Cell Biology Protocols

Protocol 6.30

311

(a)

(b)

+ Nocodazole

Figure 6.27	Golgi elements and vimentin filaments interact	in vivo. MFT-6 cells were untreated
(a) or treated	with 10 µg/ml nocodazole for 5 h at 37 ◦C to	disrupt microtubules (b). Cells were

then labelled with polyclonal anti-GM130 and monoclonal anti-vimentin antibodies. Extended Golgi elements align with vimentin filaments in untreated cells (arrowheads in (a)). In cells with disrupted microtubules, Golgi elements associate with vimentin filaments (arrowheads in B). Arrow points to a mass of collapsed vimentin. Reproduced from The Journal of Cell Biology, 2001, 152(5), 877– 893, by copyright permission of the Rockefeller University Press.

312 IN VITRO TECHNIQUES

4.Follow immunofluorescence procedure

as described (check procedure part 1/1,

Protocol 6.26). 3

Notes

1 Nocodazole (or similar reagents) disrupt microtubules, resulting in the collapse of the majority of vimentin filaments to a peri-nuclear region. However, a fraction of peripheral vimentin filaments remains. Immunofluorescence with antibodies against Golgi proteins allows the visualization of individual Golgi fragments aligned on the vimentin filaments (Figure 6.27).

2 Alternative fixatives (3% formaldehyde) and permeabilization reagents (0.07% Triton X-100 in PBS) can be used.

3 Individual filaments (or filament bundles) have weak fluorescence, and

anti-vimentin antibodies must be used at high concentrations. In addition, images may need to be enhanced with software to desired brightness [6].

References

1.Fuchs, E. and Weber, K. (1994) Ann. Rev. Biochem., 63, 345–382.

2.Gao, Y.-S. and Sztul, E. (2001) J. Cell Biol., 152, 877–893.

3.Taylor, R. S., Jones, S. M., Dahl, R. H., Nordeen, M. H. and Howell, K. E. (1997) Mol. Biol. Cell, 8, 1911–1931.

4.Gao, Y.-S., Vrielink, A., MacKenzie, R. and Sztul, E. (2002) Euro. J. Cell Biol., 81, 391–401.

5.Gurland, G. and Gundersen, G. G. (1995) J. Cell Biol., 131, 1275–1290.

6.Franke, W. W., Grund, C., Kuhn, C., Jackson, B. W. and Illmensee, K. (1982) Differentiation, 23, 43–59.

PROTOCOL 6.31

Microtubule peroxisome interaction

Meinolf Thiemann and H. Dariush Fahimi

Introduction

The microtubular system plays a vital role for the distribution, translocation and determination of the shape of cell organelles. Peroxisomes are ubiquitous membr- ane-bounded organelles with essential functions in lipid metabolism and detoxification of reactive oxygen species. Their association to microtubules has been shown and the motor-dependent transport of peroxisomes along microtubules demonstrated [1–4]. The molecular mechanisms of these binding and motility events, however, are far from being understood. Hence we have developed a semi-quantitative In vitro peroxisome–microtubule binding assay for investigating the physicochemical characteristics of this binding process and to screen for the proteins involved [5].

The peroxisome–microtubule binding assay presented here could also be adapted to investigate the interaction of other membrane-bounded organelles with microtubules and to identify the proteins mediating it.

Reagents

MAP-free tubulin from bovine brain (Tebu, Frankfurt/Main, Germany)

Taxol (= paclitaxel) was obtained from Sigma (Taufkirchen, Germany)

(All chemicals should be of analytical grade and be prepared with high quality deionized water)

Equipment

Microcentrifuge

Shaker

Standard equipment for SDS-PAGE, Western blotting and enhanced chemiluminescence

Scanner and software for densitometric quantitation of immunoreactive protein bands

Water bath

96-Well microtiterplates (flat bottom microtiterplates with high binding capacity from Costar, Cambridge, MA, USA, proved to be well suited for the attachment of microtubules)

Procedure

The protocol of the microtubule–per- oxisome binding assay is summarized in Figure 6.28.

