F actin bundles in Drosophila bristles. I. Two filament cross-links are involved in bundling.

Transverse sections though Drosophila bristles reveal 7-11 nearly round, plasma membrane-associated bundles of actin filaments. These filaments are hexagonally packed and in a longitudinal section they show a 12-nm periodicity in both the 1.1 and 1.0 views. From earlier studies this periodicity is attributable to cross-links and indicates that the filaments are maximally cross-linked, singed mutants also have 7-11 bundles, but the bundles are smaller, flattened, and the filaments within the bundles are randomly packed (not hexagonal); no periodicity can be detected in longitudinal sections. Another mutant, forked (f36a), also has 7-11 bundles but even though the bundles are very small, the filaments within them are hexagonally packed and display a 12-nm periodicity in longitudinal section. The singed-forked double mutant lacks filament bundles. Thus there are at least two species of cross-links between adjacent actin filaments. Hints of why two species of cross-links are necessary can be gleaned by studying bristle formation. Bristles sprout with only microtubules within them. A little later in development actin filaments appear. At early stages the filaments in the bundles are randomly packed. Later the filaments in the bundles become hexagonally packed and maximally cross-linked. We consider that the forked proteins may be necessary early in development to tie the filaments together in a bundle so that they can be subsequently zippered together by fascin (the singed gene product).

F-actin bundles in Drosophila bristles are assembled from modules composed of short filaments.

The actin bundles in Drosophila bristles run the length of the bristle cell and are accordingly 65 microns (microchaetes) or 400 microns (macrochaetes) in length, depending on the bristle type. Shortly after completion of bristle elongation in pupae, the actin bundles break down as the bristle surface becomes chitinized. The bundles break down in a bizarre way; it is as if each bundle is sawed transversely into pieces that average 3 microns in length. Disassembly of the actin filaments proceeds at the "sawed" surfaces. In all cases, the cuts in adjacent bundles appear in transverse register. From these images, we suspected that each actin bundle is made up of a series of shorter bundles or modules that are attached end-to-end. With fluorescent phalloidin staining and serial thin sections, we show that the modular design is present in nondegenerating bundles. Decoration of the actin filaments in adjacent bundles in the same bristle with subfragment 1 of myosin reveals that the actin filaments in every module have the same polarity. To study how modules form developmentally, we sectioned newly formed and elongating bristles. At the bristle tip are numerous tiny clusters of 6-10 filaments. These clusters become connected together more basally to form filament bundles that are poorly organized, initially, but with time become maximally cross-linked. Additional filaments are then added to the periphery of these organized bundle modules. All these observations make us aware of a new mechanism for the formation and elongation of actin filament bundles, one in which short bundles are assembled and attached end-to-end to other short bundles, as are the vertical girders between the floors of a skyscraper.

Formation of actin filament bundles in the ring canals of developing Drosophila follicles.

Growing the intracellular bridges that connect nurse cells with each o ther and to the developing oocyte is vital for egg development. These ring canals increase from 0.5 microns in diameter at stage 2 to 10 microns in diameter at stage 11. Thin sections cut horizontally as you would cut a bagel, show that there is a layer of circumferentially oriented actin filaments attached to the plasma membrane at the periphery of each canal. By decoration with subfragment 1 of myosin we find actin filaments of mixed polarities in the ring such as found in the "contractile ring" formed during cytokinesis. In vertical sections through the canal the actin filaments appear as dense dots. At stage 2 there are 82 actin filaments in the ring, by stage 6 there are 717 and by stage 10 there are 726. Taking into account the diameter, this indicates that there is 170 microns of actin filaments/canal at stage 2 (pi x 0.5 microns x 82), 14,000 microns at stage 9 and approximately 23,000 microns at stage 11 or one inch of actin filament! The density of actin filaments remains unchanged throughout development. What is particularly striking is that by stages 4-5, the ring of actin filaments has achieved its maximum thickness, even though the diameter has not yet increased significantly. Thereafter, the diameter increases. Throughout development, stages 2-11, the canal length also increases. Although the density (number of actin filaments/micron2) through a canal remains constant from stage 5 on, the actin filaments appear as a net of interconnected bundles. Further information on this net of bundles comes from studying mutant animals that lack kelch, a protein located in the ring canal that has homology to the actin binding protein, scruin. In this mutant, the actin filaments form normally but individual bundles that comprise the fibers of the net are not bound tightly together. Some bundles enter into the ring canal lumen but do not completely occlude the lumen. all these observations lay the groundwork for our understanding of how a noncontractile ring increases in thickness, diameter, and length during development.

