Tracking Single Serotonin Transporter Molecules at the Endoplasmic Reticulum and Plasma Membrane

Transmembrane proteins are synthesized and folded in the endoplasmic reticulum (ER), an interconnected network of flattened sacs or tubes. Up to now, this organelle has eluded a detailed analysis of the dynamics of its constituents, mainly due to the complex three-dimensional morphology within the cellular cytosol, which precluded high-resolution, single-molecule microscopy approaches. Recent evidences, however, pointed out that there are multiple interaction sites between ER and the plasma membrane, rendering total internal reflection microscopy of plasma membrane proximal ER regions feasible. Here we used single-molecule fluorescence microscopy to study the diffusion of the human serotonin transporter at the ER and the plasma membrane. We exploited the single-molecule trajectories to map out the structure of the ER close to the plasma membrane at subdiffractive resolution. Furthermore, our study provides a comparative picture of the diffusional behavior in both environments. Under unperturbed conditions, the majority of proteins showed similar mobility in the two compartments; at the ER, however, we found an additional 15% fraction of molecules moving with 25-fold faster mobility. Upon degradation of the actin skeleton, the diffusional behavior in the plasma membrane was strongly influenced, whereas it remained unchanged in the ER.Live-cell microscopy and three-dimensional electron tomography has boosted our understanding of endoplasmic reticulum (ER) dynamics and morphology. Proteins have been identified which regulate the formation of cisternae versus tubelike membranes, and the contacts between ER and the various cellular organelles have been studied in detail (1). Little information, however, is available when it comes to protein dynamics and organization within the ER membrane. Its complex three-dimensional topology hampers standard diffraction-limited fluorescence microscopy approaches: in fluorescence recovery after photobleaching, for example, the obtained diffusion coefficients can be several-folds off, if the ER morphology is not correctly taken into account (2). A method is therefore needed which allows for resolving molecular movements on length scales below the typical dimensions of the ER structures.In principle, single-molecule tracking would provide the required spatial resolution due to the high precision in localizing the moving point emitters: localization errors of <40 nm can be easily achieved (3). This technique has given rise to multiple studies, in which the paths of the diffusing objects were used to make conclusions on the properties of the environment; particularly, the plasma membrane has become a favorite target for such investigations, yielding precise determinations of the diffusion coefficients of a variety of membrane proteins or lipids (4).Here, we report what is, to our knowledge, the first application of single-molecule tracking for a comparative study of the diffusion dynamics of a membrane protein at the ER versus the plasma membrane. As the protein of interest, we chose the human serotonin transporter (SERT): it is a polytopic membrane protein containing 12 transmembrane domains, with both C- and N-termini residing in the cytoplasm. Stable SERT oligomers of various degrees were observed to coexist in the plasma membrane (5). Functionally, SERT (6) is a pivotal element in shaping serotonergic neurotransmission: SERT-mediated high-affinity uptake of released serotonin clears the synaptic cleft and supports refilling of vesicular stores (7). Wild-type SERT (SERT-wt) is efficiently targeted to the presynaptic plasma membrane, whereas the truncation of its C-terminus (SERT-ΔC30) retains the mutant protein in the ER (8). The N-terminal mGFP- and eYFP-fusion constructs of the two versions of SERT thus allowed us to specifically address SERT located at the ER (eYFP-SERT-ΔC30) or at the plasma membrane (mGFP-SERT-wt (7)).Our experiments were performed at 37°C on proteins heterologously expressed in CHO cells. Total internal reflection (TIR) illumination afforded a reduction in background fluorescence and allowed for selective imaging of single mGFP-SERT-wt molecules at the cells’ plasma membrane or single eYFP-SERT-ΔC30 molecules at plasma membrane-proximal ER (Fig. 1 and see the Supporting Material). TIR was particularly crucial for single-molecule imaging of the ER-retained mutant, where out-of-focus background would surpass the weak single-molecule signals in epi-illumination.Open in a separate windowFigure 1Schematics of the plasma membrane (PM) and a part of the ER containing mGFP-SERT-wt or the ER-retained eYFP-SERT-ΔC30 mutant, respectively. Both can be excited by total internal reflection fluorescence (TIRF) excitation. Experiments were carried out either on cells expressing mGFP-SERT-wt or eYFP-SERT-ΔC30.For both mutants, the majority of molecules were mobile: in fluorescence-recovery-after-photobleaching experiments we observed a mobile fraction of 82 ± 8% for mGFP-SERT-wt and 91 ± 4% for eYFP-SERT-ΔC30. For single-molecule tracking, the high surface density of signals was reduced by completely photobleaching a rectangular part of the cell in epi-illumination; after a brief recovery period, a few single-molecule signals had entered the bleached area and could be monitored and tracked at high signal/noise using TIR excitation. Samples were illuminated stroboscopically for t_ill = 2 ms, and movies of 500 frames were recorded with a delay of t_del = 6 ms; the short delay times ensured that even rapidly diffusing molecules hardly reached the borders of the ER tubes between two consecutive frames. This illumination protocol was run for 20 times per cell, yielding ∼2500 trajectories per cell.The single-molecule localizations were first used to map those areas that are accessible to the diffusing