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Current Opinion in Cell Biology Oct 2016Apico-basal polarity is a hallmark of epithelial tissues. The integrated activity of several evolutionarily conserved protein complexes is essential to control... (Review)
Review
Apico-basal polarity is a hallmark of epithelial tissues. The integrated activity of several evolutionarily conserved protein complexes is essential to control epithelial polarity during development and homeostasis. Many components of these protein complexes were originally identified in genetic screens performed in Drosophila or Caenorhabditis elegans due to defects in cell polarity. With time, it became obvious that these protein complexes not only control various aspects of apico-basal polarity, but also perform a plethora of other functions, such as growth control, organization of endocytic activity, regulation of signaling and asymmetric cell division, to mention just a few. Here we summarize some results mostly obtained from studies in Drosophila to elucidate how variation in protein composition and modification of individual components contribute to make polarity complexes versatile platforms to fulfill a variety of functions.
Topics: Animals; Cell Polarity; Drosophila Proteins; Drosophila melanogaster; Epithelial Cells; Models, Biological
PubMed: 27085003
DOI: 10.1016/j.ceb.2016.03.018 -
ELife Apr 2022The ability to control transgene expression, both spatially and temporally, is essential for studying model organisms. In , spatial control is primarily provided by the...
The ability to control transgene expression, both spatially and temporally, is essential for studying model organisms. In , spatial control is primarily provided by the GAL4/UAS system, whilst temporal control relies on a temperature-sensitive GAL80 (which inhibits GAL4) and drug-inducible systems. However, these are not ideal. Shifting temperature can impact on many physiological and behavioural traits, and the current drug-inducible systems are either leaky, toxic, incompatible with existing GAL4-driver lines, or do not generate effective levels of expression. Here, we describe the auxin-inducible gene expression system (AGES). AGES relies on the auxin-dependent degradation of a ubiquitously expressed GAL80, and therefore, is compatible with existing GAL4-driver lines. Water-soluble auxin is added to fly food at a low, non-lethal, concentration, which induces expression comparable to uninhibited GAL4 expression. The system works in both larvae and adults, providing a stringent, non-lethal, cost-effective, and convenient method for temporally controlling GAL4 activity in .
Topics: Animals; Animals, Genetically Modified; Drosophila; Drosophila Proteins; Gene Expression; Indoleacetic Acids; Transcription Factors
PubMed: 35363137
DOI: 10.7554/eLife.67598 -
Current Biology : CB Sep 2023The needs fulfilled by sleep are unknown, though the effects of insufficient sleep are manifold. To better understand how the need to sleep is sensed and discharged,...
The needs fulfilled by sleep are unknown, though the effects of insufficient sleep are manifold. To better understand how the need to sleep is sensed and discharged, much effort has gone into identifying the neural circuits involved in regulating arousal, especially those that promote sleep. In prevailing models, the dorsal fan-shaped body (dFB) plays a central role in this process in the fly brain. In the present study we manipulated various properties of the dFB including its electrical activity, synaptic output, and endogenous gene expression. In each of these experimental contexts we were unable to identify any effect on sleep that could be unambiguously mapped to the dFB. Furthermore, we found evidence that sleep phenotypes previously attributed to the dFB were caused by genetic manipulations that inadvertently targeted the ventral nerve cord. We also examined expression of two genes whose purported effects have been attributed to functions within a specific subpopulation of dFB neurons. In both cases we found little to no expression in the expected cells. Collectively, our results cast doubt on the prevailing hypothesis that the dFB plays a central role in promoting sleep.
Topics: Animals; Drosophila; Drosophila melanogaster; Drosophila Proteins; Sleep; Sleep Deprivation
PubMed: 37552985
DOI: 10.1016/j.cub.2023.07.043 -
Current Topics in Developmental Biology 2022Synapse development is coordinated by intercellular communication between the pre- and postsynaptic compartments, and by neuronal activity itself. In flies as in... (Review)
Review
Synapse development is coordinated by intercellular communication between the pre- and postsynaptic compartments, and by neuronal activity itself. In flies as in vertebrates, neuronal activity induces input-specific changes in the synaptic strength so that the entire circuit maintains stable function in the face of many challenges, including changes in synapse number and strength. But how do neurons sense synapse activity? In several studies carried out using the Drosophila neuromuscular junction (NMJ), we demonstrated that local BMP signaling provides an exquisite sensor for synapse activity. Here we review the main features of this exquisite sensor and discuss its functioning beyond monitoring the synapse activity but rather as a key controller that operates in coordination with other BMP signaling pathways to balance synapse growth, maturation and function.
