Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018, USA
* Author for correspondence (e-mail: gguild{at}sas.upenn.edu)
Accepted 10 March 2004
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Summary |
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Key words: Microvilli, Bristles, Drosophila, Jasplakinolide, Actin, Microvillar formation, Filament assembly
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Introduction |
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In this paper we present evidence that during Drosophila bristle formation, microvilli are also secondarily modified to provide the basis of large cortical bundles. In a recent review (DeRosier and Tilney, 2000) we speculated that such a modification might exist. We also show that the crossbridge used in the generation of the core bundles in microvilli is neither the forked protein nor fascin, two known crossbridges required for the generation of large cortical bundles in bristles. Additionally, we show that it cannot be villin, a crossbridging protein found in nurse cells of developing Drosophila egg chambers (Mahajan-Miklos and Cooley, 1994
). Armed with the fact that microvilli are found at bristle tips and that the lateral aggregation of the core bundles in these microvilli account for the intermediate steps in mature bundle morphogenesis, we can now interpret results presented earlier for which we then had no explanation. We show that the formation of microvilli in situ occurs by filament elongation from small densities located on the plasma membrane, not by bundling of filaments in the cell cortex as recently suggested for microspike formation in motile cells (Svitkina et al., 2003
). Although descriptive, a format viewed by some as passé, this study is paramount because it answers in-depth questions furthering our understanding of the formation of bristles and pattern development in this model system. We now have to search for a third, as yet unidentified crossbridge as well as try to determine exactly how and by which proteins the precursor stage in the formation of microvilli in this as well as other model systems are orchestrated.
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Materials and Methods |
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Confocal and electron microscopy
The procedures for fixation, antibody and phalloidin staining, and confocal microscopy were described previously (Guild et al., 2002). The rabbit polyclonal antibody directed against the forked proteins was also described previously (Guild et al., 2003
). The mouse monoclonal antibody (6B9) directed against the Drosophila quail protein (Drosophila villin) was developed by Mahajan-Miklos and Cooley (Mahajan-Miklos and Cooley, 1994
) and obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa. The procedures used for thin-section transmission EM have been described previously (Tilney et al., 1998
).
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Results |
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We next examined thin sections of newly emerging bristles. Numerous short protrusions or pimples are found at the tips of these bristle shafts (Fig. 2a). Similar pimples are present on the apical surface of the socket cell (Fig. 2a) as well as along the apical surface of the epithelial cells that separate adjacent bristles (Fig. 3a,b). All of these pimples are characterized by having some electron-dense material attached to the cytoplasmic surface of the plasma membrane (Figs 2 and 3). In some pimples short actin filaments (approximately 0.1 µm in length) extend from the dense material into the cortex. From this morphology and from what we know about other systems (see Discussion) we suspected that the pimples might represent a precursor stage in microvillar formation. To test this idea, we incubated cultured pupal thoraces with the sponge toxin jasplakinolide before fixation (Fig. 2b-e). Jasplakinolide is a membrane-permeant phalloidin-like compound (Bubb et al., 1994; Bubb et al., 2000
) that binds to actin filaments and prevents their disassembly. If these pimples are indeed microvillar precursors then in the presence of jasplakinolide the filaments in the pimples might elongate as any filament that forms would fail to disassemble. Accordingly, the pimples seen in the tip of newly emerging bristles should elongate. In fact this is exactly what we found when we examined thin sections cut through newly emerging bristle shafts and adjacent epithelial cells from preparations that had been treated with jasplakinolide prior to fixation (Fig. 2b-c). We found large numbers of microvilli at the bristle tips. Furthermore these microvillar-like extensions from the newly emerging bristle tips all possess an internal core of actin filaments that extend from dense material attached to the cytoplasmic surface of the microvillar tips (Fig. 2b-e). This is demonstrated particularly clearly when two serial sections of a microvillar-like extension seen on a bristle tip (Fig. 2c,d) were observed at a greater magnification (Fig. 2e). Here the core microfilaments extend from the dense material at the microvillus tip all the way into the cortical cytoplasm of the bristle proper. There are transverse stripes on this core bundle which indicate the presence of a crossbridge.
