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Morphology and cellular origins of substrate-attached material from mouse fibroblasts
Printed in Sweden Copyright @ 1977 by Academic Press, Inc. All rights of reproduction in any form reserved ISSN 0014-4827
Experimental Cell Research 107 (1977) 139-149
MORPHOLOGY
AND CELLULAR
SUBSTRATE-ATTACHED MOUSE
ORIGINS
MATERIAL
OF
FROM
FIBROBLASTS
J. J. ROSEN* and Ll. A. CULP’ ‘Department of Microbiology, School of Medicine, and *Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH44Io6, USA
SUMMARY Scanning electron microscopy @EM) has been used to determine the pattern of reorganization of substrate-bound cell surface material durinn attachment of Swiss 3T3 mouse tibroblasts to fresh serum-coated glass substrates after prior-detachment with the Ca*+-specific chelator EGTA (ethylene&s-(oxyethylenenitrilo)-tetraacetic acid). A limited number of filonodia make contact with the substrate surface and branch into finger-like extensions. A thin web-like membrane organizes between these filopodial extensions to eventually form mature footpads which appear to have a limited size. Footpads appear to be the principal morphological entities mediating cellsubstrate adhesion. The mechanism of EGTA-mediated detachment of cells and the morphology of the substrateattached material (SAM) left adherent to the substrate after complete removal of cells have also been examined. EGTA treatment results in cell rounding, with minimal effects on the morphology of the adhesive footpads which remain linked to the cell body via stressed retraction fibers. These stressed fibers eventually break, leaving footpads on the substrate as SAM and liberating the cell body into suspension. This evidence suggests that EGTA treatment minimally affects adhesive materials in the footpad site but disorganizes the cytoskeleton of the cell with resultant shearing of the cell body away from the footnads. The size, mornhologv, and pattern of SAM left on the substrate establish-that it is essentially footpad material: SAdfrom highly-spread cells appears to be virtually-intact footpads, while SAM from attaching cells appears to be disrupted footpad material. These results are discussed in terms of the morphological and biochemical organization of the footpad adhesion sites of cells.
The cellular origin and molecular organization of the substrate-attached material (SAM), left tightly bound to the tissue culture substrate after EGTA (ethylene-bis(oxyethylenenitrilo)-tetraacetic acid)-mediated removal of normal and virus-transformed mammalian cells, have been of considerable interest because of the prospective function of this material in mediating cell-substrate adhesion [7]. Biochemical ’ To whom reprint requests should be addressed.
analyses [6, 201 have established that SAM is composed of hyaluronate proteoglycans, the LETS (large-extemal-transformationsensitive) glycoprotein [ 121, microfilamentassociated proteins, and a number of unidentified proteins. These components are present in SAM in similar relative proportions after study of a variety of cellular attachment and growth conditions [6, 71 and are coordinately resistant to removal from the substrate with a number of dissociative reagents, suggesting that these Expfl
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components exist as a tightly-associated cell ‘surface’ complex which mediates adhesion. The presence of microfilament-associated actin and a myosin-like protein in SAM [6] indicated that treatment of substrate-bound cells with the Ca2+-specific chelator EGTA was not merely dissociating external cell surface macromolecules, with resultant separation of the intact surface membrane from the substrate, but that a more complicated process was occurring, perhaps with ‘pinching-off of a membrane vesicle. Autoradiographic evidence [5] has confirmed that radioactive SAM protein and polysaccharide are topographically arranged in focal patterns very similar in density to the footpads by which the cell adheres to the substrate [ 171. In order to further analyse the cellular origin of SAM, we have utilized scanning electron microscopy @EM) to determine the morphology and cellular origins of this material. Revel and his colleagues f17, 181 have demonstrated that cells adhere to the substrate at focal regions of the surface membrane called footpads and that trypsin treatment for short periods minimally affects footpad morphology, while causing rounding-up of the cell body and detachment via breakage of the elastically-labile retraction fibers of the surface membrane. Similarly, SEM has been used to study the morphological events of re-attachment of trypsinized cells to serum-free [16] or serum-coated [9] substrates. In this study, we report the morphological re-organization of substrate-attached cell surface processes during attachment of EGTA-subcultured cells to fresh serumcoated glass substrates. Interest has also been focused on the mechanism by which EGTA-mediated detachment of cells occurs, as well as the morphology and cellular Expti Cell Res 107 (1977)
origin of SAM which resists EGTA action MATERIALS
AND METHODS
Cell growth Swiss 3T3 mouse fibroblasts were utilized for these studies and were routinely grown as described previously [4] in Eagle’s minimal essential medium supplemented with four times the normal concentration of amino acids and vitamins (MEMx4), 10% donor calf serum, penicillin (250 U/ml) and streptomycin (0.25 mg/ml); in Brockway glass bottles at 37°C; and in an incubator with humidified air and a 5 % CO, concentration. These cells were assayed for and were found to be free of Mycuplusma contamination. When these cells reached approx. 70% confluency (prior to becoming density-inhibited), they were removed for experimental use by gentle shaking at 37°C with 0.5 mM EGTA in PBS. These cells were enumerated with a hemocytometer, pelleted by centrifugation, and resuspended in MEMx4 to a final concentration of 2.0x l@ cells/ml. This suspension was inoculated into tissue culture dishes (containing glass coverslips) for experiments.
Preparation of glass coverslips Glass coverslips, I5 mm diameter, were acid-washed, rinsed in distilled water eight times, rinsed in 95% ethanol three times and flamed before being placed in 10 cm Falcon plastic tissue culture dishes (five coverslips/dish). At least 2 h before an attachment experiment was initiated, the dishes were filled with 15 ml of MEMX4 with 10% donor calf serum and equilibrated at 37°C. The MEMx4 was replaced with fresh medium before cell inoculation.
Cell attachment Two million cells, prepared as indicated above, were inoculated into coverslip-containing dishes. After gentle shaking to uniformly disperse the cells, the coverslips were repositioned around the dish. At times varying from 15 min to 24 h after inoculation of cells and incubation at 37”C, coverslips were carefully removed from the dish, gently dipped twice in each of three batches of PBS-II, drained, and placed in buffered 0.1% glutaraldehyde solution in preparation for electron microscopic analysis. The kinetics of attachment of these cells to serum-coated substrates have been established [4,8].
Cell removal by EGTA treatment After cells had been allowed to attach to serum-coated glass substrates for times varying from 15 min to 24 h at 37”C, the detachment process was studied by exposing the cells to EGTA. The medium was first decanted from the dishes. and the cells were rinsed twice with PBS. A 0.5 mM EGTA solution in PBS was added to the dishes, followed by gentle shaking at 37°C for times varying from 5 to 25 min. When com-
Morphology
of substrate-attached
material
141
Fig. 1. The morphology of Swiss 3T3 fibroblasts during attachment and spreading on glass coverslips. Cells which had been subcultured by EGTA treatment were inoculated into tissue culture dishes containing coverslips in medium with 10% donor calf serum. After attachment had proceeded for various times, the
cells were fixed with glutaraldehyde, and prepared for SEM as described in Materials and Methods. The high tilt angle (80”) permits more effective visualization of cell-substrate interactions. Cells are shown after (a) 30 min; (b) 60 min; (c) 2 h; (d) 24 h of attachment. Original magnification x 2 800.
plete removal of all cells was desired, the dishes were gently pipetted after 25 min of exposure to EGTA to examine the SAM left adherent to the substrate [6, 7, 203. The coverslips were gently rinsed twice in each of three batches of PBS-II, drained, and placed in a buffered 0.1% ghttaraldehyde solution in preparation for electron microscopic analysis.
followed by graded Freon 13 solutions in ethanol with three changes in 100% Freon 13 (all incubations at 4°C). Sampies were then critical point dried in Freon 113, snutter-coated with gold-palladium, and desiccated until observation in a Cambridge S4-10 Stereoscan scanning electron microscope.
