Diphthamide modification of eEF2 is required for gut tumor-like hyperplasia induced by oncogenic Ras.

Eukaryotic elongation factor 2 (eEF2) undergoes a unique post-translational modification called diphthamidation. Although eEF2 diphthamidation is highly conserved, its pathophysiological function is still largely unknown. To elucidate the function of diphthamidation in tumor, we examined the involvement of diphthamidation pathway enzyme Dph5 in tumor progression in Drosophila adult gut. Expression of oncogenic RasV12 in gut intestinal stem cells (ISCs) and enteroblasts (EBs) causes hypertrophy and disruption of gut epithelia, and shortened life span. Knockdown of Dph5 ameliorated these pathogenic phenotypes. Dph5 is required for gross translation activation and high dMyc protein level in RasV12 tumor-like hyperplasia. Transcriptome analysis revealed that Dph5 is involved in the regulation of ribosome biogenesis genes. These results suggest that diphthamidation is required for translation activation partly through the regulation of ribosome biogenesis in Ras-induced tumor-like hyperplasia model in Drosophila gut.

Activatable Photosensitizer for Targeted Ablation of lacZ-Positive Cells with Single-Cell Resolution.

To achieve highly selective ablation of lacZ-positive cells in a biological milieu in vivo, we developed an activatable photosensitizer, SPiDER-killer-ßGal, targeted to ß-galactosidase encoded by the lacZ reporter gene. Hydrolysis of SPiDER-killer-ßGal by ß-galactosidase simultaneously activates both its photosensitizing ability and its reactivity to nucleophiles, so that the phototoxic products generated by light irradiation are trapped inside the lacZ-positive cells. The combination of SPiDER-killer-ßGal and light irradiation specifically killed lacZ-positive cells in coculture with cells without lacZ expression. Furthermore, ß-galactosidase-expressing cells in the posterior region of cultured Drosophila wing discs and in pupal notum of live Drosophila pupae were selectively killed with single-cell resolution. This photosensitizer should be useful for specific ablation of targeted cells in living organisms, for example, to investigate cellular functions in complex networks.

The hidden nature of protein translational control by diphthamide: the secrets under the leather.

The protein translation elongation factor eEF2 undergoes a unique posttranslational modification called diphthamidation. eEF2 is an essential factor in protein translation, and the diphthamide modification has been a famous target of the diphtheria toxin for a long time. On the other hand, the physiological function of this rare modification in vivo remains unknown. Recent studies have suggested that diphthamide has specific functions for the cellular stress response and active proliferation. In this review, we summarize the history and findings of diphthamide obtained to date and discuss the possibility of a specific function for diphthamide in regulating protein translation.

Red-Shifted Fluorogenic Substrate for Detection of lacZ-Positive Cells in Living Tissue with Single-Cell Resolution.

The Escherichia coli lacZ gene encoding ß-galactosidase is a widely used reporter, but few synthetic substrates are available for detecting its activity with single-cell resolution in living samples. Our recently reported fluorogenic substrate SPiDER-ßGal is suitable for this purpose, but its hydrolysis product shows green fluorescence emission, and a red-shifted analogue is therefore required for use in combination with green fluorescent protein (GFP) markers. Herein, we describe the development of a red-shifted fluorogenic substrate for ß-galactosidase, SPiDER-Red-ßGal, based on a silicon rhodol scaffold and a carboxylic group as the intramolecular nucleophile. LacZ-positive cells were successfully labeled with SPiDER-Red-ßGal at single-cell resolution in living samples, which enabled us to visualize different cell types in combination with GFP markers.

Nutritional Control of Stem Cell Division through S-Adenosylmethionine in Drosophila Intestine.

The intestine has direct contact with nutritional information. The mechanisms by which particular dietary molecules affect intestinal homeostasis are not fully understood. In this study, we identified S-adenosylmethionine (SAM), a universal methyl donor synthesized from dietary methionine, as a critical molecule that regulates stem cell division in Drosophila midgut. Depletion of either dietary methionine or SAM synthesis reduces division rate of intestinal stem cells. Genetic screening for putative SAM-dependent methyltransferases has identified protein synthesis as a regulator of the stem cells, partially through a unique diphthamide modification on eukaryotic elongation factor 2. In contrast, SAM in nutrient-absorptive enterocytes controls the interleukin-6-like protein Unpaired 3, which is required for rapid division of the stem cells after refeeding. Our study sheds light upon a link between diet and intestinal homeostasis and highlights the key metabolite SAM as a mediator of cell-type-specific starvation response.

The plant homeodomain finger protein MESR4 is essential for embryonic development in Drosophila.

Misexpression Suppressor of Ras 4 (MESR4), a plant homeodomain (PHD) finger protein with nine zinc-finger motifs has been implicated in various biological processes including the regulation of fat storage and innate immunity in Drosophila. However, the role of MESR4 in the context of development remains unclear. Here it is shown that MESR4 is a nuclear protein essential for embryonic development. Immunostaining of polytene chromosomes using anti-MESR4 antibody revealed that MESR4 binds to numerous bands along the chromosome arms. The most intense signal was detected at the 39E-F region, which is known to contain the histone gene cluster. P-element insertions in the MESR4 locus, which were homozygous lethal during embryogenesis with defects in ventral ectoderm formation and head encapsulation was identified. In the mutant embryos, expression of Fasciclin 3 (Fas3), an EGFR signal target gene was greatly reduced, and the level of EGFR signal-dependent double phosphorylated ERK (dp-ERK) remained low. However, in the context of wing vein formation, genetic interaction experiments suggested that MESR4 is involved in the EGFR signaling as a negative regulator. These results suggested that MESR4 is a novel chromatin-binding protein required for proper expression of genes including those regulated by the EGFR signaling pathway during development. genesis 53:701-708, 2015. © 2015 Wiley Periodicals, Inc.

