Dynamic visualization of membrane-inserted fraction of pHluorin-tagged channels using repetitive acidification technique
© Khiroug et al; licensee BioMed Central Ltd. 2009
Received: 14 July 2009
Accepted: 30 November 2009
Published: 30 November 2009
Changes in neuronal excitability, synaptic efficacy and generally in cell signaling often result from insertion of key molecules into plasma membrane (PM). Many of the techniques used for monitoring PM insertion lack either spatial or temporal resolution.
We improved the imaging method based on time-lapse total internal reflection fluorescence (TIRF) microscopy and pHluorin tagging by supplementing it with a repetitive extracellular acidification protocol. We illustrate the applicability of this method by showing that brief activation of NMDA receptors ("chemical LTP") in cultured hippocampal neurons induced a persistent PM insertion of glutamate receptors containing the pHluorin-tagged GluR-A(flip) subunits.
The repetitive acidification technique provides a more accurate way of monitoring the PM-inserted fraction of fluorescently tagged molecules and offers a good temporal and spatial resolution.
The ability to monitor changes in the amount of key proteins residing in PM is crucial for understanding neuronal function and synaptic plasticity, but existing methods have several restrictions. Total internal reflection fluorescence (TIRF) microscopy has been employed to monitor protein trafficking to and from PM [1–4]. The TIRF method uses the so called evanescent field to excite fluorescence within 100-200 nm above the glass bottom of a culture dish. However, the visibility of a fluorescent molecule in a TIRF image does not necessarily mean that the molecule is inserted in the PM, because many intracellular organelles located near the PM are well within the evanescent field. Indeed, TIRF microscopy readily visualizes unfused secretory vesicles , lysosomes , mitochondria [7, 8] and the endoplasmic reticulum (ER) [9, 10].
Another technique for monitoring PM insertion of fluorescent molecules is based on pHluorin tagging [11–13]. Fluorescence of ecliptic pHluorin, the multiple-point mutant of EGFP, is completely quenched at pH below 6.0 . The key assumption of this technique is that pHluorin is fully quenched while in the lumen of secretory organelles , whereas upon PM insertion the tagged molecules pHluorin regains fluorescence due to exposure to extracellular milieu (pHo~7.4). In practice, this assumption is incorrect because the lumen of many intracellular organelles, notably the ER, is not acidic but has pH around 7.2 . Hence, pHluorin-tagged molecules located in the ER exhibit bright fluorescence, which may add a strong background and thus "contaminate" the fluorescent signal of the PM-inserted molecules. Since fluorescence of intracellular and extracellular pHluorin-tagged molecules often overlaps, the imaging results are prone to misinterpretation.
Two groups have recently employed TIRF imaging to monitor PM insertion of pHluorin-tagged AMPA receptors [17, 18]. While offering a greatly improved sensitivity, this assay is not, in principle, devoid of image contamination caused by the unquenched pHluorin residing in non-acidic intracellular compartments. In the present study, we solved this problem by using repetitive acidification tests in combination with TIRF imaging and pHluorin tagging of GluR-A-containing AMPA receptors.
Results and Discussion
While in some secretory organelles the fluorescence of pHluorin molecules is fully quenched by acidic intralumenal milieu [14, 15], quenching of the ER-residing pHluorin may not occur because pH in the ER is not acidic . To test directly whether the ER-residing pHluorin retains its fluorescence, we overexpressed in cultured hippocampal neurons a pHluorin-encoding construct engineered for expression in the lumen of endoplasmic reticulum (pHluorin-ER). This protein is retained in the ER via retrograde transport based on KDEL receptor (see Methods). We observed bright fluorescence of the pHluorin-containing ER (Additional file 2: Figure S2). Importantly, pHluorin-ER was detectable not only in epifluorescence but also in TIRF mode (Additional file 2: Figure S2), consistent with our previous findings [5, 7]. This observation lead us to conlcude that i) fluorescence of the ER-resident pHluorin molecules is not quenched, and that ii) TIRF imaging mode does not fully exclude the fluorescence of intracellular pHluorin-tagged molecules.
