Direct electrochemical observation of glucosidase activity in isolated single lysosomes from a living cell

Abstract

The protein activity in individual intracellular compartments in single living cells must be analyzed to obtain an understanding of protein function at subcellular locations. The current methodology for probing activity is often not resolved to the level of an individual compartment, and the results provide an extent of reaction that is averaged from a group of compartments. To address this technological limitation, a single lysosome is sorted from a living cell via electrophoresis into a nanocapillary designed to electrochemically analyze internal solution. The activity of a protein specific to lysosomes, β-glucosidase, is determined by the electrochemical quantification of hydrogen peroxide generated from the reaction with its substrate and the associated enzymes preloaded in the nanocapillary. Sorting and assaying multiple lysosomes from the same cell shows the relative homogeneity of protein activity between different lysosomes, whereas the protein activity in single lysosomes from different cells of the same type is heterogeneous. Thus, this study for the analysis of protein activity within targeted cellular compartments allows direct study of protein function at subcellular resolution and provides unprecedented information about the homogeneity within the lysosomal population of a single cell.

Keywords: electrochemical observation; glucosidase activity; nanocapillary; single lysosomes; subcellular analysis.

Fig. 1.

(A) The scheme of electrochemical setup for the detection of glucosidase activity in isolated single lysosomes from a single cell. The capillary coated with a Pt layer (dark shading) and an Ag/AgCl wire (light shading) inserted in the capillary is connected with an electrochemical station. Circle: amplified view of capillary tip with a Pt layer at the edge of the inner surface and the outer surface of the capillary to sort one lysosome (labeled in red). The arrow exhibits the flow direction of buffer with the lysosome. Cross-section view is used to illustrate Pt layer at the inner capillary and kit reaction. (Right) Displaying the release of glucosidase after the lysis of lysosome, the generation of hydrogen peroxide from kit reactions and the following electrochemical detection of hydrogen peroxide at the tip (dark shading). The arrow exhibits the flow direction of previously loaded glucosidase and generated reaction debris outside the capillary. (B) Reaction process for the determination of glucosidase activity using β-d-glucopyranoside as the substrate and glucose oxidase as the coenzyme.

Fig. 2.

(A) Fabrication of the nanoelectrode deposited with a Pt layer in the initial part of the inner capillary and at the entire outer wall of the capillary. (B) Cyclic voltammetry data of the nanoelectrode containing 10 mM PBS (pH 7.4) with 5 mM [Fe(CN)₆]⁴⁻. The scan rate was 0.1 V/s.

Fig. 3.

(A) Charge traces of nanoelectrodes containing 10 mM PBS (pH 7.4) with 5 mM β-d-glucopyranoside, 1 U/mL glucose oxidase, 1% Triton X-100 (curve a), and 0.1 (curve b), 0.15 (curve c), 0.2 (curve d), and 0.3 (curve e) U/mL glucosidase. (B) Traces of the charge increase from the nanoelectrode before and after the loading of 0.1 (curve a), 0.15 (curve b), 0.2 (curve c), and 0.3 (curve d) U/mL glucosidase. (C) Correlation between the charge increase from the nanoelectrode and the protein activity. (D) Background charge and the charge before and after the loading of 0.15 U/mL glucosidase in five runs using one electrode. (E) The charge increase from the nanoelectrode at the first minute with different concentrations of β-d-glucopyranoside before and after the loading of 0.15 U/mL glucosidase. (F) Plot of 1/m and 1/conc. The value of m was the slope of [H₂O₂] generated versus t² at the first minute, and conc. was the corresponding initial substrate (β-d-glucopyranoside) concentration. All of the measurements were performed under a potential of 600 mV in air. The error bar shows the SD calculated from four independent measurements.

Fig. 4.

(A) Bright-field image of the insertion of a nanoelectrode into a living HeLa cell. (B) Fluorescence image of the living cell stained with LysoTracker for the visualization of lysosomes. (C) Overlapping of image A and B. (D) Overlapping image of the nanoelectrode and the cell immediately after the sorting of one lysosome into the capillary. The insets in image C and D are the amplified display of the rectangular region in image C and D. The fluorescence at the lysosomes was false-colored into green for better visualization. The fluorescence spot arrowed in image C (Inset) was the lysosome sorted.

Fig. 5.

(A) Traces of the charge increase from the nanoelectrode containing 10 mM PBS (pH 7.4) with 5 mM β-d-glucopyranoside, 1 U/mL glucose oxidase, and 1% Triton X-100 before and after the loading of one lysosome into the capillary (trace a). Trace b represents the charge difference before and after the loading of one lysosome into the capillary in the absence of Triton X-100. (B) Charge increases from the nanoelectrode after the sorting of single lysosomes in 20 individual cells. Each column presents one charge measurement from one lysosome in one individual cell. (C) Charge increase from four individual lysosomes in three cells, numbered as 1, 2, and 3. (D) Plot of 1/m and 1/conc. All of the measurements were performed under a potential of 0.6 V in air. The error bar presents the SD calculated from three independent measurements.