Iozzo Renato V. Proteoglycan Protocols

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490 Gribbon et al.

wide range of conditions, including high concentrations, and in the presence of mul-

tiple interacting components. Confocal FRAP also has the major advantage that

interactions are analyzed under near-equilibrium conditions, which allows the analysis

of the weak interactions that are potentially important in defining matrix properties.

Fig. 1. Translational diffusion coefficient of FITC-G1 (80 µg/mL) as a function of increasing

concentration of hyaluronan (800 kDa). The self-diffusion of G1 is 3.3 × 10

-8

/s. All

measurements in PBS (pH 7.4) at 25°C

Fig. 2. Inverse Scatchard type plot of data from Fig. 1. Analysis of binding between FITC-

G1 (80 µg/mL) and hyaluronan (800 kDa). The dissociation constant was calculated as 4 × 10

-8

and n =1 (see Eq. 6).

Carbohydrate–Protein Interactions 491

2. Materials

2.1. Protein Labeling

1. Protein labeling buffer, 0.2 M: NaHCO

(1.48 g /100 mL), Na

(0.24 g/100 mL), pH

9.0. Filter 0.2 µm before use. Should be prepared fresh, keeps at 4°C for 2 wk.

2. PBS: 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na

HPO

∞• 2H

O, 0.2 g of KH

, made up to

1 L, pH 7.4. Filter 0.2 µm before use, keep at 4°C.

2.2. Photobleaching Experiments

1. Upright confocal laser scanner and fluorescence microscope: The authors use a 100-mW

argon-ion laser with a MRC-1000 scanner (Bio-Rad, Hemel Hempstead, UK). Macros

written in a simple programming language (MPL) control data acquisition and analysis.

The majority of confocal microscopes include similar facilities for user application

development.

2. Microscope objectives of numerical aperture < 0.5 (magnification: ↔10). A low numerical

aperture gives a more uniform and parallel bleach volume.

3. Cavity microscope slides, 12-mm cavity diameter, 30 µL volume (Scientific Lab Supplies,

Nottingham, UK).

3. Methods

3.1. Sample Preparation and Fluorescence Labeling

For studies of interactions involving carbohydrate components of the extra cellular

matrix (ECM), glycosaminoglycans such as chondroitin sulfate and hyaluronan are

commercially available (e.g., Sigma, Seikagaku) from several tissue sources (e.g., bac-

teria, cock’s comb, umbilical cord). Commercially available FITC-labeled proteins

are sometimes unsuitable for binding studies, as they can be excessively substituted

Fig. 3. Competition between high-molecular-weight hyaluronan (800 kDa, (25 µg/mL) and

hyaluronan oligosaccharides (4mer to 14mer, 21 µM) for binding to FITC-G1 (88 µg/mL). All

measurements in PBS (pH 7.4) at 25°C.

492 Gribbon et al.

(>10 mol FITC per mole protein) and their binding and diffusion properties may differ

significantly from those of the native protein. Many important matrix proteins, such as

the G1 domain of aggrecan, are not commercially available and will have to be extracted

from tissues using standard protocols (14). Functional binding properties of proteins

can normally be preserved if they are labeled with a low degree of FITC substitution,

as outlined next.

3.1.2. Protein Labeling

1. Dissolve protein at 0.5 mg/mL in carbonate-labeling buffer (pH 9.0) by mixing with gentle

rotation for 24 h at 4°C.

2. Add 0.1 mg of FITC to 1 mL of labeling buffer. Immediately, add the cold FITC solution

dropwise until the final molar ratio of FITC to protein is 20/1 and incubate overnight at 4°C

with gentle rotation.

3. Remove unconjugated FITC by exhaustive dialysis against de-ionized water at 4°C and

freeze-dry to constant mass.

3.1.3. Preparation of Samples for Confocal FRAP Analysis

FITC concentration in final solutions for analysis should be kept below 1 mM to

avoid fluorescence self-quenching effects.

