Cell Biology Protocols

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Procedure	Fill the microchamber with 5 µl	of
	mock 3.

1.Prepare a microchamber by drilling a hole of 2.5 mm diameter into the bottom of a tissue culture dish and gluing a cover slide to the bottom of the dish.

2.Manually isolate and purify a nucleus

from a Xenopus oocyte. Remove a stage VI oocyte ( 1.2 mm diameter, Figure 6.5(a)) from a piece of Xenopus

(a)

(b)

(c)

(d)

Bottom of

dish

Coverslip

Microchamber

1.0 mm

Nucleus

2.5 mm

Figure 6.5 Specimen for assaying nucleocytoplasmic transport in isolated Xenopus oocyte nuclei. The nucleus of a stage VI Xenopus oocyte (a) was isolated manually (b) and, after manual purification with a fine glass needle, deposited in a microchamber (c). The microchamber (d) has a volume of 5 µl. (From ref. 4)

242 IN VITRO TECHNIQUES

ovary, kept in amphibian Ringer’s solution, and transfer it into a small glass dish containing mock 3. Using a stereomicroscope at 16× total magnification and fine forceps open the oocyte and set free the nucleus (Figure 6.5(b)). Transfer the nucleus into another small glass dish containing fresh mock 3. Further purify the nucleus from adhering yolk particles by repeatedly touching the nucleus with a microcapillary. Transfer the nucleus into the microchamber (Figure 6.5(c)), strictly avoiding the nucleus touching the air– water interface.

3. Mount the microchamber on the stage of a confocal laser scanning microscope. Visualize the nucleus in through-light at low magnification (10× or 16× objective). Focus onto the largest perimeter of the nucleus. Adjust laser power, 3 wavelength, multiplier voltages, pinhole, etc. so that both the transport substrate (NLS protein) and the control substrate (Texas-red labelled 70 kDa dextran) will be optimally imaged.

4.Use a GELoader Tip to inject 10 µl of the transport solution into the microchamber. Start timer and scanner

(a)	Import substrate	Control substrate	(b)
	P4K	TRD70	(a.u.)Fluorescence
2 min

e.s rim

intranuclear space

rim e.s

a

150

30 min

Fe

100

Fi(∞)

50

2 min

5 min

(c)

10 min

Fluorescence (a.u.)

30 min

1000 mm

0 −400	−200	0	200	400
		Distance (µm)

bcontrol, extranuclear substrate, nuclear rim substrate, extranuclear substrate, intranuclear control, intranuclear

200

160

120

80

40

0

0

10

20

30

Time (min)

Figure 6.6 Example of a transport measurement. (a) A Xenopus oocyte nucleus was incubated with a solution containing a NLS-protein (P4K) and a control substrate (TRD70). Scans were taken at indicated times after transport solution addition. (b) Profiles through the nucleus showing the time development of the transport substrate fluorescence. (c) Time development of mean intraand extranuclear fluorescence of transport and control substrate. (From ref. 4)

(t = 0). Continue with scanning at intervals properly resolving transport kinetics (Figure 6.6(a)).

5.Evaluate the image series (Figure 6.6(b)), using an image processing program such as Image J (public domain Java version by W. Rasband, http://rbs.

info.nih.gov/ij/) to derive Fsi, Fse, Fci, Fce, the mean corrected intraand extranuclear fluorescence intensities of transport or control substrate, as well as the nuclear radius R (Figure 6.6(c)). The

number Ni(t ) of imported substrate molecules can then be calculated according to:

Ni(t ) = (Fi/Fe) Ce VN L

where Ce is the substrate concentration in the transport solution, VN is the nuclear volume (= 4/3π R3) and L is Avogadro’s number. In addition, intensity profiles (Figure 6.6(b)) may be obtained.

Notes

This procedure will take approximately 1 h.

1 Mock 3 is an intracellular mock medium adjusted, however, to 3 µM free Ca++. Prepare a fresh batch every day.

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2 BSA serves to balance intranuclear osmotic pressure. Dialyse stock BSA solution (100 g/l) against mock 3.