1.Polymerize microtubules by incubating glycerol stabilized MAP-free tubu-

lin in P/G/T-buffer (35 mM Pipes, 5 mM MgSO4, 1 mM EGTA, 0.5 mM

EDTA, 1 mM	DTT,	1 mM GTP,
20 µM taxol,	pH 7.4)	at 37 ◦C for

15 min.

2.Perform the binding assay (Figure 6.28) at a constant temperature of 25 ◦ C in 96well microtiterplates.

3.Coat wells with microtubules by incubating 50 µl of the microtubule

314 IN VITRO TECHNIQUES

Microtubule–peroxisome

binding assay

Binding of peroxisomes to microtubules immobilized to a microtiterplate

coat wells with microtubule solution block with casein solution

1 × washing with buffer incubation with purified peroxisome solution

3 × washing with buffer

elution of bound proteins with SDS-sample buffer

Detection and quantitation

of peroxisomes bound to microtubules

SDS-PAGE

Western blotting

peroxisome specific primary antibody secondary antibody (HRP-labelled) detection of binding by ECL quantitation by densitometry

Figure 6.28 Schematic summary of the protocol of the microtubule–peroxisome binding assay which is carried out in microtiter plates. The binding of peroxisomes to microtubules is quantitated by the detection of a peroxisomal marker protein in rat liver (e.g. urate oxidase) by Western blotting

solution per well (final concentration 0.25 mg protein/ml) for 45 min. For controls take 50 µl P/G/T-buffer per well.

1 mM EDTA, 0.2 mM PMSF, 1 mM

6-aminocaproic acid, 5 mM benzamidine, 10 µg/ml leupeptin, 0.2 mM DTT, pH 7.4) – to each well and incubate for 30 min.

7.Wash three times with 50 µl/well P/ G/T-buffer.

8.Elute bound proteins and organelles in 20 µl SDS-sample buffer by pipetting five times up and down.

9.Perform SDS-PAGE with the samples by using 10% polyacrylamide gels.

10.Transfer proteins from the gel electrophoretically to a PVDF-membrane.

11.Perform immunocomplexing by using

an antibody to a specific peroxisomal protein, e.g. urate oxidase. 2

12.Detect immunocomplexes with a horseradish peroxidase-labelled secondary antibody, employing enhanced chemiluminescence.

13.Quantitate the intensity of immunoreactive bands by densitometry.

Examples of data

In vitro binding of peroxisomes to microtubules can be demonstrated by neg-

4.Block non-specific binding to the ative staining electron microscopy [7] microtiterplate surface by 45 min (Figure 6.29). Purified rat liver perox-

incubation with 0.3 ml/well casein (2.5 mg protein/ml in P/G/T-buffer).

5.Wash once with 0.3 ml/well P/G/T- buffer.

isomes specifically associate to taxolstabilized microtubules reconstituted from bovine brain tubulin. Binding was observed at the entire length of microtubules and only very few unbound peroxisomes were

6.Isolate purified peroxisomes by difseen. Although this morphological assay ferential cell fractionation of rat liver employs different experimental condi-

homogenate according to a protocol described previously [5, 6]. Add 40 µl of the peroxisomal preparation1 – adjusted to 0.1 mg protein/ml with homogenization buffer (250 mM sucrose, 5 mM MOPS, 0.1% ethanol,

tions than the biochemical binding assay described here, both assay formats point out the specificity of the In vitro binding of peroxisomes to microtubules. The biochemical assay was saturable for both, the concentrations of peroxisomes and

PROTOCOL 6.31

315

(a)(b)