1. Make up solutions of labeled protein and matrix macromolecules by weight and equili-

brate at 4°C for 48 h. In competition studies, add competitor species and equilibrate for a

further 24 h at 4°C.

2. Pipet 30 uL of sample onto a cavity slide and seal under a 20-mm-diameter circular cover

slip using nail polish; allow 10 min to dry. Place slide on microscope stage and allow to

reach thermal equilibrium.

3.2. Microscope Setup and Data Acquisition

In a confocal FRAP experiment a prebleach image is acquired, the sample is

bleached, and a recovery image series is collected and analyzed. Scanning confo-

cal microscopes excite fluorescence with a laser and detect emitted light with a

photomultiplier (PMT). The essential elements of a protocol, based on a square

bleach and a moments recovery analysis will be given (10,11). The analysis is not

dependent on the shape of the bleach as long as it is symmetrical about the bleach centre.

1. Determine instrument settings for bleach and prebleach/recovery and save as separate

setup configurations to be accessed by the bleaching software. Typically, for prebleach/

recovery, scan time = 2 s, laser intensity = 1% of maximum, PMT gain = 1000 V, iris size

= 75% of maximum. Typical bleach settings are scan time = 2 s, laser intensity = 100%,

PMT gain = 0, iris size = minimum, (see Notes 1 and 2).

2. At the start of the bleaching and data acquisition program, a square region for bleaching

is defined at the center of the field of view (see Note 3).

3. The microscope is set for recovery and one or more prebleach images are scanned, centered

on the proposed bleach. Scan dimensions are 4↔ the bleach width, optical zoom = 1. Several

prebleach images may be averaged to reduce noise.

4. The microscope switches to bleach configuration and sample bleaching is performed at

100% laser power.

Carbohydrate–Protein Interactions 493

5. The microscope resets to the prebleach setup, and a series of recovery images (up to 50)

is collected. For slowly moving species or where there is strong binding, two or more

images should be taken consecutively and averaged to reduce noise (see Note 4).

6. Final images should be taken at two long time points and compared to determine whether

recovery approaches completeness. This is important, as incomplete recovery will indi-

cate the presence of irreversible binding.

7. The image series is processed and analyzed. This operation is ideally carried out on a

separate computer (see Notes 1–4).

8. Less than 0.01% of the total sample volume is typically included in the bleach. This allows

a number of repeat experiments to be made on a single sample. The authors normally

perform 6 measurements per sample.

3.3. Data Analysis

The data analysis strategy for confocal FRAP is based on the variance method of

Kubitscheck et al. for determining the diffusion properties of monodisperse popula-

tions of macromolecules (10,11) (see Note 5). This method can be applied to binding

studies, provided the validity of Eq. 3 is ensured (see Notes 6 and 7). The redistribu-

tion of fluorescence is measured over the whole imaged area, which includes the area

inside and immediately outside the bleached region (see Notes 3 and 4). The straight-

forward computing routine developed by the authors combines the image analysis

tools in the MRC-1000 Microscope Programming Language (MPL) and the data

analysis capabilities found in Excel.

4. Notes

1. Each recovery series is checked to ensure that no convective motion of the solution has

occurred.

2. The effects of nonuniformity in illumination and detector response are corrected for by

dividing each recovery image by the prebleach image.

3. Pixels contained within three bleach widths of the center are quantified and a normalized radial

intensity distribution is determined for each image, with the bleach center set as the origin.

4. Intensity profile data is passed from MPL to Excel. The radial distribution of bleached

fluorophores is determined by subtracting radial intensity values from the image back-

ground intensity.

5. The variance of the radial distribution of bleached fluorophores is calculated and plotted as

a function of time (see ref. 11 for background details of how to calculate the variance

values). The lateral translational diffusion coefficient is calculated from the gradient deter-

mined from a linear least-squares fit to the data.

