Myosin II is not required for Drosophila tracheal branch elongation and cell intercalation

The Drosophila tracheal system consists of an interconnected network of monolayered epithelial tubes that ensures oxygen transport in the larval and adult body. During tracheal dorsal branch (DB) development, individual DBs elongate as a cluster of cells, led by tip cells at the front and trailing cells in the rear. Branch elongation is accompanied by extensive cell intercalation and cell lengthening of the trailing stalk cells. Although cell intercalation is governed by Myosin II (MyoII)-dependent forces during tissue elongation in the Drosophila embryo that lead to germ-band extension, it remained unclear whether MyoII plays a similar active role during tracheal branch elongation and intercalation. Here, we have used a nanobody-based approach to selectively knock down MyoII in tracheal cells. Our data show that, despite the depletion of MyoII function, tip cell migration and stalk cell intercalation (SCI) proceed at a normal rate. This confirms a model in which DB elongation and SCI in the trachea occur as a consequence of tip cell migration, which produces the necessary forces for the branching process

Amplification of neural stem cell proliferation by intermediate progenitor cells in Drosophila brain development

BACKGROUND: In the mammalian brain, neural stem cells divide asymmetrically and often amplify the number of progeny they generate via symmetrically dividing intermediate progenitors. Here we investigate whether specific neural stem cell-like neuroblasts in the brain of Drosophila might also amplify neuronal proliferation by generating symmetrically dividing intermediate progenitors. RESULTS: Cell lineage-tracing and genetic marker analysis show that remarkably large neuroblast lineages exist in the dorsomedial larval brain of Drosophila. These lineages are generated by brain neuroblasts that divide asymmetrically to self renew but, unlike other brain neuroblasts, do not segregate the differentiating cell fate determinant Prospero to their smaller daughter cells. These daughter cells continue to express neuroblast-specific molecular markers and divide repeatedly to produce neural progeny, demonstrating that they are proliferating intermediate progenitors. The proliferative divisions of these intermediate progenitors have novel cellular and molecular features; they are morphologically symmetrical, but molecularly asymmetrical in that key differentiating cell fate determinants are segregated into only one of the two daughter cells. CONCLUSION: Our findings provide cellular and molecular evidence for a new mode of neurogenesis in the larval brain of Drosophila that involves the amplification of neuroblast proliferation through intermediate progenitors. This type of neurogenesis bears remarkable similarities to neurogenesis in the mammalian brain, where neural stem cells as primary progenitors amplify the number of progeny they generate through generation of secondary progenitors. This suggests that key aspects of neural stem cell biology might be conserved in brain development of insects and mammals

Spatio-temporally separated cortical flows and spindle geometry establish physical asymmetry in fly neural stem cells

Asymmetric cell division, creating sibling cells with distinct developmental potentials, can be manifested in sibling cell size asymmetry. This form of physical asymmetry occurs in several metazoan cells, but the underlying mechanisms and function are incompletely understood. Here we use Drosophila neural stem cells to elucidate the mechanisms involved in physical asymmetry establishment. We show that Myosin relocalizes to the cleavage furrow via two distinct cortical Myosin flows: at anaphase onset, a polarity induced, basally directed Myosin flow clears Myosin from the apical cortex. Subsequently, mitotic spindle cues establish a Myosin gradient at the lateral neuroblast cortex, necessary to trigger an apically directed flow, removing Actomyosin from the basal cortex. On the basis of the data presented here, we propose that spatiotemporally controlled Myosin flows in conjunction with spindle positioning and spindle asymmetry are key determinants for correct cleavage furrow placement and cortical expansion, thereby establishing physical asymmetry

A cis-regulatory element promoting increased transcription at low temperature in cultured ectothermic Drosophila cells

Background Temperature change affects the myriad of concurrent cellular processes in a non-uniform, disruptive manner. While endothermic organisms minimize the challenge of ambient temperature variation by keeping the core body temperature constant, cells of many ectothermic species maintain homeostatic function within a considerable temperature range. The cellular mechanisms enabling temperature acclimation in ectotherms are still poorly understood. At the transcriptional level, the heat shock response has been analyzed extensively. The opposite, the response to sub-optimal temperature, has received lesser attention in particular in animal species. The tissue specificity of transcriptional responses to cool temperature has not been addressed and it is not clear whether a prominent general response occurs. Cis-regulatory elements (CREs), which mediate increased transcription at cool temperature, and responsible transcription factors are largely unknown. Results The ectotherm Drosophila melanogaster with a presumed temperature optimum around 25 °C was used for transcriptomic analyses of effects of temperatures at the lower end of the readily tolerated range (14–29 °C). Comparative analyses with adult flies and cell culture lines indicated a striking degree of cell-type specificity in the transcriptional response to cool. To identify potential cis-regulatory elements (CREs) for transcriptional upregulation at cool temperature, we analyzed temperature effects on DNA accessibility in chromatin of S2R+ cells. Candidate cis-regulatory elements (CREs) were evaluated with a novel reporter assay for accurate assessment of their temperature-dependency. Robust transcriptional upregulation at low temperature could be demonstrated for a fragment from the pastrel gene, which expresses more transcript and protein at reduced temperatures. This CRE is controlled by the JAK/STAT signaling pathway and antagonizing activities of the transcription factors Pointed and Ets97D. Conclusion Beyond a rich data resource for future analyses of transcriptional control within the readily tolerated range of an ectothermic animal, a novel reporter assay permitting quantitative characterization of CRE temperature dependence was developed. Our identification and functional dissection of the pst_E1 enhancer demonstrate the utility of resources and assay. The functional characterization of this CoolUp enhancer provides initial mechanistic insights into transcriptional upregulation induced by a shift to temperatures at the lower end of the readily tolerated range

