0 1992 Wiley-Liss,


Cytometry 13:l-8 (1992)

Simple Method for Quantification of Fast Plasma Membrane Movements’ Nicolas A.F. van Larebeke,2 Marc E. Bracke, and Marc M. Mareel Laboratory of Experimental Cancerology, Department of Radiotherapy and Nuclear Medicine, University of Gent, B 9000 Gent, Belgium Received for publication August 3, 1990; accepted July 30, 1991

We present a method for the quantification of the fast plasma membrane movements that are involved in ruffling, blebbing, fast shape change, and fast translocation. The method is based on the Kontron Vidas image analysis computer program. Video images from cells viewed through an inverted microscope were transmitted to the computer. The procedure was as follows: 4 consecutive video images were averaged (imagel);28 s later a second set of 4 video images was averaged (image 2); image 2 was subtracted from image 1and the grey level of each pixel of the resulting image was increased with 128 grey level units, resulting in the subtraction image, showing a uniform grey background speckled with brighter and darker spots corresponding

Direct analysis of cell motility in vivo is problematic because of the lack of transparency of three-dimensional living tissues. There is evidence indicating that the study of cell motility on a flat surface in vitro is relevant for the situation in vivo because cells have an inherent motility program (11,12,21). Various aspects of cell motility (see Methods for terminology used in this paper) can be assessed in vitro. Several methods have been described to quantify translocation (7,15,23), directional migration including chemokinesis and chemotaxis (13,251, as well as shape change (17,24). Quantification of fast plasma membrane movements involved in ruffling, blebbing, formation or movement of cytopodia, and fast shape change or fast translocation has received less attention. Partin et al. (17) have described an elaborate Fourier analysis for quantification of the various aspects of cell motility, including ruffling. Recently Tatsuka et al. (22) described a method for quantification of “momentary alterations of cell shape” reflecting ruff ling, pseudopodal activity, and fast cell translocation, using automatic image analysis on Allen video-enhanced contrast-differential

to areas of movement. These spots were discriminated and turned into white objects against a black background. Interactive editing was used to delete artefacts that resulted from floating debris. The total area of the discriminated objects was measured, and the parameter motile area in pm2 per cell was calculated. We have applied our method to the study of motility induced in epithelial cell lines by the tumor promoter 12-0tetradecanoyl-phorbol-13-acetate and by epidermal growth factor. Key terms: Cell motility, cell membrane, cell-surface, ruffling, blebbing, cellshape, translocation, image analysis, TPA, EGF

interference contrast images. Here, we report on a simple and rapid method for quantification of fast plasma membrane movements. It is entirely based on the Vidas automatic grey level image analysis program that is commercially available from Kontron (Eching, West Germany). We have tested our method on cell cultures differing in their type or intensity of motility as estimated from time-lapse video films. As an application of our method we analysed the effect of the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA) and of epidermal growth factor (EGF) on the motility of epithelial cell lines.

‘Supported by the Flemish Advisory Commission on Cancer Prevention, by the Fondation Philippe et Thbrese Lefevre and by Actie Kom Op Tegen Kanker 1989. ‘Address reprint requests to N. van Larebeke, Laboratory of Experimental Cancerology, Department of Radiotherapy, University Hospital, De Pintelaan 185, B 9000 Gent, Belgium.



