OSTR Measurement of Nucleocytoplasmic Transport Using Isolated Intact Nuclei Perforated Nuclei or Nuclear Envelopes of Xenopus Oocytes

For OSTR experiments, various protocols are available (see Note 7). Here, a particularly convenient, fast, and robust procedure for Xenopus oocytes (13) employing comparatively large TCs is described. Alternative protocols and a discussion of singletransporter analysis may be found in refs. 11,12, and 14.

3.2.1. Isolation and Purification of Xenopus Oocyte Nuclei

Isolate a nucleus as described in Subheading 3.1.1., but take special care to remove yolk particles from the nucleus.

3.2.2. Preparation of OSTR Chambers

1. Drill a 3.5-mm diameter hole into the bottom of a tissue culture dish and clean the dish of debris.

2. Cut a preformed TC array (Subheading 2., item 15) into pieces approx 5 x 5 mm using a scalpel or sharp scissors. Apply Eastman Instant Adhesive no. 910 around the edges of the hole in the culture dish with a fine brush. Use forceps to place the array on the hole, with TCs facing the chamber volume.

3. Alternatively, TC arrays may be created from track-etched membrane filters (Subheading 2., item 18). Apply a piece the optically clear, double-sticky tape (adhesive 8141, Subheading 2., item 19) to a clean cover slip (15 x 15 mm). Cut a track-etched membrane filter into small pieces (5 x 5 mm) and place a filter piece, shiny side up (see Note 8), on the tape. Attach the cover slip and track-etched filter assembly to the culture dish such that the membrane filter forms the bottom of the hole.

3.2.3. Attachment of Nuclei to TC Arrays and Preparation of Nuclear Envelopes

1. Fill an OSTR chamber with 10 ||L mock 3. Place the OSTR chamber on the water surface in an ultrasonic bath and sonicate for a few seconds. This fills the TCs with mock 3, which does not occur spontaneously. Remove the OSTR chamber from the ultrasonic bath and wipe off water droplets from the bottom.

2. Under the stereomicroscope, using the transfer pipet, deposit an isolated nucleus in the OSTR chamber as indicated in Fig. 3. Employ the blunt-end glass capillary (Subheading 2., item 9) to press the nucleus against the TC array. The nucleus will adopt a domelike shape. Its lower part will attach firmly to the TC array. For parameters that may affect attachment, see Notes 9 and 10.

3. If desired, repeatedly perforate the TC-attached nucleus using the sharp steel pin (Subheading 2., item 21).

4. Alternatively, employ the steel pin to completely open the nucleus, to fold back the freefloating parts of nuclear envelope, and to remove the nuclear contents. Employ a GELoader tip to rinse the nuclear envelope three times with 15 |L mock 3.

5. Prevent drying out of the OSTR chamber (which has a small volume) by including in the dish containing the OSTR chamber a wet "collar" of filter paper and putting on a lid during measurements.

Fig. 3. Preparation of Xenopus oocyte nuclei and nuclear envelopes for nucleocytoplasmic transport measurements by optical single transporter recording (OSTR). An isolated X. oocyte nucleus is deposited in an OSTR chamber. The bottom of the OSTR chamber consists of a thin transparent foil containing an array of small cavities (test compartments, TCs). The nucleus is firmly attached to the TC array and may be perforated or completely dissected. A fluorescent transport substrate is added to the OSTR chamber and the appearance of transport substrate in the cavities is monitored by confocal fluorescence microscopy. From ref. 14.

Fig. 3. Preparation of Xenopus oocyte nuclei and nuclear envelopes for nucleocytoplasmic transport measurements by optical single transporter recording (OSTR). An isolated X. oocyte nucleus is deposited in an OSTR chamber. The bottom of the OSTR chamber consists of a thin transparent foil containing an array of small cavities (test compartments, TCs). The nucleus is firmly attached to the TC array and may be perforated or completely dissected. A fluorescent transport substrate is added to the OSTR chamber and the appearance of transport substrate in the cavities is monitored by confocal fluorescence microscopy. From ref. 14.

3.2.4. Recording of Export and Import Kinetics

1. Place the OSTR chamber on the stage of the confocal microscope. Visualize the nucleus or nuclear envelope in through-light at low magnification (10-fold objective lens) and position it in the center of the field of vision.

