Cell Biology Protocols

solution in ethanol. Minimize precipitation of PMSF on dilution into aqueous solution, by slowly expelling the aliquot of PMSF stock solution from a pipette tip submersed in vigorously stirring buffer solution. PMSF has a half-life as short as 30 min in some aqueous solutions; add to the buffer solution immediately before use.

2 Chill the gizzards on ice as soon as possible after acquisition. Keep the smooth muscle tissue cold throughout the initial steps of the protocol to minimize actin contamination of the purified smitin–myosin II preparation. For optimal purity of smitin–myosin II preparations, use the gizzards within 2 h of sacrifice. Cleaned and diced gizzard smooth muscle that has been rapidly frozen in liquid N2 and stored at −20 ◦C until use also provides a suitable source of smitin for purification.

3 Use of Sephacryl S-500 instead of S- 1000 yields similar results. Minimize actin filament contamination of the smitin–myosin II Sephacryl S-1000 fractions by loading the column with a 50 ml front of 0.6 M KI (made in buffer C) immediately ahead of the protein load. For convenience, this column can be run overnight.

4 SDS-PAGE: To maximize protein concentration in gel samples of the column fractions, make gel samples using 8 × or10 × Laemmli sample buffer. Heating the samples to 90 ◦C for 3 min minimizes the amount of myosin heavy chain trapped in the smitin band on

the gel. Overheating can cause loss of full-length smitin from the sample. Samples can be analysed in large or mini gel formats. Using an acrylamide stock with a 75 : 1 ratio of acrylamide to bis-acrylamide to form the running and stacking gels enhances migration of smitin into the gel. Proteins with polypeptide molecular weights ranging from greater than 3 MDa to 15 kDa, including smitin, myosin heavy and light chains, and actin, can be fractionated in lanes on a 4–20% acrylamide gradient running gel with a 3% stacking gel. Allowing the gel to run for an additional 25–33% of the time it takes the dye front to reach the bottom of the gel increases migration of smitin into the gel and increases fractionation resolution of the proteins in the sample.

5 Prepare dialysis tubing according to the manufacturer’s recommendations. Select the dimensions of the tubing to maximize the surface to volume ratio of the bag. The rate of coassembly formation is inversely related to the protein concentration of the sample. At high protein concentration, smitin–myosin coassembly aggregates may be visible in the dialysis bag as white flocculent material in less than 6 h.

References

1.Kim, K. and Keller, T. C. (2002) Smitin, a novel smooth muscle titin-like protein, interacts with myosin filaments in vivo and in vitro,

J. Cell Biol., 156, 101–112.

Reagents

pQE30 expression vector (QIAGEN), enzymes for RT-PCR and cloning, E. coli M15 competent cells (QIAGEN), IPTG (1 M in H2O), 50% slurry of NiNTA resin (QIAGEN), buffer A (6 M GuHCl, 0.1 M Na-phosphate, 0.01 M TrisHCl pH 8.0), buffer B (8 M urea, 0.1 M Na-phosphate, 0.01 M Tris-HCl pH 8.0), buffer C (8 M urea, 0.1 M Na-phosphate, 0.01 M Tris-HCl pH 6.3), 1 M imidazole, 5 M NaCl, 1 M DTT, glycerol, 1 M MES pH 6.2, acetic acid glacial, 0.5 M EDTA, 1 M CaCl2, Protein assay reagent (Pierce), Ezblue gel staining reagent (Sigma)

Procedures

A. Isolation of the recombinant C-terminal fragment of the heavy chain of non-muscle myosin IIA

1.Clone cDNA corresponding to the C- terminal fragment of the heavy chain of non-muscle myosin IIA in BamHI

site of pQE30 bacterial expression vector 1 and use it for the transformation of E. coli M15 cells.

2.Prepare overnight culture and use it for the preparation of large-scale culture (0.5 l) according to the QIAGEN protocol.

3.Perform IPTG induction (1 mM) of culture for 5 h. Harvest cells by centrifugation at 3000g for 20 min.

4. Store cell pellet at −80 ◦C or process immediately purification of the myosin fragment in denaturing conditions according to the QIAGEN recommendations.

5.Briefly, resuspend pellet in buffer A (5 ml/g wet weight) and stir cells at room temperature until lysis of cells is completed.

6. Centrifuge the lysate at 20 000g for

20 min and mix supernatant with 4 ml of 50% slurry of Ni-NTA resin, which was previously equilibrated in buffer A.

7.After 1 h of stirring at room temperature (RT), apply resin to the column and wash consequently with buffers A, B and C until the A280 of the flowthrough is lower than 0.01 at each step.

8.Perform refolding of the myosin frag-

ment with the 30 ml of the linear

6M–1 M urea gradient in 0.5 M NaCl, 20% glycerol, 10 mM Tris pH 7.4.