Materials Preparation of samples for scanning electron microscopy Single cells attached to substrates are extremely fragile and must be treated with the utmost care if the fine ultrastructural details are to be accurately preserved. After rinsing in PBS-II, the coverslips were transferred to a 0.1% glutaraldehyde solution buffered with sodium cacodylate (0.06 M) in Ringer’s solution (uH=7.35) and osmotically balanced with sucrose a.12 M).‘After 1 h incubation at room temperature, the coverslins were transferred to a 3 % glutaraldehyde solution (incacodylate buffer) and stored at 4°C overnight. Dehydration was accomplished by a series of graded ethanols with three changes in 100% ethanol,
Plastic tissue culture dishes were purchased from Lux Scientific Co.; round glass coverslips (15 mm 0) from A. H. Thomas Co.; EGTA from Eastman Organic Chemicals; SDS from Matheson, Coleman & Bell; MEMx4 from Gibco; donor calf serum from Flow Laboratories.
RESULTS The cellular origins of substrate attached material (SAM) have been investigated using scanning electron microscopic (SEM) Exptl Cell Res 107 (1977)
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Fig. 2. Higher magnification SEMs of substrate interaction sites. Cells were inoculated into coverslipcontaining dishes after EGTA subculturing as described in tig. 1. Initial contact (a) of cells with the substrate involves extension of a limited number of lilopodia (F) onto the substrate and away from the cell body (CB). Within 1 h (b), primative pads (P) begin to
form on the substrate around the periphery of the cell body at the contact point of tilopodia with the substrate. These pads continue to grow in size and to extend filopodia from their outer margins during the next few hours (c), developing into a mature ‘footpad within the first day after initial attachment (4. Original magnification X 13000; 20” tilt.
techniques. The properties of this material are dependent on the duration of cell attachment and spreading, but in all cases it appears to be the remains of ‘footpads’ or cellular extensions tightly bound to the substrate during the attachment process. The significant morphological heterogeneity of substrate-bound cell surface material was difficult to document photographically at any given point during the attachment processes. The photomicrographs presented here were selected to illustrate generally observed patterns of cellular morphology felt to be characteristic of these processes.
Substrate interaction during attachment of EGTA-subcultured cells A cell in suspension after EGTA treatment and prior to attaching to a fresh serumcoated substrate is covered with blebs and a few microvillous structures. Within 1.5min of attaching to the serum-covered glass, a few filipodial extensions have already extended along the substrate beyond the radius of the cell body (fig. la). These extensions are approx. 0.1 pm in diameter and up to 20 pm long (fig. 2a). They emerge from the cell body near the cell surface closest to the substrate.
Exprl Cell Res 107 (1977)
Morphology of substrate-attached material
Fig. 3. Filopodial and footpad morphology. SEMs taken at high magnification (original magnification X26000) and a high angle of tilt (80”) illustrate the heterogeneity of cell attachment regions on glass. These cells have both been in contact with the substrate for 1 h. In (a), fllopodial extensions (FE) stretch between the cell body (CB) and the substrate and appear to be under tension. In (b), the complex ‘cording’ of the extensions from the cell body (CB) to the footpad (P) are apparent; this footpad morphology is more typical of cells during later stages of attachment and cell spreading.
During the first half hour of attachment, small pads (approx. 2 pm across) start forming on the substrate, often with filopodia projecting beyond these pads across the substrate (fig. 2b, c). The cell body after 1 h has lost many of its surface blebs and now has begun to actively spread across the substrate (fig. 1b). By the end of the 2nd h, most cells are well-spread (fig. 1c). Fig. Id shows a typical fully-spread cell 24 h after initial attachment. The cell surface is almost completely devoid of blebs or microvilli as demonstrated previously [9], lo-771818
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and only a few attachment filopodia extend from the cell border to the substrate (the substrate attachment regions are often hidden from view). The limited number of discrete contacts with the substrate that are evident are made through broad membranous extensions to mature footpads, often 15 km or more in diameter (fig. 2d). The substrate contact points during later times of attachment, called footpads [17], are web-like (up to 10 pm across) and are often connected to the cell body by broader regions of membrane (3-5 pm) contiguous with the cell body (fig. 2d). It was very common to see raised ridges, or ribs, in footpads which appeared to be extensions of the filipodial processes extending from the cell body to the footpad. The leading edge of web-like material that became organized between branching filopodia on the substrate was extremely thin (less than 0.05 pm). These webs appear to organize and spread over the substrate away from the branching point of two or more substratebound filopodia (see right side of fig. 2~). The substrate interaction during early attachment begins with tentative contact between thin attachment fibers or filopodial extensions (fig. 3a, FE) and the substrate in the close vicinity of the newly attached cell. These fibers appear to coalesce into bundles (fig. 3 b) or bifurcate along the substrate surface (fig. 2a). In general, bifurcation of filopodia on the substrate occurred early in the attachment process, while broadening of the membrane linking the footpad and the cell body via coalescence occurred later in the attachment process. After the first hour of attachment, the attachment tilopodia that are present begin to thicken, and increasing amounts of membranous material appear to spread over the substrate as contact pads. These contact pads (fig. 2b) gradually mature into fully ExptlCellRes157 (1977)
Fig. 4. Morphological changes during EGTA-mediated detachment. After 2 h of attachment to glass coverslius. the cell medium was reulaced with a 0.5 mM soiution of EGTA, as described in the Materials and Methods (after gentle rinsing with PBS), for 10 min prior to fixation with glutaraldehyde. Cell retraction away from the substrate as a result of cell body (CB) rounding ((a) (orig. magnification x6800; 80” tilt) puts retraction fibers in tension; (b) (orig. magnification x6800; 20” tilt) shows fibers in tension, fibers that have broken (II), and early signs of disruption of attachment footpads (D); (c) (orig. magnification x26000; 80” tilt) illustrates fiber breaks and the footpad material that remains bound to the substrate.
