Robert Farkaš

Autophagy occurs upstream or parallel to the apoptosome during histolytic cell death

Histolysis refers to a widespread disintegration of tissues that is morphologically distinct from apoptosis and often associated with the stimulation of autophagy. Here, we establish that a component of the apoptosome, and pivotal regulator of apoptosis, is also required for histolytic cell death. Using in vivo and ex vivo assays, we demonstrate a global apoptogenic requirement for dark, the fly ortholog of Apaf1, and show that a required focus of dark- organismal lethality maps to the central nervous system. We further demonstrate that the Dark protein itself is a caspase substrate and find that alterations of this cleavage site produced the first hypermorphic point mutation within the Apaf1/Ced-4 gene family. In a model of `autophagic cell death, dark was essential for histolysis but dispensable for characteristic features of the autophagic program, indicating that the induction of autophagy occurs upstream or parallel to histolytic cell death. These results demonstrate that stimulation of autophagy per se is not a `killing event and, at the same time, establish that common effector pathways, regulated by the apoptosome, can underlie morphologically distinct forms of programmed cell death.

Wide mutational spectrum of a gene involved in hormone action and insecticide resistance in Drosophila melanogaster

The Methoprene-tolerant (Met) bHLH-PAS gene in Drosophila melanogaster is involved in the molecular action of juvenile hormone (JH), and mutants result in resistance to the toxic and morphogenetic effects of JH and JH agonist insecticides such as methoprene. A detailed study of Met mutants can shed light on the poorly understood action of JH as well as the molecular basis of Met resistance to JH insecticides. Nine mutant alleles bearing point mutations in Met were examined for penetrance and expressivity of three phenotypic characters: resistance, defective oogenesis, and a novel eye defect. The collection ranged from two weak alleles having less severe phenotypes to strong alleles with severe phenotypes similar to that of a null allele. The point mutations were located in both conserved and nonconserved domains. Both the eye defect, seen as severely malformed ommatidial facets in the posterior margin of the compound eye, and the oogenesis phenotype are nonconditional, whereas expression of the resistance phenotype requires treatment with JH or JH analogs (JHAs) during early metamorphosis. A proposed basis for all the phenotypic characters centers on MET action as a transcriptional regulator of ecdysone secondary-response target genes during metamorphosis. Disruption of MET function either by mutation or by JHA presence during early metamorphosis results in transcriptional misregulation of different target genes, resulting in the pathology seen in either instance. The variety of amino acid changes in MET that resulted in resistance may portend a rapid rise in resistance in response to increased use of JH insecticides in field insect populations.

ULTRASTRUCTURAL CHANGES OF DROSOPHILA LARVAL AND PREPUPAL SALIVARY GLANDS CULTURED IN VITRO WITH ECDYSONE

SummaryAlterations in the ultrastructure of in vitro cultured larval salivary glands of Drosophila melanogaster in response to the steroid hormone ecdysone were studied in relation to complex changes in puffing patterns. We found that the changes in the fine structure of cultured glands reflected progression of the puffing pattern, and they paralleled those seen in vivo. We observed that glue secretion by exocytosis, the main function of salivary glands, took place between puff stage 5 (PS5) and PS7. Glue could not be expectorated under culture conditions but was slowly released from the lumen through a duct into the medium. After the cultured glands reached PS13/PS14, further progress of puffing and fine structural alterations required that the ecdysteroid titer be transiently extremely low or absent. Under in vitro conditions we did not observe the putative new secretory program(s) described for glands in vivo after PS12. However, ultrastructural changes which unambiguously indicated that an autohistolytic process had begun in vitro started to appear after PS17. Many salivary gland cells developed numerous features of progressive self-degradation between PS18 and PS21. Actual degradation of salivary glands in vivo seemed to be rapid, but in vitro degradation was never completed, probably due to a lack of exogenous factors from the hemolymph. Manipulations of ecdysone titer in vitro in the culture medium, known during the larval puffing cycle to cause premature induction of developmentally specific puffing patterns, did not affect the normal development of ultrastructural features of the cytoplasm and nucleus.