3 Set laser power as low as compatible with a sufficient image quality to avoid photobleaching.

References

1.Adam, S. A., Stern-Marr, R. and Gerace, L. (1990) Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors. J. Cell Biol., 111, 807–816.

2.Forbes, D. J. Kirschner, M. W. and Newport, J. W. (1983) Spontaneous formation of nucleus-like structures around bacteriophage DNA microinjected into Xenopus eggs. Cell, 34, 13–23.

3.Lohka, M. J and Masui, Y. (1983) Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components. Science, 220, 719–721.

4.Radtke,T., Schmalz,D., Coutavas,E., Soliman, T. M. and Peters,R. (2001) Kinetics of protein import into isolated Xenopus oocyte nuclei.

Proc. Natl. Acad. Sci. USA, 98, 2407–2412. 5. Keminer, O., Siebrasse, J. P. Zerf, K. and

Peters. R. (1999) Optical recording of signalmediated protein transport through single nuclear pore complexes. Proc. Natl. Acad. Sci. USA, 96, 11 842–11 847.

PROTOCOL 6.11

Transport measurements in microarrays of nuclear envelope patches by optical single transporter recording

Reiner Peters

Introduction

A most powerful approach to the experimental analysis of membrane transport processes is single transporter recording. However, previously this was possible only in the case of ion channels using the electrophysiological patch clamp method [1]. In optical single transporter recording (OSTR) [2, 3] membranes are attached to microarrays of cylindrical test compartments and transport across membrane patches that may contain single transporter or transporter populations is recorded by confocal microscopy. OSTR features a very high sensitivity, single-transporter resolution, multiplexing of transport substrates and parallel data acquisition (for review, see ref. 4). Here, a protocol for the measurement of nuclear export [5, 6] is given. For a complementary method, employing isolated Xenopus oocyte nuclei, see Protocol 6.10.

Reagents and materials

Tissue culture dishes, e.g. Falcon no. 353004

3.5 mm diameter drill

Polycarbonate track etched (PCTE) membrane filters, type Cyclopore Transpar-

ent (Whatman, Maidstone, Kent, UK), available in pore sizes of 0.1 to 8.0 µm

Eastman Instant Adhesive no. 910

Amphibian Ringer’s solution: 88 mM NaCl, 1 mM KCl, 0.8 mM MgSO4, 1.4 mM CaCl2, 5 mM Hepes (pH 7.4)

Mock 3 intracellular medium: 1 90 mM KCl, 10 mM NaCl, 2 mM MgCl2, 0.1 mM CaCl2, 1.0 mM N -(2-hydroxyethyl) ethylene-diaminetriacetic acid (HEDTA), 10 mM Hepes (pH 7.3)

Transport solution: mock 3 containing 1 µM of a fluorescent protein containing a nuclear export signal (NES) such as GG-NES [5], 1 µM of the nuclear export receptor CRM1, 1 µM Ran-GTP, 10 g/l BSA 2

Piece of Xenopus laevis ovary

Equipment

Forceps, e.g. Dumont no. 5

GELoader Tips (i.e. very fine pipette tips made by Eppendorf, Hamburg, Germany)

Stereomicroscope

Confocal laser scanning fluorescence microscope

Image processing program (e.g. Image J)

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(a) Intact nucleus

(b) Perforated nucleus

(c) Isolated nuclear envelope

		(e)
	(d) Transport substrate	NPC
	and confocal microscopy
		Isoporous
		filter
	Objective	Confocal plane
		Oil
		Coverslip

Figure 6.7 Preparation of a specimen for OSTR measurements of nuclear transport

Procedure

1.Prepare an OSTR chamber by drilling a hole of 3.5 mm diameter into the bottom of a tissue culture dish. Apply a piece

of optically clear double-sticky tape to a clean coverslip (15 mm×15 mm). Cut a

PCTE membrane filter into small pieces (5 mm×5 mm) and place a filter piece, shiny side up, on the tape. Attach the cover slip-filter assembly to the culture dish such that the filter forms the bottom of the hole. Fill the microchamber with 12 µl of mock 3. If the pores of the filter do not fill spontaneously with mock 3, swim the chamber on an ultrasonic bath and apply a short pulse (some seconds) of ultrasound. Put a moistened piece of laboratory tissue into the chamber. Close the chamber lid.

