- Email: [email protected]
Determination of plasma membrane fluidity with a fluorescent analogue of sphingomyelin by FRAP measurement using a standard confocal microscope
Brain Research Protocols 11 (2003) 46–51 www.elsevier.com / locate / brainresprot
Protocol
Determination of plasma membrane fluidity with a fluorescent analogue of sphingomyelin by FRAP measurement using a standard confocal microscope Christophe Klein a , *, Thierry Pillot b , Jean Chambaz c , Beatrice Drouet c a
´ ´ ´ des Cordeliers, 15 Rue de l’ Ecole de Medecine , 75006 Paris, Service Commun d’ Imagerie et Cytometrie, INSERM IFR-58, Institut Biomedical France b INSERM E-0014, Universite´ de Nancy I, 54000 Nancy, France c ´ ´ de Medecine , 75006 Paris, France INSERM U-505, Universite´ Pierre et Marie Curie, IFR58, 15 Rue de l’ Ecole Accepted 8 January 2003
Abstract Membrane perturbing effects have been described in neurodegenerative process like Alzheimer’s disease and prion disorders. For example, non fibrillar amyloid-b peptides (Ab) implicated in Alzheimer’s disease may exert its toxicity via membrane perturbation [9]. Membrane organisation can be evaluated by its influence on lateral diffusion of lipids, which itself can be measured by FRAP (fluorescence recovery after photobleaching). We used this technique to study the effects of Ab on membrane fluidity (Pillot et al., manuscript in preparation). We propose here a simple adaptation of FRAP using standard confocal laser scanning microscopy (CLSM). As a test experiment, we analysed the lateral diffusion of a fluorescent analogue of sphingomyelin and were able to demonstrate its increase upon cholesterol depletion induced by methyl-b-cyclodextrin (cdx). 2003 Elsevier Science B.V. All rights reserved. Theme: Cellular and molecular biology Topic: Membrane composition and cell-surface macromolecules Keywords: Plasma membrane; Fluidity; Lateral diffusion; FRAP; CLSM; Sphingomyelin; Neuroblastoma cells
1. Type of research
3. Materials
Evaluation of membrane fluidity of adherent living cells by measurement of the lateral diffusion of NBD-sphingomyelin by FRAP with a confocal laser scanning microscope. Effect of cholesterol depletion.
3.1. Cell culture • Human neuroblastoma cell line SH-SY5Y (ATCC number: CRL-2266).
3.2. Special equipment 2. Time required For each condition, FRAP acquisition and kinetics analyses were, respectively performed in 40 min.
• Lab-Tek (borosilicate-coverglass chambers) (Nalge Nunc). • Zeiss LSM510 confocal microscope. • Personal computer, Microsoft Excel software.
3.3. Chemical and reagents *Corresponding author. Tel.: 133-1-4234-6905; fax: 133-1-44413717. E-mail address: [email protected] (C. Klein).
• HBSS (Hanks’ balanced salt solution) (Gibco BRL). • NBD-C6-sphingomyelin h6-[(N-(7-nitrobenz-2-oxa-
1385-299X / 03 / $ – see front matter 2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S1385-299X(03)00016-3
C. Klein et al. / Brain Research Protocols 11 (2003) 46–51
1,3-diazol-4-yl)amino)hexanoyl]sphingosyl cholinej (Molecular Probes). • Methyl-b-cyclodextrin (Sigma).
phospho-
4. Detailed procedure
4.1. Cell labelling and cyclodextrin treatment
area as function of time, F(0) is the fluorescence intensity of the bleached area immediately after the bleach and F(`) is the fluorescence intensity of the bleached area after fluorescence recovery. F(0), F(`) and t d are determined by fitting the experimental plot [i.e., F(t)] to the recovery kinetics equation, described as: F(t) 5 f F(`) 2 F(0) g f(t) 1 F(0)
SH-SY5Y cells, a human neuroblastoma cell line (ATCC number: CRL-2266), were plated on Lab-Tek at a density of 20 000 cells per cm 2 and used for FRAP measurement 3 days after plating. Cells were rinsed with HBSS and stained with NBD-SM at 4 mM for 10 min at room temperature. Cells were then rinsed and observed in HBSS. Before FRAP experiments, NBD-SM was prepared from a stock solution (1 mg / ml chloroform). An aliquot was evaporated, resuspended in ethanol, and mixed to HBSS at a final working concentration of 3 mg / ml (ethanol 2%). For cholesterol depletion, cells were treated with 1, 2 and 5 mg / ml cyclodextrin during 1 h at 37 8C before NBD-SM labelling.
47
(3)
In addition, the mobile fraction M, which indicates the fraction of probe available for diffusion, and the percentage of bleaching B can be defined as follows: F(`) 2 F(0) M 5 ]]]] F0 2 F(0)
F
F(0) B 5 1 2 ]] F0
G
3 100
(4) (5)
where: F0 is the fluorescence intensity of the area before bleaching.