developed footpads (fig. 3b) connected to the cell by broader areas of surface membrane. Mechanism of EGTA-mediated detachment of cells When cells which have been attached to a serum-coated glass substrate are exposed to the Ca2+ specific chelating agent EGTA, two distinct morphological changes occur which lead to eventual detachment of the cell from the substrate. If a cell was wellspread on the substrate prior to the EGTA treatment, it rounds up assuming a spherical shape and large sheets of cell membrane lift off the glass during the retraction process, as shown in fig. 4a. The foreground of this micrograph shows many mature footpads which cannot be observed under the spread cell. The retraction fibers [17] of ExptlCellRes
107 (1977)
stressed membrane are put under considerable tension with one end anchored at the footpad, which appears to be minimally affected by early EGTA treatment, and the other end at the surface of the cell body (fig. 4b). Eventually this tensile force is great enough to break the retraction fibers during agitation of cultures, leaving the footpad and a piece of the fiber behind on the substrate (fig. 4b, c). During routine cell removal, shearing forces generated by shaking and pipetting of the culture probably cause the breakage of the cell body away from the substrate, leaving footpads virtually intact. Although the utmost care was taken to preserve the fine structure of membranous processes during the experiments, there is no doubt that some of the observed breaks of tilopodia in attaching cells (e.g., fig. 2a, fig. 3a) and broader
Morphology of substrate-attached material
145
during detachment. The specific nature of this degradation process is not understood but will be considered in analyses of the SAM left adherent to the substrate due to its resistance to EGTA treatment. Morphology of SAM When cells are completely removed from the substrate by EGTA treatment, the material that remains behind is morphologically organized into patterns that strongly suggest their cellular origins. Fig. 5a shows mature footpads and filopodia associated with an attaching cell in the process of spreading out onto the substrate. Fig. 5 b, taken at the same magnification and angle of tilt as fig. 5 a, shows the pattern of the SAM left on the substrate when cells grown under the same conditions are removed by EGTA treatment. This treatment produces a roughened, nodular array of membranous-looking vesicles and appears Fig. 5. Morphology of footpads and substrate-attached to remove any traces of filopodia on the material (SAM). EGTA-subcultured cells were persubstrate; but the size, shape, and distribumitted to reattach to glass coverslips in serum-containing medium for 1 h to allow formation of footpad tion of SAM corresponds closely to the (8’) attachments extending from the cell body (CB); general morphology of the footpad structypical footpads are shown in (a). A second set of coverslips with cells which had been attaching for 1 h tures observed around the periphery of were then treated with EGTA as described in the attaching cells. Most discrete areas of SAM Materials and Methods to completely remove cells for subsequent observation of SAM; two typical pools of also displayed a larger nodule that probably SAMs (S) are shown in (b) with nubs of membranis the remainder of broken retraction fibers ous material where presumably the retraction fibrils had broken away. Original magnification x 13000; like those shown in fig. 4c. 20” tilt. The SAM from well-spread cells (in conretraction fibers in EGTA-treated cells tact with the substrate for time periods of were due to handling the samples while 24 h or more) appears to be virtually-intact these weakened fibers were in tension. footpads of somewhat larger size than SAM However, control experiments using buf- from attaching cells; two examples of this fered saline instead of EGTA showed no type of SAM are given in fig. 6a, b. This is cell retraction and very few fractures. consistent with the larger size of footpads in The other primary effect of EGTA during well-spread cells when compared to those removal of attaching cells (but not well- of attaching cells. The SAM left on the substrate by cells spread cells) appears to be partial disruption of the immature footpads. One pad in fig. removed after short periods of attachment 4b (0) illustrates the start of this process appeared to be highly-disrupted footpad which was also seen on many other cells material (fig. 7a), i.e., small membranous Exptl Cell Res 107 (1977)
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peared to be a remnant of the retraction fiber (fig. 6). However, the upper surface of SAM pools appeared to be highly disorganized (fig. 6), perhaps as a result of relaxation of membrane after breakage of the stressed retraction fiber (the upper surface of cellular footpads consistently appeared as a smooth and well-organized surface). If the cells were initially inoculated at low density so that individual cells were wellisolated on the substrate, the SAM left after cell removal was often observed to form patterns that corresponded in size and distribution to the initial point of attachment
Fig. 6. Morphology of SAM from highly-spread cells. (a) (Orig. magnification x 13000; 80” tilt) shows a SAM pool remaining after 24 h of cell attachment and spreading followed by EGTA-mediated detachment as described in Materials and Methods to completely remove cells; (b) (orig. magnification x24000; 71” tilt) is an example at higher magnification of the disorganized membrane on the ‘upper’ surface of SAM pools as a result of relaxation of the remnant of the broken retraction fiber; the only consistent morphological structure observed on the ‘upper’ surface of these footpad SAM vesicles was a nub of membrane where the retraction fiber had presumably broken.
vesicles (fig. 7a, S) appeared on the substrate in a pattern suggesting that the ,periphery of the footpad was more tightly adherent than regions in the interior of the footpad (fig. 7a, S-) where little membranous material could be seen. On the other hand, SAM left after detachment of well-spread and growing cells retained the morphology of virtuahy-intact footpads (figs 6, 7b); little disruption of the interior of the footpad was observed and a nodule of membrane was frequently observed which apExptl Cell Res 107 (1977)
fig. 7. Comparison of SAM from attaching and highly-spread cells. 3T3 cells were allowed to attach to glass coverslips for (a) 2 or (b) 24 h before EGTAmediated detachment for examination of SAM (orig. magnification x24ooO; 20” tilt). The SAM from attaching cells (a) has much more of the central region free of membranous material (S-) than SAM from cells which were well-spread over the substrate (6), in which membranous material (S) covered virtually the entire SAM pool.
Morphology of substrate-attached material
Fig. 8. A pattern of SAM pools. Cells which had been attaching to a glass coverslip for 2 h were detached by EGTA treatment, and the resistant SAM was observed at low magnification with the SEM. The arrows indicate discrete, annular regions of SAM, each region of which corresponds in size and morphology to those described in (b). These regions frequently surround a diffuse’ region like the one shown in the upper right of the micrograph where very little morphologically intact material could be found. At this original magnitication (or&. magnification x2300; 20” tilt), the size of the discrete regions is identical with the average size of observed footpads of 2 h attaching cells.