In spite of the rapid initial quenching, a significant fraction of the pHluorin-GluR-A receptors retained their fluorescence during the acidification tests (Figure 2B). On average, in 39 neurons the fluorescence fraction that remained unquenched after 30 seconds of the acidification test constituted 42 ± 3%. The unquenched fluorescence was observed throughout the neuronal soma and neurites (Figure 2B) and often had a reticular appearance resembling that of the ER. To visualize spatial distribution of those pHluorin-GluR-A molecules that were quenched during the acidification test, we digitally subtracted the image obtained during the test (Figure 2B) from the control image (Figure 2A). The resulting image (Figure 2C) showed fluorescence that was distributed throughout neuronal soma and dendritic tree, a pattern consistent with the PM-inserted fraction of pHluorin-GluR-A .
When the acidification test was terminated by returning to the control perfusion solution (pHo = 7.4), the fluorescence level was completely restored to its pre-test value within few minutes (time course of a representative experiment is illustrated in Figure 2D). Repetitive 1- or 2-minute acidification tests did not have any irreversible effects on the overall pHluorin fluorescence, because complete fluorescence recovery was reliably observed when we repeated these tests for up to one hour with the 5 minute inter-test intervals (data not shown). It is conceivable that repetitive acid exposure could have caused some subtle changes in those physiological parameters of neurons that were not monitored in our experiments. However, reproducibility of acidification tests argues against accumulating detrimental effects.
Fractional contributions of the fast and slow phases varied widely between different cells and different subcellular regions. We hypothesized that larger fractional contributions by the slow phase were due to stronger "contamination" of the "signal" (i.e., fluorescence of the PM-inserted pHluorin-GluR-A receptors) by "noise" (i.e., fluorescence of the receptors residing in non-acidic organelles such as the ER). If this is true, then the slow phase of fluorescence quenching (red circles in Figure 3A) should reflect passive acidification of these organelles, which is secondary to the decrease in cytosolic pH caused, in turn, by extracellular acidification. To test this hypothesis, we transfected neurons with a construct encoding for soluble pHluorin expressed in the cytosol (pHluorin-CS). A two-minute perfusion with acidic solution (pHo = 5.4) produced a slow, gradually developing decrease in pHluorin-CS fluorescence (Figure 3B). This decrease was well fitted with a linear regression (R2 = 0.994), with the coefficient of 0.47%/s. This result indicates that, at a later stage of the pH test, the partial collapse of the proton gradient across the PM caused gradual acidification of the cytosol.
The cytosolic acidification should then lead to acidification of those intracellular organelles that lack active proton transport. We transfected cells with the ER-trapped pHluorin (pHluorin-ER), performed acidification tests and compared the time course of fluorescence changes in neurons expressing pHluorin-ER (Figure 3C) to those expressing pHluorin-CS (Figure 3B) or pHluorin-GluR-A (red circles in Figure 3A). Similarly to pHluorin-CS, the pHluorin-ER fluorescence exhibited a slow decrease during the acidification test and was well fitted with a linear regression (R2 = 0.991), yielding the linear coefficient of 0.45%/s. Importantly, the near-linear time course of the fluorescence changes both in the cytosol and ER was very similar to the slow phase of pHluorin-GluR-A fluorescence quenching (red circles in Figure 3A). Indeed, the linear coefficients were close for all three constructs. To compare them, one should take into account the fractional contribution of the pHluorin-GluR-A slow phase that was, on average, 42% (Figure 3A). Thus, the corrected linear coefficient for the slow phase of pHluorin-GluR-A quenching is 0.17%/s/0.42 = 0.40%/s, which corresponds well to the linear coefficients of both pHluorin-CS (0.47%/s) and pHluorin-ER (0.45%/s). Taken together, these results strongly suggest that the slow phase of pHluorin-GluR-A fluorescence quenching was due to intracellular acidification, and that this slow phase developed after the fast phase was largely complete.