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Core bundles in microvilli aggregate into cortical bundles, a process that requires actin crossbridging proteins
Forked protein
In an earlier publication (Tilney et al., 1998) we used mutants to show that the forked crossbridges were instrumental in aggregating tiny core bundles of actin filaments into larger bundles. These aggregations differentiate into the cortical bundles present in fully elongated bristles. Subsequently the actin filaments are crossbridged into a paracrystallic array by the fascin crossbridge. Much to our initial surprise when we examined transverse sections through the newly emerging or emerged tips of forked mutants we found not fewer pimples or microvilli, but many more than the wild type (Tilney et al., 1998
). This was a surprise for two reasons. First, we assumed that the actin filaments making up the core filament bundles in microvilli would be crosslinked by the same crossbridge used to aggregate the bundle laterally (the forked crossbridge) reasoning that if the forked proteins were missing (in the forked mutant), microvilli would not appear. As microvilli do form it follows that the actin filaments in the core bundles are not crosslinked together by the forked proteins. Second, why the increased number of microvilli? We assume the answer to this question is that lateral aggregation of the core bundles is less likely in the absence of the forked protein and this role must now be taken over by another crossbridge, perhaps fascin. Thus, the emerging tips would elongate more slowly and at the same time have an increased number of microvilli. This hypothesis is born out. The bristles of this mutant are 40% shorter than the wild type and contain, in mature bristles, threefold smaller bundles than the wild type (Tilney et al., 2000a
).
Fascin protein
A mutant lacking the other major crossbridge, fascin, but expressing normal amounts of the forked protein, now becomes very revealing. First of all, pimples and microvilli in this mutant appear not only along the epithelial cells between the bristle shafts but are also prominent on the new emerging bristle. This fact means that fascin, like the forked proteins, cannot account for the bundling of actin filaments together in the microvilli and/or pimple precursors. Second, and what is particularly interesting, is that in the fascin-less mutant (singed) we find linear arrays of tiny actin bundles attached to the plasma membrane (Fig. 4a) (Tilney et al., 1998). Each of these contains approximately the same number of filaments (Fig. 4b) as those in the microvilli on the bristle tip (Fig. 2e). We presume that the reason why we see these linear aggregates is that in the absence of fascin these aggregates mature slowly or not at all into normal round cortical bundles. By removing fascin we have frozen a stage in the differentiation of the round cortical bundles. If this interpretation is correct then longitudinal sections through a bristle tip should reveal parallel core bundles. This is indeed the case (Fig. 4c,d).
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If pimples, as hypothesized earlier, are microvillar precursors, then the core actin bundles in those microvilli would be aggregated by the forked proteins to form the large actin bundles present in the bristle shaft. Assuming this to be true, then one wonders why large actin bundles do not form in socket cells which as shown in Fig. 2a display pimples on their apical surfaces. Perhaps the reason why socket cells do not form large actin bundles is that they lack the forked proteins. To test this possibility we stained socket cells and bristle shafts with an antibody prepared against the forked proteins. The socket cells are not recognized by this antibody, in contrast to the adjacent shaft that stains heavily (Fig. 5), possibly explaining why the socket cells do not form large actin bundles.
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Pimples are microvillar precursors
In the presence of jasplakinolide not only are true microvilli present on the tips of newly emerging bristle shafts (Fig. 2c,d) but also microvilli extend from the epithelial cells adjacent to the bristle shaft (right side of these two panels). To prove that the pimples are a first step in microvillar formation requires real-time observations of individual pimples growing into microvilli; this is just not possible with current technology. We do have, however, data from examination of the pimples from epithelial cells consistent with this idea.
Tiny pimples are present on the apical surface of epithelial cells prior to emergence of a bristle, e.g. 32-hour pupae. In thin section these pimples (Fig. 3a) appear as minute protuberances within which is a small accumulation of electron-dense material. Actin filaments infrequently extend from these densities into the cell. In older pupae actin filaments are often found extending from these pimple densities into the cortical cytoplasm. If isolated thoraces of pupae are incubated in jasplakinolide then fixed and examined instead of pimples with only a few actin filaments attached to the densities, the majority have a core bundle of actin filaments that extend into the cortical cytoplasm as a rootlet (Fig. 3c,d) and in many cases the pimples elongate and appear as short microvilli (Fig. 3d). Within the latter is a core bundle of actin filaments that extends into the cortical cytoplasm as a rootlet. These core bundles then appear to be units or the tiny bundles present in the bristle (Fig. 4d) that aggregate together to form the cortical bundles. Thus jasplakinolide treatment has allowed us to concentrate on the steps of bundle formation, namely how the core bundles with their rootlets are formed from the pimples and how aggregating these with the forked proteins and their subsequent differentiation by fascin could be the first step in the formation of the large cortical bundles. Further studies are necessary to see what influence other actin binding proteins, e.g. profilin, capping protein or the Arp 2/3 complex, have on pimple formation, elongation and microvillar aggregation.
