Cell Motility and the Cytoskeleton 23:45-60 (1992)

Distribution of Detyrosinated Microtubules in Motile NRK Fibroblasts Is Rapidly Altered Upon Cell-Cell Contact: Implications for Contact Inhibition of Locomotion T. Nagasaki, C.J. Chapin, and G.G. Gundersen Department of Anatomy and Cell Biology, Columbia University, College of Physicians and Surgeons, New York, New York Fibroblasts migrating into an experimental wound contain an extensive array of detyrosinated microtubules (Glu MTs) oriented in the direction of migration, whereas nonmotile cells in the interior of a monolayer contain Glu MTs that are primarily coiled around the nucleus. To determine the role of cell-cell contact in the formation of these distinct arrays of Glu MTs, we studied the distribution of Glu MTs by immunofluorescence in NRK fibroblasts that had been fixed at different intervals after they had established contact with other cells. Time-lapse video recordings were made of the contacting cells to provide a record of cellular behavior. In motile cells that became completely surrounded by virtue of contact with other cells, Glu MTs were found mostly coiled around the nucleus. The proportion of cells whose Glu MTs extended to the original leading edge decreased dramatically after the cells had been surrounded for 10 min or more. At earlier times, when the contact was confined to a portion of the cell margin, Glu MTs were absent from the area behind the contact site, yet were still oriented toward the noncontacting and ruffling margins. The contact-induced alteration of Glu MTs was not due to the cessation of forward locomotion of cells per se, since immobilization of cells with cytochalasin D did not cause a dramatic change in Glu MTs. That cell-cell contact also specifies the type of Glu MTs formed in cells was shown by experiments in which MTs were regrown following complete depolymerization with nocodazole. The remodeling of Glu MTs during cell-cell contact may be involved in cellular repolarization during contact inhibition of locomotion and will be a useful marker for further dissecting the molecular events of contact inhibition of motility. 0 1992 wiley-Liss, Inc. Key words: cell motility, cytoskeleton, microtubule dynamics, wound healing, leading edge, ruming

INTRODUCTION The term “contact inhibition of locomotion” was coined by Abercrombie and Heaysman [1954] to describe a series of events initiated by motile cells when they collide with each other. The colliding cells stop locomotion in the direction of the movement and reestablish a new direction. Abercrombie, Heaysman, and others have accumulated evidence that contact inhibition of locomotion is a specific and widespread biological phenomenon in cultured cells [reviewed in Abercrombie, 1970; Heaysman, 19781. It has also been suggested that 0 1992 Wiley-Liss, Inc.

contact inhibition plays an important role in motility of both normal and tumor cells in vivo [Abercrombie, 19791. Transformed cells are generally defective in the contact inhibition reaction and this property may contribute to the invasive nature of some tumor cells [Aber

Received September 27, 1991; accepted February 13, 1992. Address reprint requests to Gregg Gundersen, Department of Anatomy and Cell Biology, Columbia University, 630 West 168th Street, New York. NY 10032.