2. Depending on the TC diameter, continue with the 10-fold objective or change to a higher magnification. A 10-fold objective (air) or a 16-fold objective (water immersion, numerical aperture 0.5) is appropriate for TCs with 10- to 100-|lm diameters. A 40-fold objective (oil immersion, numerical aperture 1.0) is required for TCs with 0.1- to 1.0-|lm diameters.

3. After choosing the appropriate objective lens, position the nucleus or nuclear envelope such that the edge of the nucleus/nuclear envelope runs through the field of vision (Fig. 4A). This yields two populations of TCs: measuring TCs, which are covered by the nuclear envelope, and reference TCs, which are not covered.

4. Focus on the bottom of the TCs (Fig. 4B). Adjust the confocal system regarding filter settings, laser power, high voltage, scan speed, and zoom factor (see Note 11). It is advantageous to do these adjustments beforehand using an OSTR chamber filled with transport solution but void of a nucleus/nuclear envelope. If the microscope system is able to execute time-lapse programs, design a program to suit the transport kinetics.

5. Start the time-lapse program or activate the scanner manually. Use a GELoader tip to inject 20 ||L transport solution 2 into the OSTR chamber between two scans (see Note 6). The transport solution, containing 100 mM sucrose, will displace mock 3, which spills over the edge of the OSTR chamber. Record transport kinetics until substrate has equilibrated between OSTR chamber and TCs. The transport solution used in OSTR measurements contains a transport substrate (e.g., NTF2) and a control substrate (e.g., TRD70). The control substrate permits following the kinetics of solution exchange and checking for a tight seal of the nuclear envelope to the TC array and the integrity of the nuclear envelope. Thus (Fig. 4B), after injection of transport solution, the measuring TCs should be filled by the transport substrate in a time-dependent manner and remain void of control substrate. In contrast, control TCs should be filled immediately with both transport substrate and control substrate.

6. After completion of export, import may be recorded by restarting the time-lapse program and injecting approx 50 |L mock 3 supplemented with 100 mM sucrose into the OSTR chamber.

7. For a second round of export and import measurements, remove the specimen from the confocal microscope. Under the stereomicroscope, wash the nucleus or nuclear envelope with a surplus of mock 3 (e.g., 3 x 50 ||L). Then, place the specimen back on the stage of the confocal microscope and start an export measurement.

3.3. Data Evaluation

1. Employ an image analysis program (see Note 12) to derive the integral time-dependent fluorescence of single intact nuclei (if performing measurements according to Subheading 3.1.) or of all the TCs in a scan series (if performing measurements according to Subheading 3.2.). Plot the fluorescence of isolated nuclei or individual TCs vs time, as illustrated in Fig. 4C,D.

2. Fit the experimental OSTR data by Eq. 6 to obtain the rate constant k, which can be used to derive P according to Eq. 8 (see Note 13).

3. With large TCs (or nuclei), the assumptions made for deriving Eq. 6 may not hold. In that case, a deconvolution method (13) can be applied to separate membrane transport from intra-TC (intranuclear) diffusion and substrate mixing and to recover, within certain limits, true k and P values.

4. Notes

1. Xenopus ovary can be used for about a week if kept at 10°C and medium is changed daily.

2. Use a freshly isolated nucleus for each experiment. Isolated nuclei will rapidly swell in mock 3. This is desirable to a certain extent because the nuclear surface is made smoother, and protusions (see Note 10) are reduced. Osmotic swelling of nuclei can be prevented by addition of approx 20% (w/v) BSA to mock 3.

Fig. 4. Measurement of nucleocytoplasmic transport by optical single transporter recording (OSTR). (A) In an OSTR chamber, a nuclear envelope was attached to an array of test compartments (TCs). The nuclear envelope covers only one of the four TCs contained in the field of view. (B) Transport solution 2, containing the nuclear transport receptor NTF2 and the control

Fig. 4. Measurement of nucleocytoplasmic transport by optical single transporter recording (OSTR). (A) In an OSTR chamber, a nuclear envelope was attached to an array of test compartments (TCs). The nuclear envelope covers only one of the four TCs contained in the field of view. (B) Transport solution 2, containing the nuclear transport receptor NTF2 and the control

3. Ease by which Xenopus oocyte nuclei can be isolated and purified varies largely from batch to batch.

4. Isolated purified nuclei are very sticky. To avoid sticking of nuclei to glass surfaces, apply a BSA solution (1 mg/mL) to the surface for a few minutes, then wash with mock 3. Also, do not wash glass dishes used for nuclear isolation extensively but rinse with distilled water only. Furthermore, use always the same "dirty" tip on the transfer pipet and never clean the blunt-end glass pipet used for cleaning isolated nuclei from yolk particles and attaching isolated nuclei to TC arrays.