on the under-surface of the cell. In fig. 8, the indicated areas of SAM generally surrounded a central region that appears in the upper right quadrant of the micrograph (the area observed in this electron micrograph is approx. one-eighth of the area occupied by a well-spread cell). The morphological organization of SAM was minimally affected by treatment with 0.5% Triton X-100 (in PBS) for 30 min at 3PC, as determined by SEM (data not included). SDS treatment (0.2%), on the other hand, quantitatively removed all morphologically-distinguishable material leaving a barren substrate. Examination of serum-coated control substrates (without cells) indicated a complete absence of morphologically-distinctive features. DISCUSSION Analysis of the cell surface material which interacts with fresh serum-coated glass sub-
147
strate during attachment of EGTA-subcultured cells has indicated a sequence of morphological events leading to the formation of footpads (perhaps the principal morphological entity which mediates cell-substrate adhesion [ 171).Filopodia make initial contact with the substrate, followed by their extension over the substrate by some unknown mechanism with splitting into two-or-more finger-like processes. These finger-like processes appear to act as a framework for development of a thin weblike membrane between them during the first hour of the attachment process. The web-like matrices between Iilopodia eventually form ‘mature’ footpads on the substrate which are readily observed during attachment of cells or subsequent to cell rounding during short periods of EGTA treatment of highly spread cells. Although there was a sizable degree of synchrony in substrate-bound process formation during attachment and spreading of cells allowing us to identify this sequence of events, there was heterogeneity in the extent of process formation around the periphery of any single attaching cell and within a population of cells. This reorganization of substrate-bound cell processes during attachment of EGTAsubcultured cells is similar to the attachment process of trypsin-subcultured cells. Erickson & Trinkaus [9] observed elastic membranous microvilli stretching between the cell body and ‘bulbous’ enlargements of substrate-bound membrane during attachment to serum-coated glass by trypsinized BHK cells. The initial substrate-exploratory function of Iilopodial extensions of attaching cells which had been previously trypsinized has also been discussed by Albrecht-Buehler and his collaborators [2, 31. These studies have emphasized the initial contact with the substrate of a limited numEsptiCellRPS to7(1977)
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ber of long narrow (approx. 0.1 pm 0) filopodia, followed by flattening and extension of the membrane initially at the substratebound end. Rajaraman et al. [16] examined by SEM the attachment of cells to serum-free glass substrates. Although tilopodial binding to the substrate appeared to be the principal early event in adhesion, footpads were never observed in this adhesion process. Instead, cytoplasmic webbing occurred with broad extensions of the cell surface across the substrate. The morphological differences in the organization of substratebound material during attachment to serumcoated or serum-free substrate [14] no doubt reflect biochemically distinct processes [8, 11, 18, 191; for example, cells attached to serum-free substrates are more resistant to detachment with trypsin or EGTA [8, 111. The mechanism of EGTA action leading to cell detachment appears to be very similar to the mechanism of trypsin-mediated detachment as found. by Revel et al. [17]. EGTA treatment results in change of shape from the flat-extended cell to a sphericallyshaped cell body linked to footpads by thin membranous retraction fibers. Footpad morphology is minimally affected during the EGTA treatment. Breakage of the labile retraction fibers by shearing forces generated during culture shaking liberates the rounded cell body into suspension, leaving virtually intact footpads on the substrate as SAM. Our studies using EGTA detachment and those of Revel et al. [17] using trypsin indicate that the adhesive materials in the footpad adhesion site are protected from trypsin action and are not associated by strictly Ca2+-mediated bonding [6, 71. Several experimental approaches consistently indicate that the substrateattached material which persists after Exprl Cell Res 107 (1977)
EGTA-mediated removal of cells is indeed footpad material. SAM possessed the size, morphology, and patterned arrangement on the substrate virtually identical to the original cellular footpads by these electron microscopic analyses. Also, many of the SAM pools examined possessed a ‘nub’ of membranous material where shearing of the stressed retraction fiber probably occurred. The membrane-enclosed nature of SAM has also been confirmed by biochemical evidence [21] showing that proteins in this material (including actin [6]) are resistant to trypsin cleavage and lactoperoxidase-catalysed iodination. It was previously shown [5] that leucine- or glucosamine-radiolabeled SAM detected autoradiographically was present on the substrate (a) in focal pools whose density was similar to that of cellular footpads; and (b) only at substrate regions of immediate contact between the cell and the substrate. SEM analysis has also been used in these studies to show that SAM can be quantitatively removed by SDS treatment (confirmed by biochemical evidence) but was so firmly bound to the substrate that it was resistant to dissociation with non-ionic detergents (confirmed by biochemical evidence; Cathcart & Culp, unpublished data). The only morphological entity observed in SAM was the footpad-like structure and not the so-called ‘sole plate’ [17] left on the substrate when glutaraldehyde-fixed cells are detached with a stream of water. Perhaps ‘sole plates’ are actually large regions of the cell undersurface which are weakly associated with the adsorbed layer of serum protein [8, 18, 191; membrane proteins in these regions may then be cross-linked to the serum layer by glutaraldehyde treatment and persist as sheets of undersurface membrane with associated cytoskeletal elements [17] during the vigorous shearing
Morphology of substrate-attached material treatment. These weak associations may be sensitive to the action of the Ca2+-specific chelating agent used in our studies. The differences in morphology of SAM from attaching versus highly spread cells were striking. Although both displayed the size and patterns of cellular footpads, SAM from highly spread cells appeared to be virtually intact footpads, while that from attaching cells appeared to be membranous remnants of the periphery of the footpad. This may indicate that adhesive materials and/or subsurface cytoskeletal microfilaments [l, 10, 13, 151 organize initially around the periphery of the footpad and secondarily across the entire footpad-substrate interface over a much longer period of time. On the other hand, perhaps the limiting process is synthesis and organization of externally oriented LETS glycoprotein and/or hyaluronate proteoglycans [7]. Biochemical analyses of SAM proteins indicated that there was as much actin and myosin-like protein in SAM from attaching cells as in SAM from long-term growth cells. Actin-containing microfilaments therefore probably play an important organizational role at the periphery of the maturing footpad, along with outer surface components as a well organized adhesion ‘complex’ at the surface of the cell [7]. Complementary utilization of a variety of molecular probes and electron microscopic analysis may provide further clues as to the precise molecular organization of the hyaluronate proteoglycans, the LETS glycoprotein, and the actin-containing microfilaments in the footpad adhesion site. This study on the mechanism of EGTA detachment of cells and a previous study of the mechanism of trypsin action [ 171emphasize the importance of surface membrane-associated cytoskeletal elements in providing a rigid lattice for strengthening the adhesion
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site. Cell-substrate adhesion may be mediated by two different levels of cell ‘surface’ organization-interaction of external surface hyaluronate proteoglycans and the LETS glycoprotein with substrateadsorbed serum proteins, followed by reorganization of membrane-bound microfilaments to form a strong surface lattice to resist abusive treatment in the environment [7]. Control of the organization of this cytoskeleton may provide the cell with an important mechanism for modulating the adhesion process. L. A. C. acknowledges funding from the NC1 (research grant 5-ROl-CA-13513) and from the ACS (research grant BC-217 with partial support from the Ohio division). J. R. R. is a postdoctoral fellow of the NIH (fellowship 1F 32-GM-0524401). L. A. C. is a Career Development Awardee of the NC1 (l-KO4-CA-70709).