Figure 6.29 In vitro binding of peroxisomes to microtubules as demonstrated by negative staining electron microscopy. Peroxisomes (0.1 mg/ml) were incubated with microtubules (0.25 mg/ml) for 1 h in Eppendorf tubes. After fixation of the peroxisome–microtubule complexes with 0.25% glutaraldehyde for 5 min, a drop of this mixture was placed on a formvar-coated 200-mesh grid for 2 min. Finally, a washing step was followed by the staining of the preparation with 2% phosphotungstic acid for 3 min. Negative staining electron microscopy (EM) reveals the specificity of the association of highly purified rat liver peroxisomes (PO) to taxol-stabilized microtubules from bovine brain (MT). Bars: 0.25 µm (a), 0.05 µm (b)

microtubules (Figure 6.30). The optimal assay conditions comprised a tubulin concentration of 0.25 mg/ml and a peroxisome concentration of 0.1 mg/ml. As an example, an immunoblot incubated with an antibody to urate oxidase and serving for the quantification of peroxisomes to microtubules is shown (Figure 6.30(c)). The binding assay showed excellent reproducibility and high specificity and provided a powerful tool for the evaluation of binding conditions and the various factors involved.

Employing this assay, the binding of peroxisomes to microtubules was characterized and a CLIP-like protein was shown to be involved in the binding process [5].

Notes

This procedure will take approximately 24 h. Store tubulin and peroxisomes at −80 ◦C and avoid repeated thawing and freezing.

1 It is essential that peroxisomes are not aggregated. To ensure the removal of peroxisomal aggregates, briefly centrifuge the peroxisomal preparation (10 s, 2000g) prior to use in the assay.

2 An antibody to urate oxidase [8] was shown to be an excellent marker for the binding of peroxisomes to microtubules in this assay. Urate oxidase is exclusively localized to the crystalline cores of rat liver peroxisomes, which are not released from the organelle by washing procedures. Furthermore, isolated urate oxidase cores do not bind to microtubules.

References

1.Schrader, M., Burkhardt, J. K., Baumgart, E., Luers,¨ G., Spring, H., Volkl,¨ A. and Fahimi,

H.D. (1996) Eur. J. Cell Biol., 69, 24–35.

2.Rapp, S., Saffrich, R., Anton, M., Jackle,¨ W., Ansorge, W., Gorgas, K. and Just, W. (1996)

J.Cell Sci., 109, 837–849.

316IN VITRO TECHNIQUES

(a)120

Specific binding (%)

100

80

60

40

20

	0
	0	0.1	0.2	0.3	0.4	0.5
		Peroxisome concentration (mg/ml)
(b)	120
(%)	100
binding	80
	60
Specific	40
	20
	0
	0	0.2		0.4	0.6	0.8
		Tubulin concentration (mg/ml)

(c)97

66

45

31

22

14

MW (kDa) 0 0.05 0.1 0.25 0.5 0.75

Tubulin Concentration (mg/ml)

Figure 6.30 The binding of peroxisomes to microtubules depends on the concentrations of peroxisomes and tubulin. The In vitro binding assay was performed and binding was analysed as described. To optimize the protocol of the binding assay, the concentrations of peroxisomes (a) and tubulin (b,c) were varied. (c) As an example, the immunoblot, generated according to the standard protocol of the binding assay and incubated with an antibody to urate oxidase, is shown. Data are presented as mean

± SE from six experiments. Reproduced by permission of the European Journal of Biochemistry [5]

3.	Wiemer, E. A. C., Wenzel, T., Deerinck, T. J.,			6.	Volkl,¨ A.,	Baumgart, E.	and	Fahimi, H. D.
	Ellisman, M. H. and Subramani, S. (1997) J.				(1996) In: Subcellular Fractionation – a Prac-
	Cell Biol., 136, 71–80.				tical Approach (J. Graham and D. Rickwood,
4.	Schrader, M., King, S. J., Stroh, T. A. and Sc-				eds), pp. 143–167. Oxford University Press,
	hroer, T. A. (2000) J. Cell Sci., 113, 3663–				Oxford, UK.
	3671.			7.	Schrader, M., Thiemann, M. and Fahimi, H. D.
5.	Thiemann, M.,	Schrader, M.,	Volkl,¨ A.,		(2003) Micr. Res. Tech., 11, 171–178.
	Baumgart, E. and Fahimi, H. D. (2000) Eur.			8.	Volkl,¨ A.,	Baumgart, E.	and	Fahimi, H. D.
	J. Biochem., 267, 6264–6275.				(1988) J. Histochem. Cytochem., 36, 329–336.