Actin filament cables in Drosophila nurse cells are composed of modules that slide passively past one another during dumping.

At a late stage in Drosophila oogenesis, nurse cells rapidly expel their cytoplasm into the oocyte via intracellular bridges by a process called nurse cell dumping. Before dumping, numerous cables composed of actin filaments appear in the cytoplasm and extend inward from the plasma membrane toward the nucleus. This actin cage prevents the nucleus, which becomes highly lobed, from physically blocking the intracellular bridges during dumping. Each cable is composed of a linear series of modules composed of approximately 25 cross-linked actin filaments. Adjacent modules overlap in the cable like the units of an extension ladder. During cable formation, individual modules are nucleated from the cell surface as microvilli, released, and then cross-linked to an adjacent forming module. The filaments in all the modules in a cable are unidirectionally polarized. During dumping as the volume of the cytoplasm decreases, the nucleus to plasma membrane distance decreases, compressing the actin cables that shorten as adjacent modules slide passively past one another just as the elements of an extension ladder slide past one another for storage. In Drosophila, the modular construction of actin cytoskeletons seems to be a generalized strategy. The behavior of modular actin cytoskeletons has implications for other actin-based cytoskeletal systems, e.g., those involved in Listeria movement, in cell spreading, and in retrograde flow in growth cones and fibroblasts.

Regulation of actin filament cross-linking and bundle shape in Drosophila bristles.

Previous studies demonstrate that in developing Drosophila bristles, two cross-linking proteins are required sequentially to bundle the actin filaments that support elongating bristle cells. The forked protein initiates the process and facilitates subsequent cross-linking by fascin. Using cross-linker-specific antibodies, mutants, and drugs we show that fascin and actin are present in excessive amounts throughout bundle elongation. In contrast, the forked cross-linker is limited throughout bundle formation, and accordingly, regulates bundle size and shape. We also show that regulation of cross-linking by phosphorylation can affect bundle size. Specifically, inhibition of phosphorylation by staurosporine results in a failure to form large bundles if added during bundle formation, and leads to a loss of cross-linking by fascin if added after the bundles form. Interestingly, inhibition of dephosphorylation by okadaic acid results in the separation of the actin bundles from the plasma membrane. We further show by thin section electron microscopy analysis of mutant and wild-type bristles that the amount of material that connects the actin bundles to the plasma membrane is also limited throughout bristle elongation. Therefore, overall bundle shape is determined by the number of actin filaments assembled onto the limited area provided by the connector material. We conclude that assembly of actin bundles in Drosophila bristles is controlled in part by the controlled availability of a single cross-linking protein, forked, and in part by controlled phosphorylation of cross-links and membrane actin connector proteins.

Why are two different cross-linkers necessary for actin bundle formation in vivo and what does each cross-link contribute?

In developing Drosophila bristles two species of cross-linker, the forked proteins and fascin, connect adjacent actin filaments into bundles. Bundles form in three phases: (a) tiny bundles appear; (b) these bundles aggregate into larger bundles; and (c) the filaments become maximally cross-linked by fascin. In mutants that completely lack forked, aggregation of the bundles does not occur so that the mature bundles consist of <50 filaments versus approximately 700 for wild type. If the forked concentration is genetically reduced to half the wild type, aggregation of the tiny bundles occurs but the filaments are poorly ordered albeit with small patches of fascin cross-linked filaments. In mutants containing an excess of forked, all the bundles tend to aggregate and the filaments are maximally crossbridged by fascin. Alternatively, if fascin is absent, phases 1 and 2 occur normally but the resultant bundles are twisted and the filaments within them are poorly ordered. By extracting fully elongated bristles with potassium iodide which removes fascin but leaves forked, the bundles change from being straight to twisted and the filaments within them become poorly ordered. From these observations we conclude that (a) forked is used early in development to aggregate the tiny bundles into larger bundles; and (b) forked facilitates fascin entry into the bundles to maximally cross-link the actin filaments into straight, compact, rigid bundles. Thus, forked aligns the filaments and then directs fascin binding so that inappropriate cross-linking does not occur.