5. Isolated nuclei will be destroyed when coming into contact with the air-water interface. Make sure that pipet tips do not contain air bubbles when aspirating a nucleus. Keep mock 3 at room temperature during experiments to avoid the formation of air bubbles in dishes and chambers.

6. Injection of transport solution during scanning is facilitated by attaching a magnifying glass to the microscope stage.

7. In OSTR experiments, transport across TC-spanning membrane patches is usually induced by changing the concentration of transport substrates in the OSTR chamber and monitored by recording the fluorescence of transport substrates in the TCs. Within this frame, a number of specific protocols exist:

a. In OSTR experiments with isolated intact nuclei, the addition of transport substrate to the microchamber will induce the import of the substrate from the chamber into the nucleus and, after a lag time, the export from the nucleus into TCs.

b. If in such experiments the substrate has equilibrated among the chamber, nucleus, and TCs, removal of substrate from the OSTR chamber will induce the import from the TCs into the nucleus and simultaneously export from the nucleus into the OSTR chamber.

c. In OSTR experiments with isolated nuclear envelopes, the addition of substrate to the chamber will induce export. After equilibration of the substrate, its removal from the chamber will induce import.

d. In case of two transport substrates with different emission wavelengths (e.g., green and red), addition to and removal from the OSTR chamber can be arranged such that either both substrates are exported or imported or one is exported and the other imported.

e. If a transport substrate has been equilibrated between OSTR chamber and TCs, transport can be induced by photobleaching of individual TCs (14). Neighboring TCs will remain essentially unaffected. Similarly, photoactivation of a substrate by an ultraviolet flash can be used to induce import from single TCs.

f. The transport of nonfluorescent substrates can be monitored by substrate-specific fluorescent indicators. Such indicators are available not only for many inorganic ions but also for certain proteins (e.g., ref. 15). For a transport measurement, the OSTR chamber is filled with a solution containing an indicator. Then, a nucleus/nuclear envelope

(caption continued from previous page) substrate TRD70, was added to the OSTR chamber, and the TCs were observed by confocal microscopy. NTR2 rapidly entered the nuclear envelope-covered TC; TRD70 was excluded. Open TCs were immediately filled with both NTF2 and TDR70. (C, D) From a series of confocal scans, the import and export kinetics were derived for NTF2 and GFP. The transport kinetics of the two compounds differ drastically, although their molecular dimensions are virtually identical. From ref. 13.

is attached to the TC array, and the residual indicator is washed out of the OSTR chamber. Transport substrate is added to the OSTR chamber, and fluorescence of the indicator in the TCs is recorded.

g. Usually, substrate fluorescence is monitored by confocal x-y sections. The confocal plane is adjusted to pass through TCs close to their bottom and parallel to the plane of the TC array. This has the advantage that many TCs can be recorded in parallel. However, it is also possible to employ vertical (x-z) sectioning. In that case, a single TC or a line of TCs can be imaged along the TC axis together with nuclear envelope and chamber volume. Thus, the fluorescence of TCs, nuclear envelope, and chamber contents can be monitored simultaneously. In principle, stacks of x-y scans can also be acquired and used to reconstruct substrate flux in three dimensions and time h. Depending on the area density of transporters and the TC diameter, TC-spanning membrane patches will contain one, few, or many transporters. By employing TC arrays with different TC diameters, it is thus possible to measure on the same membrane the flux through single transporters or transporter populations. In the Xenopus oocyte nuclear envelope, the area density of NPCs is approx 50/|lm2, and the single NPC occupies approx 0.02 |m2, corresponding to the cross section of a cylindrical TC with a 0.160-|m diameter. Therefore, for the single-transporter analysis of the Xenopus oocyte NPC, TCs with approx 0.2-|m diameter are appropriate.

8. When employing track-etched membrane filters to create TC arrays, it is important to chose filters designated as transparent. In this filter type, the pore density is small, and all pores are oriented perpendicular to the filter plane. It is also essential to note that filters have a shiny, smooth side and a less-shiny, rough side. When creating OSTR chambers, the smooth side of the filter should face the chamber volume.