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crombie, 19791. Through these studies, contact inhibition has come to be defined as a sequence of morphologically distinct events, including adhesion, paralysis, contraction, and repolarization [reviewed in Heaysman, 19781. Nevertheless, the molecular basis of these morphological events is poorly understood, in large part because they are difficult to quantify in an objective manner. Without a quantifiable marker, the relationship between the morphological events and the underlying biochemical reactions cannot be obtained in a straightforward manner. Therefore, measurable biochemical markers for contact inhibition, which can be linked to the morphologically defined events, would greatly facilitate the process of dissecting the molecular events that comprise contact inhibition. It has been shown previously that proliferating cultured cells contain subsets of microtubules (MTs) that are distinguishable by their content of post-translationally detyrosinated or-tubulin [Gundersen et al., 19841. A number of experiments have shown that microtubules enriched in detyrosinated tubulin (Glu MTs) are stabilized; they do not undergo the rapid turnover exhibited by most cellular MTs [Schulze et al., 1987; Webster et al., 19871 and are relatively resistant to agents that depolymerize MTs [Kreis, 1987; Khawaja et al., 19881. Currently, the factors that regulate the stability of Glu MTs are not known. We have reported earlier that the Glu MTs in 3T3 fibroblasts migrating into an experimental wound were specifically oriented toward the active leading edge of the cell, which is the direction of migration [Gundersen and Bulinski, 19881. The oriented distribution of Glu MTs appeared before translocation of the cells into the wound and closely paralleled the reorientation of the microtubule organizing center (MTOC) . The polarized array of Glu MTs seemed to be specific to cells at the wound edge that were either motile or becoming motile, since Glu MTs in nonmotile cells within the monolayer were primarily coiled around the nucleus. In contrast, Tyr MTs, which are enriched in tyrosinated tubulin and contain levels of detyrosinated tubulin undetectable by immunofluorescence, were found throughout the cytoplasm, whether the cell was motile or nonmotile. The factors that specify the type of Glu MT array, whether polarized or juxtanuclear, are currently unknown. The motile state of the cell itself is not likely to be responsible for the production of these arrays, since polarized arrays of Glu MTs are found in cells at the wound edge prior to the onset of motility [Gundersen and Bulinski, 19881. However, since cells at the wound edge have a free cell margin while cells inside the monolayer are completely surrounded by other cells, it seems likely that the state of cell-cell contact regulates the interconversion between these arrays. In order to examine the relationship between the

state of cell-cell contact and the displacement of the array of Glu MTs, we have extended our original study by determining the effect of nascent cell-cell contacts on the polarized arrays of Glu MTs in motile NRK fibroblasts. Results described here show that the spatial distribution of Glu MTs in these cells is indeed dependent on cell-cell contact, and suggest that the oriented Glu MTs present at the leading edge of motile NRK cells are rapidly cleared from the leading lamellae after cell-cell collision. The close temporal correlation between cell-cell collision and the loss of polarized Glu MTs suggest that these events may represent an integral step in contact inhibition of locomotion. MATERIALS AND METHODS Materials

Rat monoclonal antibody (YL 1/2) against Tyr tubulin was obtained from Dr. J. Kilmartin (MRC, Cambridge). Rabbit polyclonal antiserum against Glu tubulin has been described earlier [Gundersen et al., 19841. Fluorescein-conjugated goat anti-rabbit IgG was from Cappel (Durham, NC). Rhodamine-conjugated goat anti-rat IgG was from Jackson Immunoresearch (West Grove, PA). Nocodazole was purchased from Aldrich Chemical (Milwaukee, WI). Cytochalasin D (CD) was from Calbiochem (San Diego, CA). NRK fibroblasts were obtained from Dr. R. Assoian (Department of Biochemistry, Columbia University), and maintained in DMEM (GIBCO) supplemented with penicillin, streptomycin, 10 mM HEPES (pH 7.4), and 10% calf serum (Hyclone, Denver, CO), in a humidified CO, incubator at 37°C. Time-Lapse Recording

For time-lapse recording, NRK cells were plated onto glass coverslips that had been mounted on the bottom of 35 mm tissue culture dishes (Corning). The “COVerslip dish” was made by first punching a hole (13 mm diameter) in a 35 mm dish and then mounting a 22 x 22 mm acid-washed glass coverslip to the bottom of the dish from underneath using a mixture of heat-melted paraffin and Vaseline (mixed at 3:l). NRK cells were grown to confluency in 10% calf serudDMEM and a “wound” was made by scraping a narrow strip of cells with the edge of a jeweler’s screw driver. The width of the wound was usually two to four cell lengths, so that migrating cells from the two edges of the wound came into contact 2 to 3 hours after wounding. The wounded monolayer was immediately rinsed twice with recording medium (10% calf serum, penicillin, streptomycin, and 20 mM HEPES, pH 7.4, in modified DMEM containing sodium bicarbonate at 1/25 the level of regular DMEM), and then 2 ml of recording medium was added to the dish, followed by 4 ml of light mineral oil (Fisher Sci-