An ecdysterone-responsive puff site in Drosophila contains a cluster of seven differentially regulated genes.

We have determined the molecular organization of an ecdysterone-responsive puff site in Drosophila melanogaster. The 71E puff site contains a tightly linked cluster of at least seven genes within a neighborhood of 10 X 10(3) base-pairs. All the genes are expressed in a tissue-specific manner in either the larval or the prepupal salivary gland. However, these genes can be divided into two groups on the basis of their temporal pattern of transcription. Six of the genes are expressed only in prepupal salivary glands and are arranged as three divergently transcribed pairs. Nestled within this region is one gene expressed primarily in late third-instar salivary glands. We conclude that this developmentally complex puff site contains six members of the ecdysterone-induced "late"-gene set and one member of the ecdysterone-regulated "intermolt" -gene set. Additional complexity is found at the transcript level: a heterogeneously sized population of RNA molecules arises from each of the seven genes.

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.

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.

Molecular characterization of the 71E late puff in Drosophila melanogaster reveals a family of novel genes.

Early metamorphic development in Drosophila melanogaster is initiated by pulses of the steroid hormone ecdysone, which are transduced into tissue-specific transcriptional cascades. This process begins with the hormone-dependent activation of a set of transcription factors (early genes) that, in turn, activate set of tissue-specific effector genes (late genes). The 71E cytogenetic region of the salivary gland polytene genome contains several ecdysone-regulated transcription units. Molecular techniques were used to analyze these genes, their transcriptional program and their evolutionary relatedness. We find that this region contains a cluster of ten coordinately regulated late genes (L71 genes) that are organized as five divergently transcribed gene pairs. Maximum parsimony analysis suggests that an ancestral L71 gene duplicated to form the first gene pair which was, in turn, duplicated to form the set of gene pairs. The L71 gene products form a family of small, chemically basic proteins with a conserved backbone of cysteine residues. In addition, the 71E region contains another gene (I71-1) with the regulatory and biochemical characteristics of the salivary gland intermolt glue proteins.

The ecdysone-induced puffing cascade in Drosophila salivary glands: a Broad-Complex early gene regulates intermolt and late gene transcription.

The steroid hormone 20-OH ecdysone triggers a classic and well-defined program of chromosome puffing that is assumed to reflect changes in transcriptional activity in Drosophila salivary glands. Mutations in each of four Broad-Complex locus (BR-C) complementation groups were analyzed for their effects on the expression of other genes that reside in several major salivary gland puffs. RNA blot analysis showed that the rbp function of the BR-C is required for the transcription of six genes in the 71E late puff and is the first demonstration that an ecdysone-induced early gene controls the transcription of late genes within the puffing cascade. In addition, the rbp function is required for the transcription of four intermolt genes (Sgs-3, Sgs-4, Sgs-5 and 71E gene VII). Mutations in the broad, l(1)2Bc and l(1)2Bd functions of the BR-C had no effect on the expression of the genes examined. We propose that the BR-C functions to control transcription at many other salivary gland loci at the beginning of metamorphosis.

The Drosophila Broad-Complex encodes a family of related proteins containing zinc fingers.

The Broad-Complex (BR-C) is essential for metamorphosis in Drosophila melanogaster. This locus is coextensive with the 2B5 ecdysone-responsive early puff and is necessary for puffing and transcription of many subsequently activated late genes in the developing salivary gland. Mapping of 31 cDNA clones indicates that approximately 100 kb of the genome is devoted to the synthesis of many BR-C RNAs. Sequence analyses of these cDNA clones show that the BR-C encodes a family of related proteins characterized by a common core amino-terminal domain fused to alternate carboxy domains each containing a pair of zinc fingers. Most proteins also contain domains rich in distinctive amino acids located between the common core and zinc finger regions. BR-C mutant alleles resulting from chromosomal rearrangements at 2B5 are associated with deletions of 5'-untranslated sequences, separation of the core coding domain from the downstream zinc finger domains, or a P element insertional disruption of a zinc finger coding sequence. We infer that the BR-C directly regulates late gene expression by specifying the synthesis of a family of proteins with DNA binding potential.

A new high-capacity cosmid vector and its use.

