Subcellular western blotting of single cells

Abstract

Although immunoassays are the de facto standard for determining subcellular protein localization in individual cells, antibody probe cross-reactivity and fixation artifacts remain confounding factors. To enhance selectivity while providing single-cell resolution, we introduce a subcellular western blotting technique capable of separately assaying proteins in the 14 pL cytoplasm and 2 pL nucleus of individual cells. To confer precision fluidic control, we describe a passive multilayer microdevice that leverages the rapid transport times afforded by miniaturization. After isolating single cells in microwells, we apply single-cell differential detergent fractionation to lyse and western blot the cytoplasmic lysate, whereas the nucleus remains intact in the microwell. Subsequently, we lyse the intact nucleus and western blot the nuclear lysate. To index each protein analysis to the originating subcellular compartment, we utilize bi-directional electrophoresis, a multidimensional separation that assays the lysate from each compartment in a distinct region of the separation axis. Single-cell bi-directional electrophoresis eliminates the need for semi-subjective image segmentation algorithms required in immunocytochemistry. The subcellular, single-cell western blot is demonstrated for six targets per cell, and successfully localizes spliceosome-associated proteins solubilized from large protein and RNA complexes, even for closely sized proteins (a 7 kDa difference). Measurement of NF-κB translocation dynamics in unfixed cells at 15-min intervals demonstrates reduced technical variance compared with immunofluorescence. This chemical cytometry assay directly measures the nucleocytoplasmic protein distribution in individual unfixed cells, thus providing insight into protein signaling in heterogeneous cell populations.

Keywords: cytometry; microfluidic design; proteomics; single-cell analysis.

Figure 1

Microfluidic subcellular western blotting reports protein localization to the cytoplasmic or nuclear compartment of single cells. (a) Photograph of the base layer and microwell array of the (sc)²WB device, with (b) insets showing the fluorescence micrograph of the subcellular western blot array (56 U373 cells) for lamin A/C (magenta) and TurboGFP (green) and for a single U373 cell with a companion intensity profile plot. (c) Rendering of the assembled (sc)²WB device. (d) Schematic cross-section in the x–z plane of (c). When placed atop the base layer, the 500 μm-thick hydrogel lid simultaneously delivers the lysis reagents via diffusion and electrically addresses the base layer for rapid transition between the lysis and electrophoresis stages. (e) Schematic of the (sc)²WB workflow: (Stage 0) Settle single cells into microwells via sedimentation; (Stage 1) cytoplasm-specific lysis buffer is diffusively applied from the lid, PAGE is performed on solubilized cytoplasmic proteins along the separation axis to the ‘east’ of the microwell, and the cytoplasmic proteins are photo-immobilized to the gel; (Stage 2) nucleus-specific lysis buffer is diffusively applied from the lid, PAGE is performed on solubilized nuclear proteins along the separation axis to the ‘west’ of the microwell, and nuclear proteins are photo-immobilized to the gel; (Stage 3) in-gel immunoprobing and image fluorescence are performed. At bottom: (Step 0) false-color fluorescence micrographs of an intact cell in a microwell; (Step 1) PAGE of cytoplasmic GFP (E=40 V cm⁻¹; Δt=10 s) with the nucleus retained in the microwell; (Step 2) western blotting after bi-directional PAGE with cytoplasmic protein ‘east’ and nuclear protein ‘west’ of the microwell. The microwells are encircled with a white dashed line for clarity; TurboGFP (green) and Hoechst DNA stain (blue). (f) Stripping and reprobing for the expression and localization of six protein targets from one mammalian cell. The relative expression (AUC/AUC_max) is reported for n=44 U373 cells. GFP, green fluorescent protein; PAGE, polyacrylamide gel electrophoresis.

Figure 2

The (sc)²WB assay detects a panel of well-described protein targets, thus validating target and localization selectivity. (a) Intensity profile and false-color fluorescence for a representative (sc)²WB assay (TurboGFP, green signal; β-tubulin, blue signal; lamin A/C, magenta signal; U373-GFP cells; lysis duration: 25 s; PAGE duration: 17 s at E=40 V cm⁻¹). Dashed lines in the intensity profile denote the microwell border. Cytoplasmic proteins are to the right (west) of the microwell and nuclear proteins are to the left (east). (b) Separation resolution of a 1-mm PAGE separation distance (n=27 cells). (c) Mean-normalized expression (AUC/AUC_mean) and (d) subcellular localization (Nuc=AUC_nuclear/AUC_total, Cyt=AUC_cytoplasm/AUC_total) as determined by the (sc)²WB for membranous organelles: mitochondria-targeted GFP, Calnexin (ER), and GRP-75 (mitochondrial matrix), cytoplasmic (TurboGFP, β-tubulin), and nuclear (lamin A/C, H3) targets. Error bars are±1 s.d. ER, endoplasmic reticulum; GFP, green fluorescent protein; PAGE, polyacrylamide gel electrophoresis; s.d., standard deviation.

Figure 3

Spliceosome protein localization and expression in single mammalian cells. (a) The (sc)²WB assigns subcellular localization to cytoplasmic (TurboGFP, green; β-tubulin, blue) and nuclear (SFPQ, magenta; PTBP1, gray) proteins, even when the targets are components of large molecular machines. The dashed line is the microwell border. A representative intensity profile and false-color micrograph are shown here. A dashed line denotes the microwell border in the intensity profile. (b) Bi-directional PAGE enhances selectivity because uni-directional PAGE cannot resolve β-tubulin and SFPQ. The dashed line indicates the position of the next row of microwells (array period). Note that in the whole-cell scWB, the TurboGFP band has overrun into the next separation lane. (c) Mean-normalized expression (AUC/AUC_mean) and (d) subcellular localization (Nuc=AUC_nuclear/AUC_total, Cyt=AUC_cytoplasm/AUC_total) of TurboGFP, β-tubulin, SFPQ, and PTBP1 (n=44 cells). Error bars are±1 s.d. GFP, green fluorescent protein; PAGE, polyacrylamide gel electrophoresis; s.d., standard deviation.

Figure 4

Monitoring dynamic changes in NF-κB localization using the (sc)²WB. (a) The nucleocytoplasmic distribution of NF-κB changes in response to stimulation with LPS. (b) The median localization of NF-κB for each time point as measured by ICC (x axis) and the (sc)²WB (y axis) correlate with ρ=0.90. Nuclear NF-κB=AUC_nuc/AUC_total. The gray region indicates the 95% confidence interval. (c) False-color fluorescence micrographs from ICC and (sc)²WB analysis of U373 cells at different times after LPS stimulation (U373 cells, 5 μg mL⁻¹ LPS). Magenta traces on the (sc)²WB intensity profiles are the raw signal and the black traces are the Gaussian fits. Dashed lines denote the microwell border. (d) The median fluorescence signal (AUC) from NF-κB in the nucleus is determined by ICC and (sc)²WB, and reports a similar time-to-peak and translocation trend. (e) Histograms of nuclear NF-κB expression over the time course by both ICC and (sc)²WB. (f) Localization distribution parameters from ICC (n=4 wells) and (sc)²WB (n=3 devices). Error bars are±1 s.d. ICC, immunocytochemistry; LPS, lipopolysaccharide; s.d., standard deviation.