4.3. Data acquisition 4.2. Method principle The measurement of lateral diffusion of molecule in membranes by FRAP has already been extensively described [1,3,6,10]. Briefly, a fluorescent probe, here a labelled lipid, is incorporated in cell membranes. When a small area of the labelled membrane is exposed to a flash of high power light, the fluorophore is photobleached. The fluorescent probe as well as endogenous membrane components undergo a constant diffusion motion through the bleached area, the fluorescence intensity of which then progressively increases (recovery of fluorescence). Thus, the kinetics of fluorescence recovery depends on the diffusion rate of the probe, measured as the diffusion coefficient, D. The diffusion coefficient D is calculated from Eq. (1):
t d 5 v 2 / 4D
(1)
where t d is the characteristic diffusion time and v the radius of the bleached area. t d is deduced from Eq. (2) which describes the fractional fluorescence recovery kinetics for a uniform circular bleach area [6,10]:
S
D F S D S DG
2 2t d 2t d 2t d f(t) 5 exp ]] ? I0 ] 1 I1 ] t t t
(2)
where t is time, and I0 and I1 are modified Bessel functions. The fractional fluorescence f(t) is defined as: F(t) 2 F(0) f(t) 5 ]]]] F(`) 2 F(0) where F(t) is the fluorescence intensity of the bleached
All experiments were made with a Zeiss LSM510 confocal laser scanning microscope. This microscope is equipped with a 25 mW air cooled argon ion laser, which was set at its maximum power. We used the 488 nm line of this laser under the control of an acousto optical tuneable filter (AOTF). We used the AIM 2.5 version of the Zeiss software. The objective used was a Zeiss C-Apochromat, 363, Numerical Aperture (NA) 1.2, water immersion. In order to obtain a sufficient speed and a suitable signal to noise ratio, image size was set to 512364 pixels (0.14 mm / pixel) and speed to 8 (1.76 ms / pixel). Those settings permitted an image to be scanned in 196.6 ms. The zoom factor was set to 2 in order to have a sufficient sampling without bleach during acquisition. For bleaching, a region of interest (ROI) was defined as a circle of 22 pixels in diameter that was centred on position x5255, y532. At zoom 2, the ROI was 3 mm in diameter. The bleaching was performed by five successive scans of this ROI at maximum speed with the AOTF transmission set to 100%. In those conditions, the bleach duration was 230 ms and the bleach amount was typically about 50 to 80%. For data acquisition, one cell was placed by moving the stage position and focus in order to have the apical cell body membrane centred on the ROI. Recovery of fluorescence was monitored at about 0.6% of the AOTF transmission to avoid bleaching during the acquisition of the fluorescence recovery. PMT (photomultiplier tube) gain and signal amplification were set in order to have a sufficiently high signal (at least a ROI mean grey level of 100 for prebleach images). The pinhole diameter was set to 1 airy unit, which corresponds in those conditions to a 1.2 mm depth of field, to reduce the contribution of cytoplasm
48
C. Klein et al. / Brain Research Protocols 11 (2003) 46–51
Fig. 1. Repartition of NBD-SM in the plasma membrane of SH-SY5Y cell. For a short time of exposure (up to 20 min) NBD-SM is exclusively localized in the plasma membrane as shown by confocal sections: (A) basal membrane, (B) mid plane, (C) apex of the cell. Note that cytoplasm is unstained.
fluorescence. As the ROI is a circular area, the analysed zone is a 331.2 mm cylinder. Before bleach, five images were recorded to define initial fluorescence. The postbleach images were scanned 10 Hz during 10 s, 2 Hz during the next 10 s and 0.5 Hz during the last 60 s, resulting in a total acquisition of 80 points during 90 s, the time required to complete the maximum recovery of fluorescence.
4.4. Data analysis The mean grey level of the ROI was computed for each image in the ‘ROI mean’ menu of AIM software. Saved tables were analysed by custom-written program, implementing the Marquardt algorithm or a grid search algorithm for least-square fit of arbitrary function [2,8]. This program computes t d by fitting Eq. (1) to experimental data with t d, F(`) and F(0) as unknown parameters. D is then calculated from t d according to Eq. (3). Results of the fit are saved as ASCII text files. Alternatively, Excel Solver can be used to fit the data with the same variable parameters. Curves of experimental and theoretical (fitted) kinetics of fluorescence recovery were plotted with Microsoft Excel.
5. Results After 10 min of incubation, NBD-SM specifically accumulated in the plasma membrane of SH-SY5Y neuroblastoma cells, and produced a strong signal which could be easily imaged. With the conditions used, confocal microscopy enabled to discriminate accurately the apical plasma membrane from inside of the cell (Fig. 1). Once the apical membrane was put in focus and centred, bleaching was performed on a 3 mm circular area (white circle, Fig. 2a). The zone analysed is shown on XZ sections (Fig. 2b). Variations in the recorded signal were only due to variations of probe concentrations in the plasma membrane. A few images of a typical acquisition are presented in Fig. 3. The fast modulation of the laser intensity allowed to obtained bleached area with sharp edges. This resulted in an uniformly circular bleach profile, suitable to analyse the data according to the theoretical model. Non homogeneous stained small membrane areas were occasionally observed. Nevertheless the small size of the bleached area allows to choose the ROI outside of these regions which are stable enough not to disturb the acquisition of data. Once the mean intensity of the ROI was recorded for all the images, data were fitted, and D was computed.
