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, Chapter 7, Unit7.11

Metabolic Labeling With Noncanonical Amino Acids and Visualization by Chemoselective Fluorescent Tagging

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Metabolic Labeling With Noncanonical Amino Acids and Visualization by Chemoselective Fluorescent Tagging

Susanne Tom Dieck et al. Curr Protoc Cell Biol.

Abstract

Fluorescent labeling of proteins by genetically encoded fluorescent protein tags has enabled an enhanced understanding of cell biological processes but is restricted to the analysis of a limited number of identified proteins. This approach does not permit, e.g., the unbiased visualization of a full proteome in situ. We describe here a fluorescence-based method to follow proteome-wide patterns of newly synthesized proteins in cultured cells, tissue slices, and a whole organism. This technique is compatible with immunohistochemistry and in situ hybridization. Key to this method is the introduction of a small bio-orthogonal reactive group by metabolic labeling. This is accomplished by replacing the amino acid methionine by the azide-bearing methionine surrogate azidohomoalanine (AHA) in a step very similar to classical radioisotope labeling. Subsequently, an alkyne-bearing fluorophore is covalently attached to the group by "click chemistry"--a copper(I)-catalyzed [3+2]azide-alkyne cycloaddition. By similar means, metabolic labeling can also be performed with the alkyne-bearing homopropargylglycine (HPG) and clicked to an azide-functionalized fluorophore.

Figures

Figure 7.11.1
Figure 7.11.1
FUNCAT strategy and protocol overview. The flow chart summarizes the steps of the protocols provided and indicates protocol choice points. Alternatives and options mentioned in the text, but not extensively described, are included to indicate potential extensions (light gray).
Figure 7.11.2
Figure 7.11.2
Special reagents and materials described in the FUNCAT protocols. (A) FUNCAT incubation plate made from a 24-well cell culture plate with paraffin drops for upside-down incubation of circular coverslips. (B) Tube-lid support filled with modeling clay and sealed with two-component epoxy glue for upside-down incubation of MatTek glass-bottom dishes. (C) Standard microfluidic chamber for compartmentalized neuron incubation. Neurons are plated on the cell body chamber side through the connected wells 1. They extend axons and dendrites into the microgrooves but only axons grow the whole 900-μm distance through the microgrooves and reach the axon chamber that is accessible via the connected wells 2. Dendrites usually stop growing at a 200- to 300-μm distance within the microgrooves. The cell body and axon chamber can be fluidically isolated; therefore, compartments can be incubated with different solutions. (D) The microfluidic local perfusion (μLP) version of the microfluidic chamber has a perfusion channel perpendicular to the microgrooves (Taylor et al., 2010). It is located at a distance from the cell body chamber where dendrites still populate the microgrooves. Thus, perfusion via the perfusion channel allows one to manipulate selectively a proportion of dendrites and axons. (E) The microfluidic chambers are assembled on coverslips where the cells attach. After metabolic labeling, the PDMS part of the chamber is removed and the cells are fixed and processed further on the coverslip. Upside-down incubation for the click reaction is performed on Parafilm with unilateral silicone spacer support in a humidified chamber.
Figure 7.11.3
Figure 7.11.3
FUNCAT: chemistry and principle. (A) The chemical structures of the noncanonical amino acids AHA (azide-bearing) and HPG (alkyne-bearing) are similar to methionine (Met). A variety of azide- or alkyne-functionalized fluorophores (A) are available to covalently ligate a fluorophore to the noncanonical amino acids by Cu(I)-catalyzed azide + alkyne [3+2]-cycloaddition (B). The Cu(I) catalyst is produced in the reaction mixture from Cu(II) and TCEP and is stabilized by the triazole ligand (TBTA). (C) Explanation of FUNCAT procedure steps during metabolic labeling and click reaction.
Figure 7.11.4
Figure 7.11.4
Example results. Representative FUNCAT experiments in (A) COS7 cells, (B) glial cells, (C) cultured hippocampal neurons, and (D) acute hippocampal slices using different fluorescent tags. (A) COS cells incubated with 4 mM AHA for 1 hr, clicked to Alexa594-alkyne, labeled for actin with Alexa488-phalloidin and DAPI to stain nuclei. In the presence of the protein synthesis inhibitor anisomycin (40 μM), the FUNCAT signal is significantly reduced (present only at low levels in the nucleus) and when AHA is replaced by Met no FUNCAT signal is visible. (B) Primary astrocytes treated with 4 mM AHA, clicked to TxRed-alkyne and stained for GFAP (two left panels) and the respective anisomycin control (two right panels). (C) Increasing the duration of 4 mM AHA incubation increases the FUNCAT signal (AHA, Tamra-alkyne and Met control) in hippocampal neurons stained for the neuron marker MAP2 and the presynaptic protein synaptophysin. FUNCAT signal (as fire lookup table in lower panel) is clearly visible in soma and dendrites after 2 hr. After 6 hr, there is also ample labeling of synaptic sites. (D) Micrograph of area CA1 from a FUNCAT experiment in an acute hippocampal slice incubated for 4 hr with 4 mM AHA (left panel) and clicked to Alexa488-alkyne and the respective Met control (right panel). For better visualization, orientation slices are stained with the neuron marker MAP2. DAPI labeling in the AHA slice shows that the FUNCAT signal in pyramidal cells is higher than in other cells dispersed in the neurophil layer. Scale bars 20 μm (A), 10 μm (B,C), 100 μm (D).
Figure 7.11.5
Figure 7.11.5
Expected results. (A) FUNCAT in whole 7 dpf zebrafish larvae after 72 hr AHA labeling was combined with antibody labeling for parvalbumin. A dorsal view of the head of larvae incubated without (ctrl) and with 4 mM AHA, clicked to Alexa594-tag and stained for parvalbumin, shows the specificity and low background of the FUNCAT labeling (A, left panel). Higher magnifications of different regions—a lateral view of the spinal cord (s, Alexa594-tag), dorsal view of the cerebellum (cb, Alexa488-tag), and a lateral view of the pectoral fin (pf, Alexa488-tag) show that within the tissue cell populations show differences in FUNCAT signal and can be identified by antibody staining. (B,C). Application of 4 mM AHA for 2 hr via the perfusion channel (pc) in a μLP chamber (B) or 1 hr via the axon chamber (ax) of a microfluidic chamber without perfusion channel (C). Both conditions lead to signal in the cell body compartment (cb) in the soma of cells (cb) that send neurites to the respective compartment indicating that axons and dendrites are capable of AHA uptake. MAP2 positive cells (B) that do not extend dendrites through the microgrooves (mg) to the perfusion channel (pc) are not intensely labeled. In a well-grown chamber culture usually ~30% to 50% of the neurons in a distance of 150 μm from the microgrooves are labeled when AHA is loaded from the perfusion channel. (D) High-resolution FISH for Rab1 mRNA combined with FUNCAT in a hippocampal neuron. After FISH the click reaction was performed for only 2 hr instead of overnight for maximal preservation of the FISH signal. (E) Fluorescent precipitates (arrow) appear in the samples and make analysis difficult when the FUNCAT click reaction is not performed overhead or grease from sealing the MatTek dishes on the incubation support during overhead click reaction spills into the sample. Scale bars 100 μm (A, left panel), 25 μm (A, other panels), 20 μm (B, D), 50 μm (C).

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