Regulation of Toll and Toll-like receptor signaling by the endocytic pathway.

The Toll/TLR receptor family plays a central role in both vertebrate and insect immunity, driving the activation of humoral immunity in response to pathogens. In Drosophila, Toll is also responsible for directing the formation of the Dorsal/NFkappaB gradient specifying dorsoventral patterning of the embryo. Two recent studies have revealed that endocytosis and elements of the molecular machinery governing endosomal progression are required for Drosophila Toll signaling in development and immunity. We demonstrated that Toll is not only present at the plasma membrane but also in a Rab5(+) early endosomal compartment in the embryo and that the distribution of constitutively active Toll(10B) is shifted towards endosomes. Localized inhibition of Rab5 function on the ventral side leads to a reduction of nuclear Dorsal levels, while locally increasing Rab5 function leads to potentiation of signaling. Independently, another laboratory identified the endosomal protein Mop as a potentiator of Toll signaling in Drosophila cell culture and fat-body tissue. Mop functions together with the ESCRT 0 component, Hrs, previously reported to stimulate endosomal progression and the signaling ability of internalized EGFR. We discuss these studies and briefly summarize the most significant findings concerning the role of intracellular localization and trafficking in mammalian TLR function.

A set of P-element transformation vectors permitting the simplified generation of fluorescent fusion proteins in Drosophila melanogaster.

We have assembled a molecular toolkit enabling the facile generation of C-terminal fluorescent protein fusion constructs for the construction of transgenic Drosophila lines in the form of a series of modified vectors for P-element transformation. These vectors contain one of a variety of fluorescent tags including GFP, mCherry, Venus, Cerulean, photoactivatable paGFP and the red-to-green photoconvertible protein Dendra and provide a range of options with respect to transcriptional regulation. The vectors have been extensively tested in vivo and can produce fluorescent chimeric proteins that are functional.

Shedding light on Toll signaling through live imaging.

The Toll receptor propagates the ventralizing signal designating dorsal/ventral cell fate in the Drosophila embryo. The application of live-imaging approaches to this classical developmental signaling pathway is yielding some surprising new insights into Toll receptor signaling. In addition to its previously known plasma membrane localization, Toll is present in Rab5+ early endosomes. Dominant, constitutively active forms of Toll preferentially partition into endosomes. Blocking endocytosis locally prevents Toll from signaling suggesting that endocytosis is required for Toll to signal. Augmenting endocytosis increases Toll signaling. Both interventions alter the shape of the Dorsal gradient globally indicating an important role of endocytosis in fixing spatial information for the Dorsal gradient.

Localization and activation of the Drosophila protease easter require the ER-resident saposin-like protein seele.

Drosophila embryonic dorsal-ventral polarity is generated by a series of serine protease processing events in the egg perivitelline space. Gastrulation Defective processes Snake, which then cleaves Easter, which then processes Spätzle into the activating ligand for the Toll receptor. seele was identified in a screen for mutations that, when homozygous in ovarian germline clones, lead to the formation of progeny embryos with altered embryonic patterning; maternal loss of seele function leads to the production of moderately dorsalized embryos. By combining constitutively active versions of Gastrulation Defective, Snake, Easter, and Spätzle with loss-of-function alleles of seele, we find that Seele activity is dispensable for Spätzle-mediated activation of Toll but is required for Easter, Snake, and Gastrulation Defective to exert their effects on dorsal-ventral patterning. Moreover, Seele function is required specifically for secretion of Easter from the developing embryo into the perivitelline space and for Easter processing. Seele protein resides in the endoplasmic reticulum of blastoderm embryos, suggesting a role in the trafficking of Easter to the perivitelline space, prerequisite to its processing and function. Easter transport to the perivitelline space represents a previously unappreciated control point in the signal transduction pathway that controls Drosophila embryonic dorsal-ventral polarity.

Endocytosis is required for Toll signaling and shaping of the Dorsal/NF-kappaB morphogen gradient during Drosophila embryogenesis.