The construction of a cosmid, MUA-3, designed for the convenient cloning of eukaryotic DNA segments up to 48 kb in length is described. The cosmid contains all of the plasmid pBR322 with approx. 400 bases of lambda DNA, including the cohesive end site, inserted at the pBR322 PstI endonuclease recognition site. Methods for using this vector to construct several types of Drosophila melanogaster genomic DNA libraries are given, and libraries made by these methods are characterized. A sheared Drosophila DNA-EcoRI linker library is shown to stably maintain average Drosophila DNA inserts of over 40 kb and up to 48 kb, and the efficiency of producing clones by a partial restriction and ligation method is shown to be over 3 X 10(5) clones/microgram of Drosophila DNA.

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.

Molecular analysis of the ecdysterone-inducible 2B5 "early' puff in Drosophila melanogaster.

The 2B5 region of the X-chromosome in Drosophila melanogaster plays a developmentally important role in the ecdysterone-triggered response of the late third instar salivary gland. Using a combination of transposon-tagging and chromosomal walking techniques, we have isolated 231 kb of contiguous genomic DNA sequences corresponding to this region. We have more precisely aligned this DNA to the 2B1,2 to 2B5-6 interval of the cytogenetic map by locating the position of three well-characterized chromosomal breakpoints by in situ hybridization and genomic DNA blotting experiments. Labeled cDNA, synthesized from poly(A)+ RNA isolated from hormone-induced salivary gland and imaginal disc tissues and hybridized to the cloned DNA, demonstrated that the ecdysterone-inducible sequences mapped to DNA segments corresponding to the 2B3,4 to 2B5-6 interval. Although some of these sequences were inducible in only one tissue type, many were found to be inducible in both salivary glands and imaginal discs. RNA blotting experiments have detected a major 4.5-kb RNA which is hormone inducible in the larval salivary gland and whose quantitative induction is not inhibited by cycloheximide. Thus, the 4.5-kb RNA represents at least one product from the ecdysterone-responsive 2B5 "early' puff.

Poly(A) shortening of coregulated transcripts in Drosophila.

The 71E ecdysterone-regulated puff of Drosophila melanogaster contains a cluster of six coregulated "late" genes which are expressed in the prepupal salivary gland. The resulting transcripts exhibit a decrease in their length during the 12-hr period in which they accumulate. Using the enzyme ribonuclease H, we show that this size decrease is a result of a progressively shorter poly(A) tract and suggest that these transcripts undergo an active sequential shortening of their poly(A) tracts in prepupal salivary glands. It is interesting to note that this shortening precedes the complete loss of these transcripts from the RNA population.

Closely linked DNA elements control the expression of the Sgs-5 glue protein gene in Drosophila.

cis-acting sequence regions involved in the regulation of Sgs-5 gene expression were mapped by testing DNA segments containing the Sgs-5 RNA coding region and various amounts of adjacent sequences for the ability to express Sgs-5 RNA. Following injection of the DNA segments into Drosophila embryos, expression of the gene was assayed in the salivary glands of the injected animals after they developed to third instar larvae, these somatically transformed individuals serving as an in vivo transient expression system. The information necessary for the expression of Sgs-5 is contained within 109 bp upstream and 69 bp downstream of the transcribed region. Somatic transformation experiments also show that some feature within the limits of a 1012-bp DNA segment containing the Sgs-5 RNA coding region derived from the Sgs-5 RNA null stock CA-2 must be responsible for the lack of transcription from this allele. The only DNA sequence differences between active and null alleles, within the 1012 bp, are seven single-base-pair substitutions between -84 bp and +175 bp relative to the RNA start site. One or a combination of these sites are likely contributors to the transcriptional inactivity of the Sgs-5CA2 allele.

Differential expression of Broad-Complex transcription factors may forecast tissue-specific developmental fates during Drosophila metamorphosis.