Protein interference applications in cellular and developmental biology using DARPins that recognize GFP and mCherry

Protein–protein interactions are crucial for cellular homeostasis and play important roles in the dynamic execution of biological processes. While antibodies represent a well-established tool to study protein interactions of extracellular domains and secreted proteins, as well as in fixed and permeabilized cells, they usually cannot be functionally expressed in the cytoplasm of living cells. Non-immunoglobulin protein-binding scaffolds have been identified that also function intracellularly and are now being engineered for synthetic biology applications. Here we used the Designed Ankyrin Repeat Protein (DARPin) scaffold to generate binders to fluorescent proteins and used them to modify biological systems directly at the protein level. DARPins binding to GFP or mCherry were selected by ribosome display. For GFP, binders with KD as low as 160 pM were obtained, while for mCherry the best affinity was 6 nM. We then verified in cell culture their specific binding in a complex cellular environment and found an affinity cut-off in the mid-nanomolar region, above which binding is no longer detectable in the cell. Next, their binding properties were employed to change the localization of the respective fluorescent proteins within cells. Finally, we performed experiments in Drosophila melanogaster and Danio rerio and utilized these DARPins to either degrade or delocalize fluorescently tagged fusion proteins in developing organisms, and to phenocopy loss-of-function mutations. Specific protein binders can thus be selected in vitro and used to reprogram developmental systems in vivo directly at the protein level, thereby bypassing some limitations of approaches that function at the DNA or the RNA level

Engineered kinases as a tool for phosphorylation of selected targets in vivo

Reversible protein phosphorylation by kinases controls a plethora of processes essential for the proper development and homeostasis of multicellular organisms. One main obstacle in studying the role of a defined kinase-substrate interaction is that kinases form complex signaling networks and most often phosphorylate multiple substrates involved in various cellular processes. In recent years, several new approaches have been developed to control the activity of a given kinase. However, most of them fail to regulate a single protein target, likely hiding the effect of a unique kinase-substrate interaction by pleiotropic effects. To overcome this limitation, we have created protein binder-based engineered kinases that permit a direct, robust, and tissue-specific phosphorylation of fluorescent fusion proteins in vivo. We show the detailed characterization of two engineered kinases based on Rho-associated protein kinase (ROCK) and Src. Expression of synthetic kinases in the developing fly embryo resulted in phosphorylation of their respective GFP-fusion targets, providing for the first time a means to direct the phosphorylation to a chosen and tagged target in vivo. We presume that after careful optimization, the novel approach we describe here can be adapted to other kinases and targets in various eukaryotic genetic systems to regulate specific downstream effectors

Ensconsin/Map7 promotes microtubule growth and centrosome separation in Drosophila neural stem cells.

International audienceThe mitotic spindle is crucial to achieve segregation of sister chromatids. To identify new mitotic spindle assembly regulators, we isolated 855 microtubule-associated proteins (MAPs) from Drosophila melanogaster mitotic or interphasic embryos. Using RNAi, we screened 96 poorly characterized genes in the Drosophila central nervous system to establish their possible role during spindle assembly. We found that Ensconsin/MAP7 mutant neuroblasts display shorter metaphase spindles, a defect caused by a reduced microtubule polymerization rate and enhanced by centrosome ablation. In agreement with a direct effect in regulating spindle length, Ensconsin overexpression triggered an increase in spindle length in S2 cells, whereas purified Ensconsin stimulated microtubule polymerization in vitro. Interestingly, ensc-null mutant flies also display defective centrosome separation and positioning during interphase, a phenotype also detected in kinesin-1 mutants. Collectively, our results suggest that Ensconsin cooperates with its binding partner Kinesin-1 during interphase to trigger centrosome separation. In addition, Ensconsin promotes microtubule polymerization during mitosis to control spindle length independent of Kinesin-1

γCOP Is Required for Apical Protein Secretion and Epithelial Morphogenesis in Drosophila melanogaster