6. Independent noninteracting multiple diffusing species with greatly differing diffusion coef-

ficients show characteristic multicomponent recovery of total intensity in the bleached area.

Typically there is fast initial recovery as the smaller components redistribute, followed by a

slower recovery phase as the mobility of larger species dominates. This also results in plots

of variance against time being nonlinear.

7. In dynamic binding equilibria, fluorescent molecules redistribute between bound and

unbound forms and fast and slow components are not distinguishable in the recovery

curve. Plots of variance vs time are essentially linear, and the equilibrium mixture behaves

similarly to a monodisperse species having an intermediate diffusion coefficient, provid-

ing the experiment is designed so that Eq. 3 is valid.

494 Gribbon et al.

References

1. Winlove, C. P. and Parker, K. H. P. (1995) The physiological functions of extracellular

matrix macromolecules, in Interstitium, Connective Tissue and Lymphatics (Reed, R. K.,

McHale, N. G., Bert, J. L., Winlove, C. P., and Laine, G. A., eds.), Portland Press, Lon-

don, UK, pp. 137–165.

2. Comper, W. D. and Laurent, T. C. (1978) Physiological functions of connective tissue

polysaccharides. Physiol. Rev. 58, 255–316.

3. Comper, W. D., ed. (1996) Extracellular Matrix. Harwood, Amsterdam, The Netherlands.

4. Maroudas, A. (1976) Balance between swelling pressure and collagen tension in normal and

degenerate cartilage. Nature 260, 808–809.

5. Grodzinsky, A. J. (1983) Electromechanical and physicochemical properties of connective

tissue. CRC Crit. Rev. Bioeng. 14, 133–199.

6. Hardingham, T. E. and Fosang, A. (1992) Proteoglycans: many forms and many functions.

FASEB. J. 6, 861–870.

7. Mould, A. P., Garratt, A. N., PuzonMcLaughlin, W., Takada. Y., and Humphries, M. J.

(1998) Regulation of integrin function: evidence that bivalent-cation-induced conformational

changes lead to the unmasking of ligand- binding sites within integrin alpha 5 beta 1.

Biochem.J. 33, 821–828.

8. Rahmoune, H., Rudland, P. S., Gallagher, J. T., and Fernig, D. G. (1998) Hepatocyte

growth factor scatter factor has distinct classes of binding site in heparan sulfate from

mammary cells. Biochemistry 37, 6003–6008.

9. Axelrod, D., Koppel, D. E., Schlessinger, J., Elson, E., and Webb, W. W. (1976) Mobility

measurements by analysis of fluorescence photobleaching recovery kinetics. Biophys. J.

16, 1055–1069.

10. Kubitscheck, H., Wedekind, P., and Peters R. (1994) Lateral diffusion measurements at high

spatial resolution by scanning microphotolysis in a confocal microscope. Biophys. J. 67,

946–965.

11. Gribbon, P., and Hardingham, T. E. (1998) Macromolecular diffusion of biological poly-

mers measured by confocal fluorescence recovery after photobleaching. Biophys. J. 75,

1032–1039.

12. Blonk, J. C. G., Don, A., Van Aalst, H., and Birmingham, J. J. (1993). Fluorescence

photobleaching in the confocal scanning light microscope. J. Microsc. 169, 363–374.

13. Kaufmann, E. N., and Jain, R. K., (1991) Measurement of mass transport and reaction

parameters in bulk solutions using photobleaching—reaction limited binding regime.

Biophys. J. 60, 596–610.

14. Hardingham, T. E., Ewins, R. J. F., and Muir, H. (1976) Cartilage proteoglycans: structure

and heterogeneity of the protein core and the effects of specific protein modifications on the

binding to hyaluronate. Biochem. J. 157, 127–143.

15. Heinegard, D. and Hascall, V. C. (1974) Aggregation of cartilage proteoglycans. 3.

Characterisation of proteins isolated from trypsin digests of aggregates. J. Biol. Chem.