PROTOCOL 6.32

Detection of cytomatrix proteins by immunogold embedment-free electron microscopy

Robert Gniadecki and Barbara Gajkowska

Introduction

The simple view of the cell as the membrane sack containing organelles floating in the amorphous cytoplasm has over the last 30 years been replaced by a concept of a complicated three-dimensional cellular structure comprising spatially separated yet functionally interconnected compartments. The basic scaffold of most cells is afforded by the cytoskeleton (comprising actin microfilaments, intermediate filaments and the microtubules) that not only determines rigidity and motility of the cells but also provides support for the organelles.

However, our understanding of cellular structure is still incomplete. No imaging technique is alone able to visualize the intrinsic structure of the cell. Immunofluorescence microscopy with labelled antibodies against various cytoskeletal components turned out to be a very powerful technique that accounts for much of the progress that has been made in the understanding of the three-dimensional cell structure. However, the major drawbacks are low resolution and the fact that only known proteins, against which the labelled probes are available, may be investigated.

Electron microscopy seems to be ideally suited for studying cell ultrastructure.

However, this technique has three major limitations:

1.The optical properties of the resins used for embedding are similar to those of proteins, hence most proteinaceous structures remain unresolved and the cytoplasm in electron microscopic images seems to be quite homogeneous.

2.In classic electron microscopy only some filaments are visualized, and only those that happen to lie at the surface of the section.

3.Aldehyde fixation, which is a necessary step during the preparation of the material, cross-links proteins and lead to the emergence of artificial structures.

Some of these limitations may be overcome by the use of the embedment-free electron microscopy (EFEM). It this technique the material is temporarily embedded in the mounting medium for cutting, but the embedding material is removed before observation. Although fixation is still necessary, no resin is present at the moment of observation in the microscope. The group of S. Penman should be credited with the development and popularization of EFEM [1, 2]. The main use of EFEM

318 IN VITRO TECHNIQUES

has been the study of the nuclear matrix. Penman et al. successfully visualized the non-chromatin nuclear matrix as a complicated three-dimensional network comprised of 10 nm ‘core’ filaments decorated with globular ribonucleoprotein structures and dense bodies [3]. The outline of the EFEM protocol is as follows:

1.The cells are extracted with non-ionic detergent that dissolves the membranes and allows the soluble proteins to diffuse away.

2.DNA is removed by digestion with nuclease.

3.The remaining structure (cellular scaffold or cytomatrix) is temporarily embedded in diethylene glycol distearate (DGD) and sectioned.

4.Before viewing in the electron microscope the DGD is removed so that intact scaffolds are observed.

A recent EFEM protocol may be found in Nickerson et al. [4]. In our laboratory we have successfully adopted this protocol for the study of diverse cell types such as normal or transformed keratinocytes and cancer cell lines (C6, U- 373, MG [glioma lines], COLO 205 [colorectal adenocarcinoma], PA-1 [ovarian cancer cells]) [5–8].

The material processed for EFEM may also be used for immunostaining with gold-labelled antibodies. This provides an elegant combination of the clarity and resolution of EFEM with the power of immunocytochemistry. It is important to underline that only proteins associated with the cytomatrix will be identified. Soluble proteins will be lost during the preparation of the scaffolds and remain undetected. We have successfully used single-, doubleand triple immunostaining in combination with EFEM for the study of the cellular distribution of apoptosis-related

proteins bax, bid, vdac-1 and caspase- 8 [5, 8].

