Cell-selective labelling of proteomes in Drosophila melanogaster

Abstract

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.

Figure 1. Cell-type-specific in situ labelling of proteins via FUNCAT.

(a–c) ANL incorporation into larval proteins is monitored via FUNCAT on targeted expression of dMetRS^L262G-EGFP in neurons (a, elav^C155-Gal4;;UAS-dMetRS^L262G-EGFP), glia cells (b, repo-Gal4/UAS-dMetRS^L262G-EGFP) and muscle cells (c, C57-Gal4/UAS-dMetRS^L262G-EGFP) at larval neuromuscular junctions (muscles 6/7, segment A2). Co-staining with the neuron-specific marker anti-HRP (a–c) reveals that, wherever nerve terminal boutons (b), glial protrusions (g) and muscles (m) are in close contact, ANL-TAMRA signals are restricted to the dMetRS^L262G-EGFP-expressing cell type (a–c). dMetRS^L262G-EGFP is predominantly found in the cytosol, whereas TAMRA-harbouring proteins are detectable throughout cells including nuclei (n) and the bouton surrounding SSR area (c, dashed line). (d) Expression of dMetRS^L262G-EGFP in a wing disc epithelium along the anterior–posterior border (ptc-Gal4;UAS-dMetRS^L262G-EGFP) is accompanied by a respective confinement of ANL-Atto647N signals. The outline represents the shape of the entire disc. Scale bars, 10 μm (a,b); 5 μm (c); and 50 μm (d). SSR, subsynaptic reticulum.

Figure 2. Tagging of ANL-labelled proteins in Drosophila larvae and flies.

L3 stage larval body walls (a) or brains of L3 stage larvae (d) were dissected after chronic ANL feeding using 4 mM ANL concentration. Heads from adult Drosophila flies (b,c) were collected 0–3 days post eclosion after chronic ANL feeding using an ANL concentration of 4 mM. ANL is efficiently incorporated into muscle (a), neuronal (b) or glial proteins (c) when Drosophila larvae (a,d) or adult flies (b,c) express mMetRS^L274G-EGFP or dMetRS^L262G-EGFP. ANL-labelled proteins were tagged by a biotin-alkyne affinity-tag, and purified via NeutrAvidin agarose. Tagged input (I, before NeutrAvidin purification), unbound (U, no ANL-containing proteins) and eluted (E, enriched ANL-labelled proteins after NeutrAvidin purification) fractions from ANL labelled and control samples were run in mirror-imaged order on SDS-PAA gels, blotted and probed with anti-biotin antibody. Effective ANL labelling and subsequent biotin tagging were verified for selected marker proteins (‘anti-candidate protein'), that is, Dlg (a) in muscles, Synapsin in neurons (b) as well as Draper I (c) and to a small amount Dlg (c) in glia cells. Furthermore, the intracellular domain (d) of the transmembrane protein Notch was found to be ANL labelled when dMetRS^L262G-EGFP is expressed ubiquitously.

Figure 3. Neuronal ANL incorporation leaves NMJ morphology and prominent NMJ marker proteins unaffected.

Motor nerve terminals at NMJs from elav^C155-Gal4;;UAS-dMetRS^L262G-3xmyc L3 larvae fed without (a,c) or with (b,d) 4 mM ANL were visualized by the surface marker HRP and assessed for either FUNCAT-mediated TAMRA signals in combination with immunofluorescent labelling of the homophilic cell adhesion molecule FasII (a,b) or co-stained for the active zone marker Brp and PMCA to visualize the postsynaptic SSR compartment (c,d). There are no obvious differences between ANL- and non-ANL-reared animals. Scale bar, 5 μm. NMJ, neuromuscular junction; SSR, subsynaptic reticulum.

Figure 4. NCAT monitoring of protein synthesis over time.

Newly synthesized proteins were monitored by FUNCAT in motor neurons of OK371-Gal4;UAS-dMetRS^L262G-EGFP third instar larvae that were exposed to ANL for 12, 24 or 48 h (a). Selective dMetRS^L262G-EGFP expression in motor neurons was confirmed by EGFP fluorescence (a, upper panel). Fluorescence intensity of TAMRA tagged newly synthesized proteins increased with increasing ANL exposure times (a, lower panel). Identical confocal settings were used for acquisition of all images, and representative images are shown. Scale bar, 20 μm. (b) Quantification of fluorescence intensities confirmed the positive correlation between FUNCAT labelling intensity and the duration of ANL exposure. Averages±s.d. relative to 24 h ANL exposure (100%) are shown. Mann–Whitney U-test, n=10–11, **P≤0.01, ***P≤0.001 12 versus 24 h: **P=0.001530673; 12 versus 48 h: ***P=1.08E−04; 24 versus 48 h: ***P=9.34E−05. s.d. (12 h ANL): 11.83; s.d. (24 h ANL): 59.28; s.d. (48 h ANL): 103.50.

Figure 5. Time course of ANL labelling of neuronal proteins in adult flies.

(a) Zero to three days old elav^C155-Gal4;;UAS-mMetRS^L274G-EGFP flies were placed either onto ONM with 4 mM ANL or without ANL. After 24 and 48 h, heads from adult Drosophila flies were collected and ANL incorporation was addressed by BONCAT. Representative immunoblots showing the extent of ANL-labelled proteins and the neuronal marker protein synapsin after NeutrAvidin purification. The signal intensities for both overall newly synthesized proteins and synapsin increases from 24 to 48 h of ANL incorporation periods. (b) The assessment of protein-incorporated ANL into proteins and into synapsin, which is transmitted from larval neurons throughout the pupal phase into adult brain proteins, is rather high compared with flies that were on ANL-containing ONM through all developmental stages. Depicted immunoblots show input (I, before NeutrAvidin purification) and eluted fractions (E, enriched ANL-labelled proteins after NeutrAvidin purification) of samples.+ represents 4 mM ANL and − represents w/o ANL.