REFERENCES
’
1. Abercrombie, M, Heaysman, J E M & Pegrum, S M, Exp cell res 67 (1971) 359. 2. Albrecht-Buehler, G, J cell bio169 (1976) 275. 3. Albrecht-Buehler, G & Goldman, R D, Exp cell res 97 (1976) 329. 4. Culp, LA, J cell bio163 (1974) 71. 5. - EXD cell res 92 (1975) 467. 6. -Biochemistry 15 (1976) 4094. - J supramol structure 5 (1976) 239. 87.Culp, LA 8zBuniel, J F, J cell physio188 (1976) 89. 9: Erickson, C A & Trinkaus, J P, Exp cell res 99 (1976) 375. 10. Goldman, R D h Knipe, D M, Cold Spring Harbor symp quant bio137 (1973) 523. 11. Grinnell, F, Exp cell res 160(1974) 304. Hynes, R 0 &Bye, J M. Cell 3 (1974) 113. i:: McNutt, N S, C&p, L A & Black, PH, J cell biol 56 (1973) 412. 14. Pegrum; S M C Maroudas, N G, Exp cell res % (1975) 416. 15. Perdue. J F. J cell biol58 (1973) 265. 16. Rajaraman,‘R, Rounds, D E, qen, S P S & Rembaum, A, Exp cell res 88 (1974) 327. 17. Revel, J P, Hoch, P & Ho, D, Exp cell res 84 (1974) 207. 18. Revel, J P & Wolken, K, Exp cell res 78 (1973) 1. Takeichi, M, EXP cell res 68 (1971) 88. :‘o: Terry, A H 8~ C&p, L A, Biochemistry 13 (1974) 414. 21. Culp, L. A. In preparation. Received November 9, 1976 Accepted December 29, 1976 Exptl Cell Res 107 (1977)
Experimental Cell Research 107 (1977) 139-149
MORPHOLOGY
AND CELLULAR
SUBSTRATE-ATTACHED MOUSE
ORIGINS
MATERIAL
OF
FROM
FIBROBLASTS
J. J. ROSEN* and Ll. A. CULP’ ‘Department of Microbiology, School of Medicine, and *Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH44Io6, USA
SUMMARY Scanning electron microscopy @EM) has been used to determine the pattern of reorganization of substrate-bound cell surface material durinn attachment of Swiss 3T3 mouse tibroblasts to fresh serum-coated glass substrates after prior-detachment with the Ca*+-specific chelator EGTA (ethylene&s-(oxyethylenenitrilo)-tetraacetic acid). A limited number of filonodia make contact with the substrate surface and branch into finger-like extensions. A thin web-like membrane organizes between these filopodial extensions to eventually form mature footpads which appear to have a limited size. Footpads appear to be the principal morphological entities mediating cellsubstrate adhesion. The mechanism of EGTA-mediated detachment of cells and the morphology of the substrateattached material (SAM) left adherent to the substrate after complete removal of cells have also been examined. EGTA treatment results in cell rounding, with minimal effects on the morphology of the adhesive footpads which remain linked to the cell body via stressed retraction fibers. These stressed fibers eventually break, leaving footpads on the substrate as SAM and liberating the cell body into suspension. This evidence suggests that EGTA treatment minimally affects adhesive materials in the footpad site but disorganizes the cytoskeleton of the cell with resultant shearing of the cell body away from the footnads. The size, mornhologv, and pattern of SAM left on the substrate establish-that it is essentially footpad material: SAdfrom highly-spread cells appears to be virtually-intact footpads, while SAM from attaching cells appears to be disrupted footpad material. These results are discussed in terms of the morphological and biochemical organization of the footpad adhesion sites of cells.
The cellular origin and molecular organization of the substrate-attached material (SAM), left tightly bound to the tissue culture substrate after EGTA (ethylene-bis(oxyethylenenitrilo)-tetraacetic acid)-mediated removal of normal and virus-transformed mammalian cells, have been of considerable interest because of the prospective function of this material in mediating cell-substrate adhesion [7]. Biochemical ’ To whom reprint requests should be addressed.
analyses [6, 201 have established that SAM is composed of hyaluronate proteoglycans, the LETS (large-extemal-transformationsensitive) glycoprotein [ 121, microfilamentassociated proteins, and a number of unidentified proteins. These components are present in SAM in similar relative proportions after study of a variety of cellular attachment and growth conditions [6, 71 and are coordinately resistant to removal from the substrate with a number of dissociative reagents, suggesting that these Expfl
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components exist as a tightly-associated cell ‘surface’ complex which mediates adhesion. The presence of microfilament-associated actin and a myosin-like protein in SAM [6] indicated that treatment of substrate-bound cells with the Ca2+-specific chelator EGTA was not merely dissociating external cell surface macromolecules, with resultant separation of the intact surface membrane from the substrate, but that a more complicated process was occurring, perhaps with ‘pinching-off of a membrane vesicle. Autoradiographic evidence [5] has confirmed that radioactive SAM protein and polysaccharide are topographically arranged in focal patterns very similar in density to the footpads by which the cell adheres to the substrate [ 171. In order to further analyse the cellular origin of SAM, we have utilized scanning electron microscopy @EM) to determine the morphology and cellular origins of this material. Revel and his colleagues f17, 181 have demonstrated that cells adhere to the substrate at focal regions of the surface membrane called footpads and that trypsin treatment for short periods minimally affects footpad morphology, while causing rounding-up of the cell body and detachment via breakage of the elastically-labile retraction fibers of the surface membrane. Similarly, SEM has been used to study the morphological events of re-attachment of trypsinized cells to serum-free [16] or serum-coated [9] substrates. In this study, we report the morphological re-organization of substrate-attached cell surface processes during attachment of EGTA-subcultured cells to fresh serumcoated glass substrates. Interest has also been focused on the mechanism by which EGTA-mediated detachment of cells occurs, as well as the morphology and cellular Expti Cell Res 107 (1977)
origin of SAM which resists EGTA action MATERIALS
AND METHODS
Cell growth Swiss 3T3 mouse fibroblasts were utilized for these studies and were routinely grown as described previously [4] in Eagle’s minimal essential medium supplemented with four times the normal concentration of amino acids and vitamins (MEMx4), 10% donor calf serum, penicillin (250 U/ml) and streptomycin (0.25 mg/ml); in Brockway glass bottles at 37°C; and in an incubator with humidified air and a 5 % CO, concentration. These cells were assayed for and were found to be free of Mycuplusma contamination. When these cells reached approx. 70% confluency (prior to becoming density-inhibited), they were removed for experimental use by gentle shaking at 37°C with 0.5 mM EGTA in PBS. These cells were enumerated with a hemocytometer, pelleted by centrifugation, and resuspended in MEMx4 to a final concentration of 2.0x l@ cells/ml. This suspension was inoculated into tissue culture dishes (containing glass coverslips) for experiments.
Preparation of glass coverslips Glass coverslips, I5 mm diameter, were acid-washed, rinsed in distilled water eight times, rinsed in 95% ethanol three times and flamed before being placed in 10 cm Falcon plastic tissue culture dishes (five coverslips/dish). At least 2 h before an attachment experiment was initiated, the dishes were filled with 15 ml of MEMX4 with 10% donor calf serum and equilibrated at 37°C. The MEMx4 was replaced with fresh medium before cell inoculation.
Cell attachment Two million cells, prepared as indicated above, were inoculated into coverslip-containing dishes. After gentle shaking to uniformly disperse the cells, the coverslips were repositioned around the dish. At times varying from 15 min to 24 h after inoculation of cells and incubation at 37”C, coverslips were carefully removed from the dish, gently dipped twice in each of three batches of PBS-II, drained, and placed in buffered 0.1% glutaraldehyde solution in preparation for electron microscopic analysis. The kinetics of attachment of these cells to serum-coated substrates have been established [4,8].
Cell removal by EGTA treatment After cells had been allowed to attach to serum-coated glass substrates for times varying from 15 min to 24 h at 37”C, the detachment process was studied by exposing the cells to EGTA. The medium was first decanted from the dishes. and the cells were rinsed twice with PBS. A 0.5 mM EGTA solution in PBS was added to the dishes, followed by gentle shaking at 37°C for times varying from 5 to 25 min. When com-
Morphology
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Fig. 1. The morphology of Swiss 3T3 fibroblasts during attachment and spreading on glass coverslips. Cells which had been subcultured by EGTA treatment were inoculated into tissue culture dishes containing coverslips in medium with 10% donor calf serum. After attachment had proceeded for various times, the
cells were fixed with glutaraldehyde, and prepared for SEM as described in Materials and Methods. The high tilt angle (80”) permits more effective visualization of cell-substrate interactions. Cells are shown after (a) 30 min; (b) 60 min; (c) 2 h; (d) 24 h of attachment. Original magnification x 2 800.
plete removal of all cells was desired, the dishes were gently pipetted after 25 min of exposure to EGTA to examine the SAM left adherent to the substrate [6, 7, 203. The coverslips were gently rinsed twice in each of three batches of PBS-II, drained, and placed in a buffered 0.1% ghttaraldehyde solution in preparation for electron microscopic analysis.
followed by graded Freon 13 solutions in ethanol with three changes in 100% Freon 13 (all incubations at 4°C). Sampies were then critical point dried in Freon 113, snutter-coated with gold-palladium, and desiccated until observation in a Cambridge S4-10 Stereoscan scanning electron microscope.