Reagents

Chemicals

Aminoethylbenzenesulfonyl fluoride (AEBSF, Pefablok SC, Roche Applied Science)

Ammonium sulfate (Sigma)

Bovine serum albumin (Sigma)

Diethylene glycol distearate (Polysciences Inc., Warrington, PA)

DNase I, RNase free (Roche Applied Science)

Donkey serum (Sigma)

EGTA (Sigma)

Ethanol (100%)

Glutaraldehyde, EM grade, 50% stock solution (Sigma)

HMDS (hexamethyldisilazane) (Sigma)

Hydrochloric acid (HCl, 1 M)

Isobutanol (Sigma)

Magnesium chloride (Sigma)

Osmium tetroxide (Sigma)

Paraformaldehyde (Merck, Hohenbrunn,

Germany)

PBS (phosphate-buffered saline, without calcium and magnesium, pH 7.2) (Sigma)

PIPES (Sigma)

Poly-L-lysine (Sigma)

Propylene oxide (Sigma)

Sodium cacodylate trihydrate, ultra pure (Sigma)

Sodium chloride (Sigma)

Sodium hydroxide (NaOH), 1 M solution

Sucrose (Sigma)

Triton X-100 (Sigma)

Tween 20 (Sigma)

Vanadyl riboside complex (VRC) (Fluka)

Water, double distilled

Consumables

Carbon-coated copper or nickel grids (can be coated with poly-L-lysine for increased adhesiveness)

Embedding capsules BEEM (LKB, USA)

Equipment

Electron microscope (e.g. JEOL 1200 EX at 80 kV)

Ultramicrotome (e.g. LKB Ultracut from Reichert, Germany) equipped with diamond knives (glass knives are reported to work for fine EFEM, but we have no experience of our own) carbon evaporator

Laboratory tabletop centrifuge with cooling

pH meter

Forceps

Pasteur and automatic pipettes

Scalpel or razor blades

1. Cell extraction

Reagents

Extraction buffer 1: 10 mM PIPES, pH6.8, 300 mM sucrose, 100 mM NaCl, 3 mM MgCl2, 1 mM EGTA,0.5% Triton X-100, 20 mM vanadyl riboside complex (VRC), 1 mM AEBSF

Remarks: PIPES is dissolved first and pH adjusted with NaOH. Then sucrose, NaCl, MgCl2 and EGTA are dissolved sequentially at room temperature (RT) under constant magnetic stirring. At this stage the buffer can be sterile-filtrated and stored for a couple of weeks in a refrigerator or frozen at −20 ◦C for up to 3 months. Other components (Triton X- 100, VRC and AEBSF) are added before use. Triton X-100 is added v/v with a 1 ml syringe under constant, slow stirring.

PROTOCOL 6.32

319

Extraction buffer 2: 10 mM PIPES, pH6.8, 250 mM ammonium sulfate, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100, 20 mM VRC, 1 mM AEBSF Remarks: Prepared as extraction buffer 1 above; in this buffer NaCl is replaced by

ammonium sulfate.

DNA digestion buffer: 10 mM PIPES, pH6.8, 300 mM sucrose, 50 mM NaCl, 3 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100, 20 mM vanadyl riboside complex, 1 mM AEBSF, 200 units/ml RNase free DNase I.

Remarks: Prepared as extraction buffer 1. DNase I is added before use.

High salt buffer: 10 mM PIPES, pH 6.8, 300 mM sucrose, 2 M NaCl, 3 mM MgCl2, 1 mM EGTA, 20 mM VRC, 1 mM AEBSF

Remarks: PIPES, sucrose, MgCl2 and EGTA are added and dissolved in this sequence. pH is adjusted with NaOH. Then add NaCl to the buffer and dissolve at room temperature. VRC and AEBSF are added before use.