2.Manually isolate and purify a nucleus from a Xenopus oocyte, as described in Protocol 6.10, step 2. Transfer the purified nucleus into the OSTR chamber. Attach the nucleus firmly to the PCTE filter by pressing the nucleus against the filter using a small glass rod (Figure 6.7(a)). Use a very fine steel needle or a glass microcapillary to open the nucleus (Figure 6.7(b)). Remove the nuclear contents and purify the nuclear side of the nuclear envelope by washing it three times with 15 µl of mock 3 (Figure 6.7(c)).

3. Use a	GELoader		Tip to inject
15 µl	of	the transport solution into
the microchamber (Figure 6.7(d)). The
transport		solution,	containing	10 g/l
BSA,	will	sink to	the bottom	of the

246 IN VITRO TECHNIQUES

(a)		(b)
Measuring field within	GG-NES	70 kD dextran
nucleus-covered area
2 min
		in/out
		F

GG-NES, CRM1, RanGTP, GTP

GG-NES, GTP

2.0GG-NES, CRM1, GTP GG-NES, RanGTP, GTP

1.5

1.0

4 min

6 min

8 min

Reference field outside nucleuscovered area

		0.5
		0.0
	(c)		GG-NES, CRM1, RanGTP
			GG-NES, CRM1,Ran(Q69L)GTP
		2.0	GG-NES, CRM1, RanGTPg S
		1.5
	in/out	1.0
	F
		0.5
62.5 m		0.0
		0	100	200	300	400	500	600

Time (s)

Figure 6.8 Example of an OSTR measurement of signal-dependent nuclear protein export. (a) Raw data. A transport solution containing an export complex (i.e. a 1 : 1 : 1 complex of GG-NES, CRM1 and RanGTP) and a control substrate (Texas-red labelled 70 kDa dextran) was added to the OSTR chamber at t = 0 min and confocal scans of the oil–filter interface of a nucleus-covered area acquired at the indicated times. After transport had reached a plateau a nucleus-free area was imaged for reference. (b) Data showing that only the complete export complex was transported. (c) Data showing that hydrolysis of Ran-bound GTP was required for export. (From ref. 5)

chamber replacing the pure mock 3. Remove surplus buffer. Start the timer (t = 0).

4.Mount the microchamber on the stage of a confocal laser scanning micro-

scope. Visualize the nucleus in throughlight at low magnification (10× objective). Place the nucleus into the centre of the field of vision. Switch to a 40fold, oil immersion objective without moving the OSTR chamber. Focus on the interface between oil and filter. Start

scanner, adjust microscope parameters, and continue with scanning at intervals properly resolving transport kinetics (Figure 6.8(a)). After recording transport kinetics in the nucleus-covered area, shift the nucleus out of the field of view and obtain scans of a free area of the filter for reference (Figure 6.8(a), bottom).

5.Evaluate the image series, using an image processing program such as Image J (public domain Java version by

W. Rasband, http://rbs.info.nih.gov/ij/). We have written a plug-in to that program which automatically finds the filter pores, measures their fluorescence intensity, subtracts the local background, and plots the time-dependent background-corrected fluorescence for each filter pore individually (examples are given in Figure 6.8(b)).

Notes

This procedure will take approximately 30 min.

1 Mock 3 is an intracellular mock medium adjusted, however, to 3 µM free Ca++. Prepare a fresh batch every day.

2 Dialyse BSA stock solution (100 g/l) against mock 3.

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References

1.Neher, E. and Sakmann, B. (1976). Singlechannel currents recorded from membrane of denervated frog fibres. Nature, 260, 779–802.