A third crossbridge is required for connecting adjacent actin filaments in microvilli
In the double mutant lacking both forked and fascin crossbridges, pimples are also present on the epithelial cells as well as on newly emerging bristles. If cultured in the presence of jasplakinolide the pimples elongate into microvilli (Fig. 3e). Transverse sections through the tips of these microvilli show actin filaments cut in cross section (Fig. 3f). As pimples and microvilli form in the absence of one or the other or both cross-linkers, we conclude that the core filaments in the microvilli must be crosslinked by a third yet to be identified crossbridge.
Additional evidence for the existence of a third crossbridge comes from recent results (Tilney et al., 2003) on sections of bristles of the singed-forked double mutant cultured in the presence of jasplakinolide. In these bristles because filament disassembly/turnover has been inhibited, we found that the bristle cytoplasm filled with actin filaments. Some of these are free filaments but there were also clusters of filaments. As in this mutant both the forked and fascin proteins are not present, a third crossbridge must exist to bundle the filaments into clusters.
Matova et al. (1999) demonstrated that Drosophila villin is essential for actin bundle formation in the nurse cells during oogenesis. As villin crossbridges actin filaments in vertebrate microvilli, we immediately suspected that this crossbridge might be the unknown crossbridge needed for bristle microvillar formation. Accordingly, we stained developing bristles with an antibody prepared against Drosophila villin. Although the antibody stains the nerve that innervates the bristle and the hairs present on the Drosophila wings, the newly emerging bristles are completely negative (data not shown). Therefore, the unknown crossbridge cannot be villin.
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Discussion |
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Two examples illustrate this last point. First, from our studies it is clear that three separate actin filament crossbridges are essential in sequentially building the actin bundles in mature bristles: fascin, the forked proteins and the so far unidentified crossbridge shown to be present here. A similar philosophy occurs in the formation of stereocilia and microvilli in intestinal epithelial cells and in the convoluted tubule cells of the kidney (DeRosier and Tilney, 2000) although in each case different crossbridging proteins are used. Second, the pimples appear to be precursors to microvilli. Microvilli are defined as stereotyped linear extensions of the cell surface containing a central core of crosslinked actin filaments that insert into some electron-dense material attached to the plasma membrane. Formation of microvilli therefore occurs by a different method to the assembly of microspikes on the pseudopodia of fibroblasts and keratocytes (Svitkina et al., 2003
).
Microvilli are key intermediates in bundle assembly
We now can understand why during bristle elongation in the absence of the forked crossbridge extra tiny actin bundles appear at the elongating bristle tip (Tilney et al., 2000b) and why bristle elongation in the absence of the fascin cross-linker produces flattened linear aggregates attached to the plasma membrane (Tilney et al., 2000b
) instead of round bundles. In both cases the lack of one crossbridge slows bundle development sufficiently so we can more easily recognize intermediate steps in bundle formation. Thus without the forked proteins to aggregate the actin filament bundles that comprise the cores of microvilli or microvillar stubs, many more cores are seen in thin section than in the wild type. Likewise, without fascin, the aggregated core bundles slowly change or fail to change from a linear aggregate of microvillar core bundles to a compact often spherical mass of filaments (Tilney et al., 1995
; Tilney et al., 1998
).
Microvillar dynamics
Jasplakinolide stabilizes actin filaments against depolymerization (Bubb et al., 2000; Tilney et al., 2003
). The fact that pimples on newly emerged bristles (and epithelial cells) elongate in the presence of jasplakinolide suggests that these pimples are likely to be microvillar precursors capable of extension induced by filament elongation, provided that filament disassembly is inhibited. This behavior is in keeping with recently published results using jasplakinolide on wild-type and mutant bristles (Tilney et al., 2003
). This interpretation further explains the elaboration of microvilli on the apical surface of the shaft cell (Fig. 2a) prior to its elongation. However, it is also true that the pimples often extend their core bundles as rootlets into the cortical cytoplasm. Rootlets exposed to the forked crossbridges induce aggregation into linear arrays, a key step in bundle formation in bristles.
