bundles is depicted at high magnification in (b). The lines indicate the 12-nm period attributable to the crosslink fascin.

View larger version (11K): [in a new window]   Fig. 11. Model for the breakdown of the modular actin bundles that support Drosophila bristles. (a) Two adjacent modules (gray boxes) are shown attached to the plasma membrane (vertical black line) of the cell. The orientation of polarized actin filaments in the modules and the orientation of the modules relative to the bristle tip are shown to the right. In these panels, only the lower module undergoes breakdown, whereas the upper module remains intact for comparison. (b) Actin filaments shorten by the loss of subunits from their barbed ends. This process can be accelerated by inhibiting protein synthesis with cycloheximide. (c) The module also begins to breakdown into submodules by longitudinal cleavages. This process can be accelerated by inhibiting actin polymerization with cytochalasin. It is not clear whether depolymerization or subbundle formation is initiated sequentially or simultaneously. (d) The module is completely split from the plasma membrane, leaving behind a membrane-bound actin `ghost'. Some submodules can become completely split off from the module proper and can be found deeper in the cytoplasm. (e) In the end, only the `ghost' of the module remains attached to the membrane.

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