Fig. 2. Position of the bleach area on the cell body membrane. (A) Bleaching is performed on a 3 mm diameter ROI at the apex of the cell. (B) Due to the image depth of field, the analyzed volume is a flat cylinder (3 mm31.2 mm) centered on the plasma membrane as shown on the XZ section.
C. Klein et al. / Brain Research Protocols 11 (2003) 46–51
49
Fig. 3. Typical images of a FRAP experiment. Cell before and just after bleaching (0 s), and 2.5, 5 and 25 s after bleaching. Notice the centripetal fluorescence recovery in the bleached area.
Although sometimes a little bit noisy, the data could be fitted without smoothing (Fig. 4). In order to evaluate the sensitivity of the method, we studied the effect of cholesterol depletion on NBD-SM lateral diffusion. Three different concentrations of cyclodextrin were tested. The membrane fluidity was not affected by 1 mg / ml cyclodextrin (D50.34760.110 mm 2 / s as compared to D50.37160.126 mm 2 / s for control cells). In contrast, cyclodextrin at a concentration of 2 and 5 mg / ml gave rise to D values of 0.49760.169 and 0.55460.229 mm 2 / s, respectively, which corresponded to a significant increase in the coefficient of diffusion of 34 and 49%, respectively (P,0.05) (Fig. 5).
Fig. 4. Examples of FRAP analyses curves from a control cell and cyclodextrin-treated cell. Experimental fractional fluorescence [i.e., f(t)] and corresponding fitted values were plotted against time. The diffusion rate of NBD-SM in the bleached area is faster for the cell treated with 5 mg / ml of cdx (D50.607 mm 2 / s) compared to the control cell (D50.254 mm 2 / s).
6. Discussion We have developed a simple protocol for FRAP analyses on Zeiss LSM confocal microscope, that we have applied to SH-SY5Y cells. We were able to measure the lateral diffusion of NBD-SM in the plasma membrane. The analysis of D reflects the general organisation of the plasma membrane taking into account the movements of membrane microdomains.
6.1. Trouble-shooting Probe internalisation The theoretical description of the process implies that it
Fig. 5. Effect of cyclodextrin on the lateral diffusion of NBD-SM in the SH-SY5Y plasma membrane. The mean and standard deviation of NBDSM mobility: D values (mm 2 / s) were obtained from three independent cell preps. The number of analyzed cells are indicated above the columns. (Statistical analyzes were carried out using a Student t-test, * P#0.05).
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C. Klein et al. / Brain Research Protocols 11 (2003) 46–51
occurs in a plane (i.e., the plasma membrane). Thus, if the probe is present outside of the membrane, its fluorescence may participate in the signal recorded and so corrupt the result. Although the confocal microscope rejects out-offocus light, the focal ‘plane’ is in fact 1.2 mm thick in the present conditions and cannot be neglected. As NBD-SM is often used to label the Golgi apparatus in living cells an important internalisation could be expected. At room temperature (2463 8C) accumulation of the probe in the Golgi apparatus needed 20 min. However this was not a problem since confocal microscopy enables one to focus on the plasma membrane and to discriminate from the Golgi. This is not the case for cytosolic fluorescence which accumulated after about 40 min of incubation. Thus acquisition had to be stopped once fluorescence is observed in the cytosol. Sample and data exclusion Cells displaying debris, heterogeneous labelling or movement during scanning were not used for FRAP measurements. Sets of scans with broadly scattered experimental points, with M less than 65% or more than 100% or B less than 35%, were discarded. Cyclodextrin treatment To check the relation between measured D values and membrane fluidity, we treated cells with cyclodextrin, a well-known chelator of membrane cholesterol. Cholesterol depletion may facilitate the lateral movement of sphingomyelin and other lipids in membrane [4]. As expected we were able to quantify an increase in diffusion of NBD-SM in plasma membrane. The results obtained with 5 mg / ml cyclodextrin treatment may have been an underestimate. This treatment caused some of the cells to round up and detach from the bottom of the chamber, thus rendering the measurement inaccurate. Since only the remaining flat cells were measured, cells which may have been less effectively treated by the drug, this may have introduced a bias into our sample. Variability in the measurement of D Determination of D was affected by some variability of the measured data from one experiment to another. This could be explained by the analyse of a small number of cells among an heterogeneous population. Furthermore, the area analysed vary in lipids or protein microdomains composition. Nevertheless, with a sufficient number of acquisition the data become statistically significant.
6.2. Alternative and support protocols Bleaching The composition in lipids and proteins is different between somatodendritic and axonal membranes [12]. Thus a difference of fluidity along the neuronal cell could
reflect heterogeneous functional area. However, as the bleached zone is on the order of a few micrometers, a higher spatial resolution must be reached to evaluate the fluidity of axon membranes. Nevertheless, the radius of the bleached area cannot be reduced infinitely. Under some limits, depending on the numerical aperture of the objective used, the bleach profile is no more uniform. Thus one alternative, which is in fact the most used in conventional FRAP experiments, is to perform the bleach with an immobile laser beam. In those conditions the bleach profile becomes gaussian and the resolution is in the micrometer range. However, the theoretical formulation is a little bit different [1] and even more complex in the case of CLSM [3,11].