9.Elute protein in 0.5 ml fractions using

5ml of the elution buffer (1 M urea, 0.5 M NaCl, 10% glycerol, 10 mM Tris pH 7.4, 250 mM imidazole).

10.Combine pick fractions and dialyse against 0.6 M NaCl, 10 mM MES pH 6.2, 1 mM DTT.

11.Centrifuge sample after dialysis for 10 min at 20 000g. Aliquot sample,

freeze in liquid N2 and store at −80 ◦C.

B.Isolation of recombinant S100A4

1.Clone cDNA of S100A4 in the pQE30 expression vector (QIAGEN) in BamHI site (2) and use for the transformation of E. coli M15 cells.

2.Prepare 1 l of bacterial culture as described for the myosin preparation.

3.Resuspend cell pellet in the sonication buffer (50 mM Tris-HCl pH 7.5, 200 mM NaCl) at 4 volumes per gram of wet weight.

4.After three times freezing (liquid N2) and thawing (RT), sonicate cell lysate on ice three times for 20 s following centrifugation for 20 min at 20 000g.

5.Mix supernatant with 6 ml of 50% slurry of Ni-NTA resin equilibrated by the sonication buffer.

6.Raise NaCl concentration in the mix-

ture up to 1 M and rotate for 1 h at +4 ◦C.

7.Load resin into the column and wash

at +4 ◦C with 50 mM Tris, pH 7.5 until the A280 of the flow-through is less than 0.01.

8.Elute the S100A4 with buffer containing 1 M NaCl, 100 mM acetic acid pH 4.0.

9.Dialyse sample against access of the buffer containing 10 mM MES pH

6.2, 50 mM NaCl, 0.5 mM EDTA, 1 mM DTT overnight at +4 ◦C and centrifuge at 20 000g for 10 min.

10.Aliquot the protein, freeze in liquid N2 and store at −80 ◦C. 3

11.Measure the concentration of the protein by using the Protein assay reagent (Pierce).

C. Preparation of myosin filaments

1 mM CaCl2 at RT.

2.Dialyse for 5 h at RT against 200 ml of the buffer containing 10 mM MES pH 6.2, 1 mM DTT, 1 mM CaCl2, 50 mM NaCl. Myosin filaments are formed during dialysis.

3.Precipitate filaments by centrifugation

D. S100A4 stimulates disassembly of preformed myosin filaments

1.Add 50 µl of the S100A4 (1 mg/ml) in low ionic strength buffer (10 mM MES pH 6.2, 1 mM DTT, 1 mM CaCl2, 50 mM NaCl) or 50 µl of high ionic strength buffer (10 mM MES pH 6.2, 1 mM DTT, 1 mM CaCl2, 600 mM NaCl) without S100A4 to the myosin pellet (10 µg).

2.Incubate tubes at RT for 1 h with gentle agitation.

3.Centrifuge 10 min at 20 000g and analyse supernatants by running the 15% SDS-PAGE. Almost 80% of the C- terminal fragment becomes soluble in the presence of S100A4, while the high ionic strength buffer is able to solubilize 40% of the precipitated myosin fragment.

374 IN VITRO TECHNIQUES

E. S100A4 prevents aggregation of myosin filaments in the presence of calcium

1mM DTT) containing 1 mM CaCl2 or

5mM EDTA.

2.Dialyse proteins for 5 h at RT against

200ml of buffer, 10 mM MES pH 6.2,

1mM DTT, 1 mM CaCl2 or 5 mM EDTA and different NaCl concentrations, 50, 100, 200, 300 and 400 mM.

3. Centrifuge samples for 10 min at

20 000g.

4.Analyse supernatant in the presence of the myosin fragment by running a 15%

SDS-PAGE and staining with Ezblue gel staining reagent (Sigma). 4

PCR using specific myosin primers, forward primer, CGGGATCCCAGAT CAACGCCGAC (+5303 to +5317), and reverse primer, CGGGATCCGG CTTATTCGGCAGG (+5894 to +5908).

2 Synthesis of the human S100A4 cDNA: prepare total RNA from human platelets and perform RTPCR with specific S100A4 primers, forward primer, CGGGATCCATGGC GTGCCCTCTG (+65 to +79) and reverse primer, CGGGATCCGAG TTTTCATTTCTTCCTGGG (+356 to +376).

3 Fresh prepared S100A4 keep at −80 ◦C in order to prevent aggregation of the protein.

4 In the presence of S100A4 and calcium, the myosin fragment remains in a soluble form in low ionic buffer.

Notes

1 Synthesis of the cDNA corresponding to the C-terminal 202 aa fragment of the heavy chain of the human nonmuscle myosin IIA: prepare total RNA from human platelets and perform RT-

Acknowledgements

This work was supported by grants from the Danish Cancer Society, Dansk Kræftforsknings Fond, Novo Nordisk Fonden and Yorkshire Cancer Research.