Dorsoventral cell fate in the Drosophila embryo is specified by activation of the Toll receptor, leading to a ventral-to-dorsal gradient across nuclei of the NF-κB transcription factor Dorsal. Toll receptor has been investigated genetically, molecularly, and immunohistologically, but much less is known about its dynamics in living embryos. Using live imaging of fluorescent protein chimeras, we find that Toll is recruited from the plasma membrane to Rab5(+) early endosomes. The distribution of a constitutively active form of Toll, Toll(10b), is shifted from the plasma membrane to early endosomes. Inhibition of endocytosis on the ventral side of the embryo attenuates Toll signaling ventrally and causes Dorsal to accumulate on the dorsal side of the embryo, essentially inverting the dorsal/ventral axis. Conversely, enhancing endocytosis laterally greatly potentiates Toll signaling locally, altering the shape of the Dorsal gradient. Photoactivation and fluorescence recovery after photobleaching studies reveal that Toll exhibits extremely limited lateral diffusion within the plasma membrane, whereas Toll is highly compartmentalized in endosomes. When endocytosis is blocked ventrally, creating an ectopic dorsal signaling center, Toll is preferentially endocytosed at the ectopic signaling center. We propose that Toll signals from an endocytic compartment rather than the plasma membrane. Our studies reveal that endocytosis plays a pivotal role in the spatial regulation of Toll receptor activation and signaling and in the correct shaping of the nuclear Dorsal concentration gradient.

Dynamics of the Dorsal morphogen gradient.

The dorsoventral (DV) patterning of the Drosophila embryo depends on the nuclear localization gradient of Dorsal (Dl), a protein related to the mammalian NF-kappaB transcription factors. Current understanding of how the Dl gradient works has been derived from studies of its transcriptional interpretation, but the gradient itself has not been quantified. In particular, it is not known whether the Dl gradient is stable or dynamic during the DV patterning of the embryo. To address this question, we developed a mathematical model of the Dl gradient and constrained its parameters by experimental data. Based on our computational analysis, we predict that the Dl gradient is dynamic and, to a first approximation, can be described as a concentration profile with increasing amplitude and constant shape. These time-dependent properties of the Dl gradient are different from those of the Bicoid and MAPK phosphorylation gradients, which pattern the anterior and terminal regions of the embryo. Specifically, the gradient of the nuclear levels of Bicoid is stable, whereas the pattern of MAPK phosphorylation changes in both shape and amplitude. We attribute these striking differences in the dynamics of maternal morphogen gradients to the differences in the initial conditions and chemistries of the anterior, DV, and terminal systems.

Embryonic expression of Drosophila IMP in the developing CNS and PNS.

Drosophila IMP (dIMP) is related to the vertebrate RNA-binding proteins IMP1-3, ZBP1, Vg1RBP and CRD-BP, which are involved in RNA regulatory processes such as translational repression, localization and stabilization. The proteins are expressed in many fetal tissues, including the developing nervous system, and IMP up-regulation in solid tumors correlates with a high metastatic potential and poor prognosis. In this study, we used immunohistochemistry and live-imaging of an endogenous promoter-driven GFP-dIMP fusion protein to reveal the expression pattern of dIMP protein throughout embryogenesis. In the cellular blastoderm, immunoreactivity was seen in the entire cell-layer, where it was localized apically to the nucleus, and in the pole cells. Later, the GFP-dIMP fusion protein appeared in the developing central nervous system, both in the brain and in the ventral nerve cord. In the peripheral nervous system, immunoreactivity was detected in both neurons and accessory cells of chordotonal and external sensory organs.

Nucleocytoplasmic shuttling mediates the dynamic maintenance of nuclear Dorsal levels during Drosophila embryogenesis.

In Drosophila, the NF-kappaB/REL family transcription factor, Dorsal, redistributes from the cytoplasm to nuclei, forming a concentration gradient across the dorsoventral axis of the embryo. Using live imaging techniques in conjunction with embryos expressing a chimeric Dorsal-GFP, we demonstrate that the redistribution of Dorsal from cytoplasm to nucleus is an extremely dynamic process. Nuclear Dorsal concentration changes continuously over time in all nuclei during interphase. While Dorsal appears to be nuclearly localized primarily in ventral nuclei, it is actively shuttling into and out of all nuclei, including nuclei on the dorsal side. Nuclear export is blocked by leptomycin B, a potent inhibitor of Exportin 1 (CRM1)-mediated nuclear export. We have developed a novel in vivo assay revealing the presence of a functional leucine-rich nuclear export signal within the carboxyterminal 44 amino acids of Dorsal. We also find that diffusion of Dorsal is partially constrained to cytoplasmic islands surrounding individual syncitial nuclei. A model is proposed in which the generation and maintenance of the Dorsal gradient is a consequence of an active process involving both restricted long-range diffusion and the balancing of nuclear import with nuclear export.