The clear temporal segregation of fast and slow quenching phases allowed us to perform accurate separation of the fluorescent signal derived from the PM-inserted pHluorin-GluR-A receptors from that of the intracellular receptors. For our repetitive acidification method, we selected the time point of 30 seconds after the beginning of the acidification test because at this point approximately 90% of the fast quenching phase is completed, whereas the contribution of the slow phase is negligible. We estimated the error due to the slow phase contribution to be below 3%. This estimate is based on two measurements: i) thirty seconds after the beginning of the acidification test, the decrease in pHluorin-ER fluorescence (black arrow in Figure 3C) was 5.8 ± 1.1% (n = 5), and ii) on average, the intracellular fraction of the pHluorin-GluR-A fluorescence was 42%. Thus, the error of our method can be calculated as 5.8% × 0.42 = 2.4%. Importantly, the acidification tests are highly reproducible and thus can be used for monitoring changes in the PM-inserted fraction of pHluorin-tagged channels, such as their insertion or internalization during synaptic plasticity.
To demonstrate applicability of the repetitive acidification method, we used it to monitor translocation of pHluorin-GluR-A receptors to the PM. Membrane insertion of GluR-A-containing AMPA receptors associated with LTP is a well-established phenomenon (for review see ). In a recent study, Yudowski and colleagues used pHluorin tagging to visualize membrane insertion of GluR-A(flop) receptors . Here, we focused on the "flip" splice variant of this receptor. On hippocampal neurons transfected with pHluorin-GluR-A, we performed repetitive acidification tests with a 10 minute interval. During each test, we measured the amplitude of the fast quenching phase 30 seconds after the onset of acidification.
The second notable advantage of the repetitive acidification method is that it allows distinguishing between PM-insertion-related changes in pHluorin fluorescence from those caused by e.g. changes in the pH of the intracellular organelles containing pHluorin-tagged molecules. In the example of Figure 5B, the neuron was monitored under control conditions without NMDA receptor activation. During the 30 minutes between the first and the second acidification tests (left and right traces, respectively) the overall pHluorin-GluR-A fluorescence increased by (112-100)/100 = 12%. In contrast, the amplitude of the fast quenching phase remained unchanged: first test 100-62 = 38, second test 112-74 = 38, change (38-38)/38 = 0%. This observation suggests that interpreting the increase in pHluorin fluorescence as an indication of membrane insertion may lead to erroneous conclusions and should, therefore, always be verified.
The use of repetitive acidification tests allows avoiding several pitfalls of the existing methods. In a number of recent studies, the increase in the brightness of the TIRF image has been attributed to insertion of additional fluorescent molecules in the PM [1, 4]; here, we demonstrated that the PM-associated TIRF image can be strongly "contaminated" by the background fluorescence of molecules located in non-acidic organelles, such as the ER. The mere visibility of pHluorin-tagged molecules has often been interpreted as evidence of their PM residence [11, 13]; we now showed that as many as half of the pHluorin molecules visible in TIRF images of cultured hippocampal neurons may actually reside in intracellular compartments with near-neutral pH (Figure 2B and Figure 3A). It is also worth noticing that the fast quenching phase amplitude was measured as percentage of the overall fluorescence, i.e. as a ratio. Like any ratiometric technique, this method allows direct comparison between different regions of the same cell or between cells expressing different constructs. Although we did not pursue detailed spatial analysis in this study, future research will greatly benefit from the repetitive acidification method because it provides information on the extent of PM insertion at different subcellular sites.
2.1 DNA constructs
Standard molecular biological techniques, including PCR-assisted mutagenesis were used to make expression plasmids. Expression plasmids for ER-retained pHluorin and N-terminally pHluorin-tagged rat GluR-A were constructed in pcDNA3.1(-) (Stratagene). Briefly, pHluorin cDNA was inserted between a signal peptide derived from rat GluR-D (residues 1-21; P19493) and cDNA encoding the mature polypeptide of GluR-A (residues 19-907; flip isoform; P19490). To make pHluorin-ER, an ER-lumen resident form of pHluorin, KDEL tetrapeptide sequence was added to the carboxyterminus of pHluorin encoded by GluR-D signal peptide - pHluorin expression cassette. The cytosolic form of pHluorin, pHluorin-CS, with no signal peptide or any other sequence modifications was constructed in pcDNA3.1. All constructs were verified by restriction mapping and by sequencing through all PCR-derived parts.
