PubMed Health⌕ Search

Biomedical subjects

Dan Hultmark

Publications and source records attributed to Dan Hultmark.

16 recordsLinked to original sources
PubMed2007

Reciprocal regulation of Rac1 and Rho1 in Drosophila circulating immune surveillance cells.

In many cell types it is evident that the small GTPases Rac and Rho regulate each other's activities. What is unclear is exactly how this regulation occurs. To further elucidate this interaction we examined the activities of Rac1 and Rho1 in Drosophila cellular immune surveillance cells. In larvae the cellular immune response involves circulating cells (hemocytes) that can be recruited from a hematopoietic organ located behind the brain, as well as a sessile population found just underneath the larval cuticle. We demonstrate for the first time that Rho-kinase activation requires both Rho1 and the Drosophila c-Jun N-terminal kinase (Basket). We also show that Rac1, via Basket, regulates Rho1 activity, possibly by inhibiting RhoGAPp190. In the reciprocal pathway, co-expression of dominant negative Rho-kinase and constitutive active Rho1 induces a Rac1-like phenotype. This induction requires the formin Diaphanous. Co-expression of dominant negative Rho-kinase and constitutive active Rho1 also induces filopodia formation, with Diaphanous enriched at the tips. The Rac1-like phenotypes, and filopodia formation, could be blocked by co-expression of dominant negative Rac1. Finally, though dominant negative Rac1 is able to block filopodia formation in the overexpression experiments, only Rac2 is necessary for filopodia formed by hemocytes after parasitization.

Animals↗
PubMed2006

Rac1 signalling in the Drosophila larval cellular immune response.

The Drosophila larval cellular immune response involves cells (hemocytes) that can be recruited from a hematopoietic organ located behind the brain, as well as a sessile population of cells found just underneath the larval cuticle arranged in a segmental pattern. By using two Rac1 GTPase effector-loop mutants together with epistasis studies, we show that Rac1 requires the Drosophila melanogaster Jun N-terminal kinase Basket (Bsk), as well as stable actin formation to recruit the sessile hemocyte population. We show that actin stabilization is necessary for Rac1-induced hemocyte activation by lowering cofilin (encoded by the twinstar gene tsr) expression in blood cells. Removing Bsk by RNAi suppressed Rac1-induced release of sessile hemocytes. RNAi against Bsk also suppressed Rac1 induction of lamellocytes, a specialized population of hemocytes necessary for the encapsulation of invading pathogens. Furthermore, Rac1 and Bsk are involved in regulating the formation of actin- and focal adhesion kinase (FAK)-rich placodes in hemocytes. Lastly, Rac1 and Bsk are both required for the proper encapsulation of eggs from the parasitoid wasp Leptipolina boulardi. From these data we conclude that Rac1 induces Bsk activity and stable actin formation for cellular immune activation, leading to sessile hemocyte release and an increase in the number of circulating hemocytes.

Actin Depolymerizing Factors↗
PubMed2006

Expression pattern of Filamin-240 in Drosophila blood cells.

The expression pattern of Filamin-240 was studied in subsets of Drosophila blood cells by means of immunofluorescent staining and Western blot analysis with use of an antibody specific to a "filamin-folding domain", a consensus motif profile generated from the 20 existing filamin repeats. Expression of Filamin-240 is restricted to lamellocytes - a special blood cell type of the cellular immune response - and is involved in the regulation of lamellocyte development. In the cher1 homozygous larvae, which lack Filamin-240 protein, a vigorous lamellocyte differentiation occurs which is further enhanced upon in vivo immune challenge by a parasitic wasp, Leptopilina boulardi. By introducing a full-length transgene encoding the Drosophila Filamin-240 protein into the cher1 Filamin-deficient homozygous mutant, the mutant blood cell phenotype was rescued. These data demonstrate that the expression of Filamin-240 is strictly lamellocyte specific in Drosophila blood cells and that the protein is a suppressor of lamellocyte development.

Animals↗
PubMed2006

Nora virus, a persistent virus in Drosophila, defines a new picorna-like virus family.

Several viruses, including picornaviruses, are known to establish persistent infections, but the mechanisms involved are poorly understood. Here, a novel picorna-like virus, Nora virus, which causes a persistent infection in Drosophila melanogaster, is described. It has a single-stranded, positive-sense genomic RNA of 11879 nt, followed by a poly(A) tail. Unlike other picorna-like viruses, the genome has four open reading frames (ORFs). One ORF encodes a picornavirus-like cassette of proteins for virus replication, including an iflavirus-like RNA-dependent RNA polymerase and a helicase that is related to those of mammalian picornaviruses. The three other ORFs are not closely related to any previously described viral sequences. The unusual sequence and genome organization in Nora virus suggest that it belongs to a new family of picorna-like viruses. Surprisingly, Nora virus could be detected in all tested D. melanogaster laboratory stocks, as well as in wild-caught material. The viral titres varied enormously, between 10(4) and 10(10) viral genomes per fly in different stocks, without causing obvious pathological effects. The virus was also found in Drosophila simulans, a close relative of D. melanogaster, but not in more distantly related Drosophila species. It will now be possible to use Drosophila genetics to study the factors that control this persistent infection.