Materials Preparation of samples for scanning electron microscopy Single cells attached to substrates are extremely fragile and must be treated with the utmost care if the fine ultrastructural details are to be accurately preserved. After rinsing in PBS-II, the coverslips were transferred to a 0.1% glutaraldehyde solution buffered with sodium cacodylate (0.06 M) in Ringer’s solution (uH=7.35) and osmotically balanced with sucrose a.12 M).‘After 1 h incubation at room temperature, the coverslins were transferred to a 3 % glutaraldehyde solution (incacodylate buffer) and stored at 4°C overnight. Dehydration was accomplished by a series of graded ethanols with three changes in 100% ethanol,
Plastic tissue culture dishes were purchased from Lux Scientific Co.; round glass coverslips (15 mm 0) from A. H. Thomas Co.; EGTA from Eastman Organic Chemicals; SDS from Matheson, Coleman & Bell; MEMx4 from Gibco; donor calf serum from Flow Laboratories.
RESULTS The cellular origins of substrate attached material (SAM) have been investigated using scanning electron microscopic (SEM) Exptl Cell Res 107 (1977)
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Fig. 2. Higher magnification SEMs of substrate interaction sites. Cells were inoculated into coverslipcontaining dishes after EGTA subculturing as described in tig. 1. Initial contact (a) of cells with the substrate involves extension of a limited number of lilopodia (F) onto the substrate and away from the cell body (CB). Within 1 h (b), primative pads (P) begin to
form on the substrate around the periphery of the cell body at the contact point of tilopodia with the substrate. These pads continue to grow in size and to extend filopodia from their outer margins during the next few hours (c), developing into a mature ‘footpad within the first day after initial attachment (4. Original magnification X 13000; 20” tilt.
techniques. The properties of this material are dependent on the duration of cell attachment and spreading, but in all cases it appears to be the remains of ‘footpads’ or cellular extensions tightly bound to the substrate during the attachment process. The significant morphological heterogeneity of substrate-bound cell surface material was difficult to document photographically at any given point during the attachment processes. The photomicrographs presented here were selected to illustrate generally observed patterns of cellular morphology felt to be characteristic of these processes.
Substrate interaction during attachment of EGTA-subcultured cells A cell in suspension after EGTA treatment and prior to attaching to a fresh serumcoated substrate is covered with blebs and a few microvillous structures. Within 1.5min of attaching to the serum-covered glass, a few filipodial extensions have already extended along the substrate beyond the radius of the cell body (fig. la). These extensions are approx. 0.1 pm in diameter and up to 20 pm long (fig. 2a). They emerge from the cell body near the cell surface closest to the substrate.
Exprl Cell Res 107 (1977)
Morphology of substrate-attached material
Fig. 3. Filopodial and footpad morphology. SEMs taken at high magnification (original magnification X26000) and a high angle of tilt (80”) illustrate the heterogeneity of cell attachment regions on glass. These cells have both been in contact with the substrate for 1 h. In (a), fllopodial extensions (FE) stretch between the cell body (CB) and the substrate and appear to be under tension. In (b), the complex ‘cording’ of the extensions from the cell body (CB) to the footpad (P) are apparent; this footpad morphology is more typical of cells during later stages of attachment and cell spreading.
During the first half hour of attachment, small pads (approx. 2 pm across) start forming on the substrate, often with filopodia projecting beyond these pads across the substrate (fig. 2b, c). The cell body after 1 h has lost many of its surface blebs and now has begun to actively spread across the substrate (fig. 1b). By the end of the 2nd h, most cells are well-spread (fig. 1c). Fig. Id shows a typical fully-spread cell 24 h after initial attachment. The cell surface is almost completely devoid of blebs or microvilli as demonstrated previously [9], lo-771818
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and only a few attachment filopodia extend from the cell border to the substrate (the substrate attachment regions are often hidden from view). The limited number of discrete contacts with the substrate that are evident are made through broad membranous extensions to mature footpads, often 15 km or more in diameter (fig. 2d). The substrate contact points during later times of attachment, called footpads [17], are web-like (up to 10 pm across) and are often connected to the cell body by broader regions of membrane (3-5 pm) contiguous with the cell body (fig. 2d). It was very common to see raised ridges, or ribs, in footpads which appeared to be extensions of the filipodial processes extending from the cell body to the footpad. The leading edge of web-like material that became organized between branching filopodia on the substrate was extremely thin (less than 0.05 pm). These webs appear to organize and spread over the substrate away from the branching point of two or more substratebound filopodia (see right side of fig. 2~). The substrate interaction during early attachment begins with tentative contact between thin attachment fibers or filopodial extensions (fig. 3a, FE) and the substrate in the close vicinity of the newly attached cell. These fibers appear to coalesce into bundles (fig. 3 b) or bifurcate along the substrate surface (fig. 2a). In general, bifurcation of filopodia on the substrate occurred early in the attachment process, while broadening of the membrane linking the footpad and the cell body via coalescence occurred later in the attachment process. After the first hour of attachment, the attachment tilopodia that are present begin to thicken, and increasing amounts of membranous material appear to spread over the substrate as contact pads. These contact pads (fig. 2b) gradually mature into fully ExptlCellRes157 (1977)
Fig. 4. Morphological changes during EGTA-mediated detachment. After 2 h of attachment to glass coverslius. the cell medium was reulaced with a 0.5 mM soiution of EGTA, as described in the Materials and Methods (after gentle rinsing with PBS), for 10 min prior to fixation with glutaraldehyde. Cell retraction away from the substrate as a result of cell body (CB) rounding ((a) (orig. magnification x6800; 80” tilt) puts retraction fibers in tension; (b) (orig. magnification x6800; 20” tilt) shows fibers in tension, fibers that have broken (II), and early signs of disruption of attachment footpads (D); (c) (orig. magnification x26000; 80” tilt) illustrates fiber breaks and the footpad material that remains bound to the substrate.