Topics: Animals; Drosophila; Drosophila Proteins; Drosophila melanogaster; Neuromuscular Junction; Signal Transduction; Synapses
PubMed: 35817503
DOI: 10.1016/bs.ctdb.2022.04.001 -
The Journal of Cell Biology Nov 2018Yurt (Yrt) and its mammalian orthologue EPB41L5 limit apical membrane growth in polarized epithelia. EPB41L5 also supports epithelial-mesenchymal transition and...
Yurt (Yrt) and its mammalian orthologue EPB41L5 limit apical membrane growth in polarized epithelia. EPB41L5 also supports epithelial-mesenchymal transition and metastasis. Yrt and EPB41L5 contain a four-point-one, ezrin, radixin, and moesin (FERM) domain and a FERM-adjacent (FA) domain. The former contributes to the quaternary structure of 50 human proteins, whereas the latter defines a subfamily of 14 human FERM proteins and fulfills unknown roles. In this study, we show that both Yrt and EPB41L5 oligomerize. Our data also establish that the FERM-FA unit forms an oligomeric interface and that multimerization of Yrt is crucial for its function in epithelial cell polarity regulation. Finally, we demonstrate that aPKC destabilizes the Yrt oligomer to repress its functions, thereby revealing a mechanism through which this kinase supports apical domain formation. Overall, our study highlights a conserved biochemical property of fly and human Yrt proteins, describes a novel function of the FA domain, and further characterizes the molecular mechanisms sustaining epithelial cell polarity.
Topics: Animals; Cell Polarity; Drosophila Proteins; Drosophila melanogaster; Epithelial Cells; Humans; Membrane Proteins; Protein Domains; Protein Multimerization
PubMed: 30082297
DOI: 10.1083/jcb.201803099 -
Developmental Biology Jan 2024The orphan nuclear receptor Tailless (Tll) exhibits conserved roles in brain formation and maintenance that are shared, for example, with vertebrate orthologous forms...
The orphan nuclear receptor Tailless (Tll) exhibits conserved roles in brain formation and maintenance that are shared, for example, with vertebrate orthologous forms (Tlx). However, the early expression of tll in two gap domains in the segmentation cascade of Drosophila is unusual even for most other insects. Here we investigate tll regulation on pair-rule stripes. With ectopic misexpression of tll we detected unexpected repression of almost all pair-rule stripes of hairy (h), even-skipped (eve), runt (run), and fushi-tarazu (ftz). Examining Tll embryonic ChIP-chip data with regions mapped as Cis-Regulatory Modules (CRMs) of pair-rule stripes we verified Tll interactions to these regions. With the ChIP-chip data we also verified Tll interactions to the CRMs of gap domains and in the misexpression assay, Tll-mediated repression on Kruppel (Kr), kni (kni) and giant (gt) according to their differential sensitivity to Tll. These results with gap genes confirmed previous data from the literature and argue against indirect repression roles of Tll in the striped pattern. Moreover, the prediction of Tll binding sites in the CRMs of eve stripes and the mathematical modeling of their removal using an experimentally validated theoretical framework shows effects on eve stripes compatible with the absence of a repressor binding to the CRMs. In addition, modeling increased tll levels in the embryo results in the differential repression of eve stripes, agreeing well with the results of the misexpression assay. In genetic assays we investigated eve 5, that is strongly repressed by the ectopic domain and representative of more central stripes not previously implied to be under direct regulation of tll. While this stripe is little affected in tll-, its posterior border is expanded in gt- but detected with even greater expansion in gt-;tll-. We end up by discussing tll with key roles in combinatorial repression mechanisms to contain the expression of medial patterns of the segmentation cascade in the extremities of the embryo.