Procedure 1

Adherent cells may be extracted in monolayers attached to thick glass coverslips or grown in chamber slides with a glass bottom. Alternatively, the cells may be gently trypsinized or scrapped by the rubber policeman. Suspended cells are washed by centrifugation at 600 g, 5 min at 4 ◦C between steps. Coverslips with attached cells are moved between different solutions. We got best results with scrapped or trypsinized cells in suspensions. Since some loss of material is inevitable due to multiple centrifugations, start with a large amount of cells (>5 million). Most steps should be performed at 4 ◦C. Precool all the buffers. After the last extraction step we are left with cellular scaffolds ready for temporary embedding and sectioning.

320 IN VITRO TECHNIQUES

1. Wash the cells with PBS, twice at 4 ◦C.

2.	Extract in	2 ml	extraction	buffer	1 at
	4 ◦C for 5 min		with occasional agita-
	tion. At this step the membranes are
	opened by Triton X-100 and soluble
	proteins diffuse from the			cell to the
	outside.
3.	Extract in	2 ml	extraction	buffer	2 at
	4 ◦C for	5 min	with occasional		agi-

tation. Ammonium sulphate in this buffer will extract some of the histones (mainly histone 1) and many cytoplasmic proteins [4].

4. Incubate in the DNA digestion buffer for 1 h at 32 ◦C. Stir gently once every 7–10 min. At this step DNA is digested and removed from the nuclei. According to Nickerson et al. [4] the completeness of DNA removal should be monitored in pilot experiments by e.g. assessment of [3H]thymidine release. This is probably necessary in the experiments where ultimate control is needed during the preservation of the structure of nuclear matrix. We found that satisfactory results are obtained for a wide range of different cells with standard conditions.

5.Extract in the high salt buffer at 4 ◦C for 5 min. High salt concentration removes the histones and most of the high mobility group proteins from the nucleus and stabilizes the cellular scaffolding.

2. Preparation for electron microscopy

Reagents

Buffered glutaraldehyde: 2.5% glutaraldehyde in extraction buffer 1

Remarks: Dissolve glutaraldehyde from the stock solution in extraction buffer 1, shortly before use. Keep in refrigerator at 4 ◦C. Fixative older than 6 h should be discarded.

Sodium cacodylate buffer: 0.1 M sodium cacodylate, pH 7.2

Remarks: Prepare stock 0.2 M solution of sodium cacodylate in distilled water and adjust pH with 0.2 M HCl. The solution is stable for several months when stored at 4 ◦C. Before use dilute 1 : 1 (vol/vol) with distilled water.

Buffered osmium tetroxide: 1% osmium tetroxide in cacodylate buffer

Remarks: Mix osmium tetroxide stock solution with 0.2 M sodium cacodylate stock solution and dilute with distilled water to bring the concentration of sodium cacodylate to 0.1 M. The solution is stable for several months at 4 ◦C.

Procedure 2

1.Fixation. We fix routinely in 2.5% buffered glutaraldehyde at 4 ◦C for 40

min. Remember that cell pellet should ideally be approximately 1 mm3 for further embedding.

2.Wash the pellet or cells on the cover-

slip with sodium cacodylate buffer for 5 min, 4 ◦C.

3.Post-fix in buffered osmium tetroxide for 30 min at 4 ◦C.

4.Wash in cacodylate buffer for 30 min.

5.Dehydrate the material by transfer through the ethanol series: 50, 70, 80, 90, 96% two changes for 5 min each. End with three changes of dehydrated 100% ethanol for 5 min each. At this

step the material may be left overnight at 4 ◦C. After transfer through 80% ethanol the pellets are hard enough to be transferred to BEEM capsules.

6.Transfer the cells to a 1 : 1 (v/v)

mixture of ethanol : isobutanol for 5 min at room temperature, and then to pure isobutanol, twice for 5 min each time. Place the samples in the oven at 60 ◦C and allow the solvents