Acquisition For a 3 mm bleach area, the lateral diffusion of lipids in cell membrane is slow enough to be analysed with a temporal resolution in the range of 100 ms. However, in the case of smaller bleach area, a higher temporal resolution is required. Temporal resolution in the range of 10 ms can be obtained by line scanning [3].
Data analysis The method used here corresponds to the integral spot method described by Kubitcheck et al. [6]. These authors also described two other methods: the distribution and the variance methods. They are more complex, yet more qualitatively informative, but did not improve the accuracy of D measurement. Thus they were not tested in the present work.
Frap measurement of molecular motion in a threedimensional environment This work focused on measurement of D for plasma membrane molecules. However, it is of general interest to measure macromolecules motion in the cell i.e., in a three-dimensional (3D) medium (for example, see Refs. [5,7]). In this case, the most used protocol is to bleach a strip covering a large part of the cell. Therefore, fluorescence recovery is mainly observed along the smallest axis of the bleached parallelogram and a one dimensional diffusion model can be applied [7]. However, a better spatial resolution can be obtained with the method of spot bleaching as well as with the method used here. In this case the bleached volume is an elongated cylinder and the axial transport of the probe can be neglected compared to lateral diffusion. Thus an approximation to a two-dimensional diffusion model can be applied in a 3D environment [3]. If higher resolution has to be obtained one could use the method proposed by Wedekind et al. using an elongated elliptical bleach profile and applying a true 3D diffusion model [11].
C. Klein et al. / Brain Research Protocols 11 (2003) 46–51
7. Quick procedure
51
postdoctoral fellowship from Aventis Pharma (Vitry-surSeine, France).
7.1. Cell labelling and cyclodextrin treatment Stain cells for 10 min at room temperature with 4 mM NBD-SM in HBSS. Cyclodextrin treatment: incubate cells for 1 h at 37 8C with 1, 2 or 5 mg / ml cdx before NBD-SM staining.
7.2. Data acquisition • Set argon ion laser power at 100%. • Set image size at 512364 pixels, scan speed at 1.76 ms / pixel and zoom factor to 2. • Set laser power for bleaching at 100%. • Set laser power for imaging at about 0.6%. • Draw a circular ROI of 22 pixels in diameter centered on position x5255, y532. • Position apical membrane of the cell in focus and centred on the ROI. • Record five prebleach images. • Bleach for five successive scans. • Record 80 images for 90 s.
7.3. Data analysis • Record mean grey level of the ROI as function of time. • Measure F0 as the mean of the prebleach points values. • Fit fluorescence recovery experimental points to Eq. (3) with F(0), F(`) and t d as variable parameters. • Calculate D, M, B according to Eqs. (1), (4), (5). • Plot curves.
8. Essential literature references Refs. [1–3,6,10].
Acknowledgements We thank C. Chalumeau, C. Clair for their help in FRAP acquisition with the LSM510. B.D. is supported by a
References [1] D. Axelrod, D.E. Koppel, J. Schlessinger, E. Elson, W.W. Webb, Mobility measurement by analysis of fluorescence photobleaching recovery kinetics, Biophys. J. 16 (1976) 1055–1069. [2] P.R. Bevington, D.K. Robinson, Data Reduction and Error Analysis For the Physical Sciences, WCB / McGraw-Hill, New York, 1992. [3] J.C.G. Blonk, A. Don, H. Van Aalst, J.J. Birmingham, Fluorescence photobleaching recovery in the confocal scanning light microscope, J. Microscopy 169 (1992) 363–374. [4] S. Ilangumaran, D.C. Hoessli, Effects of cholesterol depletion by cyclodextrin on the sphingolipid microdomains of the plasma membrane, Biochem. J. 335 (1998) 433–440. [5] J. Ellenberg, J. Lippincott-Scwartz, Dynamics and mobility of nuclear envelope proteins in interphase and mitotic cells revealed by green fluorescent protein chimeras, Methods 19 (1999) 362–372. [6] U. Kubitscheck, P. Wedekind, R. Peters, Lateral diffusion measurement at high spatial resolution by scanning microphotolysis in a confocal microscope, Biophys. J. 67 (1994) 948–956. [7] R.D. Phair, T. Misteli, High mobility of proteins in the mammalian cell nucleus, Nature 404 (2000) 604–609. [8] W.H. Press, S.A. Teukolsky, W.T. Vetterling, B.P. Flannery, Numerical Recipes in C, Cambridge University Press, Cambridge, 1992. [9] T. Pillot, B. Drouet, S. Queille, C. Labeur, J. Vandekerchkhove, M. Rosseneu, M. Pincon-Raymond, J. Chambaz, The nonfibrillar amyloid beta-peptide induces apoptotic neuronal cell death: involvement of its C-terminal fusogenic domain, J. Neurochem. 73 (1999) 1626–1634. [10] D.M. Soumpasis, Theoretical analysis of fluorescence photobleaching recovery experiments, Biophys. J. 41 (1983) 95–97. [11] P. Wedekind, U. Kubitscheck, O. Heinrich, R. Peters, Line-scanning microphotolysis for diffraction-limited measurements of lateral diffusion, Biophys. J. 71 (1996) 1621–1632. [12] B. Winckler, P. Forscher, I. Mellman, A diffusion barrier maintains distribution of membrane proteins in polarized neurons, Nature 397 (1999) 698–701.