2.Tschodrich¨-Rotter, M. and Peters, R. (1998) An optical method for recording the activity of single transporters in membrane patches. J. Microsc., 192, 114–125.

3.Peters, R., Sauer, H., Tschopp, J. and Fritzsch, G. (1990) Transients of perforin pore formation observed by fluorescence microscopic single channel recording. EMBO J., 9, 2447–2451.

4.Peters, R. (2003) Optical single transporter recording: transport kinetics in microarrays of membrane patches. Ann. Rev. Biophys. Biomol. Struct., 32, 47–67.

5.Siebrasse J. P., Coutavas, E. and Peters, R. (2002) Reconstitution of nuclear protein export in isolated nuclear envelopes. J. Cell Biol., 158, 849–854.

6.Siebrasse J. P. and Peters, R. (2002) Rapid translocation of NTF2 through the nuclear pore of isolated nuclei and nuclear envelopes. EMBO Rep., 3, 887–892.

PROTOCOL 6.12

Cell permeabilization with Streptolysin O

Ivan Walev

Introduction

Delivery of macromolecules into the cytosol, with retention of cell viability, is increasingly being employed by cell biologists. Most of the existing methods for introducing macromolecules into the cell require considerable methodological expertise [1]. Pore-forming bacterial toxins have found widespread application as tools to study protein trafficking in eukaryotic cells because, under appropriate experimental conditions, the plasma membrane can be selectively permeabilized while the membranes of internal compartments remain intact [2, 3]. With these approaches, bacterial toxin attack is generally lethal, so the possibilities of conducting cell biological studies after permeabilization have been restricted. Recently a simple method for reversible cell permeabilization using the bacterial toxin Streptolysin O (SLO) has been described [4]. SLO is the prototype of a toxin family that have been termed oxygen-labile or sulfydryl-activatable toxin (also termed cholesterol-dependent toxins). This is because the toxin spontaneously loses activity in the presence of atmospheric oxygen, and regains activity upon reduction (e.g. with DTT). The reactivity of the single cysteine residue in the SLO-molecule is responsible for this property. However, the single cysteine residue in SLO can be replaced by alanine, without loss of activity [5]. This mutagenized SLO can be iso-

lated from E. coli and is no longer prone to oxygen-dependent inactivation [6]. SLO generates very large transmembrane pores, readily allowing delivery of molecules with mass up to 150 kDa into cytosol. Reversible permeabilization of adherent and non-adherent cells requires use of low SLO concentrations. Resealed cells remain viable for days and retain their capacity to endocytose and to proliferate [4].

Reagents

Streptolysin-O (SLO): available from several biochemical suppliers 1

Hepes buffered saline solution (HBSS): 30 mM Hepes, 150 mM NaCl (pH 7.2)

Procedure

1.Suspend the cells in HBSS without Ca2+ and Mg2+, for 5–10 min. 2

2.Permeabilization step: discard the su-

pernatants and add SLO dissolved in the same medium for 10 min at 37 ◦C. 3 The protein or agent to be delivered to the cytosol is included at the permeabilization step.

3.Resealing step: add surplus ice-cold

medium (without washing) containing Ca2+ and Mg2+ (final Ca2+ concentra-

tion 1–2 mM) and incubate the cells for at least 60 min at 37 ◦C. 4

Notes

1 SLO preparations may contain contaminating proteases or DNases. Such contaminants may create artefacts. Overall, it is therefore worthwhile to check whether a given SLO preparation is contaminant-free. Our procedure produces contaminant-free recombinant SLO (Email to: <[e-mail]>).

Because this SLO mutant does not contain a cysteine residue, activation by reduction is not necessary.

2 Cells: adherent and non-adherent cells can be used. Mouse cells are more resistant to SLO. Some adherent cells do not tolerate the 5–10 min without Ca2+ and detach from plastic. Permeabilization can be measured light microscopically by Trypan Blue or propidium iodide staining.

