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.
To achieve highly selective ablation of lacZ-positive cells in a biological milieu in vivo, we developed an activatable photosensitizer, SPiDER-killer-βGal, targeted to β-galactosidase encoded by the lacZ reporter gene. Hydrolysis of SPiDER-killer-βGal by β-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-βGal and light irradiation specifically killed lacZ-positive cells in coculture with cells without lacZ expression. Furthermore, β-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 Escherichia coli lacZ gene encoding b-galactosidase is awidely used reporter,but few synthetic substrates are available for detecting its activity with single-cell resolution in living samples.O ur recently reported fluorogenic substrate SPiDER-bGal is suitable for this purpose,b ut its hydrolysis product shows green fluorescence emission, and ar ed-shifted analogue is therefore required for use in combination with green fluorescent protein (GFP) markers.Herein, we describe the development of ar ed-shifted fluorogenic substrate for bgalactosidase,S PiDER-Red-bGal, based on as ilicon rhodol scaffold and ac arboxylic group as the intramolecular nucleophile.L acZ-positive cells were successfully labeled with SPiDER-Red-bGal at single-cell resolution in living samples, which enabled us to visualized ifferent cell types in combination with GFP markers.The Escherichia coli lacZg ene,w hich encodes b-galactosidase,i sapowerful reporter in combination with synthetic substrates, [1] and many lacZ-expressing animals have been established for experimental use.H owever,a lthough several substrates are available for detecting b-galactosidase activity, [2] selective labeling of lacZ-positive cells with single-cell resolution in living tissues or organisms is not feasible with most existing probes.W er ecently developed an ew class of functionalized fluorogenic substrate for b-galactosidase, SPiDER-bGal, [3] which is suitable for this purpose;w e controlled the fluorescence through intramolecular spirocyclization and achieved cellular retention by application of quinone methide chemistry.However,the utility of SPiDER-bGal is limited because it emits green fluorescence at around 550 nm, and therefore it is difficult to use in combination with green fluorescent protein (GFP), [4] one of the most widely used fluorescent tags for biological studies.A lthough red fluorescent proteins have been widely used as reporters in the red wavelength region, their tendency to form aggregates when expressed in neurons in mouse brain may limit their applicability. [5] Therefore,d evelopment of ar ed fluorescent probe for b-galactosidase with single-cell resolution should extend the utility of the lacZreporter.Herein, we describe the development of an ew fluorogenic substrate for b-galactosidase,w hich emits red fluorescence at above 600 nm without loss of the advantageous properties of SPiDER-bGal. We show that the new probe,SPiDER-Red-bGal, can label lacZpositive cells in combination with GFP markers and can successfully visualize multiple cell types at single-cell resolution in living samples.In order to develop red-shifted b-galactosidase probes,we first selected 2'Me-Sirhodol (Supporting Information, Scheme S1) as the core fluorophore,i nw hich the oxygen atom at the 10th position of rhodol is replaced with as ilicon atom;t his replacement has been reported to extend the absorption and fluorescence wavelengths by about 100 nm. [6] Indeed, 2'Me-Sirhodol seemed to have favorable properties for our purpose;i ts hows absorption/emission maxi...
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.
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 C 2 H 2 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.
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