MATERIALS AND METHODS Cell Lines and Culture Media Two variants from the human MCF-7 breast cancer cell family (20) coined MCF-71AZ and MCF-716 were used. The origin of these cells and their method of culture were described by Bracke et al. (3). Human A431 vulva carcinoma cells (8) were obtained from Dr. De Brabander (Janssen Pharmaceutica, Beerse, Belgium). They were cultured in Eagle’s minimum essential medium (modified) with Earle’s salts and non-essential amino acids (Gibco BRL, Gent, Belgium) supplemented with 10% (viv) fetal bovine serum (Gibco), 0.05% (wiv) L-glutamine, and 250 IU1ml penicillin. Rat G8-111-1 rhabdomyosarcoma cells (9) were obtained from Dr. C. Gerharz and Dr. H. Gabbert (Pathologisches Institut, Johannes Gutenberg University, Mainz, FRG). The culture medium was Dulbecco’s modification of Eagle’s medium supplemented with 10% (v1v) FBS, 0.05 %(w/v) L-glutamine, 250 IU1ml penicillin, 100 Fgiml streptomycin, and 1% of a 1M Hepes buffer solution. In order to reduce debris, the culture medium was filtered through a non-adsorbent acrodisc filter with 0.45 Fm pores (Gelman, Ann Arbor, MI). Drugs MCF-7/6 cells were treated with lop6M dexamethasone (Diosynth, Oss, Holland, cat No. 91225141), known to inhibit ruffling in these cells (3). FBS was substituted by 5% Ultroser G (IBF, Villeneuve-La-Garonne, France, cat. No .250902) because this substitution inhibited ruffling of MCF-716 cells (3) and caused the appearance of many vesicles (our unpublished (TPA; results); 12-0-tetradecanoyl-phorbol-13-acetate Consolidated Midland Corporation, Brewster, New York) was dissolved in DMSO a t 10 Fgiml and cell cultures were treated with 10 ngiml (final concentration). Final concentration of DMSO used as solvent in culture medium was 0.1%. Epidermal growth factor (EGF) (Sigma, St Louis, Missouri, cat. No. E4127) was dissolved in a 9 g/l NaCl solution at 10 Fgiml and cell cultures were treated with 50 ngiml.

settings were 32 x bf, 32 x ph, 20 x bf and 20 x ph, each used with illumination of a given, fixed intensity.

Video A light-sensitive high resolution WV-185OC camera (National Panasonic, Tokyo, Japan), with the automatic gain control switched off, transmitted video images to the frame grabber unit of the image analysis computer. Video films were made using a Umatic VO5850P videorecorder (Sony, Tokyo, Japan) equipped with a n animation control unit AC-580 (EOS, Barry, U.K.), a time date generator, and a n impulse generator. For time-lapse films, two video images were recorded every 15 s. The video films were viewed at the normal play-back speed of 25 images per s.

Cell Motility Terminology In the present paper, terms describing different aspects of cell motility have been used according to the following definitions: Motility describes any form of active cell movement. Translocation indicates movement of the geometric center of the horizontal projection of the cell on the substrate. Mzgration means translocation over a longer distance so that the displacement can be assessed without determination of the geometric center of the cell. Directional migration points to migration th a t occurs preferentially in a certain direction and that is measured in that direction, regardless the cause of directionality (13). Shape change is any change in the shape of the horizontal projection of the cell on the substrate. Ruffling is the formation, retraction, or movement (1,5) of sheetlike cytopodia, called ruffles or lamellipodia (2). Blebbing indicates the appearance, disappearance, or movement of spherical or hemispherical cytopodia ranging in diameter from 1 Fm to 10 Fm (2). Pseudopodal activity means the formation, retraction, or movement of pseudopodia, the latter being larger cytopodia of variable widths (from 0.5 to more than 3 Fm) (2).We use the term fastplasma membrane movements (FPMM) for any movement of the plasma membrane noticeable within a short period (28 s in our present observations). Image Analysis A Vidas image analysis computer with frame grabber, EGA board, and overlay board was loaded with software packages Vidas release 1.3 and Videoplan release 2.1 provided by Kontron (Eching, West Germany). A suitable scale factor was introduced, depending upon the magnification of the objective used. In digitizing a n image, the Vidas discriminates between 256 grey levels, numbered from 0 (=black) to 255 (=white).

