Click Chemistry (CuAAC) and Detection of Tagged de novo Synthesized Proteins in Drosophila.

Copper-catalyzed azide-alkyne-cycloaddition (CuAAC), also known as 'click chemistry' serves as a technique for bio-orthogonal, that is, bio-compatible labeling of macromolecules including proteins or lipids. Click chemistry has been widely used to covalently, selectively, and efficiently attach probes such as fluorophores or biotin to small bio-orthogonal chemical reporter groups introduced into macromolecules. In bio-orthogonal non-canonical amino acid tagging (BONCAT) and fluorescent non-canonical amino acid tagging (FUNCAT) proteins are metabolically labeled with a non-canonical, azide-bearing amino acid and subsequently CuAAC-clicked either to an alkyne-bearing biotin (BONCAT) for protein purification, Western blot, or mass spectrometry analyses or to an alkyne-bearing fluorophore (FUNCAT) for immunohistochemistry. In combination with mass spectrometry, these kinds of labeling and tagging strategies are a suitable option to identify and characterize specific proteomes in living organisms without the need of prior cell sorting. Here, we provide detailed protocols for FUNCAT and BONCAT click chemistry and the detection of tagged de novo synthesized proteins in Drosophila melanogaster.

Cell Type-specific Metabolic Labeling of Proteins with Azidonorleucine in Drosophila.

Advanced mass spectrometry technology has pushed proteomic analyses to the forefront of biological and biomedical research. Limitations of proteomic approaches now often remain with sample preparations rather than with the sensitivity of protein detection. However, deciphering proteomes and their context-dependent dynamics in subgroups of tissue-embedded cells still poses a challenge, which we meet with a detailed version of our recently established protocol for cell-selective and temporally controllable metabolic labeling of proteins in Drosophila. This method is based on targeted expression of a mutated variant of methionyl-tRNA-synthetase, MetRSL262G, which allows for charging methionine tRNAs with the non-canonical amino acid azidonorleucine (ANL) and, thus, for detectable ANL incorporation into nascent polypeptide chains.

Impaired protein translation in Drosophila models for Charcot-Marie-Tooth neuropathy caused by mutant tRNA synthetases.

Dominant mutations in five tRNA synthetases cause Charcot-Marie-Tooth (CMT) neuropathy, suggesting that altered aminoacylation function underlies the disease. However, previous studies showed that loss of aminoacylation activity is not required to cause CMT. Here we present a Drosophila model for CMT with mutations in glycyl-tRNA synthetase (GARS). Expression of three CMT-mutant GARS proteins induces defects in motor performance and motor and sensory neuron morphology, and shortens lifespan. Mutant GARS proteins display normal subcellular localization but markedly reduce global protein synthesis in motor and sensory neurons, or when ubiquitously expressed in adults, as revealed by FUNCAT and BONCAT. Translational slowdown is not attributable to altered tRNA(Gly) aminoacylation, and cannot be rescued by Drosophila Gars overexpression, indicating a gain-of-toxic-function mechanism. Expression of CMT-mutant tyrosyl-tRNA synthetase also impairs translation, suggesting a common pathogenic mechanism. Finally, genetic reduction of translation is sufficient to induce CMT-like phenotypes, indicating a causal contribution of translational slowdown to CMT.

Cell-selective labelling of proteomes in Drosophila melanogaster.

The specification and adaptability of cells rely on changes in protein composition. Nonetheless, uncovering proteome dynamics with cell-type-specific resolution remains challenging. Here we introduce a strategy for cell-specific analysis of newly synthesized proteomes by combining targeted expression of a mutated methionyl-tRNA synthetase (MetRS) with bioorthogonal or fluorescent non-canonical amino-acid-tagging techniques (BONCAT or FUNCAT). Substituting leucine by glycine within the MetRS-binding pocket (MetRS(LtoG)) enables incorporation of the non-canonical amino acid azidonorleucine (ANL) instead of methionine during translation. Newly synthesized proteins can thus be labelled by coupling the azide group of ANL to alkyne-bearing tags through 'click chemistry'. To test these methods for applicability in vivo, we expressed MetRS(LtoG) cell specifically in Drosophila. FUNCAT and BONCAT reveal ANL incorporation into proteins selectively in cells expressing the mutated enzyme. Cell-type-specific FUNCAT and BONCAT, thus, constitute eligible techniques to study protein synthesis-dependent processes in complex and behaving organisms.

Müller glial cell-provided cellular light guidance through the vital guinea-pig retina.

In vertebrate eyes, images are projected onto an inverted retina where light passes all retinal layers on its way to the photoreceptor cells. Light scattering within this tissue should impair vision. We show that radial glial (Müller) cells in the living retina minimize intraretinal light scatter and conserve the diameter of a beam that hits a single Müller cell endfoot. Thus, light arrives at individual photoreceptors with high intensity. This leads to an optimized signal/noise ratio, which increases visual sensitivity and contrast. Moreover, we show that the ratio between Müller cells and cones-responsible for acute vision-is roughly 1. This suggests that high spatiotemporal resolution may be achieved by each cone receiving its part of the image via its individual Müller cell-light guide.

Expression and function of P2Y receptors on Müller cells of the postnatal rat retina.

In the postnatal and mature retina, many processes are controlled by the action of nucleotides. Their effects are partly mediated via activation of metabotropic P2Y receptors. However, little is known about the developmental regulation and cellular localization of P2Y receptor subtypes. Combining immunohistochemical and neurophysiological methods, we investigated the developmental expression of P2Y receptors on Müller cells, the principal macroglial cells of the retina. The P2Y(1) and the P2Y(4) receptors, but no other subtypes, were unequivocally localized on Müller cells. P2Y(1) was expressed from postnatal day 5 (P5) on and mediated a calcium response to ATP in Müller cells as well as a volume regulatory signaling cascade preventing Müller cells from swelling under hypotonic conditions. Differentiation of Müller cells was accompanied by a change of the calcium response pattern; the calcium responses in Müller cell endfeet persisted, but ATP responsiveness of Müller cell somata disappeared. P2Y(4) immunoreactivity was observed in Müller cell endfeet and synaptic terminals of rod bipolar cells from P20 on. Activated protein kinases were detected by immunohistochemistry; p-ERK occurred in Müller cells and amacrine cells, whereas p-Akt was detected in bipolar cells. Our data indicate that purinergic signaling via P2Y(1) and P2Y(4) receptors might contribute to differentiation processes in the postnatal retina.