The steroid hormone ecdysone initiates metamorphosis in Drosophila melanogaster by activating a cascade of gene activity that includes primary response transcriptional regulators and secondary response structural genes. The Broad-Complex (BR-C) primary response gene is composed of several distinct genetic functions and encodes a family of related transcription factor isoforms. Our objective was to determine whether BR-C isoforms were components of the primary ecdysone response in all tissues and whether tissue-specific isoform expression is associated with tissue-specific metamorphic outcomes. We used specific antibody reagents that recognize and distinguish among the Z1, Z2 and Z3 BR-C protein isoforms to study protein expression patterns during the initial stages of metamorphosis. Western blot analyses demonstrated that BR-C isoforms are induced at the onset of metamorphosis, each with unique kinetics of induction and repression. Whole-mount immunostaining showed that the BR-C proteins accumulate in the nuclei of all larval and imaginal tissues indicating that the BR-C is induced as a primary response in many tissues. Several tissues express different levels and combinations of the BR-C isoforms suggesting that the BR-C is important in determining the tissue-specific outcome of many parallel ecdysone response cascades. For example, prepupal salivary glands (destined for histolysis during metamorphosis) express Z1 isoforms while imaginal discs (destined for cell differentiation and morphogenesis) shift from the synthesis of Z2 isoforms to the synthesis of Z1 isoforms. The prepupal central nervous system (destined for tissue remodeling) expresses all isoforms, with Z3 predominating. Salivary gland chromosome immunostaining indicated that BR-C proteins interact directly with numerous loci in the polytene genome. Finally, western blot analyses showed that distinct BR-C genetic functions can be correlated with single and specific BR-C protein isoforms.

The Drosophila Broad-Complex plays a key role in controlling ecdysone-regulated gene expression at the onset of metamorphosis.

During Drosophila third instar larval development, one or more pulses of the steroid hormone ecdysone activate three temporally distinct sets of genes in the salivary glands, represented by puffs in the polytene chromosomes. The intermolt genes are induced first, in mid-third instar larvae; these genes encode a protein glue used by the animal to adhere itself to a solid substrate for metamorphosis. The intermolt genes are repressed at puparium formation as a high titer ecdysone pulse directly induces a small set of early regulatory genes. The early genes both repress their own expression and activate more than 100 late secondary-response genes. The Broad-Complex (BR-C) is an early ecdysone-inducible gene that encodes a family of DNA binding proteins defined by at least three lethal complementation groups: br, rbp, and l(1)2Bc. We have found that the BR-C is critical for the appropriate regulation of all three classes of ecdysone-inducible genes. Both rbp and l(1)2Bc are required for glue gene induction in mid-third instar larvae. In addition, the l(1)2Bc function is required for glue gene repression in prepupae; in l(1)2Bc mutants the glue genes are re-induced by the late prepupal ecdysone pulse, recapitulating a mid-third instar regulatory response at an inappropriate stage in development. The l(1)2Bc function is also required for the complete ecdysone induction of some early mRNAs (E74A, E75A, and BR-C) and efficient repression of most early mRNAs in prepupae. Like the intermolt secondary-response genes, the late secondary-response genes are absolutely dependent on rbp for their induction. An effect of l(1)2Bc mutations on late gene activity can also be detected, but is most likely a secondary consequence of the submaximal ecdysone-induction of a subset of early regulatory products. Our results indicate that the BR-C plays a key role in dictating the stage-specificity of the ecdysone response. In addition, the ecdysone-receptor protein complex alone is not sufficient for appropriate induction of the early primary-response genes, but requires the prior expression of BR-C proteins. These studies define the BR-C as a key regulator of gene activity at the onset of metamorphosis in Drosophila.

The Broad-Complex directly controls a tissue-specific response to the steroid hormone ecdysone at the onset of Drosophila metamorphosis.

In Drosophila, all of the major metamorphic transitions are regulated by changes in the titer of the steroid hormone ecdysone. Here we examine how a key regulator of metamorphosis and primary ecdysone response gene, the Broad-Complex, transmits the hormonal signal to one of its targets, the Sgs-4 glue gene. We show that Broad-Complex RNAs accumulate in mid third instar larval salivary glands prior to Sgs-4 induction, as expected for the products of a gene that regulates the timing of Sgs-4 activation. The Broad-Complex codes for a family of zinc finger transcriptional regulators. We have identified a number of binding sites for these proteins in sequences known to regulate the timing of Sgs-4 induction, and have used these sites to derive a binding consensus for each protein. Some of these binding sites are required in vivo for Sgs-4 activity. In addition, rbp+, a genetically defined Broad-Complex function that is required for Sgs-4 induction, acts through these Broad-Complex binding sites. Thus, the Broad-Complex directly mediates a temporal and tissue-specific response to ecdysone as larvae become committed to metamorphosis.