Microscopy Cells in 25 cm2 plastic tissue culture flasks with 5 ml medium were placed on a Zeiss IM inverted microscope (Zeiss, Oberkochen, FRG) that was included in a thermostatted chamber at 37CO.l0C, equipped with a n electronic thermosensor. Continuous gassing was provided with humidified air containing 5% or 10% CO,, depending on the culture medium used. F-LD 3210.4 The Macroprogram for Quantification of Fast and F-LD 2010.25 objectives were used in combination Plasma Membrane Movements with a long working distance condensor (Zeiss No. 465224). We use the term optical setting for the comOur procedure consists of a sequence of functions bination of a particular objective with either bright each of which is provided by the Vidas program. From field (bf) or phase contrast (ph) illumination. Optical each microscopic field, 4 consecutive video images,



FIG. 1. Some essential steps of the quantification of fast plasma membrane movements. A group of MCF-7IAZ cells is analysed starting from digitized video images obtained through an F-LD 32/0.4 objective with bright field illumination. a: Image IL. b: Image 2. E: Subtraction image. d: Discriminated image. Bars = 40 pm.

taken within 0.16 s, were digitized and the corresponding averaged image was computed (image 1; Fig. la). After a time interval (At) of 28 s a second set of 4 video images was taken and the corresponding averaged image was computed (image 2; Fib. lb). The number of cells under study was counted on one of those images. Then image 2 was subtracted from image 1 and the grey level of each pixel of the resulting image was increased with 128 grey level units to give the subtraction image (Fig. lc). This subtraction image showed a uniform grey field with brighter and darker spots where grey values had diminished or increased respectively in the time interval from image 1 to image 2, corresponding to areas where fast movements had taken place. Brighter spots in the subtraction image, corresponding to pixels or groups of pixels with a grey

value above a chosen fixed level, and darker spots, corresponding to pixels or groups of pixels with a grey level below a chosen fixed level, were discriminated and turned into white objects with grey value 255 in the discriminated image (Fig. Id). Discriminatory grey values were chosen as to exclude detection of pixel to pixel video camera noise. A contour function was used to project the contour of the discriminated objects in overlay on the original subtraction image; then, by rapidly switching back and forth between the resulting image and the two original averaged images, the subtraction image was carefully screened by the observer for artefacts due to floating debris. An edit function allowed the interactive deletion of any artefact before the measurements were done. Then, a field measurement program was used t o count the number of dis-



criminated objects in the field and to measure their total surface area. In some experiments a scrap function was used to eliminate all discriminated objects smaller than 2 Fm2. The parameter motile area in pm2 per cell was obtained by dividing the surface of the total area detected a s involved in movement (sum of the areas of all discriminated objects in the field) by the number of cells in the field. The parameter number ofmotile spotsper cell was obtained by dividing the total number of discriminated objects in the field by the number of cells in the field. So, for each field under study the method produced two numerical values, reflecting changes in grey level (corresponding to cellular movements, mainly FPMM) over a 28 s period.

Experiments and Statistics Measurements were done 1 to 3 d after seeding 200,000 cells per flask. To evaluate the effect of the length of At, real time video films of groups of cells were made; on video images taken from films, FPMM was measured with images 1 and 2 separated by a A t of increasing length, which means measurements were made by subtracting a n image 2, taken at different times after image 1,from one and the same image 1. In a second type of experiment, FPMM of a population of cells in a flask was compared to that of a cell population in another flask. Results are presented a s scatter graphs, in which each point represents the result of a measurement of the motile area in pm2 per cell on a group of cells in one video-microscopic field. For statistical analysis of the significance of observed differences between groups of measurements (treated vs. control) we used the one-tailed non-parametrical Mann-Whitney U test. In a third type of experiment the FPMM of one and the same group of cells, in one microscopic field, was measured a t different points in time, before and after treatment. The 32 x bf optical setting was used. Results of measurements of motile are in p,m2 per cell are presented in function of time in a line graph. RESULTS The Method Is Sensitive to Ruffling and Other Fast Plasma Membrane Movements With a culture of MCF-716 cells that were intensively ruffling, as judged from time-lapse video films, the subtraction image showed lighter and darker patches a t places of ruff ling. By looking a t the image screen of the Vidas computer while switching continuously back and forth from image 1 to image 2, separated in time by only 28 s, we observed some change in areas of ruffling, whereas other parts of the cells remained unchanged. Apparently, ruffling was responsible for most of the differences between images 1 and 2, and, consequently, for most of the lighter and darker spots in the subtraction image and for most of the discriminated objects produced by the program. The parameter number of motile spots per cell does in no way give a n indication of