Protocol
Determination of plasma membrane fluidity with a fluorescent analogue of sphingomyelin by FRAP measurement using a standard confocal microscope Christophe Klein a , *, Thierry Pillot b , Jean Chambaz c , Beatrice Drouet c a
´ ´ ´ des Cordeliers, 15 Rue de l’ Ecole de Medecine , 75006 Paris, Service Commun d’ Imagerie et Cytometrie, INSERM IFR-58, Institut Biomedical France b INSERM E-0014, Universite´ de Nancy I, 54000 Nancy, France c ´ ´ de Medecine , 75006 Paris, France INSERM U-505, Universite´ Pierre et Marie Curie, IFR58, 15 Rue de l’ Ecole Accepted 8 January 2003
Abstract Membrane perturbing effects have been described in neurodegenerative process like Alzheimer’s disease and prion disorders. For example, non fibrillar amyloid-b peptides (Ab) implicated in Alzheimer’s disease may exert its toxicity via membrane perturbation [9]. Membrane organisation can be evaluated by its influence on lateral diffusion of lipids, which itself can be measured by FRAP (fluorescence recovery after photobleaching). We used this technique to study the effects of Ab on membrane fluidity (Pillot et al., manuscript in preparation). We propose here a simple adaptation of FRAP using standard confocal laser scanning microscopy (CLSM). As a test experiment, we analysed the lateral diffusion of a fluorescent analogue of sphingomyelin and were able to demonstrate its increase upon cholesterol depletion induced by methyl-b-cyclodextrin (cdx). 2003 Elsevier Science B.V. All rights reserved. Theme: Cellular and molecular biology Topic: Membrane composition and cell-surface macromolecules Keywords: Plasma membrane; Fluidity; Lateral diffusion; FRAP; CLSM; Sphingomyelin; Neuroblastoma cells
1. Type of research
3. Materials
Evaluation of membrane fluidity of adherent living cells by measurement of the lateral diffusion of NBD-sphingomyelin by FRAP with a confocal laser scanning microscope. Effect of cholesterol depletion.
3.1. Cell culture • Human neuroblastoma cell line SH-SY5Y (ATCC number: CRL-2266).
3.2. Special equipment 2. Time required For each condition, FRAP acquisition and kinetics analyses were, respectively performed in 40 min.
• Lab-Tek (borosilicate-coverglass chambers) (Nalge Nunc). • Zeiss LSM510 confocal microscope. • Personal computer, Microsoft Excel software.
3.3. Chemical and reagents *Corresponding author. Tel.: 133-1-4234-6905; fax: 133-1-44413717. E-mail address: [email protected] (C. Klein).
• HBSS (Hanks’ balanced salt solution) (Gibco BRL). • NBD-C6-sphingomyelin h6-[(N-(7-nitrobenz-2-oxa-
1385-299X / 03 / $ – see front matter 2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S1385-299X(03)00016-3
C. Klein et al. / Brain Research Protocols 11 (2003) 46–51
1,3-diazol-4-yl)amino)hexanoyl]sphingosyl cholinej (Molecular Probes). • Methyl-b-cyclodextrin (Sigma).
phospho-
4. Detailed procedure
4.1. Cell labelling and cyclodextrin treatment
area as function of time, F(0) is the fluorescence intensity of the bleached area immediately after the bleach and F(`) is the fluorescence intensity of the bleached area after fluorescence recovery. F(0), F(`) and t d are determined by fitting the experimental plot [i.e., F(t)] to the recovery kinetics equation, described as: F(t) 5 f F(`) 2 F(0) g f(t) 1 F(0)
SH-SY5Y cells, a human neuroblastoma cell line (ATCC number: CRL-2266), were plated on Lab-Tek at a density of 20 000 cells per cm 2 and used for FRAP measurement 3 days after plating. Cells were rinsed with HBSS and stained with NBD-SM at 4 mM for 10 min at room temperature. Cells were then rinsed and observed in HBSS. Before FRAP experiments, NBD-SM was prepared from a stock solution (1 mg / ml chloroform). An aliquot was evaporated, resuspended in ethanol, and mixed to HBSS at a final working concentration of 3 mg / ml (ethanol 2%). For cholesterol depletion, cells were treated with 1, 2 and 5 mg / ml cyclodextrin during 1 h at 37 8C before NBD-SM labelling.
47
(3)
In addition, the mobile fraction M, which indicates the fraction of probe available for diffusion, and the percentage of bleaching B can be defined as follows: F(`) 2 F(0) M 5 ]]]] F0 2 F(0)
F
F(0) B 5 1 2 ]] F0
G
3 100
(4) (5)
where: F0 is the fluorescence intensity of the area before bleaching.
