Live-cell imaging of single receptor composition using zero-mode waveguide nanostructures

Abstract

We exploit the optical and spatial features of subwavelength nanostructures to examine individual receptors on the plasma membrane of living cells. Receptors were sequestered in portions of the membrane projected into zero-mode waveguides. Using single-step photobleaching of green fluorescent protein incorporated into individual subunits, the resulting spatial isolation was used to measure subunit stoichiometry in α4β4 and α4β2 nicotinic acetylcholine and P2X2 ATP receptors. We also show that nicotine and cytisine have differential effects on α4β2 stoichiometry.

Figure 1. Membrane projection into ZMWs

Schematic of a cell plated directly on the array with extensions of small portions of the membrane protruding into ZMWs. The schematic exaggerates the well size relative to spacing, in order to illustrate the entry of the cell membrane into the wells. An individual cell typically covers 75-100 wells. The excitation volume extends ~ 50 nm into the wells creating a ~ 250 zl excitation volume for a 120 nm ZMW, as depicted by the red squares (see supplemental material). Only a few nAChRs in the plasma membrane enter wells, probably in filopodia. Under many circumstances, the ER and other organelles contain a majority of the cell’s nAChR; but these do not enter wells. The diagram of the receptors illustrates that nicotinic receptors can potentially assemble into two stoichiometries: α₃β₂ or α₂β₃.

Figure 2. Isolation of membrane receptors in ZMWs

A. N2a cells plated on a glass coverslip transfected with a plasma membrane localized monomeric cherry marker (PM-mcherry) imaged with TIRF microscopy illustrating the extent of membrane expression in both the soma and the processes. B. N2a cells cultured directly on the ZMW array and transfected with the same membrane marker as in (a), and imaged from the glass side of the array. Fluorescent spots indicate the portions of the membrane containing the PM-mcherry marker that have entered the ZMW and lie within the excitation volume. C. N2a cells plated on a glass coverslip transfected with α4-GFP β4-wt and imaged with TIRF microscopy. The image shows that α4β4 nAChRs are primarily localized on the plasma membrane and also shows the presence of many filopodia (5 – 15 μm in length) containing fluorescent nAChRs. D. N2a cells cultured directly on the array and transfected in the same manner as in (c). Fluorescent spots indicate that portions of the membrane, probably filopodia, containing fluorescently labeled α4β4 nAChRs extend into the ZMWs. The size of the pattern made by the fluorescent wells is consistent with the size of a typical cell.

Figure 3. Single step bleaching of labeled subunits in N2a cells cultured on ZMW arrays

A. Time course of fluorescence intensity from a ZMW in a dish containing N2a cells transfected with α4-GFP + β4-wt. The trace shows three bleaching steps as indicated by the arrows. The different fluorescence levels are separated by clear steps until all the GFP molecules bleach to the background level. B. Number of three-step and two-step bleaching events observed for α4-GFP + β4-wt showing predominantly three bleaching steps. This indicates primarily (α4)₃(β4)₂ stoichiometry. C. Time course of fluorescence intensity from a well in a dish transfected with α4-wt + β4-GFP. The time trace shows two bleaching steps as indicated by the arrows. D. Number of three-step and two-step bleaching events observed for α4-wt + β4-GFP showing predominantly two bleaching steps. This also indicates primarily (α4)₃(β4)₂ stoichiometry.

Figure 4. Stoichiometry of α4β2 receptors

Fraction of the informative wells exhibiting 2 bleaching steps for α4-GFP + β2-wt exposed to no drug (n= 81), nicotine (n=179), and cytisine (n=117). The error bars show the relative standard error of the mean of the wells exhibiting 2-step bleaching. *, significant at P < 0.05.