Drosophila spag is the homolog of RNA polymerase II-associated protein 3 (RPAP3) and recruits the heat shock proteins 70 and 90 (Hsp70 and Hsp90) during the assembly of cellular machineries

Background: Mammalian RNA polymerase II-associated protein 3 (RPAP3) recruits heat shock protein 90 (Hsp90) to assemble cellular machineries such as RNA polymerases. Results: Spaghetti encodes the Drosophila homolog of RPAP3. Spaghetti is essential for development. Spag protein binds and stimulates Hsp90 and Hsp70. Conclusion: RPAP3 function is conserved among metazoans. Significance: Our data suggest that Hsp70 assists RPAP3 in complex assembly. The R2TP is a recently identified Hsp90 co-chaperone, composed of four proteins as follows: Pih1D1, RPAP3, and the AAA+-ATPases RUVBL1 and RUVBL2. In mammals, the R2TP is involved in the biogenesis of cellular machineries such as RNA polymerases, small nucleolar ribonucleoparticles and phosphatidylinositol 3-kinase-related kinases. Here, we characterize the spaghetti (spag) gene of Drosophila, the homolog of human RPAP3. This gene plays an essential function during Drosophila development. We show that Spag protein binds Drosophila orthologs of R2TP components and Hsp90, like its yeast counterpart. Unexpectedly, Spag also interacts and stimulates the chaperone activity of Hsp70. Using null mutants and flies with inducible RNAi, we show that spaghetti is necessary for the stabilization of snoRNP core proteins and target of rapamycin activity and likely the assembly of RNA polymerase II. This work highlights the strong conservation of both the HSP90/R2TP system and its clients and further shows that Spag, unlike Saccharomyces cerevisiae Tah1, performs essential functions in metazoans. Interaction of Spag with both Hsp70 and Hsp90 suggests a model whereby R2TP would accompany clients from Hsp70 to Hsp90 to facilitate their assembly into macromolecular complexes.

Effect of bisacylhydrazine ecdysteroid mimics (RH-5849 and RH-5992) on chromosomal puffing, imaginal disc proliferation and pupariation in larvae of Drosophila melanogaster.

Two bisacylhydrazine insecticides with ecdysone-mimetic action, RH-5849 and RH-5992, have been subjected to several bioassay procedures that are prerequisites for ecdysone action in Drosophila larvae: (a) induction of early ecdysone-specific puffs on the polytene chromosomes of the larval salivary glands; (b) secretion of glycoprotein glue into the lumen of the salivary glands; (c) evagination of imaginal discs of adult wings and legs; and (d) partial rescue of wild-type phenotypic expression in ecdysone-deficient mutants (ecdysoneless1 (ecd1) and suppressor of forkedts67g (su(f)ts67g). In all these bioassays on Drosophila larvae, the two purely synthetic hydrazines exhibited similar dose-response relationships as did the natural steroid hormone, 20-hydroxyecdysone. In assays involving induction of early chromosomal puffs (74EF, 75B) or regression of the preexisting puffs (25AC, 68C), the dosages required for induction of standard ED-50 effects were one order of magnitude larger for the hydrazines in comparison with 20-hydroxyecdysone. In the assays related to glycoprotein glue secretion, evagination of imaginal discs, or rescue of phenotypic expression in ecdysone-deficient mutants, 20-hydroxyecdysone was two orders of magnitude more active than RH-5849 and RH-5992. We conclude that, in spite of these quantitative differences, the two hydrazine compounds studied are able to duplicate in Drosophila larvae the complex of qualitative biological effects that are a prerequisite for ecdysteroid hormones. The hormonomimetic stimulus of RH-compounds has been given at very low, intracellular, chromosomal level.

DEVELOPMENTAL REGULATION OF GRANULE SIZE AND NUMBERS IN LARVAL SALIVARY GLANDS OF DROSOPHILA BY STEROID HORMONE ECDYSONE

The size and number of secretory granules in late larval salivary glands of Drosophila melanogaster have been related to interecdysial and early metamorphic development represented by well‐known puffs in polytene chromosomes. Interecdysial period (puff stage 1 (PS1)) is characterized by presence of numerous small granules (11,000 per cell). The transition from PSI to early metamorphic phase (PS2 and upwards), induced by rapid elevation in endogenous steroid hormone ecdysone, is accompanied by continuous growth of granule diameter with concomitant reduction in their number per cell. In the PS4, just prior to secretion, ∼3000 mature granules occur per cell. The mature state is associated with the change from hyperbolic to Gaussian distribution of granule number over their size range. Similar changes in secretory granule parameters were observed in interecdysial salivary glands explanted from 3rd instar larvae and cultured in vitro in medium containing 5×10−6m ecdysone.

Apocrine secretion: New insights into an old phenomenon.

