Ralf Pflanz

Perlecan participates in proliferation activation of quiescent Drosophila neuroblasts

Drosophila neuroblasts act as stem cells. Their proliferation is controlled through cell cycle arrest and activation in a spatiotemporal pattern. Several genes have been identified that control the pattern of neuroblast quiescence and proliferation in the central nervous system (CNS), including anachronism (ana), even skipped (eve) and terribly reduced optic lobes (trol). eve acts in a non–cell‐autonomous manner to produce a transacting factor in the larval body that stimulates cell division in the population of quiescent optic lobe neuroblasts. ana encodes a secreted glial glycoprotein proposed to repress premature proliferation of optic lobe and thoracic neuroblasts. trol was shown to act downstream of ana to activate proliferation of quiescent neuroblasts either by inactivating or bypassing ana‐dependent repression. Here, we show that trol codes for Drosophila Perlecan, a large multidomain heparan sulfate proteoglycan originally identified in extracellular matrix structures of mammals. The results suggest that trol acts in the extracellular matrix and binds, stores, and sequesters external signals and, thereby, participates in the stage‐ and region‐specific control of neuroblast proliferation.

αPS2 integrin-mediated muscle attachment in Drosophila requires the ECM protein Thrombospondin

During Drosophila embryogenesis, the attachment of somatic muscles to epidermal tendon cells requires heterodimeric PS-integrin proteins (alpha- and beta-subunits). The alpha-subunits are expressed complementarily, either tendon cell- or muscle-specific, whereas the beta-integrin subunit is expressed in both tissues. Mutations of beta-integrin cause a severe muscle detachment phenotype, whereas alpha-subunit mutations have weaker or only larval muscle detachment phenotypes. Furthermore, mutations of extracellular matrix (ECM) proteins known to act as integrin binding partners have comparatively weak effects only, suggesting the presence of additional integrin binding ECM proteins required for proper muscle attachment. Here, we report that mutations in the Drosophila gene thrombospondin (tsp) cause embryonic muscle detachment. tsp is specifically expressed in both developing and mature epidermal tendon cells. Its initial expression in segment border cells, the tendon precursors, is under the control of hedgehog-dependent signaling, whereas tsp expression in differentiated tendon cells depends on the transcription factor encoded by stripe. In the absence of tsp activity, no aspect of muscle pattern formation as well as the initial contact between muscle and tendon cells nor muscle-to-muscle attachments are affected. However, when muscle contractions occur during late embryogenesis, muscles detach from the tendon cells. The Tsp protein is localized to the tendon cell ECM where muscles attach. Genetic interaction studies indicate that Tsp specifically interacts with the alphaPS2 integrin and that this interaction is needed to withstand the forces of muscle contractions at the tendon cells.

Drosophila tao Controls Mushroom Body Development and Ethanol-Stimulated Behavior through par-1

In both mammalian and insect models of ethanol-induced behavior, low doses of ethanol stimulate locomotion. However, the mechanisms of the stimulant effects of ethanol on the CNS are mostly unknown. We have identified tao, encoding a serine-threonine kinase of the Ste20 family, as a gene necessary for ethanol-induced locomotor hyperactivity in Drosophila. Mutations in tao also affect behavioral responses to cocaine and nicotine, making flies resistant to the effects of both drugs. We show that tao function is required during the development of the adult nervous system and that tao mutations cause defects in the development of central brain structures, including the mushroom body. Silencing of a subset of mushroom body neurons is sufficient to reduce ethanol-induced hyperactivity, revealing the mushroom body as an important locus mediating the stimulant effects of ethanol. We also show that mutations in par-1 suppress both the mushroom body morphology and behavioral phenotypes of tao mutations and that the phosphorylation state of the microtubule-binding protein Tau can be altered by RNA interference knockdown of tao, suggesting that tao and par-1 act in a pathway to control microtubule dynamics during neural development.