249, 4250–4256.

16. Nieduszynski, I. A., Sheehan, J. K., Phelps, C. F., Hardingham, T. E., and Muir, H. (1980)

Equilibrium binding studies of pig laryngeal aggrecan with hyaluronate fractions.

Biochem. J. 185, 927–934.

17. Cowman, M. K., Balazs, E. A., Bergmann, C. W., and Meyer, K. (1981) Preparation and

circular-dichroism analysis of sodium hyaluronate oligosaccharides and chondroitin.

Biochem. 20, 1379–1385

Syndecans in Cell Adhesion and Invasion 495

495

From:

Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols

Edited by: R. V. Iozzo © Humana Press Inc., Totowa, NJ

Regulatory Roles of Syndecans in Cell Adhesion

and Invasion

J. Kevin Langford and Ralph D. Sanderson

1. Introduction

Evidence accumulated over the last decade demonstrates that proteoglycans pro-

foundly influence many cell behaviors such as growth factor binding, apoptosis, cell

adhesion, and cell invasion. The major family of transmembrane proteoglycans is the

syndecans, all of which have heparan sulfate chains that interact with various soluble

and insoluble molecules within the extracellular matrix (1). Our lab has developed

two experimental techniques to examine the biological role of the syndecans. One

assay examines syndecan-1–mediated cell–cell adhesion (i.e., aggregation assay). The

other examines the syndecan-1–mediated inhibition of cell invasion.

The aggregation assay has the advantage of simplicity. Cells suspended in buffer

are allowed to interact by gravity and then counted on a hemocytometer for the number

of cells in aggregates (2,3). This assay is rapid, yields highly reproducible results, and

is easily manipulated by the inclusion of promoters or inhibitors of cell aggregation.

To investigate the invasive potential of myeloma cells, we employ a hydrated native

type I collagen gel. First, the distance, or depth of invasion is measured by phase micros-

copy. Next, protease digestions facilitate the harvest of noninvasive cells from the top

of the gel, followed by subsequent digestion of the gel to remove the invasive cells.

After counting cells on a hemocytometer or Coulter counter, a simple calculation

determines the percentage of invasive cells. The major advantage of this model is that

it allows quantification of both the distance and extent of cell invasion. For an exten-

sive review of in vitro and in vivo techniques used to examine cell migration or cell

invasion, see Mareel et al. (4).

Both of the above assays have proven to be powerful tools to probe the biology of

the syndecans as they relate to tumor cell adhesion and invasion. We have used these

assays to demonstrate that, following transfection with the cDNA for syndecan-1,

ARH-77 cells gain the ability to form cellular aggregates and are rendered noninvasive

in collagen gels (2,5). These assays have been further used to examine the behavior of

496 Langford and Sanderson

ARH-77 cells expressing mutant or chimeric proteoglycans in order to investigate the

molecular mechanisms of syndecan-1 function.

2. Materials

2.1. Cell Aggregation Assay

1. Aggregation buffer: For 500 mL of aggregation buffer add: 0.2 g of KCl ; 0.03 g of

; 4.0 g of NaCl; 0.046 g of Na

HPO

· 7H

O; 5 mL of a 1.0 M HEPES solution;

0.175 g of NaHCO

; 5.0 g of bovine serum albumin (BSA). Next, add water to 500 mL and

adjust pH to 7.0. Then, sterile filter and store at 4°C.

2. Heparin solution (1 mg/mL porcine heparin (Sigma; St. Louis, MO) in sterile deionized water).

3. Hemocytometer or Coulter counter.

4. Disposable transfer pipets (5-mL capacity; Fisher, Pittsburgh, PA).

2.2. Invasion Assay

1. Rat tail type I collagen stock (Collaborative Biochemical Products; Bedford, MA).

2. Sodium bicarbonate solution, 7.5% (w/v) (Gibco BRL; Grand Island, NY).

3. Complete medium: Final concentration of each component is as follows: 1 × RPMI solution;

2mM L-glutamine; and 1 × antibiotic-antimycotic solution (Mediatech, Herndon, VA); 5%

fetal calf serum (Atlanta Biologicals, Norcross, GA).