9. Parameters influencing the tightness of seal between nuclear envelope and TC arrays are (1) clean surfaces of both nuclei and TC arrays and (2) Ca2+ concentration. Prepare a fresh charge of mock 3 every day. Take care that the Ca2+ concentration of all solutions is 3 ||M. Dialyze BSA stock solution (100 g/L) against mock 3 overnight.

10. The oocyte nuclear envelope carries numerous large protusions (cf, Fig. 4A). The protrusions collapse in an onionlike manner when the nuclear envelope is attached to a TC array. For transport measurements use, only TCs void of protusions.

11. Recording of transport kinetics by confocal microscopy may induce fluorescence photobleaching. Reduce laser power and number of scans as much as possible. If necessary, increase transport substrate concentration or the labeling ratio. Check for absence of photobleaching by making confocal scans on TC arrays that are filled with transport solution and sealed with immersion oil as described in ref. 16.

12. We have developed (13) a plug-in for ImageJ (public domain Java version by W. Rasband; available at http://rsb.info.nih.gov/ij/) that permits automatic localization of TCs in con-focal scans to derive their average fluorescence intensities and to correct the readings for local background. In the case of export experiments (i.e., net transport from chamber into TCs), the initial TC fluorescence, measured before addition of the transport solution to the chamber, is subtracted from the data set; for import, all measurements are subtracted from the initial bright level, thus creating standardized curves rising with time from near-zero initial values, as illustrated in Fig. 4C,D.

13. To evaluate the experimental data in terms of transport coefficients, both the isolated-nucleus system and the OSTR system are considered two-compartment systems in which a "large" compartment (microchamber, OSTR chamber) and one or many "small" compartments (isolated nucleus, TCs) are separated by a thin membrane (the nuclear enve lope) containing discrete transporters (the NPCs). It is furthermore assumed, as a first approximation, that the transport substrate is instantaneously added to or removed from the chamber at zero time, that the chamber is well stirred so that the transport substrate concentration in the chamber CCh is constant, and that the TC (or nucleus) is so small that the transport substrate is rapidly dissipated by diffusion and the intranuclear TC concentration CTC is a function of time only. For a purely passive, diffusional membrane transport process in which the substrate is not absorbed or produced anywhere in the system and its equilibration across the membrane is very fast (thin membrane approximation), the flux of substrate per unit membrane area (mol/|m2/s) is quasi stationary at any time and can be expressed as

where P is the permeability coefficient (| m/s). The total flux through a TC-spanning membrane patch is the sum of the unitary fluxes through individual NPCs, each having the average single-transporter permeability PNPC (| m3/s). For a membrane of surface area S containing a large number n of NPCs at an average density o (NPC/|m2)

For a constant concentration difference AC across the membrane (e.g., 1 |M), the flux and permeability described by Eq. 1 can be linked to the single-transporter permeability and expressed using Avogadro's number NA as a unitary flux (molecules/s/NPC)

OSTR chamber and TC shall have volumes VCh and VTC, respectively; the membrane patches covering the TCs shall have surface area S. The mean time of substrate diffusion inside the TC is proportional to (TC dimension)2/D where D is the diffusion coefficient of the substrate. If the TC is a few micrometers only in size, the intra-TC diffusion time is very small, the TC can be considered well stirred, and the concentration CTC will be a function of time only. Then, the kinetics of substrate accumulation in the TC caused by the flux through the membrane patch is given by dCTC(t) + PS Vch + Vtc CTc(f) = PSM(t) (4)

dt VchVTc VchVTc where M(t) = VChCCh(t) + VTCCTC(t) is the total amount of substrate. In the typical OSTR specimen, the volume of the chamber is large (VCh >> VTC), and the amount of substrate in the TC is negligible, so that Eq. 4 simplifies to dCCt) = _PS [Cch(t) - CTc(t)] = k[Cch(t) - CTc(t)] (5)

dt V TC

For a constant concentration of the substrate in the chamber CCh(t)= Cmax, the solution of Eq. 5 is

The rate constant k is given by k = PS/Vtc = nPNPc/VTc (7)

For cylindrical TCs with length L and VTC = SL, the rate constant is k = P/L = oPNPC/L (8)


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