Impaired retrograde transport by the Dynein/Dynactin complex contributes to Tau-induced toxicity

The gene mapt codes for the microtubule-associated protein Tau. The R406W amino acid substitution in Tau is associated with frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) characterized by Tau-positive filamentous inclusions. These filamentous Tau inclusions are present in a group of neurodegenerative diseases known as tauopathies, including Alzheimers disease (AD). To gain more insights into the pathomechanism of tauopathies, we performed an RNAi-based large-scale screen in Drosophila melanogaster to identify genetic modifiers of Tau[R406W]-induced toxicity. A collection of RNAi lines, putatively silencing more than 7000 genes, was screened for the ability to modify Tau[R406W]-induced toxicity in vivo. This collection covered more than 50% of all protein coding fly genes and more than 90% of all fly genes known to have a human ortholog. Hereby, we identified 62 genes that, when silenced by RNAi, modified Tau-induced toxicity specifically. Among these 62 modifiers were three subunits of the Dynein/Dynactin complex. Analysis on segmental nerves of fly larvae showed that pan neural Tau[R406W] expression and concomitant silencing of Dynein/Dynactin complex members synergistically caused strong pathological changes within the axonal compartment, but only minor changes at synapses. At the larval stage, these alterations did not cause locomotion deficits, but became evident in adult flies. Our data suggest that Tau-induced detrimental effects most likely originate from axonal rather than synaptic dysfunction and that impaired retrograde transport intensifies detrimental effects of Tau in axons. In conclusion, our findings contribute to the elucidation of disease mechanisms in tauopathies like FTDP-17 or AD.

TEV protease-mediated cleavage in Drosophila as a tool to analyze protein functions in living organisms.

Drosophila provides a powerful experimental system to analyze gene functions in a multi-cellular organism. Here we describe an in vivo method that interferes with the integrity of selected proteins through site-specific cleavage in Drosophila. The technique is based on the highly specific seven-amino-acid recognition site of the tobacco etch virus (TEV) protease. We established transgenic fly lines that direct TEV protease expression in various tissues without affecting fly viability. The insertion of the TEV protease recognition site in defined positions of target proteins mediates their sequence-specific cleavage after controlled TEV protease expression in the fly. Thereby, this technique is a powerful tool that allows the in vivo manipulation of selected proteins in a time- and tissue-specific manner.

The fatty acyl-CoA reductase Waterproof mediates airway clearance in Drosophila.

The transition from a liquid to a gas filled tubular network is the prerequisite for normal function of vertebrate lungs and invertebrate tracheal systems. However, the mechanisms underlying the process of gas filling remain obscure. Here we show that waterproof, encoding a fatty acyl-CoA reductase (FAR), is essential for the gas filling of the tracheal tubes during Drosophila embryogenesis, and does not affect branch network formation or key tracheal maturation processes. However, electron microscopic analysis reveals that in waterproof mutant embryos the formation of the outermost tracheal cuticle sublayer, the envelope, is disrupted and the hydrophobic tracheal coating is damaged. Genetic and gain-of-function experiments indicate a non-cell-autonomous waterproof function for the beginning of the tracheal gas filling process. Interestingly, Waterproof reduces very long chain fatty acids of 24 and 26 carbon atoms to fatty alcohols. Thus, we propose that Waterproof plays a key role in tracheal gas filling by providing very long chain fatty alcohols that serve as potential substrates for wax ester synthesis or related hydrophobic substances that ultimately coat the inner lining of the trachea. The hydrophobicity in turn reduces the tensile strength of the liquid inside the trachea, leading to the formation of a gas bubble, the focal point for subsequent gas filling. Waterproof represents the first enzyme described to date that is necessary for tracheal gas filling without affecting branch morphology. Considering its conservation throughout evolution, Waterproof orthologues may play a similar role in the vertebrate lung.

Bark beetle controls epithelial morphogenesis by septate junction maturation in Drosophila.