4. 10 × RPMI solution (Mediatech, Herndon, VA).

5. Sterile distilled deionized water (Mediatech Inc., Herndon, VA).

6. 24-well cell culture plate.

7. Trypsin (0.25%) EDTA (0.1%) solution (Mediatech, Herndon, VA).

8. Collagenase solution (0.5 mg/mL of collagenase type 2 (Worthington Biochemical,

Freehold, NJ) in 1 × RPMI).

9. Phosphate-buffered saline (PBS) + EDTA (0.5 mM) solution.

10. Hemocytometer or Coulter counter.

3. Methods

3.1. Cell Aggregation Assay (see Fig. 1)

1. Prewarm aggregation buffer to 37°C (see Note 1).

2. Collect 5.0 × 10

cells in an eppendorf tube.

3. Centrifuge the cells at 300g for 5 min and remove the supernatant.

4. Wash the cell pellet in 1 mL of aggregation buffer and centrifuge again.

5. Using a transfer pipet, vigorously resuspend the cells in 1 mL of aggregation buffer.

During initial experiments, visually inspect the cells using a phase microscope to ensure

that the cells are in a single-cell suspension.

6. Split the sample into two equal volumes (i.e., 500 µL).

7. To one tube, add 65 µL of a heparin solution (see Note 2).

8. To the other tube, add 65 µL of sterile deionized water.

9. Incubate the cells undisturbed at 37°C for 1 h.

10. Resuspend the cells by gently pipetting the solution 5 times. This is a critical step in that

pipetting must be uniformly gentle or aggregates may be disrupted (see Notes 3 and 4).

11. Cells are counted in both the heparin-containing and heparin-free samples using a hemocy-

tometer. A single cell or aggregates containing more than 3 cells are counted as 1. However,

in aggregates consisting of 2 or 3 cells, each cell is counted (see Note 5).

Syndecans in Cell Adhesion and Invasion 497

12. The percentage of cells in aggregates can now be determined as follows:

total cells – (single cells + aggregates)

× 100 = % cells in aggregates

total cells

total cells = number of single cells in heparin-containing sample

single cells + aggregates = number of single cells plus number of aggregates in the heparin-

free sample

Fig. 1. Diagram of the aggregation assay. Cells from each sample are separated into two

tubes containing equal numbers of cells. Heparin is added to one tube (to block aggregation),

while the other cells are allowed to aggregate. Following gentle pipetting, the number of cells

in each tube is determined. Single cells and aggregates containing more than three cells are

counted as 1. Cells within groups of 2 or 3 are counted individually.

498 Langford and Sanderson

3.2. Invasion Assay

3.2.1. Preparing and Seeding Collagen Gels

1. Determine the total volume of collagen gel solution required. Use 1 mL of collagen gel

solution for each well of the 24-well plate (example: 10 wells will require 10 mL of

collagen gel solution).

2. Prepare the collagen gel solution: Add cell-culture grade deionized water to approxi-

mately one-half of the total collagen gel solution required to an appropriately sized cul-

ture tube (see Note 6).

Next, add the collagen stock solution to the final working (see Notes 7–9). Mix by invert-

ing the tube several times and place on ice. Then add 1/10 volume of 10 × RPMI dropwise

while gently shaking the tube. After addition of 10 × RPMI, mix by inverting the tube

several times and place on ice. Now add sodium bicarbonate solution dropwise while

gently shaking the tube to achieve a pH of approximately 7.2–7.4 (see Note 10). Again,

mix by inverting the tube several times and place on ice.

3. Degas the solution by allowing it to sit undisturbed for approximately 20 min on ice (see

Note 11).