4.3. Data acquisition 4.2. Method principle The measurement of lateral diffusion of molecule in membranes by FRAP has already been extensively described [1,3,6,10]. Briefly, a fluorescent probe, here a labelled lipid, is incorporated in cell membranes. When a small area of the labelled membrane is exposed to a flash of high power light, the fluorophore is photobleached. The fluorescent probe as well as endogenous membrane components undergo a constant diffusion motion through the bleached area, the fluorescence intensity of which then progressively increases (recovery of fluorescence). Thus, the kinetics of fluorescence recovery depends on the diffusion rate of the probe, measured as the diffusion coefficient, D. The diffusion coefficient D is calculated from Eq. (1):
t d 5 v 2 / 4D
(1)
where t d is the characteristic diffusion time and v the radius of the bleached area. t d is deduced from Eq. (2) which describes the fractional fluorescence recovery kinetics for a uniform circular bleach area [6,10]:
S
D F S D S DG
2 2t d 2t d 2t d f(t) 5 exp ]] ? I0 ] 1 I1 ] t t t
(2)
where t is time, and I0 and I1 are modified Bessel functions. The fractional fluorescence f(t) is defined as: F(t) 2 F(0) f(t) 5 ]]]] F(`) 2 F(0) where F(t) is the fluorescence intensity of the bleached
All experiments were made with a Zeiss LSM510 confocal laser scanning microscope. This microscope is equipped with a 25 mW air cooled argon ion laser, which was set at its maximum power. We used the 488 nm line of this laser under the control of an acousto optical tuneable filter (AOTF). We used the AIM 2.5 version of the Zeiss software. The objective used was a Zeiss C-Apochromat, 363, Numerical Aperture (NA) 1.2, water immersion. In order to obtain a sufficient speed and a suitable signal to noise ratio, image size was set to 512364 pixels (0.14 mm / pixel) and speed to 8 (1.76 ms / pixel). Those settings permitted an image to be scanned in 196.6 ms. The zoom factor was set to 2 in order to have a sufficient sampling without bleach during acquisition. For bleaching, a region of interest (ROI) was defined as a circle of 22 pixels in diameter that was centred on position x5255, y532. At zoom 2, the ROI was 3 mm in diameter. The bleaching was performed by five successive scans of this ROI at maximum speed with the AOTF transmission set to 100%. In those conditions, the bleach duration was 230 ms and the bleach amount was typically about 50 to 80%. For data acquisition, one cell was placed by moving the stage position and focus in order to have the apical cell body membrane centred on the ROI. Recovery of fluorescence was monitored at about 0.6% of the AOTF transmission to avoid bleaching during the acquisition of the fluorescence recovery. PMT (photomultiplier tube) gain and signal amplification were set in order to have a sufficiently high signal (at least a ROI mean grey level of 100 for prebleach images). The pinhole diameter was set to 1 airy unit, which corresponds in those conditions to a 1.2 mm depth of field, to reduce the contribution of cytoplasm
48
C. Klein et al. / Brain Research Protocols 11 (2003) 46–51
Fig. 1. Repartition of NBD-SM in the plasma membrane of SH-SY5Y cell. For a short time of exposure (up to 20 min) NBD-SM is exclusively localized in the plasma membrane as shown by confocal sections: (A) basal membrane, (B) mid plane, (C) apex of the cell. Note that cytoplasm is unstained.
fluorescence. As the ROI is a circular area, the analysed zone is a 331.2 mm cylinder. Before bleach, five images were recorded to define initial fluorescence. The postbleach images were scanned 10 Hz during 10 s, 2 Hz during the next 10 s and 0.5 Hz during the last 60 s, resulting in a total acquisition of 80 points during 90 s, the time required to complete the maximum recovery of fluorescence.
4.4. Data analysis The mean grey level of the ROI was computed for each image in the ‘ROI mean’ menu of AIM software. Saved tables were analysed by custom-written program, implementing the Marquardt algorithm or a grid search algorithm for least-square fit of arbitrary function [2,8]. This program computes t d by fitting Eq. (1) to experimental data with t d, F(`) and F(0) as unknown parameters. D is then calculated from t d according to Eq. (3). Results of the fit are saved as ASCII text files. Alternatively, Excel Solver can be used to fit the data with the same variable parameters. Curves of experimental and theoretical (fitted) kinetics of fluorescence recovery were plotted with Microsoft Excel.
5. Results After 10 min of incubation, NBD-SM specifically accumulated in the plasma membrane of SH-SY5Y neuroblastoma cells, and produced a strong signal which could be easily imaged. With the conditions used, confocal microscopy enabled to discriminate accurately the apical plasma membrane from inside of the cell (Fig. 1). Once the apical membrane was put in focus and centred, bleaching was performed on a 3 mm circular area (white circle, Fig. 2a). The zone analysed is shown on XZ sections (Fig. 2b). Variations in the recorded signal were only due to variations of probe concentrations in the plasma membrane. A few images of a typical acquisition are presented in Fig. 3. The fast modulation of the laser intensity allowed to obtained bleached area with sharp edges. This resulted in an uniformly circular bleach profile, suitable to analyse the data according to the theoretical model. Non homogeneous stained small membrane areas were occasionally observed. Nevertheless the small size of the bleached area allows to choose the ROI outside of these regions which are stable enough not to disturb the acquisition of data. Once the mean intensity of the ROI was recorded for all the images, data were fitted, and D was computed.