2.2 Preparation and transfection of rat hippocampal neurons
All experimental procedures were approved by the Animal Care and Use Committee, University of Helsinki. Cultured neurons were prepared from embryonic day 18 rat hippocampi. Hippocampi were dissociated with Papain solution (10 U/mL). The cells were plated at a density of 3 × 104 cells cm2 on glass-bottomed Petri dishes (MatTek) pre-coated with poly-L-lysine and laminin (1-2 μg/cm2). Cultures were maintained in the 5% CO2/95% air atmosphere at 37°C in Neurobasal medium (Invitrogen; pH = 7.4) supplemented with B27 (Invitrogen), 0.5 mM L-glutamine, 100 units/mL penicilline and 100 μg/mL streptomycine. Medium was changed twice per week. Neurons were transfected after 6-10 days in vitro with constructs using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Lipofectamine 2000 was removed after 6-8 hours. Cells were analyzed 3-5 days after transfection.
2.3 Fluorescence imaging
For TIRF imaging experiments, cell-containing MatTek dishes were transferred to the CellR imaging system (Olympus Europe, Hamburg, Germany). The system was equipped with an automated filter wheel for excitation filters and with a 488 nm (20 mW) DPSS laser (Melles Griot, CA, USA) for TIRF imaging. The microscope frame and all optical elements were maintained at 34°C using the temperature control incubator (Solent Scientific, Segensworth, UK). Images were collected with a CCD camera (Orca, Hamamatsu, Japan). In TIRF mode fluorescence was excited by the thin evanescent field formed above the glass substrate due to total internal reflection of the laser beam (attenuated to 5-10%). Frames were acquired every 3 to 10 seconds.
2.4 Drug application and acidification tests
During the experiments, cells were continuously perfused using a peristaltic pump with a standard solution containing (mM): NaCl 127, KCl 3, CaCl2 2, MgCl2 1.3, HEPES 20, glucose 10; pH was adjusted to 7.4 with NaOH. During the acidification test, the standard solution was replaced with one that had pH adjusted to 5.4 with HCl. All drugs were applied by bath application via the peristaltic pump perfusion. For NMDA receptor activation ("chemical LTP"), cells were perfused for 5 minutes with the extracellular solution lacking Mg2+ and supplemented with 200 μM glycine, 0.5 μM TTX and 200 μM picrotoxin. Time course of solution exchange was estimated by imaging fluorescence changes during wash-in and wash-out of the water soluble fluorescent dye quinacrine. The T10-90 of solution exchange was measured as 22 ± 3 s (n = 3).
2.5 Data analysis
Images were quantified and processed using Olympus Biosystems AnalySIS software. Background fluorescence was subtracted prior to calculations. The n values refer to the number of data points obtained from individual cells in separate culture dishes. Data are presented as mean ± standard error. Plots and figures were constructed using Origin 6.0 software (Microcal) and PowerPoint (Microsoft). For statistical analysis, Student's t-test and ANOVA test were used. Curve fitting was performed in Origin 6.0 software using first-order exponential decay fitting and linear fitting procedures.
total internal reflection fluorescence
enhanced green fluorescent protein
Lys-Asp-Glu-Leu sequence in the amino acid structure of a protein which keeps it from secreting from the ER
complementary deoxyribonucleic acid
- DPSS laser:
diode-pumped solid-state laser.
The study was supported by grants from the Academy of Finland and the Center for International Mobility (CIMO). The authors are grateful to Drs. Rashid Giniatullin, Mikhail Paveliev and Dmitri Molotkov for their helpful remarks on the manuscript.
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