the number of ruffles or other zones of FPMM as observed directly with the microscope o r on time-lapse video films. Each visible ruffle usually leads to the detection of many small motile spots distributed over one ruffle. Fast pseudopodal activity and fast translocation also gave rise to a signal. With the G8-111-1 rhabdomyosarcoma cells chosen for intense blebbing, we observed that blebbing also gave rise to discriminated objects. Measurements of FPMM on groups of MCF-716 or other cells with images 1 and 2 separated by a A t of increasing length show that motile area in pm2 per cell was always zero for At=0, increased quasi linearly with A t for values of A t between 10 and 40 s or more, and increased less than linearly afterwards. For groups of cells showing intensive FPMM the slope corresponding to the linear increase was steeper than for cells showing very little FPMM, but the latter show a quasi linear increase up to At’s of greater length (data not shown).

Moving Intracellular Structures Sometimes Also Induce a Signal By comparing a Vidas image showing the discriminated objects with one of the original images (image 1 or image 2) of MCF-716 cells treated with Ultroser G, it appeared that the method not only revealed FPMM, but detected, to a lesser extent, also rapidly moving intracellular vesicles. A signal was produced occasionally by nuclei during nuclear rotation or rapid translocation and by chromosomes during mitosis. Interactive correction for these movements was not included in the proposed method because it was time-consuming and introduced a subjective element. Effects of Optical Setting The kind of optical setting had a strong influence on the part of the cells that was detected as motile area (Fig. 2), the type of detected movement, and the sensitivity of detection. Using phase contrast or bright field illumination, with both the 20 x and the 32 x objectives, we measured groups of MCF-716 cells with few intracellular vesicles, chosen after videomicroscopic observation for intense, moderate, or little ruffling activity, and a group of vesicle-rich MCF-716 cells displaying little ruffling activity. These groups of cells were selected respectively from untreated cultures and from cultures treated with dexamethasone or with U1troser G (see Materials and Methods). As can be seen from Figure 3 and 4,the method allowed one to discriminate between cells differing in ruffling activity. Also, intense or moderate ruffling could be detected in MCF-7/6 cells under conditions in which movement of intracellular vesicles remained to a large extent (32 x bf; 20 x ph) or almost completely (20 x bf) undetected as measured by the parameter motile area in pm2 per cell. As compared to cells with many moving vesicles and little ruffling, intensely ruffling cells showed 5.47 (32 x bf), 8.67 (20 x ph), or 108.38 (20 x bf)





ccc1 I

I1A Ill I V


I1 111 I V



I1 I0 IV






FIG.3. Motile area in pm2 per cell (top) and number of motile spots per cell (bottom) obtained with optical settings 32 x ph (I), 32 X bf (111, 20 x ph (III), and 20 x bf (IV) for the following groups of MCF-7/6 cells. A: Intensely ruffling cells ( n = 8 ) 24 h after seeding. B: Moderately ruffling cells (n=48) 3 d after seeding. C: Cells with little ruffling (n = 25) treated with 1 O P M dexamethasone during 3 d. D: Cells with many intracellular vesicles and little ruffling (n = 13) from a culture treated with 5%Ultroser G 24 h after seeding and measured 2 d later. Measurement on the same group of cells were done within 1h, with at least 3 measurements per optical setting. Mean values are shown; coefficients of variation (not shown) were about 10%.

