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.2010 Feb 9;107(6):2693-8.
doi: 10.1073/pnas.0907915107. Epub 2010 Jan 20.

Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules

Affiliations

Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules

Jonathan A Hern et al. Proc Natl Acad Sci U S A..

Abstract

G-protein-coupled receptors (GPCRs) are the largest family of transmembrane signaling proteins in the human genome. Events in the GPCR signaling cascade have been well characterized, but the receptor composition and its membrane distribution are still generally unknown. Although there is evidence that some members of the GPCR superfamily exist as constitutive dimers or higher oligomers, interpretation of the results has been disputed, and recent studies indicate that monomeric GPCRs may also be functional. Because there is controversy within the field, to address the issue we have used total internal reflection fluorescence microscopy (TIRFM) in living cells to visualize thousands of individual molecules of a model GPCR, the M(1) muscarinic acetylcholine receptor. By tracking the position of individual receptors over time, their mobility, clustering, and dimerization kinetics could be directly determined with a resolution of approximately 30 ms and approximately 20 nm. In isolated CHO cells, receptors are randomly distributed over the plasma membrane. At any given time, approximately 30% of the receptor molecules exist as dimers, and we found no evidence for higher oligomers. Two-color TIRFM established the dynamic nature of dimer formation with M(1) receptors undergoing interconversion between monomers and dimers on the timescale of seconds.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
TIRFM recording of a single CHO cell, transfected with M1 muscarinic receptors and labeled with the fluorescent antagonist, Cy3B–telenzepine. (A) Structural formula of Cy3B–telenzepine. (B) A single video frame (33-ms exposure) at the start of a recording. The insert shows a 5 × 5-μm2 expanded region containing 15 objects, 4 having intensity consistent with two fluorophores (attributed to receptor dimers) and 11 with intensity similar to a single fluorophore. A pseudocolor look-up table is shown inset. The first 100 frames of the recording are shown inMovie S1. (C) A total of 2,659 tracks of moving M1 receptors identified from the entire recording of the cell shown inB. The insert shows nine trajectories that illustrate the random nature of the diffusive process.Movie S2 shows the identification of tracks by the tracking algorithm. (D) Plot of average mean square displacement (MSD) versus the time interval (δt) for the trajectories shown inC. The plot is linear (r2 = 0.99), over 1 μm in length, with a 5-s timescale, which is consistent with receptor movement following a random walk, and shows no evidence for anomalous diffusive behavior. (E) Histogram of the intensity of 911 objects identified in the single video frame shown inB. The shape of the distribution is due to the presence of two populations, attributed to receptor dimers (red arrowhead, intensity ~120 counts/pixel) and monomers (black arrowhead, ~60 counts/pixel). The spread of the data arises from a combination of photon noise and intensity variation between spots that are located at different regions within the specimen.
Fig. 2.
Fig. 2.
Representative examples of some of the different intensity changes shown by individual tracks of Cy3B-labeled M1 receptors. (A) Intensity trajectory corresponding to a single molecule (upper trace), compared to the background intensity of a neighboring region containing no fluorescently labeled receptors (lower trace). (B) Intensity trajectory of a single moving object with an intensity corresponding to two molecules of Cy3B–telenzepine, i.e., a dimer. (C) Intensity trajectory of a single moving object with an intensity corresponding to two Cy3B fluorophores (level 2), which changes level abruptly to that of a single fluorophore (level 1), which is due to either photobleaching or dissociation of the dimer into two monomers. (D) Intensity trajectory that changes abruptly from level 1 to level 2 (i.e., 1 → 2). This might arise if the initially tracked monomer forms a dimer with another Cy3B-labeled receptor. (E) Intensity trajectory that exhibits abrupt changes from level 1 → 2 → 1. (monomer → dimer → monomer). (F) A rare and more complex intensity track that switches from 2 → 1 → 2 (dimer → monomer → dimer). This track could be interpreted as the transient dissociation of a dimer. This behavior was confirmed by dual-color imaging (see Fig. 4G: record 3). Automatic assignment of intensity changes is described in theSI Text andFig. S5. The signal fluctuations observed in each record are mainly due to photon noise whereas the differences in average intensity level attributed to one or two fluorophores found for the different objects are caused by spatial heterogeneity in the laser illumination and object position within the depth of the evanescent field.
Fig. 3.
Fig. 3.
Illustration of two molecules of Cy3B-labeled M1 receptors reversibly forming a dimer. One hundred sequential frames (30-ms exposure time/frame) of two molecules of Cy3B-labeled M1 receptors diffusing on the surface of a CHO cell. These molecules are initially monomers (about the first 21 frames), form a dimer (single red spot) for ∼40 frames (∼1 s), and then dissociate into monomers, possibly for the rest of the recording. There may be a transient reformation of short lifetime dimers for about three frames in the latter part of the recording, but it is not possible to exclude this being a chance overlap of the images of two molecules that are not interacting but diffusing independently close to each other. (Lower) A color look-up table. Two-color imaging (see text and Fig. 4) was used to study the details of M1 receptor dimerization.
Fig. 4.
Fig. 4.
Two-color TIRFM of labeled M1 receptors moving on a single CHO cell. (A) A 33-ms frame of Alexa488–telenzepine-labeled M1 receptors (green). (B) Image of the same cell recorded 33 ms later with the Cy3B–telenzepine-labeled receptors (red). Scale bar (5 μm) inB applies toA–F. (C) Superposition of images inA andB. Yellow spots indicate candidate dimers or chance colocalization. (D) A total of 825 trajectories of Alexa488–telenzepine-labeled M1 receptors identified during the 44-s recording. (E) A total of 970 trajectories of Cy3B–telenzepine-labeled M1 receptors. (F) A total of 241 trajectories of M1 dimers labeled with both green and red fluorophores. (G) Different tracks showing dimer formation and dissociation. Thex andy coordinates are shown for five examples in the upper and lower sections of each numbered plot. Coincidence of the tracks in both thex andy dimensions for a period >660 ms was taken as evidence of dimer formation (separation <160 nm). The green and red objects are not viewed simultaneously but 33 ms apart so the objects may move during that time. The five examples shown demonstrate different behaviors: (1) trajectory of a two-color dimer formation' (2) trajectory of a two-color dimer dissociation; (3) trajectory of a two-color dimer that dissociates after ∼1.5 s and then reforms a dimer; (4, 5) formation and dissociation of two two-color dimers. (Inset) A histogram of the lifetimes of 84 M1 receptor dimers taken from trajectories similar to those in examples and and collected in 0.5-s bins. The solid line is a monoexponential fit for a mean lifetime of 0.5 s.
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