Animals↗
PubMed2005

Inhibitor of apoptosis 2 and TAK1-binding protein are components of the Drosophila Imd pathway.

The Imd signaling cascade, similar to the mammalian TNF-receptor pathway, controls antimicrobial peptide expression in Drosophila. We performed a large-scale RNAi screen to identify novel components of the Imd pathway in Drosophila S2 cells. In all, 6713 dsRNAs from an S2 cell-derived cDNA library were analyzed for their effect on Attacin promoter activity in response to Escherichia coli. We identified seven gene products required for the Attacin response in vitro, including two novel Imd pathway components: inhibitor of apoptosis 2 (Iap2) and transforming growth factor-activated kinase 1 (TAK1)-binding protein (TAB). Iap2 is required for antimicrobial peptide response also by the fat body in vivo. Both these factors function downstream of Imd. Neither TAB nor Iap2 is required for Relish cleavage, but may be involved in Relish nuclear localization in vitro, suggesting a novel mode of regulation of the Imd pathway. Our results show that an RNAi-based approach is suitable to identify genes in conserved signaling cascades.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Invertebrate immunity and the limits of mechanistic immunology.

Rapid progress is being made in elucidating the molecular mechanisms involved in invertebrate immunity. This search for molecules runs the risk of missing important phenomena. In vertebrates, acquired protection and pathogen-specific responses were demonstrated experimentally long before the mechanisms responsible were elucidated. Without analogous experiments, mechanism-driven work may not demonstrate the full richness of invertebrate immunity.

Animals↗
PubMed2005

Drosophila melanogaster Rac2 is necessary for a proper cellular immune response.

It has been reported that during Drosophila embryonic development, and in cell culture, that the Rac GTPases are redundant. To better elucidate Rac function in Drosophila, we decided to study the role of Rac2 in larval cellular defense reactions against the parasitiod Leptopilina boulardi. Here we show a dramatic effect in the context of cellular immunity where, unlike embryonic development, Rac2 appears to have a non-redundant function. When an invading parasitoid is recognized as foreign, circulating hemocytes (blood cells) should recognize and attach to the egg chorion. After attachment the hemocytes should then spread to form a multilayered capsule surrounding the invader. In Rac2 mutants this process is disrupted. Immune surveillance cells, known as plasmatocytes, adhere to the parasitoid egg but fail to spread, and septate junctions do not assemble, possibly due to mislocalization of the Protein 4.1 homolog Coracle. Finally, larger cells known as lamellocytes attach to the capsule but also fail to spread, and there is a lack of melanization. From these results it appears that Rac2 is necessary for the larval cellular immune response.

Animals↗
PubMed2004

A directed screen for genes involved in Drosophila blood cell activation.

An attack by a parasitic wasp activates a vigorous cellular immune response in Drosophila larvae. This response is manifested by an increased number of circulating cells, the hemocytes, and by the appearance of a specialized class of hemocyte, the lamellocytes, which participate in the encapsulation and killing of the parasite. To study the molecular mechanisms of this response, we have overexpressed different genes in the hemocytes, by using the GAL4-upstream activating sequence system and a hemocyte-specific Hemese-GAL4 driver. Multiple transgenes were tested, representing several important signaling pathways. We found that the proliferation response and the activation of lamellocyte formation are independent phenomena. A drastic increase in the number of circulating hemocytes is caused by receptor tyrosine kinases, such as Egfr, Pvr, and Alk, as well as by the downstream signaling components Ras85D and pointed, supporting the notion that the Ras-mitogen-activated protein kinase pathway regulates hemocyte numbers. In the case of Pvr and Alk, this phenotype also is accompanied by lamellocyte formation. By contrast, constitutively active hopscotch and hemipterous give massive activation of lamellocyte formation with little or no increase in total hemocyte numbers. This finding indicates that both the Jak/Stat and the Jun kinase pathways affect lamellocyte formation. Still other signals, mediated by aop(ACT), Toll(10b), and Rac1 expression, cause a simultaneous increase in lamellocyte and total cell numbers, and the same effect is seen when WNT signaling is suppressed. We conclude that the activation of a cellular response is complex and affected by multiple signaling pathways.

Animals↗
PubMed2004

A rapid rosetting method for separation of hemocyte sub-populations of Drosophila melanogaster.

Hemocytes, cellular elements of the innate immune system in insects, play a crucial role in the cellular and humoral immune response. Although a significant amount of information has been collected on their differentiation and function, our understanding of hemocyte development is far from complete. Their characterisation is mostly based on morphological criteria. However, molecular markers were recently identified, defining functional subsets by the aid of monoclonal antibodies. Isolated subsets of hemocytes, in sufficient quantity and purity could help to analyse their development in vitro and also to further define their molecular characteristics. Here we describe an antibody-based rosetting technique for the physical separation of Drosophila hemocyte sub-populations. We have applied anti-hemocyte antibodies coupled to sheep red blood cells for separation. The method relies on the formation of rosettes between hemocytes and sheep erythrocytes, sensitised with discriminative anti-hemocyte monoclonal antibodies. Using this method the rosetting and non-rosetting hemocytes can be separated from each other by gradient centrifugation. Rosette-forming cells from the pellet and non-rosetting cells from the interface can be isolated in high recovery. The method can be used for functional and molecular characterisation of hemocyte sub-populations. The procedure is sensitive, reproducible and easy to perform.