BACKGROUNDnWhile apocrine secretion was among the earliest secretory mechanisms to be identified, its underlying basis remains poorly understood.nnnSCOPE OF REVIEWnThis review reappraises our understanding of apocrine secretion using insights about apocrine secretion from the salivary glands of Drosophila, in which molecular genetic analyses have provided a glimmer of hope for elucidating the mechanistic aspects of this fundamental process.nnnMAJOR CONCLUSIONSnIn contrast to the well-defined process of exocytosis, apocrine secretion is non-vesicular transport and secretory pathway that entails the loss of part of the cytoplasm. It often involves apical protrusions and generates cytoplasmic fragments inside a secretory lumen. In its most intense phase this process is accompanied by the release of large fragments of cellular structures and entire organelles that include mitochondria, Golgi, and portions of the endoplasmic reticulum, among others. Proteomic analyses revealed that the secretion is composed of hundreds to thousands of membranous, cytoskeletal, microsomal, mitochondrial, ribosomal, and even nuclear as well as nucleolar proteins. Strikingly, although many nuclear proteins are released, the nuclear deoxyribonucleic acid itself remains intact. In spite of this complexity, it appears that several protein components of apocrine secretion are identical, regardless of the location of the apocrine gland.nnnGENERAL SIGNIFICANCEnThis type of secretion appears to be common to many, if not all, barrier epithelial tissues including skin derivatives and the epididymis, and is implicated also in lung/bronchi and intestinal epithelium. Apocrine secretion is a mechanism that provides the en masse delivery of a very complex proteinaceous mixture from polarized epithelial tissues to allow for communication at exterior interfaces.

Apocrine secretion in Drosophila salivary glands: subcellular origin, dynamics, and identification of secretory proteins.

In contrast to the well defined mechanism of merocrine exocytosis, the mechanism of apocrine secretion, which was first described over 180 years ago, remains relatively uncharacterized. We identified apocrine secretory activity in the late prepupal salivary glands of Drosophila melanogaster just prior to the execution of programmed cell death (PCD). The excellent genetic tools available in Drosophila provide an opportunity to dissect for the first time the molecular and mechanistic aspects of this process. A prerequisite for such an analysis is to have pivotal immunohistochemical, ultrastructural, biochemical and proteomic data that fully characterize the process. Here we present data showing that the Drosophila salivary glands release all kinds of cellular proteins by an apocrine mechanism including cytoskeletal, cytosolic, mitochondrial, nuclear and nucleolar components. Surprisingly, the apocrine release of these proteins displays a temporal pattern with the sequential release of some proteins (e.g. transcription factor BR-C, tumor suppressor p127, cytoskeletal β-tubulin, non-muscle myosin) earlier than others (e.g. filamentous actin, nuclear lamin, mitochondrial pyruvate dehydrogenase). Although the apocrine release of proteins takes place just prior to the execution of an apoptotic program, the nuclear DNA is never released. Western blotting indicates that the secreted proteins remain undegraded in the lumen. Following apocrine secretion, the salivary gland cells remain quite vital, as they retain highly active transcriptional and protein synthetic activity.

Recent Progress in Juvenile Hormone Analogs (JHA) Research

Abstract Insect juvenile hormones (JHs) are important regulators of development and reproduction. Perturbation in the homeostasis at inappropriate times in the various developmental and reproductive stages of an insect, which normally requires controlled release and metabolism of this and other hormones, results in abnormal development or lethal consequences. Such deliberate consequences have been possible by the discovery and use of Juvenile Hormone Analogs (JHAs), the synthetic chemicals that mimic JH action, which have also been utilised as insecticides for several decades. With the availability of molecular tools and improvement in pharmacophore analyses in the recent years, there has been considerable advancement in the understanding of the mode of action of JHAs and development of methods for discovery of novel JHAs. Although there is limited use of JHAs for insect pest control, the list of new insect species susceptible to these compounds has been expanding revealing the potential for future use of this class of insecticides. The relatively fewer effects of JHAs on non-target insects and animals and favourable environmental fate of these compounds make them attractive insecticides for inclusion in integrated pest management programmes. In this review, we provide a comprehensive overview of JHAs, as endocrinological and insecticidal tools covering all the relevant research topics on JHAs research during the past three decades.

Temporal regulation of Drosophila salivary gland degeneration by the Broad-Complex transcription factors.

The destruction of obsolete larval tissues at the onset of insect metamorphosis is a complex process triggered by the steroid hormone ecdysone. Among the genes required for the implementation of salivary gland (SG) degeneration the reduced bristles on palpus (rbp) gene of the Broad-Complex (BR-C) locus plays a critical role. This gene encodes the BR-C Z1 transcription factor and its expression is directly regulated by ecdysone through the ecdysone receptor (EcR/Usp). The BR-C locus encodes four major protein isoforms, including BR-C Z1, Z2, Z3, and Z4. With the exceptions of mutations in BR-C Z1 all mutations affecting the other BR-C isoforms produce pupal lethality. To gain insight into the function of the different BR-C isoforms on the process of SG degeneration, we used transgenes expressing each of the four major BR-C isoform proteins. This study revealed that, depending upon the period of expression relative to the major peak of ecdysone production, BR-C Z1, Z2, and Z4 first inhibited and then stimulated the process of SG degeneration. In contrast, BR-C Z3 exerted all time points an inhibition on SG degeneration.