Epithelial tissues separate body compartments with different compositions. Tight junctions (TJs) in vertebrates and septate junctions (SJs) in invertebrates control the paracellular flow of molecules between these compartments. This epithelial barrier function of TJs and SJs must be stably maintained in tissue morphogenesis during cell proliferation and cell movement. Here, we show that Bark beetle (Bark), a putative transmembrane scavenger receptor-like protein, is essential for the maturation but not the establishment of SJs in Drosophila. Embryos that lack bark establish functional SJs, but due to rudimentary septae formation during subsequent embryonic development, these become non-functional. Furthermore, cell adhesion is impaired at the lateral cell membrane and the core protein complexes of SJs are mis-localised, but appear to form otherwise normally in such embryos. We propose a model in which Bark acts as a scaffold protein that mediates cell adhesion and mounting of SJ core complexes during cell rearrangement in tissue morphogenesis.

Follicle separation during Drosophila oogenesis requires the activity of the Kinesin II-associated polypeptide Kap in germline cells

Cellular localization of organelles, protein complexes and single mRNAs depends on the directed transport along microtubule tracks, a process mediated by ATP‐driven molecular motor proteins of the dynein and kinesin superfamilies. Kinesin II is a heterotrimeric protein complex composed of two motor subunits and a unique nonmotor Kinesin‐associated protein (Kap). Kap was shown to transport both particulate cargo, as axoneme components in rafts, and membrane‐bounded organelles such as melanosomes. Drosophila Kinesin II was shown to be essential for the axonal transport of choline acetyltransferase in a specific set of neurons. We have generated Kap mutants and show that gene activity is not only required for neuronal function but also for separation of follicles during early oogenesis. The data suggest that Kap participates in the transport of signalling components required for instructive interactions between germline and soma cells.

Drosophila gene tao-1 encodes proteins with and without a Ste20 kinase domain that affect cytoskeletal architecture and cell migration differently.

Tao-1, the single representative of the Sterile 20 kinase subfamily in Drosophila, is best known for destabilizing microtubules at the actin-rich cortex, regulating the cytoskeletal architecture of cells. More recently, Tao-1 was shown to act in the Salvador–Warts–Hippo pathway by phosphorylating Hippo, regulating cell growth as well as cell polarity. Here, we show that tao-1 encodes two proteins, one with the Sterile 20 kinase domain (Tao-L) and one without it (Tao-S), and that they act in an antagonistic manner. Tao-L expression causes lamellipodia-like cell protrusions, whereas Tao-S expression results in filopodia-like structures that make cells stick to the surface they attach to. Ectopic Tao-1 expression in the anterior region of Drosophila embryos results in pole cell formation as normally observed at the posterior end. Tao-S expression causes primordial germ cells (PGCs) to adhere to the inner wall of the gut primordia and prevents proper transepithelial migration to the gonads. Conversely, RNAi knockdowns of Tao-1 cause disordered migration of PGCs out of the gut epithelium, their dispersal within the embryo and cell death. The results reveal a novel function of Tao-1 in cell migration, which is based on antagonistic activities of two proteins encoded by a single gene.

dtrap-1 encodes a novel member of the heat shock super family of proteins and is expressed in derivatives of all three germ layers during Drosophila embryogenesis.

Heat shock proteins (Hsps) comprise a highly conserved superfamily of proteins that are required for stress tolerance in living cells. At physiological conditions, these proteins act as chaperones during protein folding and protein assembly processes (Parsell and Lindquist, 1993. Annu. Rev. Genet. 27, 427-496). Members of the subfamily of Hsp90 proteins were shown to be additionally involved in the presentation and structural modification of components of diverse cellular signal transduction pathways including steroid hormone reception and regulatory kinase activities (Pratt, 1993. J. Biol. Chem. 268, 21455-21458); Cutforth et al., 1994. Cell 77, 1027-1036; van der Straten et al., 1997. EMBO J. 16, 1961-1997; Hunter and Poon, 1997. Trends Cell Biol. 7, 157-161). We have identified a Drosophila gene, called dtrap-1, which encodes a Hsp of a novel subfamily that is related to the Hsp90 family of proteins. During oogenesis dtrap-1 is expressed in nurse cells and its transcripts accumulate in the oocyte. The maternal transcripts remain in the egg but rapidly degrade during early embryogenesis, except in the posterior pole region. Zygotic expression is initiated after the onset of gastrulation showing dynamic patterns of transcripts in the developing mid- and hindgut as well as a subset of mesoderm derivatives.