BackgroundIn Drosophila, the transport regulator Klar displays tissue-specific localization: In photoreceptors, it is abundant on the nuclear envelope; in early embryos, it is absent from nuclei, but instead present on lipid droplets. Differential targeting of Klar appears to be due to isoform variation. Droplet targeting, in particular, has been suggested to occur via a variant C-terminal region, the LD domain. Although the LD domain is necessary and sufficient for droplet targeting in cultured cells, lack of specific reagents had made it previously impossible to analyze its role in vivo.ResultsHere we describe a new mutant allele of klar with a lesion specifically in the LD domain; this lesion abolishes both droplet localization of Klar and the ability of Klar to regulate droplet motion. It does not disrupt Klar's function for nuclear migration in photoreceptors. Using a GFP-LD fusion, we show that the LD domain is not only necessary but also sufficient for droplet targeting in vivo; it mediates droplet targeting in embryos, in ovaries, and in a number of somatic tissues.ConclusionsOur analysis demonstrates that droplet targeting of Klar occurs via a cis-acting sequence and generates a new tool for monitoring lipid droplets in living tissues of Drosophila.
During bidirectional transport, individual cargoes move continuously back and forth along microtubule tracks, yet the cargo population overall displays directed net transport. How such transport is controlled temporally is not well understood. We analyzed this issue for bidirectionally moving lipid droplets in Drosophila embryos, a system in which net transport direction is developmentally controlled. By quantifying how the droplet distribution changes as embryos develop, we characterize temporal transitions in net droplet transport and identify the crucial contribution of the previously identified, but poorly characterized, transacting regulator Halo. In particular, we find that Halo is transiently expressed; rising and falling Halo levels control the switches in global distribution. Rising Halo levels have to pass a threshold before net plus-end transport is initiated. This threshold level depends on the amount of the motor kinesin-1: the more kinesin-1 is present, the more Halo is needed before net plus-end transport commences. Because Halo and kinesin-1 are present in common protein complexes, we propose that Halo acts as a rate-limiting co-factor of kinesin-1.
During development, extracellular signals are integrated by cells to induce the transcriptional circuitry that controls morphogenesis. In the fly epidermis, Wingless (Wg)/Wnt signaling directs cells to produce either a distinctly shaped denticle or no denticle, resulting in a segmental pattern of denticle belts separated by smooth, or 'naked', cuticle. Naked cuticle results from Wg repression of shavenbaby (svb), which encodes a transcription factor required for denticle construction. We have discovered that although the svb promoter responds differentially to altered Wg levels, Svb alone cannot produce the morphological diversity of denticles found in wild-type belts. Instead, a second Wg-responsive transcription factor, SoxNeuro (SoxN), cooperates with Svb to shape the denticles. Coexpressing ectopic SoxN with svb rescued diverse denticle morphologies. Conversely, removing SoxN activity eliminated the residual denticles found in svb mutant embryos. Furthermore, several known Svb target genes are also activated by SoxN, and we have discovered two novel target genes of SoxN that are expressed in denticle-producing cells and that are regulated independently of Svb. We conclude that proper denticle morphogenesis requires transcriptional regulation by both SoxN and Svb.
KEy WoRDS enhancer of rudimentary, CG15352, P element mutagenesis ABBREVIATIoNS e(r) enhancer of rudimentary r rudimentary AcKNoWlEDGEMENTSThis work was supported by grants from the National Institutes of Health (R15 GM64364), Saint Louis University, the Monsanto Company (Monsanto Scholars Program) and SUNY Brockport. ABSTRAcTThe enhancer of rudimentary gene, e(r), encodes a 104-amino-acid, highly conserved transcription cofactor. Hypomorphic mutations of e(r) show an enhancement of a hypomorphic rudimentary mutant wing phenotype. These mutants in a wild-type background are viable, fertile, and morphologically wild-type. Since the only mutant alleles were hypomorphic, it was important to isolate null mutations to determine if any other phenotypes might be associated with a loss-of-function of e(r). We utilized a marked P element, P{SUPor-P, y + }, located 895 bp upstream of the start of transcription of e(r) to generate nineteen deficiencies in the region. Deficiencies of e(r) enhance the mutant wing phenotype of a hypomorphic rudimentary allele, r hd1 . In a wild-type background, the deficiencies of e(r), unlike the hypomorphic alleles, have a low viability and females have low fertility. The expression of e(r) in the nurse cells of the ovary is consistent with the low fertility, and suggests an ovarian function for e(r). Deficiencies of CG15352, the gene directly upstream of e(r), are not associated with any obvious mutant phenotypes and present the possibility that it encodes a nonvital or redundant function.
Scrutiny of bacterial genomes has led to the identification of a core set of genes that are essential for cell growth: the minimal genome. Many of the genes represented in the minimal genome have well defined and unambiguous functions. The function of some of the highly conserved bacterial GTPases, in particular the Era‐subfamily (Der/EngA and Era), is not completely understood. These GTPases are the topic of this investigation. Recently we have reported that over‐expression of Der caused a defect in cell wall structure and that Der could bind specifically to membrane protein. The binding reaction was reconstituted in vitro and we developed an electrophoresis based co‐migration binding assay to detect the Der‐membrane interaction. We have used that assay to assess that OmpC, and not OmpF is necessary for Der to form a high molecular weight complex when combined with membrane proteins. Based on these results, and crystal structures of OmpC and Der, we propose that domain 3 of Der binds to loop 4 on OmpC, and that this interaction is necessary for the proper regulation of cell wall assembly in Escherichia coli.
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