Background: There is increasing evidence that tissue-specific modifications of basic cellular functions play an important role in development and disease. To identify the functions of COPI coatomer-mediated membrane trafficking in Drosophila development, we were aiming to create loss-of-function mutations in the γCOP gene, which encodes a subunit of the COPI coatomer complex. Principal Findings: We found that γCOP is essential for the viability of the Drosophila embryo. In the absence of zygotic γCOP activity, embryos die late in embryogenesis and display pronounced defects in morphogenesis of the embryonic epidermis and of tracheal tubes. The coordinated cell rearrangements and cell shape changes during tracheal tube morphogenesis critically depend on apical secretion of certain proteins. Investigation of tracheal morphogenesis in γCOP loss-of-function mutants revealed that several key proteins required for tracheal morphogenesis are not properly secreted into the apical lumen. As a consequence, γCOP mutants show defects in cell rearrangements during branch elongation, in tube dilation, as well as in tube fusion. We present genetic evidence that a specific subset of the tracheal defects in γCOP mutants is due to the reduced secretion of the Zona Pellucida protein Piopio. Thus, we identified a critical target protein of COPI-dependent secretion in epithelial tube morphogenesis. Conclusions/Significance: These studies highlight the role of COPI coatomer-mediated vesicle trafficking in both general and tissue-specific secretion in a multicellular organism. Although COPI coatomer is generally required for protein secretion, we show that the phenotypic effect of γCOP mutations is surprisingly specific. Importantly, we attribute a distinct aspect of the γCOP phenotype to the effect on a specific key target protein

deGradFP: A System to Knockdown GFP-Tagged Proteins

Protein depletion by genetic means, in a very general sense including the use of RNA interference [1, 2] or CRISPR/Cas9-based methods, represents a central paradigm of modern biology to study protein functions in vivo. However, acting upstream the proteic level is a limiting factor if the turnover of the target protein is slow or the existing pool of the target protein is important (for instance, in insect embryos, as a consequence of a strong maternal contribution). In order to circumvent these problems, we developed deGradFP [3, 4]. deGradFP harnesses the ubiquitin-proteasome pathway to achieve direct depletion of GFP-tagged proteins. deGradFP is in essence a universal method because it relies on an evolutionarily conserved machinery for protein catabolism in eukaryotic cells; see refs. 5, 6 for review. deGradFP is particularly convenient in Drosophila melanogaster where it is implemented by a genetically encoded effector expressed under the control of the Gal4 system. deGradFP is a ready-to-use solution to perform knockdowns at the protein level if a fly line carrying a functional GFP-tagged version of the gene of interest is available. Many such lines have already been generated by the Drosophila community through different technologies allowing to make genomic rescue constructs or direct GFP knockins: protein-trap stock collections [7, 8] ( http://cooley.medicine.yale.edu/flytrap/ , http://www.flyprot.org/ ), P[acman] system [9], MiMIC lines [10, 11], and CRISPR/Cas9-driven homologous recombination.Two essential controls of a protein knockdown experiment are easily achieved using deGradFP. First, the removal of the target protein can be assessed by monitoring the disappearance of the GFP tag by fluorescence microscopy in parallel to the documentation of the phenotype of the protein knockdown (see Note 1 ). Second, the potential nonspecific effects of deGradFP can be assessed in control fly lacking a GFP-tagged target protein. So far, no nonspecific effects of the deGradFP effector have been reported [3]

deGradFP: A System to Knockdown GFP-Tagged Proteins

Protein depletion by genetic means, in a very general sense including the use of RNA interference [1, 2] or CRISPR/Cas9-based methods, represents a central paradigm of modern biology to study protein functions in vivo. However, acting upstream the proteic level is a limiting factor if the turnover of the target protein is slow or the existing pool of the target protein is important (for instance, in insect embryos, as a consequence of a strong maternal contribution). In order to circumvent these problems, we developed deGradFP [3, 4]. deGradFP harnesses the ubiquitin-proteasome pathway to achieve direct depletion of GFP-tagged proteins. deGradFP is in essence a universal method because it relies on an evolutionarily conserved machinery for protein catabolism in eukaryotic cells; see refs. 5, 6 for review. deGradFP is particularly convenient in Drosophila melanogaster where it is implemented by a genetically encoded effector expressed under the control of the Gal4 system. deGradFP is a ready-to-use solution to perform knockdowns at the protein level if a fly line carrying a functional GFP-tagged version of the gene of interest is available. Many such lines have already been generated by the Drosophila community through different technologies allowing to make genomic rescue constructs or direct GFP knockins: protein-trap stock collections [7, 8] ( http://cooley.medicine.yale.edu/flytrap/ , http://www.flyprot.org/ ), P[acman] system [9], MiMIC lines [10, 11], and CRISPR/Cas9-driven homologous recombination.Two essential controls of a protein knockdown experiment are easily achieved using deGradFP. First, the removal of the target protein can be assessed by monitoring the disappearance of the GFP tag by fluorescence microscopy in parallel to the documentation of the phenotype of the protein knockdown (see Note 1 ). Second, the potential nonspecific effects of deGradFP can be assessed in control fly lacking a GFP-tagged target protein. So far, no nonspecific effects of the deGradFP effector have been reported [3]