developed footpads (fig. 3b) connected to the cell by broader areas of surface membrane. Mechanism of EGTA-mediated detachment of cells When cells which have been attached to a serum-coated glass substrate are exposed to the Ca2+ specific chelating agent EGTA, two distinct morphological changes occur which lead to eventual detachment of the cell from the substrate. If a cell was wellspread on the substrate prior to the EGTA treatment, it rounds up assuming a spherical shape and large sheets of cell membrane lift off the glass during the retraction process, as shown in fig. 4a. The foreground of this micrograph shows many mature footpads which cannot be observed under the spread cell. The retraction fibers [17] of ExptlCellRes
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stressed membrane are put under considerable tension with one end anchored at the footpad, which appears to be minimally affected by early EGTA treatment, and the other end at the surface of the cell body (fig. 4b). Eventually this tensile force is great enough to break the retraction fibers during agitation of cultures, leaving the footpad and a piece of the fiber behind on the substrate (fig. 4b, c). During routine cell removal, shearing forces generated by shaking and pipetting of the culture probably cause the breakage of the cell body away from the substrate, leaving footpads virtually intact. Although the utmost care was taken to preserve the fine structure of membranous processes during the experiments, there is no doubt that some of the observed breaks of tilopodia in attaching cells (e.g., fig. 2a, fig. 3a) and broader
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during detachment. The specific nature of this degradation process is not understood but will be considered in analyses of the SAM left adherent to the substrate due to its resistance to EGTA treatment. Morphology of SAM When cells are completely removed from the substrate by EGTA treatment, the material that remains behind is morphologically organized into patterns that strongly suggest their cellular origins. Fig. 5a shows mature footpads and filopodia associated with an attaching cell in the process of spreading out onto the substrate. Fig. 5 b, taken at the same magnification and angle of tilt as fig. 5 a, shows the pattern of the SAM left on the substrate when cells grown under the same conditions are removed by EGTA treatment. This treatment produces a roughened, nodular array of membranous-looking vesicles and appears Fig. 5. Morphology of footpads and substrate-attached to remove any traces of filopodia on the material (SAM). EGTA-subcultured cells were persubstrate; but the size, shape, and distribumitted to reattach to glass coverslips in serum-containing medium for 1 h to allow formation of footpad tion of SAM corresponds closely to the (8’) attachments extending from the cell body (CB); general morphology of the footpad structypical footpads are shown in (a). A second set of coverslips with cells which had been attaching for 1 h tures observed around the periphery of were then treated with EGTA as described in the attaching cells. Most discrete areas of SAM Materials and Methods to completely remove cells for subsequent observation of SAM; two typical pools of also displayed a larger nodule that probably SAMs (S) are shown in (b) with nubs of membranis the remainder of broken retraction fibers ous material where presumably the retraction fibrils had broken away. Original magnification x 13000; like those shown in fig. 4c. 20” tilt. The SAM from well-spread cells (in conretraction fibers in EGTA-treated cells tact with the substrate for time periods of were due to handling the samples while 24 h or more) appears to be virtually-intact these weakened fibers were in tension. footpads of somewhat larger size than SAM However, control experiments using buf- from attaching cells; two examples of this fered saline instead of EGTA showed no type of SAM are given in fig. 6a, b. This is cell retraction and very few fractures. consistent with the larger size of footpads in The other primary effect of EGTA during well-spread cells when compared to those removal of attaching cells (but not well- of attaching cells. The SAM left on the substrate by cells spread cells) appears to be partial disruption of the immature footpads. One pad in fig. removed after short periods of attachment 4b (0) illustrates the start of this process appeared to be highly-disrupted footpad which was also seen on many other cells material (fig. 7a), i.e., small membranous Exptl Cell Res 107 (1977)
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peared to be a remnant of the retraction fiber (fig. 6). However, the upper surface of SAM pools appeared to be highly disorganized (fig. 6), perhaps as a result of relaxation of membrane after breakage of the stressed retraction fiber (the upper surface of cellular footpads consistently appeared as a smooth and well-organized surface). If the cells were initially inoculated at low density so that individual cells were wellisolated on the substrate, the SAM left after cell removal was often observed to form patterns that corresponded in size and distribution to the initial point of attachment
Fig. 6. Morphology of SAM from highly-spread cells. (a) (Orig. magnification x 13000; 80” tilt) shows a SAM pool remaining after 24 h of cell attachment and spreading followed by EGTA-mediated detachment as described in Materials and Methods to completely remove cells; (b) (orig. magnification x24000; 71” tilt) is an example at higher magnification of the disorganized membrane on the ‘upper’ surface of SAM pools as a result of relaxation of the remnant of the broken retraction fiber; the only consistent morphological structure observed on the ‘upper’ surface of these footpad SAM vesicles was a nub of membrane where the retraction fiber had presumably broken.
vesicles (fig. 7a, S) appeared on the substrate in a pattern suggesting that the ,periphery of the footpad was more tightly adherent than regions in the interior of the footpad (fig. 7a, S-) where little membranous material could be seen. On the other hand, SAM left after detachment of well-spread and growing cells retained the morphology of virtuahy-intact footpads (figs 6, 7b); little disruption of the interior of the footpad was observed and a nodule of membrane was frequently observed which apExptl Cell Res 107 (1977)
fig. 7. Comparison of SAM from attaching and highly-spread cells. 3T3 cells were allowed to attach to glass coverslips for (a) 2 or (b) 24 h before EGTAmediated detachment for examination of SAM (orig. magnification x24ooO; 20” tilt). The SAM from attaching cells (a) has much more of the central region free of membranous material (S-) than SAM from cells which were well-spread over the substrate (6), in which membranous material (S) covered virtually the entire SAM pool.
Morphology of substrate-attached material
Fig. 8. A pattern of SAM pools. Cells which had been attaching to a glass coverslip for 2 h were detached by EGTA treatment, and the resistant SAM was observed at low magnification with the SEM. The arrows indicate discrete, annular regions of SAM, each region of which corresponds in size and morphology to those described in (b). These regions frequently surround a diffuse’ region like the one shown in the upper right of the micrograph where very little morphologically intact material could be found. At this original magnitication (or&. magnification x2300; 20” tilt), the size of the discrete regions is identical with the average size of observed footpads of 2 h attaching cells.