3 In all cases, selection of an appropriate toxin concentration is pivotal to success. Thus, the required SLO concentration will vary depending on cell target and density, and must be determined immediately before the delivery

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experiment by titration. The goal is to identify the toxin concentration that causes permeabilization of 60–80% of the total cell population under the same conditions.

4 HBSS: some deviation of pH may help. Addition of serum to the medium is good, but not absolutely necessary.

References

1.Lauer, J. L. and Fields, G. B. (1997) Methods Enzymol., 289, 564–571.

2.Bhakdi, S., Weller, U., Walev, I., Martin, E., Jonas, D. and Palmer, M. (1993) Med. Microbiol. Immunol., 182, 167–175.

3.Ahnert-Hilger, G., Bader, M. F., Bhakdi, S. and Gratzl, M. (1988) J. Neurochem., 52, 1751–1758.

4.Walev, I., Bhakdi, S. C., Hofmann, F., Djonder, N., Valeva, A., Aktories, K. and Bhakdi,S. (2001) Proc. Natl. Acad. Sci. USA, 98, 3185–3190.

5.Pinkney, M., Beachey, E. and Kehoe, M. (1989) Infect. Immun., 57, 2553–2558.

6.Weller, U., Muller,L¨., Messner,M., Palmer,M., Valeva, A., Tranum-Jensen, J., Agrawal, P., Biermann, C., Dobereiner,¨ A., Kehoe, M. A. and Bhakdi, S. (1996) Eur. J. Biochem., 236, 34–39.

PROTOCOL 6.13

Nanocapsules: a new vehicle for intracellular delivery of drugs

Anton I. P. M. de Kroon, Rutger W. H. M. Staffhorst, Ben de Kruijff and Koert N. J. Burger

Introduction

Liposomes, aqueous compartments surrounded by lipid bilayers, have been widely used as transport vectors to deliver (impermeant) substances into the cytoplasm of cells. Examples include the delivery of chemotherapeutic agents, DNA for transfection, and fluorescent probe molecules (for reviews see refs 1 and 2). The molecular mechanism of the liposome–cell interaction leading to uptake of the liposome contents depends on the lipid composition of the liposome membrane and the cell type involved. Liposome–plasma membrane fusion has been reported in a number of cases (e.g. [3]). Endocytosis is considered the major route of entry for liposomes into cells (reviewed in ref. 4).

The efficiency of encapsulation of the compound of interest in the liposomes is an important determinant of the efficiency of uptake by the cell. The structural properties of liposomes allow for highly efficient encapsulation of hydrophilic compounds and lipophilic compounds in the aqueous interior and in the lipid bilayer of the liposome, respectively [1]. Compounds that do not meet either of these criteria were so far not amenable to efficient encapsulation in a lipid bilayer coat.

One such compound is the poorly water-soluble anti-cancer drug cisplatin,

cis-diamminedichloroplatinum (II), which is commonly used in the treatment of a variety of solid tumours, including genito-urinary, head and neck, and lung tumours [5]. Encapsulation of this drug into liposomes has many advantages including the reduction of premature inactivation of this highly reactive molecule upon entry in the blood and the reduction of deleterious side-effects such as nephro-, otoand neurotoxicity [6].

The liposomal formulations of cisplatin developed so far (see e.g. ref 7) suffer from a limited bioavailability of the drug in the tumour [8]. A key factor is likely the low water solubility (7 mM at 37 ◦C) and low lipophilicity of cisplatin, leading to liposomal formulations with low drug-to-lipid molar ratios (of the order of 0.02). Serendipitously, an alternative method was recently discovered, enabling the encapsulation of cisplatin in a lipid formulation with superior efficiency [9]. Our method takes advantage of the limited solubility of the drug in water, and produces cisplatin nanocapsules, nanoprecipitates of cisplatin surrounded by a single lipid bilayer, which exhibit an unprecedented drug-to-lipid ratio and an unprecedented in vitro cytotoxicity. The cisplatin nanocapsules may turn out to be the paradigm for encapsulating compounds