Fig. 2. Position of the bleach area on the cell body membrane. (A) Bleaching is performed on a 3 mm diameter ROI at the apex of the cell. (B) Due to the image depth of field, the analyzed volume is a flat cylinder (3 mm31.2 mm) centered on the plasma membrane as shown on the XZ section.
C. Klein et al. / Brain Research Protocols 11 (2003) 46–51
49
Fig. 3. Typical images of a FRAP experiment. Cell before and just after bleaching (0 s), and 2.5, 5 and 25 s after bleaching. Notice the centripetal fluorescence recovery in the bleached area.
Although sometimes a little bit noisy, the data could be fitted without smoothing (Fig. 4). In order to evaluate the sensitivity of the method, we studied the effect of cholesterol depletion on NBD-SM lateral diffusion. Three different concentrations of cyclodextrin were tested. The membrane fluidity was not affected by 1 mg / ml cyclodextrin (D50.34760.110 mm 2 / s as compared to D50.37160.126 mm 2 / s for control cells). In contrast, cyclodextrin at a concentration of 2 and 5 mg / ml gave rise to D values of 0.49760.169 and 0.55460.229 mm 2 / s, respectively, which corresponded to a significant increase in the coefficient of diffusion of 34 and 49%, respectively (P,0.05) (Fig. 5).
Fig. 4. Examples of FRAP analyses curves from a control cell and cyclodextrin-treated cell. Experimental fractional fluorescence [i.e., f(t)] and corresponding fitted values were plotted against time. The diffusion rate of NBD-SM in the bleached area is faster for the cell treated with 5 mg / ml of cdx (D50.607 mm 2 / s) compared to the control cell (D50.254 mm 2 / s).
6. Discussion We have developed a simple protocol for FRAP analyses on Zeiss LSM confocal microscope, that we have applied to SH-SY5Y cells. We were able to measure the lateral diffusion of NBD-SM in the plasma membrane. The analysis of D reflects the general organisation of the plasma membrane taking into account the movements of membrane microdomains.
6.1. Trouble-shooting Probe internalisation The theoretical description of the process implies that it
Fig. 5. Effect of cyclodextrin on the lateral diffusion of NBD-SM in the SH-SY5Y plasma membrane. The mean and standard deviation of NBDSM mobility: D values (mm 2 / s) were obtained from three independent cell preps. The number of analyzed cells are indicated above the columns. (Statistical analyzes were carried out using a Student t-test, * P#0.05).
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occurs in a plane (i.e., the plasma membrane). Thus, if the probe is present outside of the membrane, its fluorescence may participate in the signal recorded and so corrupt the result. Although the confocal microscope rejects out-offocus light, the focal ‘plane’ is in fact 1.2 mm thick in the present conditions and cannot be neglected. As NBD-SM is often used to label the Golgi apparatus in living cells an important internalisation could be expected. At room temperature (2463 8C) accumulation of the probe in the Golgi apparatus needed 20 min. However this was not a problem since confocal microscopy enables one to focus on the plasma membrane and to discriminate from the Golgi. This is not the case for cytosolic fluorescence which accumulated after about 40 min of incubation. Thus acquisition had to be stopped once fluorescence is observed in the cytosol. Sample and data exclusion Cells displaying debris, heterogeneous labelling or movement during scanning were not used for FRAP measurements. Sets of scans with broadly scattered experimental points, with M less than 65% or more than 100% or B less than 35%, were discarded. Cyclodextrin treatment To check the relation between measured D values and membrane fluidity, we treated cells with cyclodextrin, a well-known chelator of membrane cholesterol. Cholesterol depletion may facilitate the lateral movement of sphingomyelin and other lipids in membrane [4]. As expected we were able to quantify an increase in diffusion of NBD-SM in plasma membrane. The results obtained with 5 mg / ml cyclodextrin treatment may have been an underestimate. This treatment caused some of the cells to round up and detach from the bottom of the chamber, thus rendering the measurement inaccurate. Since only the remaining flat cells were measured, cells which may have been less effectively treated by the drug, this may have introduced a bias into our sample. Variability in the measurement of D Determination of D was affected by some variability of the measured data from one experiment to another. This could be explained by the analyse of a small number of cells among an heterogeneous population. Furthermore, the area analysed vary in lipids or protein microdomains composition. Nevertheless, with a sufficient number of acquisition the data become statistically significant.
6.2. Alternative and support protocols Bleaching The composition in lipids and proteins is different between somatodendritic and axonal membranes [12]. Thus a difference of fluidity along the neuronal cell could
reflect heterogeneous functional area. However, as the bleached zone is on the order of a few micrometers, a higher spatial resolution must be reached to evaluate the fluidity of axon membranes. Nevertheless, the radius of the bleached area cannot be reduced infinitely. Under some limits, depending on the numerical aperture of the objective used, the bleach profile is no more uniform. Thus one alternative, which is in fact the most used in conventional FRAP experiments, is to perform the bleach with an immobile laser beam. In those conditions the bleach profile becomes gaussian and the resolution is in the micrometer range. However, the theoretical formulation is a little bit different [1] and even more complex in the case of CLSM [3,11].