proteins. eYFP-SERT-ΔC30 showed distinct hotspots, representing plasma membrane-proximal ER, excitable by the evanescent field (Fig. 2 A). These hotspots hardly moved within the timescale of an experiment (tens of minutes, see Fig. S1 in the Supporting Material); indeed, remarkable ER stability was previously observed using superresolution microscopy (9). In contrast, a rather homogeneous distribution was observed for mGFP-SERT-wt in the plasma membrane (Fig. 2 B).Open in a separate windowFigure 2Superresolution and tracking data at the ER and the plasma membrane. Superresolution images are shown for the ER-retained SERT mutant eYFP-SERT-ΔC30 (A) and for mGFP-SERT-wt in the plasma membrane (B). (C and D) Diffusion coefficients of eYFP-SERT-ΔC30 (C) and mGFP-SERT-wt (D) are shown as normalized histograms before (blue) and after (red) Cytochalasin D treatment. Data were fitted by Gaussian mobility distributions (see Table S1 in the Supporting Material for the fit results).Next, we compared the mobility of the observed proteins. Single-molecule localizations were linked to trajectories as described in Gao and Kilfoil (10), and the apparent diffusion coefficient, D, of each molecule was estimated from the first two points of the mean-square displacement membrane. The distribution of log₁₀ D showed a pronounced single peak (Fig. 2 D). It could be well fitted by a linear combination of two Gaussian functions, with the major fraction (85%) characterized by D_wt = 0.30 μm²/s; a broad shoulder to the left indicates the presence of proteins that are immobilized during the observation period. In contrast, the mobility of the ER-retained mutant showed a substantially different distribution, containing two clearly visible peaks (Fig. 2 C). We fitted the data with a three-component Gaussian model: the main fraction (82%) behaved similar to SERT at the plasma membrane, with D_ΔC30 = 0.32 μm²/s. In addition, a large fraction (15%) with high mobility of D_ΔC30 = 7.8 μm²/s and a minor fraction (3%) with low mobility was observed. The proteins responded as expected to degradation of the actin membrane skeleton (red bars in Fig. 2, C and D): at the plasma membrane, the mobility of mGFP-SERT-wt increased 4.6-fold (mean values), whereas at the ER membrane there was only a minor change for eYFP-SERT-ΔC30 mobility (1.06-fold increase; note that the ER is not connected to actin filaments (11)).The observation of a high mobility subfraction at the ER membrane is surprising. In general, the presence of obstacles—irrespective of whether randomly distributed or clustered, mobile or immobile—reduces the diffusivity of mobile tracers in a membrane (12). It is generally assumed that the high protein density in cell membranes is responsible for the rather low fluidity when compared to synthetic membranes (compare, e.g., Saxton and Jacobson (13) with Weiss et al. (14)). Interestingly, the observed diffusion constant of 7.8 μm²/s is of similar order as the mobility determined for various proteins in synthetic lipid membranes (14). It is thus tempting to hypothesize the presence of extended protein-depleted regions of higher fluidity within the ER membrane; such membrane domains were indeed observed already at the plasma membrane (15). We were also concerned, however, that protein degradation fragments could have contributed to our data: the three-dimensional mobility of an 85-kDa protein is ∼10 μm²/s (16), similar to the high mobility diffusion constant of eYFP-SERT-ΔC30.We tested the two explanations by analyzing the spatial distribution of fast (D_ΔC30 > 1 μm²/s) versus slow trajectories (D_ΔC30 < 1 μm²/s) of eYFP-SERT-ΔC30 (Fig. 3). Both types of trajectories clustered in the same regions, and no segregation into ER subdomains was observable at the resolved length scales. This finding—on the one hand—disfavors freely diffusing protein fragments as the origin of the high mobility fraction. On the other hand, it calls for further experiments to identify the origin of the fast and the slow mobility subfraction. Interestingly, when analyzing all eYFP-SERT-ΔC30 trajectories we found that 80% of the molecules showed diffusion confined to domains of 230-nm radius (see Fig. S2). This size is clearly smaller than the lateral extensions of the visible ER regions observed in Fig. 3. The finding indicates domain formation at the ER membrane; domains are averaged out in Fig. 3 due to the long recording times. Note that free diffusion was observed for mGFP-SERT-wt at the plasma membrane (5).Open in a separate windowFigure 3Ripley’s K function analysis of the different mobility fractions in the ER. For the cell presented in Fig. 2, the first position of every slow (D < 1 μm²/s; red) and fast (D > 1 μm²/s; blue) trajectory was plotted in panel A. Contour lines indicate regions of ER attachment to the plasma membrane. In panel B, the point-correlation function L(r)−r is plotted for the slow (red) and fast (blue) fraction. Furthermore, the correlation between fast versus slow is plotted (green). All three curves show a peak at ∼450 nm, which agrees with the extensions of the ER attachment zones.In conclusion, we have shown that single-molecule tracking is feasible for constituents of the ER membrane. We found a surprising diffusion behavior of SERT resulting in the following:

• 1.A slow fraction showing mobility reminiscent of protein diffusion in the plasma membrane, likely reflecting SERT diffusing in protein-crowded regions of the ER membrane; and
• 2.A fast fraction showing 25-fold faster diffusion kinetics.

This likely represents diffusion in altered ER membrane environments, possibly of different lipid or protein composition. Given the fact that synthesis of virtually all membrane proteins and most lipids proceeds at the ER membrane, ER heterogeneity at the nanoscale due to the continuous synthesis activity and selection for correct folding appears highly plausible.