on the under-surface of the cell. In fig. 8, the indicated areas of SAM generally surrounded a central region that appears in the upper right quadrant of the micrograph (the area observed in this electron micrograph is approx. one-eighth of the area occupied by a well-spread cell). The morphological organization of SAM was minimally affected by treatment with 0.5% Triton X-100 (in PBS) for 30 min at 3PC, as determined by SEM (data not included). SDS treatment (0.2%), on the other hand, quantitatively removed all morphologically-distinguishable material leaving a barren substrate. Examination of serum-coated control substrates (without cells) indicated a complete absence of morphologically-distinctive features. DISCUSSION Analysis of the cell surface material which interacts with fresh serum-coated glass sub-
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strate during attachment of EGTA-subcultured cells has indicated a sequence of morphological events leading to the formation of footpads (perhaps the principal morphological entity which mediates cell-substrate adhesion [ 171).Filopodia make initial contact with the substrate, followed by their extension over the substrate by some unknown mechanism with splitting into two-or-more finger-like processes. These finger-like processes appear to act as a framework for development of a thin weblike membrane between them during the first hour of the attachment process. The web-like matrices between Iilopodia eventually form ‘mature’ footpads on the substrate which are readily observed during attachment of cells or subsequent to cell rounding during short periods of EGTA treatment of highly spread cells. Although there was a sizable degree of synchrony in substrate-bound process formation during attachment and spreading of cells allowing us to identify this sequence of events, there was heterogeneity in the extent of process formation around the periphery of any single attaching cell and within a population of cells. This reorganization of substrate-bound cell processes during attachment of EGTAsubcultured cells is similar to the attachment process of trypsin-subcultured cells. Erickson & Trinkaus [9] observed elastic membranous microvilli stretching between the cell body and ‘bulbous’ enlargements of substrate-bound membrane during attachment to serum-coated glass by trypsinized BHK cells. The initial substrate-exploratory function of Iilopodial extensions of attaching cells which had been previously trypsinized has also been discussed by Albrecht-Buehler and his collaborators [2, 31. These studies have emphasized the initial contact with the substrate of a limited numEsptiCellRPS to7(1977)
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ber of long narrow (approx. 0.1 pm 0) filopodia, followed by flattening and extension of the membrane initially at the substratebound end. Rajaraman et al. [16] examined by SEM the attachment of cells to serum-free glass substrates. Although tilopodial binding to the substrate appeared to be the principal early event in adhesion, footpads were never observed in this adhesion process. Instead, cytoplasmic webbing occurred with broad extensions of the cell surface across the substrate. The morphological differences in the organization of substratebound material during attachment to serumcoated or serum-free substrate [14] no doubt reflect biochemically distinct processes [8, 11, 18, 191; for example, cells attached to serum-free substrates are more resistant to detachment with trypsin or EGTA [8, 111. The mechanism of EGTA action leading to cell detachment appears to be very similar to the mechanism of trypsin-mediated detachment as found. by Revel et al. [17]. EGTA treatment results in change of shape from the flat-extended cell to a sphericallyshaped cell body linked to footpads by thin membranous retraction fibers. Footpad morphology is minimally affected during the EGTA treatment. Breakage of the labile retraction fibers by shearing forces generated during culture shaking liberates the rounded cell body into suspension, leaving virtually intact footpads on the substrate as SAM. Our studies using EGTA detachment and those of Revel et al. [17] using trypsin indicate that the adhesive materials in the footpad adhesion site are protected from trypsin action and are not associated by strictly Ca2+-mediated bonding [6, 71. Several experimental approaches consistently indicate that the substrateattached material which persists after Exprl Cell Res 107 (1977)
EGTA-mediated removal of cells is indeed footpad material. SAM possessed the size, morphology, and patterned arrangement on the substrate virtually identical to the original cellular footpads by these electron microscopic analyses. Also, many of the SAM pools examined possessed a ‘nub’ of membranous material where shearing of the stressed retraction fiber probably occurred. The membrane-enclosed nature of SAM has also been confirmed by biochemical evidence [21] showing that proteins in this material (including actin [6]) are resistant to trypsin cleavage and lactoperoxidase-catalysed iodination. It was previously shown [5] that leucine- or glucosamine-radiolabeled SAM detected autoradiographically was present on the substrate (a) in focal pools whose density was similar to that of cellular footpads; and (b) only at substrate regions of immediate contact between the cell and the substrate. SEM analysis has also been used in these studies to show that SAM can be quantitatively removed by SDS treatment (confirmed by biochemical evidence) but was so firmly bound to the substrate that it was resistant to dissociation with non-ionic detergents (confirmed by biochemical evidence; Cathcart & Culp, unpublished data). The only morphological entity observed in SAM was the footpad-like structure and not the so-called ‘sole plate’ [17] left on the substrate when glutaraldehyde-fixed cells are detached with a stream of water. Perhaps ‘sole plates’ are actually large regions of the cell undersurface which are weakly associated with the adsorbed layer of serum protein [8, 18, 191; membrane proteins in these regions may then be cross-linked to the serum layer by glutaraldehyde treatment and persist as sheets of undersurface membrane with associated cytoskeletal elements [17] during the vigorous shearing
Morphology of substrate-attached material treatment. These weak associations may be sensitive to the action of the Ca2+-specific chelating agent used in our studies. The differences in morphology of SAM from attaching versus highly spread cells were striking. Although both displayed the size and patterns of cellular footpads, SAM from highly spread cells appeared to be virtually intact footpads, while that from attaching cells appeared to be membranous remnants of the periphery of the footpad. This may indicate that adhesive materials and/or subsurface cytoskeletal microfilaments [l, 10, 13, 151 organize initially around the periphery of the footpad and secondarily across the entire footpad-substrate interface over a much longer period of time. On the other hand, perhaps the limiting process is synthesis and organization of externally oriented LETS glycoprotein and/or hyaluronate proteoglycans [7]. Biochemical analyses of SAM proteins indicated that there was as much actin and myosin-like protein in SAM from attaching cells as in SAM from long-term growth cells. Actin-containing microfilaments therefore probably play an important organizational role at the periphery of the maturing footpad, along with outer surface components as a well organized adhesion ‘complex’ at the surface of the cell [7]. Complementary utilization of a variety of molecular probes and electron microscopic analysis may provide further clues as to the precise molecular organization of the hyaluronate proteoglycans, the LETS glycoprotein, and the actin-containing microfilaments in the footpad adhesion site. This study on the mechanism of EGTA detachment of cells and a previous study of the mechanism of trypsin action [ 171emphasize the importance of surface membrane-associated cytoskeletal elements in providing a rigid lattice for strengthening the adhesion
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site. Cell-substrate adhesion may be mediated by two different levels of cell ‘surface’ organization-interaction of external surface hyaluronate proteoglycans and the LETS glycoprotein with substrateadsorbed serum proteins, followed by reorganization of membrane-bound microfilaments to form a strong surface lattice to resist abusive treatment in the environment [7]. Control of the organization of this cytoskeleton may provide the cell with an important mechanism for modulating the adhesion process. L. A. C. acknowledges funding from the NC1 (research grant 5-ROl-CA-13513) and from the ACS (research grant BC-217 with partial support from the Ohio division). J. R. R. is a postdoctoral fellow of the NIH (fellowship 1F 32-GM-0524401). L. A. C. is a Career Development Awardee of the NC1 (l-KO4-CA-70709).
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1. Abercrombie, M, Heaysman, J E M & Pegrum, S M, Exp cell res 67 (1971) 359. 2. Albrecht-Buehler, G, J cell bio169 (1976) 275. 3. Albrecht-Buehler, G & Goldman, R D, Exp cell res 97 (1976) 329. 4. Culp, LA, J cell bio163 (1974) 71. 5. - EXD cell res 92 (1975) 467. 6. -Biochemistry 15 (1976) 4094. - J supramol structure 5 (1976) 239. 87.Culp, LA 8zBuniel, J F, J cell physio188 (1976) 89. 9: Erickson, C A & Trinkaus, J P, Exp cell res 99 (1976) 375. 10. Goldman, R D h Knipe, D M, Cold Spring Harbor symp quant bio137 (1973) 523. 11. Grinnell, F, Exp cell res 160(1974) 304. Hynes, R 0 &Bye, J M. Cell 3 (1974) 113. i:: McNutt, N S, C&p, L A & Black, PH, J cell biol 56 (1973) 412. 14. Pegrum; S M C Maroudas, N G, Exp cell res % (1975) 416. 15. Perdue. J F. J cell biol58 (1973) 265. 16. Rajaraman,‘R, Rounds, D E, qen, S P S & Rembaum, A, Exp cell res 88 (1974) 327. 17. Revel, J P, Hoch, P & Ho, D, Exp cell res 84 (1974) 207. 18. Revel, J P & Wolken, K, Exp cell res 78 (1973) 1. Takeichi, M, EXP cell res 68 (1971) 88. :‘o: Terry, A H 8~ C&p, L A, Biochemistry 13 (1974) 414. 21. Culp, L. A. In preparation. Received November 9, 1976 Accepted December 29, 1976 Exptl Cell Res 107 (1977)