Introduction

It has long been known that native-like collagen fibrils can be produced in vitro by self-assembly by incubation of extracted and purified type I collagen molecules in warm, neutral solution conditions [1, 2]. The individual fibrils have a polarized arrangement of molecules with an axial D-periodicity of 67 nm and are of nearuniform diameter. Collagen fibrils are stabilized in tissue by the formation of intermolecular cross-links involving specific lysine/hydroxylysine residues and aldimine derivatives of these. Collagen is commonly extracted from young tissues (typically calf skin or rat tail tendon) using acetic acid solution [3] which breaks these intermolecular cross-links. Alternatively collagen can be released by pepsin treatment which cleaves the telopeptides (terminal non-triple-helical domains) where crosslink sites are located. Fibril assembly is, however, critically dependent on the intactness of the telopeptides [2, 4]. Partial loss can lead to a changed morphology and loss of polarity. Extensive loss can completely inhibit fibril formation [4].

The fibrils in the final reconstituted gel of type I collagen have a fairly broad diameter distribution, compared with those in tissue, and this is dependent on the solution conditions at pH 7.4 (temperature and ionic strength, as well as ion type). Optimal conditions for the formation of compact, uniformdiameter, native-like fibrils have generally

involved a temperature of 34 ◦C or less and a phosphate buffer [5, 6].

Different assembly pathways have been observed in the reconstitution of collagen fibrils [5, 7]. The transfer route used to raise pH, temperature and ionic strength from the initial conditions (cold, acid solution) to the reconstitution conditions (warm, neutral solution) has been found to be a major determinant of the assembly pathway [5]. Warming before neutralization (‘warm start initiation’) leads to abundant short ‘early fibrils’ with tapered tips. Similar ‘early fibrils’ are also observed

lowed by warming (‘neutral start initiation’) leads to the early accumulation of filaments [5, 7].

The procedure described here is based on a set of ‘optimal’ reconstitution conditions: 200 µg/ml collagen, I 0.2, pH 7.4 phosphate buffer and a temperature of 34 ◦C. These conditions are established using the ‘warm start’ initiation (Figure 6.45(a)). The kinetics of assembly can be monitored by turbidimetry (Figure 6.45(b)) and the assembly products can be studied by transmission electron microscopy (Figure 6.45(c)).

Reagents

Extracted and purified type I collagen is

available from biochemical suppliers.

1 2

(d)

(b)

Turbidity

Incubation time (min)

Suitable acetic-acid-soluble type I collagen can alternatively be extracted and purified by well-established procedures [3] with modifications to ensure minimal loss of the telopeptides [4]. Laboratory reagents should be of high purity.

Equipment

Temperature-controlled water bath

Spectrophotometer with temperature-con- trolled cell holder

Transmission electron microscope

necessary) at 4 ◦C.

2.Dilute 3 the collagen to 400 µg/ml

with 10 mM acetic acid solution and keep at 4 ◦C.

3.Warm equal volumes of collagen solution and double strength phosphate

buffer (124 mM Na2HPO4, 29.2 mM KH2PO4) separately to 34 ◦C for about 5 min. This yields final solution conditions of I 0.2, pH 7.4.

4.Mix the two volumes (acid collagen solution and double strength buffer) to

start fibril reconstitution and incubate at 34 ◦C for several hours. 4

5.A uniform, cloudy gel will form. This can be pulled from the tube with forceps.

6.Prepare TEM specimens by wiping a small portion of gel over a carbonfilmed grid, washing with several drops of water and negatively staining with 1–2% sodium phosphotungstate, pH

The fibrils should be compact with a polarized, D-periodic stain pattern and uniform diameter along individual fibrils (Figures 6.45(d) and 6.46). Fibril diameters are typically in the range 30–90 nm.

Notes

1 Suppliers include BD Biosciences, USA and Elastin Products Company, USA.

2 The fibril reconstitution procedure described here is appropriate for ace- tic-acid-extracted collagen rather than pepsin-extracted.

3 Collagen concentration can be determined by a direct colorimetric assay (‘Sircol’ kit available from Biocolor Ltd, Northern Ireland).

4 The kinetics of assembly can be simply monitored by turbidimetry measurements using a spectrophotometer

Borate buffer

Boric acid H3BO3

May be harmful by inhalation, ingestion and skin absorption. Causes irritation. Vapour causes irritation to eyes, mucous membranes and upper respiratory tract

Harmful by inhalation, in contact with skin and if swallowed. Irritating to eyes, respiratory system and skin. Possible teratogen. Reproductive hazard