Cell-type-specific quantification of protein synthesis in vivo

Abstract

Although protein synthesis is a conserved and essential cellular function, it is often regulated in a cell-type-specific manner to influence cell fate, growth and homeostasis. Most methods used to measure protein synthesis depend on metabolically labeling large numbers of cells with radiolabeled amino acids or amino acid analogs. Because these methods typically depend on specialized growth conditions, they have been largely restricted to yeast, bacteria and cell lines. Application of these techniques to investigating protein synthesis within mammalian systems in vivo has been challenging. The synthesis of O-propargyl-puromycin (OP-Puro), an analog of puromycin that contains a terminal alkyne group, has facilitated the quantification of protein synthesis within individual cells in vivo. OP-Puro enters the acceptor site of ribosomes and incorporates into nascent polypeptide chains. Incorporated OP-Puro can be detected through a click-chemistry reaction that links it to a fluorescently tagged azide molecule. In this protocol, we describe how to administer OP-Puro to mice, obtain cells of interest (here, we use bone marrow cells) just 1 h later, and quantify the amount of protein synthesized per hour by flow cytometry on the basis of OP-Puro incorporation. We have used this approach to show that hematopoietic stem cells (HSCs) exhibit an unusually low rate of protein synthesis relative to other hematopoietic cells, and it can be easily adapted to quantify cell-type-specific rates of protein synthesis across diverse mammalian tissues in vivo. Measurement of protein synthesis within bone marrow cells in a cohort of six mice can be achieved in 8-10 h.

Fig. 1 ∣. Molecular structure of OP-Puro.

OP-Puro is an analog of puromycin with a terminal alkyne group.

Fig. 2 ∣. Overview of key steps in the procedure to measure protein synthesis in vivo on the basis of OP-Puro incorporation.

OP-Puro is administered directly to mice via an i.p. injection (49.5 mg/kg; Steps 3–5). Exactly 1 h after injection, the mice are euthanized and the cells of interest (in this case, bone marrow cells) are obtained (Steps 6–24). Isolated cells are immunostained with a cocktail of antibodies against cell-surface proteins that enable detection of cell types of interest, in this case HSCs and restricted myeloid progenitor cells (CMPs/GMPs/MEPs; Steps 25–33). Appropriate single-color controls must be made (Steps 26 and 30). After immunostaining, cells are fixed and permeabilized before an azide–alkyne cycloaddition is performed to fluorescently label peptides with incorporated OP-Puro (Steps 34–49). Finally, OP-Puro incorporation is assessed by flow cytometry, and the MFI of OP-Puro in each cell is used as a relative measurement of protein synthesized per hour (Steps 50–65). BM, bone marrow.

Fig. 3 ∣. Obtaining bone marrow cells from mouse long bones.

a, Mouse limbs are pinned to a Styrofoam board with the stomach facing upward, and the fur/skin is sprayed with 70% (vol/vol) ethanol. b, The skin is cut away from the hind limbs. The tibia is removed by cutting just above the ankle and below the knee (blue arrows). The femur is removed by cutting just above the knee and at the pelvis (yellow arrows). c, The tibia (left) and femur (right). d, A 23-gauge needle and a 3-ml syringe are used to flush bone marrow from the femurs and tibias into a 14-ml Falcon tube containing 5 ml of staining medium. All procedures in this protocol involving mice were approved by the UC San Diego Institutional Animal Care and Use Committee.

Fig. 4 ∣. HSCs synthesize less protein per hour than unfractionated bone marrow cells.

Representative flow cytometry plots showing the gating strategy for analyzing CD150⁺CD48⁻LSK HSCs. A representative histogram shows OP-Puro incorporation into HSCs (blue) compared to unfractionated bone marrow cells from the same mouse (red) and HSCs from a PBS-injected control mouse (gray). Cell numbers and frequencies are shown in Supplementary Tables 1 and 3, respectively.BM, bone marrow; FITC, fluorescein isothiocyanate; FSC-A, front-scatter area; SSC-A, side-scatter area. All procedures in this protocol involving mice were approved by the UC San Diego Institutional Animal Care and Use Committee.

Fig. 5 ∣. Restricted myeloid progenitors have much higher protein synthesis rates than HSCs.

(Top) Representative flow cytometry plots showing the gating strategy for CMPs (left), GMPs (center) and MEPs (right). (Bottom) Representative histograms show OP-Puro incorporation into CMPs (left; blue), GMPs (middle; blue) and MEPs (right; blue) compared to HSCs from the same mouse (red) and progenitors from a PBS-injected control mouse (gray). Cell numbers and frequencies are shown in Supplementary Tables 2 and 4, respectively. All procedures in this protocol involving mice were approved by the UC San Diego Institutional Animal Care and Use Committee.

Fig. 6 ∣. OP-Puro incorporation by HSCs and myeloid progenitor cells in vivo.

MFI of OP-Puro in unfractionated bone marrow cells (BM), HSCs, CMPs, GMPs and MEPs (n = 4). Background fluorescence has been subtracted from each population. The raw data are shown in Tables 5 and 6. Data represent mean ± standard deviation. Statistical significance relative to HSCs was assessed using a one-way ANOVA followed by Dunnett’s test for multiple comparisons; ***P < 0.001. All procedures in this protocol involving mice were approved by the UC San Diego Institutional Animal Care and Use Committee.