4. Gently, so as not to introduce bubbles, pipet 1 mL of the collagen gel solution into each well

of the 24-well plate.

5. Place the plate containing the collagen gel solution into a tissue culture incubator at 37°C and

5% CO

for at least 1 h. Polymerization can be assessed by tilting the plate 45°C. If the

solution remains liquid, discard.

6. To equilibrate the gel, carefully pipet 1 mL of complete medium to the surface of each

collagen gel, and place back into the incubator for at least 1 h (see Note 12). For conve-

nience, the gels may be equilibrated overnight.

7. To each gel, carefully add cells diluted in 1 mL of complete medium and culture at 37°C

in 5% CO

for 48 h (see Notes 13–15).

3.2.2. Quantification of Maximum Invasive Depth

The depth of cell invasion is determined by measuring the distance from the top of

the gel to the leading front of migrating cells using the calibrated micrometer present

on the fine-focus dial of an inverted phase microscope (see Fig. 2). The leading front

is defined as the point at which two of the leading cells within a given field are in the

same focal plane under 200× magnification.

1. Measurements are taken in five fields within each well; using the center of the well as a

landmark and as the first point (center point), the four additional measurements are taken in

fields selected by moving the stage to points north, south, east, and west of center. The distance

to each field is defined as half the distance from the center point to the edge of the well.

2. Starting near the bottom of the gel, turn the fine-focus dial so the focal plane moves toward

the surface of the gel. When two cells appear in the same focal plane, begin counting

revolutions until you reach the surface of the gel.

3. To calculate the distance from the gel surface to cells at the leading front, multiply the

number of revolutions of the fine-focus dial by the number of µm/revolution. For the Nikon

Diaphot used in our lab, 1 revolution moves the focal plane 100 µm.

3.2.3. Quantification of the Percentage of Invasive Cells

The percentage of invading cells is determined by using a limited trypsin and colla-

genase treatment to remove the noninvasive cells (i.e., cells attached at or near the gel

Syndecans in Cell Adhesion and Invasion 499

surface). This is followed by complete digestion of the gel with collagenase and recovery

of the invasive cells (see Fig. 3). Once the numbers of invading and no+ninvading cells

are determined, the percentage of invading cells can be calculated.

1. Collection of the noninvasive cell population:

a. Aspirate the medium from the surface of each gel and discard.

b. Carefully wash the gel with 0.5 mL of PBS + 5 mM EDTA solution and collect by

aspiration into the appropriate tube (see Note 12). Repeat this wash/collection step

two additional times. Place all solutions (i.e., washes, trypsin, and collagenase solu-

tions) collected from each well into the same 15-mL culture tube.

c. Incubate the collagen with 0.5 mL of trypsin/EDTA solution at room temperature

for 30 min and collect the medium and released cells by aspiration. Although this

digestion does not release many cells, it does prepare the collagen for the collagenase

treatment.

d. Wash the gels twice and collect the solution two times as in step b.

e. Incubate the gels with 0.5 mL of collagenase solution (0.5 mg/mL of collagenase in

1 × RPMI, prewarmed to 37°C) for approximately 10 min at 37°C (see Notes 16–17).

f. Quickly but carefully remove the collagenase solution and wash the gels 3 times as

in step b.

Fig. 2. Measuring depth of cell invasion. Following the incubation of the cells on the col-

lagen gels, the distance from the surface of the gel to the cells of the leading front is determined

using the calibrated micrometer (on the fine-focus dial) of an inverted phase microscope. Illus-

trations of collagen gels with an invasive or noninvasive cell line are drawn on the left side of

the figure. Photomicrographs taken of cells within collagen gels are shown on the right. In this

example, for the invasive cell culture, invading cells are visible 300 µm below the surface of

the gel. However, for noninvasive cells, the field at 300 µm below the surface of the gel is

devoid of cells.