FIG.2. Photographs from the image screen of the Vidas computer, taken within 10 min, showing (left column) the same group of MCF7/6 cells, viewed using different optical settings and (right column) the respective discriminated images. a and e: Phase contrast. x 32. b and f: Bright field. x 32. c: and g: Phase contrast. x 20. d and h Bright field. ~ 2 0Bars . = 40 bm.

times more motile area in pm2 per cell. It could also be seen that, for MCF-716 cells, the parameter motile area in pm2 per cell was more discriminatory than the parameter number of motile spots per cell when intensely ruffling cells were compared to less ruffling cells, and when intensely or moderately ruffling cells were compared to vesicle-rich cells. The parameter number of motile spots per cell was more sensitive to the presence of vesicles than the parameter motile area in km2 per cell. Eliminating (by the Vidas scrap function) discriminated objects smaller than 2 pm2 did not increase the specificity of the method for ruffling (data not shown). Measurements done with the 32 x ph optical setting were less sensitive to differences in ruffling activity in MCF-7/6 cells than those done with the 32 x bf setting, but were on the contrary very sensitive to the presence of vesicles (Fig. 4). The 20xbf setting was not very Sensitive and resulted in lower numerical values, detecting only movements involving somewhat larger areas; this setting had very good discriminatory power as to the difference between and less intensely ruffling MCF-716 cells, and was almost completely insensitive to the presence of moving intracel-





1 12 1 4 1

.= rn

I 0

i TPA -treated




















Solvent Control

FIG.5. FPMM of MCF-7/AZ cells, 3 d after seeding, between 12 and 45 min after addition of 10 ngIml TPA or of 0.1% DMSO, measured with a 32 x bf optical setting. Each symbol represents 1 randomly chosen field. TPA-treated f l a s k 10 fields with a total of 473 cells were measured; solvent control flask: 10 fields with a total of 401 cells. Median values are indlcated by a horizontal line (P=0.001).


FIG.4. Ratios for 2 groups of cells between the values from Figure 3 of the parameters motile area i n km2 per cell (top) and number of motile spots per cell (bottom) obtained with optical settings 32 x ph (I), 32 x bf (111, 20 x ph (III),and 20 x bf (IV).

lular vesicles. The 20 x ph setting had comparable discriminatory power as the 3 2 x b f setting, and was slightly less sensitive to the movement of vesicles.

35 30


Fast Plasma M e m b r a n e Movements Induced b y T P A and by EGF From time-lapse video films we observed that TPA induced ruffling in MCF-7IAZ cells. Quantification of FPMM allowed objective confirmation of this induction of ruffling (Fig. 5). Motile area in Fm'per cell was 6.64 Fm2 k 2.81 pm2 (mean value standard deviation) for MCF-7IAZ cells between 12 and 45 min after receiving 10 ng TPAIml(10 randomly chosen fields with a total of 473 cells were measured) as opposed to 2.77 pm2 k 2.02 pm2 for cells treated with 0.1% DMSO alone (10 randomly chosen fields with a total of 401 cells were measured). This difference was statistically significant (P = 0.001). Our method offers the possibility to follow FPMM as a function of time (Fig. 6). Our observations demonstrated a steep increase in FPMM within 40 min after addition of TPA. EGF is known to induce ruffling in A431 cells (4). We monitored induction of FPMM by EGF (Fig. 7): after 6 rnin a n increase in FPMM was detectable and peak activity was reached after 19 min; after 40 min FPMM had decreased to a steady state level that still exceeded the prestimulation values. By visually comparing digitized images taken with a time interval of












Time in minutes from addition of TPA or solvent

FIG.6. FPMM of MCF-7IAZ cells ( n = 15) before and after addition of 10 ngiml TPA).( and of MCF-7IAZ (n = 8) before and after receiving 0.1% DMSO (0).

28 s we could ascertain that ruffling was responsible for most of the FPMM detected in EGF-treated A431 cells. EGF also caused a n increase in FPMM in MCF-7IAZ cells (Fig. 7). Here peak activity was measured after 4




-20 0 20 40 60 Time 13minuies from addition of EGF

FIG.7. FPMM of A431 (n=4) (A)and MCF-7IAZ ( n = 15) ( 0 ) cells, before and after addition of 50 ng/ml EGF.

min, and after 8 min activity had returned to prestimulation levels. Ruffling was responsible for most of the FPMM also in MCF-7IAZ cells.