Topics: Animals; Drosophila; Drosophila Proteins; Homeodomain Proteins; Repressor Proteins; Transcription Factors
PubMed: 37879494
DOI: 10.1016/j.ydbio.2023.09.014 -
Archives of Biochemistry and Biophysics Mar 2021Cryptochromes, FAD-dependent blue light photoreceptors, undergo a series of electron transfer reactions after light excitation. Time-resolved optical spectroscopy was...
Cryptochromes, FAD-dependent blue light photoreceptors, undergo a series of electron transfer reactions after light excitation. Time-resolved optical spectroscopy was employed to investigate the pH dependence of all light-dependent reactions in the cryptochrome from fruit flies. Signal state formation experiments on a time scale of seconds were found to be strongly pH dependent, and formation of both anionic and neutral FAD radicals could be detected, with reaction rates increasing by a factor of ~2.5 from basic to neutral pH values. Additionally, the influence of the amino acid His378 was investigated in further detail: Two protein variants, DmCry H378A and H378Q, showed significantly reduced rate constants for signal state formation, which again differed at neutral and alkaline pH values. Hence, His378 was identified as an amino acid responsible for the pronounced pH dependence; however, this amino acid can be excluded as a proton donor for the protonation of the anionic FAD radical. Other conserved amino acids appear to alter the overall polarity of the binding pocket and thus to be responsible for the pronounced pH dependence. Furthermore, the influence of pH and other experimental parameters, such as temperature, glycerol or ferricyanide concentrations, on the intermediately formed FAD-tryptophan radical pair was explored, which deprotonates on a microsecond time scale with a clear pH dependence, and subsequently recombines within milliseconds. Surprisingly, the latter reaction showed no pH dependence; potential reasons are discussed. All results are reviewed in terms of the photoreceptor and potential magnetoreceptor functions of Drosophila cryptochrome.
Topics: Amino Acid Substitution; Animals; Cryptochromes; Drosophila Proteins; Drosophila melanogaster; Eye Proteins; Hydrogen-Ion Concentration; Mutation, Missense; Oxidation-Reduction; Protein Stability
PubMed: 33545100
DOI: 10.1016/j.abb.2021.108787 -
The International Journal of... 2022Secreted wingless-interacting protein (Swim) is the ortholog gene of the mammalian Tubulointerstitial Nephritis Antigen like 1 (TINAGL1), also known as lipocalin-7...
Secreted wingless-interacting protein (Swim) is the ortholog gene of the mammalian Tubulointerstitial Nephritis Antigen like 1 (TINAGL1), also known as lipocalin-7 (LCN7), or adrenocortical zonation factor 1 (AZ-1). Swim and TINAGL1 proteins share a significant homology, including the somatomedin B and the predictive inactive C1 cysteine peptidase domains. In mammals, both TINAGL1 and its closely related homolog TINAG have been identified in basement membranes, where they may function as modulators of integrin-mediated adhesion. In , Swim was initially identified in the eggshell matrix and was subsequently detected in the culture medium of S2 cells. Further biochemical analysis indicated that Swim binds to wingless (wg) in a lipid-dependent manner. This observation, together with RNAi-knockdown studies, suggested that Swim is an essential cofactor of wg-signalling. However, recent elegant genetic studies ruled out the possibility that Swim is required alone to facilitate wg-signalling in , because flies without Swim are viable and fertile. Here, we use the UAS/Gal4 expression system together with confocal imaging to analyze the localization of a chimeric Swim-GFP in the developing embryo. Our data fully support the notion that Swim is an extracellular matrix component that is secreted upon ectopic expression and preferentially associates with the basement membranes of various organs and with the specialized tendon matrix at the muscle attachment sites (MAS). Interestingly, the accumulation of Swim at the MAS does not require integrins. In conclusion, Swim is an extracellular matrix component, and Swim may exhibit overlapping functions in concert with other undefined components.