From the literature we know that precursors of microvilli resemble the pimples described here. Examples include (1) the microvilli on the apical surface of developing intestinal epithelial cells that elongate from tiny precursors (Stidwill and Burgess, 1986); (2) the `short papillae' or microvillar precursors (Schroeder, 1978
) that elongate following sea urchin egg fertilization (Eddy and Shapiro, 1976
); (3) de novo formation of microvilli following the removal of existing microvilli such as occurs following treatment with hydrostatic pressure in intestinal epithelial cells (Tilney and Cardell, 1970
) and fertilized sea urchin eggs (Begg et al., 1983
; Henson and Begg, 1988
).
Particularly relevant to our studies on cortical bundle formation in elongating bristles is the fact that the rootlets of microvilli elongate from their precursor pimples or papillae, e.g. after treatment with jasplakinolide (this study) or in sea urchin eggs following fertilization (Wong et al., 1997). Another example is during microvillar formation in intestinal epithelial cells which begins in the crypts of Lieberkuln and continues on the villus before its cells are sloughed away from the villus tip 2-3 days later (Fath et al., 1990
; Mamajiwalla et al., 1992
). In this last system the rootlets form first and subsequently zipper the plasma membrane down around them to form microvilli. As the total length of the core actin bundle (including the microvillus and the rootlet) is approximately the same as the length of the rootlet in the crypt cell, it was proposed that the microvillus elongates by membrane addition at the microvillus base (Mamajiwalla et al., 1992
; Fath et al., 1990
). The reason we dwell on this is that it is the core bundles in the rootlets of the microvilli that aggregate together under the influence of the forked proteins to produce the cortical bundles in bristles (Fig. 5) (Tilney et al., 2000b
). Likewise it is the rootlets of the microvilli that associate like extension ladders to form the actin cage in nurse cells (Guild et al., 1997
). Our experiments on bristles treated with jasplakinolide (Fig. 2) emphasize that microvilli can elongate from the bristle tip, but what is key to cortical bundle formation is the aggregation of the rootlets by the forked proteins and their subsequent crosslinking by fascin that accounts for the final shape of the cortical bundles in the bristles.
We should also mention that microvilli are dynamic and can shorten or lengthen. Perhaps the most-studied examples are the microvilli of intestinal epithelial cells that shorten during starvation (Misch et al., 1980), after treatment with cyclohexamide (Lecount and Grey, 1972
) or lectins (Weinman et al., 1989
) and thereafter re-elongate provided the stimulus is removed. Furthermore these microvilli elongate when G-actin is added to the membrane intact `brush borders' derived from intestinal epithelial cells (Mooseker et al., 1982
). Interestingly the elongation of the core bundle in each microvillus occurs at the tips of the microvilli, insertion occurring between the electron-dense material at the tips and the ends of the filaments in core bundles.
Formation of microspikes and microvilli seem to occur by different mechanisms
Several investigators questioned whether microvilli are the same as microspikes. We think they are different for the following reasons. First, in contrast to microvilli, microspikes found on neuron growth cones and pseudopodia like those present on the surface of fibroblasts and keratocytes are generally not of uniform diameter but often are volcano shaped. Within them is a core or cores of actin filaments and sometimes microtubules, but unlike microvilli the actin filaments in microspikes are less regularly crossbridged and the filaments vary in length. Furthermore these two types of extensions tend to be found on different categories of cells although the distinction is far from perfect. Microspikes tend to be located on motile cells rather than on cells that are attached to others by junctions and are thus less motile. Second, microvilli seem to be more stable and less likely to turnover than microspikes, as for example, the microvillus brush borders can be isolated intact, although they can shorten if traumatized. In contrast, growth cones in neurons and keratocytes form, retract completely, and regrow within short time courses.
This study has pointed out a third difference, namely in their formation, which to our thinking is paramount. As already mentioned, pimples seem to be precursors to microvilli, similar to the papillae noted on the surface of unfertilized sea urchin eggs (Henson and Begg, 1988; Schroeder, 1978
) and in intestinal epithelial cells that are reforming microvilli after treatment with hydrostatic pressure (Tilney and Cardell, 1970
). In both cases the pimples seem to elongate after fertilization (Begg et al., 1982
) or cessation of pressure treatment. Thus in systems where microvilli elongate, the filaments within them elongate downward from some electron-dense material at their tips, which in turn can lead to an elongation of the microvillus or the formation of long rootlets that extend into the cell cortex. In contrast, in recent work on microspike formation in keratocytes it has been proposed that microspikes form by the zippering together of cortical actin filaments that act to form a microvillus by pushing the membrane from the cortex upwards (Svitkina et al., 2003
). This suggests that the control of these processes is different, although we must be open to the suggestion that process extension may result from a combination of both of these methods.
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Acknowledgments |
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References |
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