DISCUSSION The proposed method allows some form of quantification of FPMM, including ruffling, blebbing, fast pseudopodal activity, fast shape change, and fast translocation. In fact, the method measures to what extent the cell surface is involved in movements that affect the microscopic image of the cell. Measurements are not absolute, allowing only comparisons between cell populations in matched experiments. In the epithelial cell lines used in this study, the main type of FPMM was ruffling. The phorbol ester tumor promoter TPA is known to induce ruff ling in several epithelial cell lines (6,10,16,18,19)as EGF does in A431 cells (4). Here we objectified this effect for TPA on MCF-7/AZ cells and for EGF on A431 cells. Our method is suited for monitoring FPMM in function of time: we showed the time course of induction of ruffling by TPA in MCF-7/AZ, and by EGF in A431 cells. Monitoring FPMM as a function of time showed that EGF also induces FPMM in MCF-7IAZ cells, an effect that could have escaped attention as it lasted only for a few minutes. The method can be adapted to particular circumstances by the appropriate choice of optics, time interval, and threshold grey level values. We now most frequently use the optical setting 32 x bf, but we prefer the 20 x bf optical setting when intense plasma membrane motility has to be measured with minimal interference from moving intracellular particles.


We have not used other systems that allow assessment of part o r all of FPMM because either they rest on subjective judgement and do not provide quantification (14),or they are based on manual tracing of cell contours and are insensitive to ruffling projecting inside these contours (171, or they require more expensive and sophisticated equipment (22). The visual grading system developed by Mohler et al. (14)is insensitive to artefacts, allows separate assessment of different types of FPMM, but is time-consuming, rests on subjective judgement, and does not provide quantification. The method proposed by Partin et al. (17) is based on mathematical analysis of cell contours that have to be traced manually. It gives a very complete picture of cell motility, allows one to distinguish “directionally persistent” from “random cell walks,” and provides quantitative information on “undulation of the cell membrane,” ruff ling, and pseudopodal activity, in addition to information on cell shape. Possible drawbacks might be that the method is quite complex; that, in our experience, manual tracing of cell contours is time-cons u i n g ; that, especially where cells are in close contact, it is insufficiently precise to permit assessment of FPMM; and that it might be less sensitive to ruffling. There is a much less than proportional increase in the Fourier ruffling index as the percentage of total cell contour occupied by ruffling increases, and the method cannot detect ruffling activity that does not affect the cell contours. Ruffles that project inside these contours (1,2,5)will not be taken into account. We assume that the method of Partin et al. (17) is not suited for the assessment of ruffling in cells enclosed by other cells such as in epithelial cell islands and in confluent monolayers. Recently Tatsuka et al. (22) described a method for quantification of “momentary alterations of cell shape” reflecting ruffling, pseudopodal activity, and fast cell translocation, using automatic image analysis on Allen video-enhanced contrast-differential interference contrast images. This method is based on quantification of the intensity of a “trace image” obtained by subtracting a digital image of cells in a video frame from a digital image of the same cells in a frame taken 20 s later. It requires a more sophisticated equipment than our method, does not provide for interactive correction of artefacts, and does not provide visual control of the type of movement that is being detected. It takes into account the number of pixels (area) in which any increase (no discriminatory levels) in grey value is detected and the magnitude of the increase, whereas our method takes into account the number of pixels (area) in which either an increase or a decrease exceeding a fixed discriminatory grey value has taken place. Our method allows visual control of the type of movement that is being measured as well as interactive correction for artefacts. Its major drawback is its sensitivity to artefacts due to movement of intracellular vesicles. Our method allows about 8 cell cultures to be compared in 1 working d; in experiments concerned with monitoring FPMM as a function of time our



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present equipment permits 1 measurement to be done every 2 min. Our method is well suited for the study of the regulation of cell motility. The method is currently used in the search for motility factors that are possibly implicated in the regulation of invasion.

Simple method for quantification of fast plasma membrane movements.

We present a method for the quantification of the fast plasma membrane movements that are involved in ruffling, blebbing, fast shape change, and fast ...
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