Animals↗
PubMed2003

Functional diversity of the Drosophila PGRP-LC gene cluster in the response to lipopolysaccharide and peptidoglycan.

The peptidoglycan recognition protein PGRP-LC is a major activator of the imd/Relish pathway in the Drosophila immune response. Three transcripts are generated by alternative splicing of the complex PGRP-LC gene. The encoded transmembrane proteins share an identical intracellular part, but each has a separate extracellular PGRP-domain: x, y, or a. Here we show that two of these isoforms play unique roles in the response to different microorganisms. Using RNA interference in Drosophila mbn-2 cells, we found that PGRP-LCx is the only isoform required to mediate signals from Gram-positive bacteria and purified bacterial peptidoglycan. By contrast, the recognition of Gram-negative bacteria and bacterial lipopolysaccharide requires both PGRP-LCa and LCx. The third isoform, LCy, is expressed at lower levels and may be partially redundant. Two additional PGRP domains in the gene cluster, z and w, are both included in a single transcript of a separate gene, PGRP-LF. Suppression of this transcript does not block the response to any of the microorganisms tested.

Alternative Splicing↗
PubMed2003

Caspase-mediated processing of the Drosophila NF-kappaB factor Relish.

The NF-kappaB-like transcription factor Relish plays a central role in the innate immune response of Drosophila. Unlike other NF-kappaB proteins, Relish is activated by endoproteolytic cleavage to generate a DNA-binding Rel homology domain and a stable IkappaB-like fragment. This signal-induced endoproteolysis requires the activity of several gene products, including the IkappaB kinase complex and the caspase Dredd. Here we used mutational analysis and protein microsequencing to demonstrate that a caspase target site, located in the linker region between the Rel and the IkappaB-like domain, is the site of signal-dependent cleavage. We also show physical interaction between Relish and Dredd, suggesting that Dredd indeed is the Relish endoprotease. In addition to the caspase target site, the C-terminal 107 aa of Relish are required for endoproteolysis and signal-dependent phosphorylation by the Drosophila IkappaB kinase beta. Finally, an N-terminal serine-rich region in Relish and the PEST domain were found to negatively regulate Relish activation.

Animals↗
PubMed2003

Hemese, a hemocyte-specific transmembrane protein, affects the cellular immune response in Drosophila.

We have identified a previously undescribed transmembrane protein, Hemese, from Drosophila melanogaster blood cells (hemocytes), by using a monoclonal pan-hemocyte antibody. Heavy glycosylation is suggested by the heterogeneous size distribution, ranging between 37 and 70 kDa. Hemese expression is restricted to the cell surfaces of hemocytes of all classes, and to the hematopoietic organs. The sequence of the corresponding gene, Hemese (He), predicts a glycophorin-like protein of 15 kDa, excluding an N-terminal signal peptide, with a single hydrophobic transmembrane region. The extracellular region consists mainly of Ser/Thr-rich sequence of low complexity, with several potential O-glycosylation sites. Hemese contains phosphotyrosine and the cytoplasmic region has potential phosphorylation sites, suggesting an involvement in signal transduction. Depletion of Hemese by RNA interference has no obvious effect under normal conditions, but the cellular response to parasitic wasps is much enhanced. This finding indicates that Hemese plays a modulatory role in the activation or recruitment of the hemocytes.

Amino Acid Sequence↗
PubMed2003

A cytokine in the Drosophila stress response.

The fruit fly, Drosophila melanogaster, has become a popular tool for studying immediate reactions to environmental hazards, such as the heat shock and innate immune responses. In mammals, protective responses to infections and other insults are coordinated by a complex network of cytokines that mediate cell-to-cell signaling. By contrast, the corresponding heat shock and innate immune responses in Drosophila have usually been regarded as cell-autonomous processes. However, in this issue of Developmental Cell, show that cytokines do play a role in mediating an acute phase response in this organism.

Animals↗
PubMed2002

Requirement for a peptidoglycan recognition protein (PGRP) in Relish activation and antibacterial immune responses in Drosophila.

Components of microbial cell walls are potent activators of innate immune responses in animals. For example, the mammalian TLR4 signaling pathway is activated by bacterial lipopolysaccharide and is required for resistance to infection by Gram-negative bacteria. Other components of microbial surfaces, such as peptidoglycan, are also potent activators of innate immune responses, but less is known about how those components activate host defense. Here we show that a peptidoglycan recognition protein, PGRP-LC, is absolutely required for the induction of antibacterial peptide genes in response to infection in Drosophila and acts by controlling activation of the NF-kappaB family transcription factor Relish.

Active Transport, Cell Nucleus↗