Topics: Animals; Drosophila; Drosophila Proteins; Embryo, Mammalian; Extracellular Matrix; Integrins; Mammals; Signal Transduction
PubMed: 34881800
DOI: 10.1387/ijdb.210205cz -
Cell and Tissue Research Sep 2014Antisera to orcokinin B, CCHamide 1, and CCHamide 2 recognize enteroendocrine cells in the midgut of the fruitfly Drosophila melanogaster and its larvae. Although the...
Antisera to orcokinin B, CCHamide 1, and CCHamide 2 recognize enteroendocrine cells in the midgut of the fruitfly Drosophila melanogaster and its larvae. Although the antisera to CCHamide 1 and 2 are mutually cross-reactive, polyclonal mouse antisera raised to the C-terminals of their respective precursors allowed the identification of the two different peptides. In both larva and adult, CCHamide 2 immunoreactive endocrine cells are large and abundant in the anterior midgut and are also present in the anterior part of the posterior midgut. The CCHamide 2 immunoreactive endocrine cells in the posterior midgut are also immunoreactive with antiserum to allatostatin C. CCHamide 1 immunoreactivity is localized in endocrine cells in different regions of the midgut; those in the caudal part of the posterior midgut are identical with the allatostatin A cells. In the larva, CCHamide 1 enteroendocrine cells are also present in the endocrine junction and in the anterior part of the posterior midgut. Like in other insect species, the Drosophila orcokinin gene produces two different transcripts, A and B. Antiserum to the predicted biologically active peptide from the B-transcript recognizes enteroendocrine cells in both larva and adult. These are the same cells as those expressing β-galactosidase in transgenic flies in which the promoter of the orcokinin gene drives expression of this enzyme. In the larva, a variable number of orcokinin-expressing enteroendocrine cells are found at the end of the middle midgut, while in the adult, those cells are most abundant in the middle midgut, while smaller numbers are present in the anterior midgut. In both larva and adult, these cells also express allatostatin C. We also made a specific polyclonal antiserum to the NPF precursor in order to determine more precisely the expression of this peptide in the midgut. Using this antiserum, we find expression in the midgut to be the same as described previously using transgenic flies, while in the adult, midgut expression appears to be concentrated in the middle midgut, thus suggesting that in the anterior midgut only minor quantities of NPF are produced.
Topics: Amino Acid Sequence; Animals; Carrier Proteins; Drosophila Proteins; Drosophila melanogaster; Enteroendocrine Cells; Gene Expression Regulation; Larva; Molecular Sequence Data; Neuropeptides
PubMed: 24850274
DOI: 10.1007/s00441-014-1880-2 -
Mechanisms of Ageing and Development Jun 2022Gut homeostasis is a dynamically balanced state to maintain intestinal health. Vacuolar ATPases (V-ATPases) are multi-subunit proton pumps that were driven by ATP...
Gut homeostasis is a dynamically balanced state to maintain intestinal health. Vacuolar ATPases (V-ATPases) are multi-subunit proton pumps that were driven by ATP hydrolysis. Several subunits of V-ATPases may be involved in the maintenance of intestinal pH and gut homeostasis in Drosophila. However, the specific role of each subunit in this process remains to be elucidated. Here, we knocked down the Drosophila gene VhaAC39-1 encoding the V0d1 subunit of V-ATPases to assess its function in gut homeostasis. Knockdown of VhaAC39-1 resulted in the loss of midgut acidity, the increase of the number of gut microbiota and the impairment of intestinal epithelial integrity in flies. The knockdown of VhaAC39-1 led to the hyperproliferation of intestinal stem cells, increasing the number of enteroendocrine cells, and activated IMD signaling pathway and JAK-STAT signaling pathway, inducing intestinal immune response of Drosophila. In addition, knockdown of VhaAC39-1 caused the disturbance of many physiological indicators such as food intake, triglyceride level and fecundity of flies, which ultimately led to the shortening of the life span of Drosophila. These results shed light on the gut homeostasis mechanisms which would help to identify interventions to promote healthy aging.
Topics: Adenosine Triphosphatases; Animals; Drosophila; Drosophila Proteins; Drosophila melanogaster; Homeostasis; Stem Cells
PubMed: 35398002
DOI: 10.1016/j.mad.2022.111673