The secretory membrane system in the Drosophila syncytial blastoderm embryo exists as functionally compartmentalized units around individual nuclei.

Drosophila melanogaster embryogenesis begins with 13 nuclear division cycles within a syncytium. This produces >6,000 nuclei that, during the next division cycle, become encased in plasma membrane in the process known as cellularization. In this study, we investigate how the secretory membrane system becomes equally apportioned among the thousands of syncytial nuclei in preparation for cellularization. Upon nuclear arrival at the cortex, the endoplasmic reticulum (ER) and Golgi were found to segregate among nuclei, with each nucleus becoming surrounded by a single ER/Golgi membrane system separate from adjacent ones. The nuclear-associated units of ER and Golgi across the syncytial blastoderm produced secretory products that were delivered to the plasma membrane in a spatially restricted fashion across the embryo. This occurred in the absence of plasma membrane boundaries between nuclei and was dependent on centrosome-derived microtubules. The emergence of secretory membranes that compartmentalized around individual nuclei in the syncytial blastoderm is likely to ensure that secretory organelles are equivalently partitioned among nuclei at cellularization and could play an important role in the establishment of localized gene and protein expression patterns within the early embryo.

Fluorescently labeled inhibitors detect localized serine protease activities in Drosophila melanogaster pole cells, embryos, and ovarian egg chambers.

Serine proteases are typically synthesized as proteolytically inactive zymogens that often become activated in a limited and highly localized manner. Consequently, determination of the spatial and temporal activation pattern of these molecules is of great importance to understanding the biological processes that they mediate. Until only recently, the tools to conveniently address the question of where and when serine proteases are active within complex tissues have been lacking. In order to detect spatially restricted serine protease activities in Drosophila embryos and ovaries we introduce a technique using fluorescent synthetic and protein-based inhibitors. With this approach we have detected a novel serine protease activity with a relative mobility of 37 kDa, localized to the surface of pole cells, the germ-line precursors, in embryos between nuclear cycles 11 and 14 in development. A second novel cell-specific protease activity was localized to the tissues of early gastrulating embryos. Microinjection of inhibitors into the perivitelline space of stage 2 embryos perturbed normal embryonic development. Fluorescein-conjugated chymotrypsin inhibitor and Bowman-Birk inhibitor labeled protease activity localized to the oocyte-somatic follicle cell interface of the developing egg chamber. Our results suggest that this technique holds promise to identify new spatially restricted activities in adult Drosophila tissues and developing embryos.

Molecular cloning, functional expression, and gene silencing of two Drosophila receptors for the Drosophila neuropeptide pyrokinin-2.

The database of the Drosophila Genome Project contains the sequences of two genes, CG8784 and CG8795, predicted to code for two structurally related G protein-coupled receptors. We have cloned these genes and expressed their coding parts in Chinese hamster ovary cells. We found that both receptors can be activated by low concentrations of the Drosophila neuropeptide pyrokinin-2 (CG8784, EC(50) for pyrokinin-2, 1x10(-9)M; CG8795, EC(50) for pyrokinin-2, 5 x 10(-10)M). The precise role of Drosophila pyrokinin-2 (SVPFKPRLamide) in Drosophila is unknown, but in other insects, pyrokinins have diverse myotropic actions and are also initiating sex pheromone biosynthesis and embryonic diapause. Gene silencing, using the RNA-mediated interference technique, showed that CG8784 gene silencing caused lethality in embryos, whereas CG8795 gene silencing resulted in strongly reduced viability for both embryos and first instar larvae. In addition to the two Drosophila receptors, we also identified two probable pyrokinin receptors in the genomic database from the malaria mosquito Anopheles gambiae. The two Drosophila pyrokinin receptors are, to our knowledge, the first invertebrate pyrokinin receptors to be identified.