Gregory M. Guild

Long continuous actin bundles in Drosophila bristles are constructed by overlapping short filaments

The actin bundles essential for Drosophila bristle elongation are hundreds of microns long and composed of cross-linked unipolar filaments. These long bundles are built from much shorter modules that graft together. Using both confocal and electron microscopy, we demonstrate that newly synthesized modules are short (1–2 μm in length); modules elongate to ∼3 μm by growing over the surface of longitudinally adjacent modules to form a graft; the grafted regions are initially secured by the forked protein cross-bridge and later by the fascin cross-bridge; actin bundles are smoothed by filament addition and appear continuous and without swellings; and in the absence of grafting, dramatic alterations in cell shape occur that substitutes cell width expansion for elongation. Thus, bundle morphogenesis has several components: module formation, elongation, grafting, and bundle smoothing. These actin bundles are much like a rope or cable, made by overlapping elements that run a small fraction of the overall length, and stiffened by cross-linking.

Molecular analysis of a developmentally regulated gene which is expressed in the larval salivary gland of Drosophila.

The polytene genome of the larval salivary gland of Drosophila undergoes dramatic alterations in localized transcriptional activity late in third-instar development. One of these changes involves the 20-OH ecdysone-mediated and coordinate repression of a dispersed set of intermolt puff sites. The DNA from one of these loci (that located within the 90BC interval) has been isolated by molecular cloning techniques. This DNA was shown to be present one time per haploid Drosophila genome and to contain a gene (designated the Group V gene) which is developmentally regulated. The Group V transcript was shown to accumulate only during third-instar larval development and to be located exclusively in the salivary gland. These results are consistent with the hypothesis that the Group V gene codes for a salivary gland glue protein.

Larval salivary gland secretion proteins in Drosophila: Identification and characterization of the Sgs-5 structural gene☆

The 90BC locus on the polytene chromosomal map of Drosophila melanogaster contains the structural gene for a third-instar, salivary gland-specific, polyadenylated RNA (the group V RNA). This also belongs to the intermolt puff set whose dispersed and co-ordinately regulated members are (1) transcriptionally active in the salivary gland during the third-instar developmental stage and (2) comprise (at least in part) the structural genes for a set of salivary gland secretion proteins. Previous developmental studies of the group V intermolt gene (located cytogenetically within the 90B3-8 interval) suggest that it controls the expression of a salivary gland secretion protein. By analyzing different D. melanogaster laboratory stocks for variation in group V gene expression, we have been able to correlate the presence of the group V RNA with the salivary gland secretion protein P4. In vitro translation experiments show that the salivary gland messenger RNA population derived from a stock that fails to synthesize the group V RNA does not direct the synthesis of a polypeptide similar in molecular weight to protein P4. In addition, cloned genomic DNA segments complementary to the group V RNA are capable of arresting the in vitro translation of this protein. Comparative two-dimensional fractionation of cysteine-labeled, protease-generated peptides shows that (1) the in vitro translation product arrested by group V gene DNA is biochemically very similar to or identical with the salivary gland secretion protein P4, and (2) protein P4 is equivalent to the salivary gland secretion protein previously designated SGS-5. Since designations of the latter type have been employed in naming the genetic loci that represent the structural genes for the salivary gland secretion protein gene set, the group V gene (previous designation) represents the SGS-5 structural gene and its appropriate genetic designation should now be Sgs-5.

Microvilli appear to represent the first step in actin bundle formation in Drosophila bristles

During bristle development the emerging bristle shaft, socket cell, and the apical surface of thoracic epithelial cells form tiny protuberances or pimples that contain electron-dense material located on the cytoplasmic surface of the pimple tip. In a few cases short actin filaments extend from this material into the cortical cytoplasm. When cultured in the presence of jasplakinolide, an agent that prevents filament disassembly, pimples elongate to form microvilli containing a core of crosslinked filaments. Emerging-bristle mutants delay cortical bundle formation and are aggregated by forked protein crossbridges. Using these mutants and enhancing core bundle formation with jasplakinolide we found that microvillar formation represents the first stage in the morphogenesis of much larger actin bundles in Drosophila bristle shaft cells. Evidence is presented showing that socket cells do not contain forked protein crossbridges, a fact that may explain why cortical bundles only appear in bristle shaft cells. Furthermore, as pimples and microvilli form in the absence of both forked and fascin crossbridges, we also conclude that neither of these crossbridges account for core bundle formation in microvilli, but there must exist a third, as yet unidentified crossbridge in this system. Immunocytochemisty suggested that this new crossbridge is not Drosophila villin. Finally, ultrastructural comparisons suggest that microspikes and microvilli form very differently.

Larval salivary gland secretion proteins in Drosophila structural analysis of the Sgs-5 gene

The structure of the Drosophila melanogaster salivary gland secretion gene Sgs-5 has been determined by DNA sequence analysis of cloned genomic DNA. This developmentally and tissue-specific gene is a member of the third instar intermolt gene set and is under control of the insect molting hormone ecdysterone. RNA protection experiments show that the RNA coding region of Sgs-5 contains 769 nucleotides and is divided into three exons by two small introns. The protein-coding region appears to begin after a short untranslated RNA leader (33 nucleotides) and to result in a protein of 163 amino acids. The first 18 amino acids give the amino-terminal end the highly hydrophobic nature characteristic of a signal peptide.