Research Article 3531 Microvilli appear to represent the first step in actin bundle formation in Drosophila bristles Lewis G. Tilney, Patricia S. Connelly and Gregory M. Guild* Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018, USA *Author for correspondence (e-mail: [email protected]) Accepted 10 March 2004 Journal of Cell Science 117, 3531-3538 Published by The Company of Biologists 2004 doi:10.1242/jcs.01215 Summary During bristle development the emerging bristle shaft, Evidence is presented showing that socket cells do not socket cell, and the apical surface of thoracic epithelial cells contain forked protein crossbridges, a fact that may explain form tiny protuberances or pimples that contain electron- why cortical bundles only appear in bristle shaft cells. dense material located on the cytoplasmic surface of the Furthermore, as pimples and microvilli form in the absence pimple tip. In a few cases short actin filaments extend from of both forked and fascin crossbridges, we also conclude this material into the cortical cytoplasm. When cultured that neither of these crossbridges account for core bundle in the presence of jasplakinolide, an agent that prevents formation in microvilli, but there must exist a third, filament disassembly, pimples elongate to form microvilli as yet unidentified crossbridge in this system. containing a core of crosslinked filaments. Emerging- Immunocytochemisty suggested that this new crossbridge bristle mutants delay cortical bundle formation and are is not Drosophila villin. Finally, ultrastructural aggregated by forked protein crossbridges. Using these comparisons suggest that microspikes and microvilli form mutants and enhancing core bundle formation with very differently. jasplakinolide we found that microvillar formation represents the first stage in the morphogenesis of much Key words: Microvilli, Bristles, Drosophila, Jasplakinolide, Actin, larger actin bundles in Drosophila bristle shaft cells. Microvillar formation, Filament assembly Introduction morphogenesis, we can now interpret results presented earlier Microvilli are common extensions of the apical surface of for which we then had no explanation. We show that the epithelial cells. Examples include intestinal epithelial cells, formation of microvilli in situ occurs by filament elongation the convoluted tubules of the kidney, as well as cells of the from small densities located on the plasma membrane, not by reproductive system such as oocytes, newly fertilized eggs, bundling of filaments in the cell cortex as recently suggested developing embryos and nurse cells (reviewed by DeRosier for microspike formation in motile cells (Svitkina et al., 2003). and Tilney, 2000). Microvillar extensions are uniform in Although descriptive, a format viewed by some as passé, this diameter and have a core of crosslinked actin filaments that study is paramount because it answers in-depth questions run from the plasma membrane, where they are often furthering our understanding of the formation of bristles and embedded into some electron-dense material, down into the pattern development in this model system. We now have to cortex of the cell. Furthermore microvilli can be modified search for a third, as yet unidentified crossbridge as well as try secondarily to form the stereocilia of hair cells of the cochlea to determine exactly how and by which proteins the precursor or the actin bundles in Drosophila nurse cells (Guild et al., stage in the formation of microvilli in this as well as other 1997). model systems are orchestrated. 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 Materials and Methods (DeRosier and Tilney, 2000) we speculated that such a Drosophila stocks, developmental staging, dissection and modification might exist. We also show that the crossbridge culture used in the generation of the core bundles in microvilli is The Oregon-R strain of Drosophila melanogaster was used as the wild 3 36a neither the forked protein nor fascin, two known crossbridges type in these studies. The singed stock (sn ), forked stock (f ) and singed-forked (sn3 f 36a) double-mutant stock were maintained as required for the generation of large cortical bundles in bristles. X2 Additionally, we show that it cannot be villin, a crossbridging viable homozygotes. The In(1)dl-49, singed chromosome was maintained over the In(1)Mud, Mud1 chromosome. Flies were protein found in nurse cells of developing Drosophila egg maintained on standard cornmeal-molasses-yeast food at 25°C, 60- chambers (Mahajan-Miklos and Cooley, 1994). Armed with 70% relative humidity, with a 12 hour/12 hour day/night cycle. the fact that microvilli are found at bristle tips and that the Complete descriptions of genes and symbols can be found elsewhere lateral aggregation of the core bundles in these microvilli (Lindsley and Zimm, 1992) and on FlyBase (Flybase Consortium, account for the intermediate steps in mature bundle 2003). Developmental staging, thoracic dissections and culturing in 3532 Journal of Cell Science 117 (16) the presence of jasplakinolide (3 µM) were as described (Tilney et al., cytoplasmic surface of the plasma membrane (Figs 2 and 3). 2003). 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 Confocal and electron microscopy (see Discussion) we suspected that the pimples might represent The procedures for fixation, antibody and phalloidin staining, and a precursor stage in microvillar formation. To test this idea, we confocal microscopy were described previously (Guild et al., 2002). The rabbit polyclonal antibody directed against the forked proteins incubated cultured pupal thoraces with the sponge toxin was also described previously (Guild et al., 2003). The mouse jasplakinolide before fixation (Fig. 2b-e). Jasplakinolide is a monoclonal antibody (6B9) directed against the Drosophila quail membrane-permeant phalloidin-like compound (Bubb et al., protein (Drosophila villin) was developed by Mahajan-Miklos and 1994; Bubb et al., 2000) that binds to actin filaments and Cooley (Mahajan-Miklos and Cooley, 1994) and obtained from the prevents their disassembly. If these pimples are indeed Developmental Studies Hybridoma Bank developed under the microvillar precursors then in the presence of jasplakinolide auspices of the NICHD and maintained by the University of Iowa. the filaments in the pimples might elongate as any filament that The procedures used for thin-section transmission EM have been forms would fail to disassemble. Accordingly, the pimples seen described previously (Tilney et al., 1998). 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 Results epithelial cells from preparations that had been treated with Microvilli are present on emerging bristle tips jasplakinolide prior to fixation (Fig. 2b-c). We found large Bristles begin to emerge in 32-hour pupae and elongate over numbers of microvilli at the bristle tips. Furthermore these the next 16 hours after which they reach their mature length microvillar-like extensions from the newly emerging bristle (Tilney et al., 2000b). Examination of thin sections cut through tips all possess an internal core of actin filaments that extend the apical surface of the shaft of a bristle cell just prior to its from dense material attached to the cytoplasmic surface of the emergence from the surface of the thorax reveals the presence microvillar tips (Fig. 2b-e). This is demonstrated particularly of numerous microvilli on this tiny surface (Fig. 1). These clearly when two serial sections of a microvillar-like extension microvilli are at least 1 µm in length. It is as if the apical seen on a bristle tip (Fig. 2c,d) were observed at a greater surface of the shaft anticipates its elongation. magnification (Fig. 2e). Here the core microfilaments extend We next examined thin sections of newly emerging bristles. from the dense material at the microvillus tip all the way into Numerous short protrusions or pimples are found at the tips of the cortical cytoplasm of the bristle proper. There are these bristle shafts (Fig. 2a). Similar pimples are present on the transverse stripes on this core bundle which indicate the apical surface of the socket cell (Fig. 2a) as well as along the presence of a crossbridge. 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 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.