Acquisition For a 3 mm bleach area, the lateral diffusion of lipids in cell membrane is slow enough to be analysed with a temporal resolution in the range of 100 ms. However, in the case of smaller bleach area, a higher temporal resolution is required. Temporal resolution in the range of 10 ms can be obtained by line scanning [3].
Data analysis The method used here corresponds to the integral spot method described by Kubitcheck et al. [6]. These authors also described two other methods: the distribution and the variance methods. They are more complex, yet more qualitatively informative, but did not improve the accuracy of D measurement. Thus they were not tested in the present work.
Frap measurement of molecular motion in a threedimensional environment This work focused on measurement of D for plasma membrane molecules. However, it is of general interest to measure macromolecules motion in the cell i.e., in a three-dimensional (3D) medium (for example, see Refs. [5,7]). In this case, the most used protocol is to bleach a strip covering a large part of the cell. Therefore, fluorescence recovery is mainly observed along the smallest axis of the bleached parallelogram and a one dimensional diffusion model can be applied [7]. However, a better spatial resolution can be obtained with the method of spot bleaching as well as with the method used here. In this case the bleached volume is an elongated cylinder and the axial transport of the probe can be neglected compared to lateral diffusion. Thus an approximation to a two-dimensional diffusion model can be applied in a 3D environment [3]. If higher resolution has to be obtained one could use the method proposed by Wedekind et al. using an elongated elliptical bleach profile and applying a true 3D diffusion model [11].
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7. Quick procedure
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postdoctoral fellowship from Aventis Pharma (Vitry-surSeine, France).
7.1. Cell labelling and cyclodextrin treatment Stain cells for 10 min at room temperature with 4 mM NBD-SM in HBSS. Cyclodextrin treatment: incubate cells for 1 h at 37 8C with 1, 2 or 5 mg / ml cdx before NBD-SM staining.
7.2. Data acquisition • Set argon ion laser power at 100%. • Set image size at 512364 pixels, scan speed at 1.76 ms / pixel and zoom factor to 2. • Set laser power for bleaching at 100%. • Set laser power for imaging at about 0.6%. • Draw a circular ROI of 22 pixels in diameter centered on position x5255, y532. • Position apical membrane of the cell in focus and centred on the ROI. • Record five prebleach images. • Bleach for five successive scans. • Record 80 images for 90 s.
7.3. Data analysis • Record mean grey level of the ROI as function of time. • Measure F0 as the mean of the prebleach points values. • Fit fluorescence recovery experimental points to Eq. (3) with F(0), F(`) and t d as variable parameters. • Calculate D, M, B according to Eqs. (1), (4), (5). • Plot curves.
8. Essential literature references Refs. [1–3,6,10].
Acknowledgements We thank C. Chalumeau, C. Clair for their help in FRAP acquisition with the LSM510. B.D. is supported by a
References [1] D. Axelrod, D.E. Koppel, J. Schlessinger, E. Elson, W.W. Webb, Mobility measurement by analysis of fluorescence photobleaching recovery kinetics, Biophys. J. 16 (1976) 1055–1069. [2] P.R. Bevington, D.K. Robinson, Data Reduction and Error Analysis For the Physical Sciences, WCB / McGraw-Hill, New York, 1992. [3] J.C.G. Blonk, A. Don, H. Van Aalst, J.J. Birmingham, Fluorescence photobleaching recovery in the confocal scanning light microscope, J. Microscopy 169 (1992) 363–374. [4] S. Ilangumaran, D.C. Hoessli, Effects of cholesterol depletion by cyclodextrin on the sphingolipid microdomains of the plasma membrane, Biochem. J. 335 (1998) 433–440. [5] J. Ellenberg, J. Lippincott-Scwartz, Dynamics and mobility of nuclear envelope proteins in interphase and mitotic cells revealed by green fluorescent protein chimeras, Methods 19 (1999) 362–372. [6] U. Kubitscheck, P. Wedekind, R. Peters, Lateral diffusion measurement at high spatial resolution by scanning microphotolysis in a confocal microscope, Biophys. J. 67 (1994) 948–956. [7] R.D. Phair, T. Misteli, High mobility of proteins in the mammalian cell nucleus, Nature 404 (2000) 604–609. [8] W.H. Press, S.A. Teukolsky, W.T. Vetterling, B.P. Flannery, Numerical Recipes in C, Cambridge University Press, Cambridge, 1992. [9] T. Pillot, B. Drouet, S. Queille, C. Labeur, J. Vandekerchkhove, M. Rosseneu, M. Pincon-Raymond, J. Chambaz, The nonfibrillar amyloid beta-peptide induces apoptotic neuronal cell death: involvement of its C-terminal fusogenic domain, J. Neurochem. 73 (1999) 1626–1634. [10] D.M. Soumpasis, Theoretical analysis of fluorescence photobleaching recovery experiments, Biophys. J. 41 (1983) 95–97. [11] P. Wedekind, U. Kubitscheck, O. Heinrich, R. Peters, Line-scanning microphotolysis for diffraction-limited measurements of lateral diffusion, Biophys. J. 71 (1996) 1621–1632. [12] B. Winckler, P. Forscher, I. Mellman, A diffusion barrier maintains distribution of membrane proteins in polarized neurons, Nature 397 (1999) 698–701.