Identification of a novel role for Drosophila MESR4 in lipid metabolism.

Drosophila provides a powerful genetic model to analyze lipid metabolism. Drosophila has an adipose-like organ called the fat body, which plays a crucial role in energy homeostasis. Here, we conducted a fat body-specific misexpression screen to identify genes involved in lipid metabolism. We found that over-expression of a nuclear protein with nine C2 H2 type zinc-finger motifs and a PHD-finger, Misexpression suppressor of ras 4 (MESR4), reduces lipid accumulation in the fat body, whereas MESR4 knockdown increases it. We further show that MESR4 up-regulates the expression of major lipases, which may account for the reduction in lipid storage in the fat body and the release of free fatty acids (FFAs) in the body. These results suggest that MESR4 acts as an important upstream regulator of energy homeostasis.

Identification of proteasome components required for apical localization of Chaoptin using functional genomics.

Abstract: the distinct localization of membrane proteins with regard to cell polarity is crucial for the structure and function of various organs in multicellular organisms. However, the molecules and mechanisms that regulate protein localization to particular subcellular domains are still largely unknown. To identify the genes involved in regulation of protein localization, the authors performed a large-scale screen using a Drosophila RNA interference (RNAi) library, by which Drosophila genes could be knocked down in a tissue- and stage-specific manner. Drosophila photoreceptor cells have a morphologically distinct apicobasal polarity, along which Chaoptin (Chp), a glycosylphosphatidylinositol (GPI)-anchored membrane protein, and the Na (+) , K(+) -ATPase are localized to the apical and basolateral domains, respectively. By examining the subcellular localization of these proteins, the authors identified 106 genes whose knockdown resulted in mislocalization of Chp and Na(+) , K(+) -ATPase. Gene ontology analysis revealed that the knockdown of proteasome components resulted in mislocalization of Chp to the basolateral plasma membrane. These results suggest that the proteasome is involved, directly or indirectly, in selective localization of Chp to the apical plasma membrane of Drosophila photoreceptor cells.

Differential control of cell affinity required for progression and refinement of cell boundary during Drosophila leg segmentation.

Domain boundary formation in development involves sorting of different types of cells into separate spatial domains. The segment boundary between tarsus 5 (Ta5) and the pretarsus (Pre) of the Drosophila leg initially appears at the center of the leg disc and progressively sharpens and expands to its final position, accompanied by down-regulation of the cell recognition molecule Capricious and Tartan and cell displacement from Ta5 to Pre across the boundary. Capricious and Tartan are controlled by transcription factor Bar and Al, and their loss of function leads to reduction of cell affinity to wild type neighbors and cell displacement activities. In addition, although the mutant cells formed Ta5/Pre boundary, its progression and sharpening were compromised. Cells overexpressing Capricious or Tartan became invasive within Ta5 and Pre, sometimes escaping the compartmental restriction of cell movement. Dynamic spatiotemporal regulation of cell affinity mediated by Capricious and Tartan is a key property of refinement of the Ta5/Pre boundary.

Regulation of layer-specific targeting by reciprocal expression of a cell adhesion molecule, capricious.

Layer-specific innervation is a major form of synaptic targeting in the central nervous system. In the Drosophila visual system, photoreceptors R7 and R8 connect to targets in distinct layers of the medulla, a ganglion of the optic lobe. We show here that Capricious (CAPS), a transmembrane protein with leucine-rich repeats (LRRs), is a layer-specific cell adhesion molecule that regulates photoreceptor targeting in the medulla. During the period of photoreceptor targeting, caps is specifically expressed in R8 and its target layer but not in R7 or its recipient layer. caps loss-of-function mutations cause local targeting errors by R8 axons, including layer change. Conversely, ectopic expression of caps in R7 redirects R7 axons to terminate in the CAPS-positive R8 recipient layer. CAPS promotes homophilic cell adhesion in transfected S2 cells. These results suggest that CAPS regulates layer-specific targeting by mediating specific axon-target interaction.

Wing-to-Leg homeosis by spineless causes apoptosis regulated by Fish-lips, a novel leucine-rich repeat transmembrane protein.

Growth, patterning, and apoptosis are mutually interactive during development. For example, cells that select an abnormal fate in a developing field are frequently removed by apoptosis. An important issue in this process that needs to be resolved is the mechanism used by cells to discern their correct fate from an abnormal fate. In order to examine this issue, we developed an animal model that expresses the dioxin receptor homolog Spineless (Ss) ectopically in the Drosophila wing. The presence of mosaic clones ectopically expressing ss results in a local transformation of organ identity, homeosis, from wing into a leg or antenna. The cells with misspecified fates subsequently activate c-Jun N-terminal kinase to undergo apoptosis in an autonomous or nonautonomous manner depending on their position within the wing, suggesting that a cell-cell interaction is, at least in some cases, involved in the detection of misspecified cells. Similar position dependence is commonly observed when various homeotic genes controlling the body segments are ectopically expressed. The autonomous and nonautonomous apoptosis caused by ss is regulated by a novel leucine-rich repeat family transmembrane protein, Fish-lips (Fili) that interacts with surrounding normal cells. These data support a mechanism in which the lack of some membrane proteins helps to recognize the presence of different cell types and direct these cells to an apoptotic fate in order to exclude them from the normal developing field.