Glu Microtubules in Contacting Cells

entific) to prevent evaporation of medium during timelapse recording. The dish was placed on the stage of Zeiss Axiovert35 microscope and movement of cells was observed with either a 20X or 40X Neofluar objective using phase contrast or differential interference contrast (DIC) optics. Temperature was maintained at 37 & 1°C with an aircurtain generated by a “Curly Top” hair dryer (Windmere Products, Miami Lakes, FL), and monitored with a YSI 2100 tele-thermometer equipped with a themoprobe (YSI-427). Phase contrast and DIC images were captured with a Newvicon video camera (DAGE-mti, 70 series), and recorded at 20 second intervals to an optical disc with an optical disc recorder (Panasonic, TQ-2028F). Both recording and playing back were controlled by an Image- 1 image analysis program (Universal Image Corp., West Chester, PA) installed on a desktop computer (Dell, System 310). At the end of the time-lapse recording, mineral oil and medium were aspirated, cells were briefly rinsed with phosphate-buffered saline at 37”C, and then the entire dish was plunged into methanol at -20°C for 5 minutes to fix the cells. For Figures 2 and 5 , selected images from the time-lapse recording were displayed on a video monitor (SONY, PVM- 1271Q) and photographed with Kodak technical pan 2415 film. lmmunofluorescence and Digital Processing

Indirect double immunofluorescence of fixed cells for Tyr and Glu MTs was done as described previously [Gundersen et al., 19871, using rhodamine- and fluorescein-conjugated secondary antibodies for Tyr MTs and Glu MTs, respectively. Stained cells were mounted in Fluoromount-G (Fisher Scientific) and observed with a Nikon Optiphot epifluorescence microscope with appropriate filter cubes. Cells of interest, which had been monitored for cell-cell contact, were located using the last image from the time-lapse recording as reference. For presentation purposes, immunofluorescence images were photographed with a 40 X Zeiss Planapochromat objective lens using hypersensitized Kodak technical pan 2415 film prepared with a hypersensitization kit (Lumicom, Livermore, CA). For routine analysis of immunofluorescence data, images were captured with a SIT camera (DAGE-mti, 65 series) and digitized with the Image-1 program. The digitized images were then printed with a video graphic printer (SONY, UP-850) and these printouts were used to analyze immunofluorescence data. The orientation of the MTOC was determined as previously described [Gundersen and Bulinski, 19881.

Inhibition of Motility with Cytochalasin D (CD) To determine the minimum concentration of CD necessary to inhibit forward translocation of NRK cells,

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motile cells were treated with CD at a final concentration of 0.1,0.2,0.5, and 1.O pM CD. Confluent monolayers of NRK cells in a coverslip dish were wounded as described above. About 30 min after wounding, the cells were switched to recording medium, overlaid with mineral oil and then placed on a prewarmed microscope stage for time-lapse recording. A wound edge was recorded for 60 min at 37°C as described above to provide a baseline of the cells’ motility. The cells were then treated with CD by adding 2 ml of prewarmed recording medium containing the appropriate concentration of CD (diluted more than 1,000 times from a stock solution prepared in dimethylsulfoxide) . The time-lapse recording was continued for at least 2 hr after CD addition. Of the concentrations tested, 0.2 p M CD was the lowest that completely inhibited forward translocation of NRK cells (see Fig. 8a); therefore this concentration was used in subsequent experiments. To determine the effect of inhibiting cell translocation on the oriented array of Glu MTs, confluent monolayers of NRK cells on glass coverslips were wounded and allowed to develop oriented Glu MTs for 90 min. At 90 min, the medium was removed and fresh medium containing 0.2 pM CD was added; controls received fresh medium without CD. Addition of dimethylsulfoxide at the concentration used for the CD delivery had no measurable effect. Cells were fixed in methanol at different intervals after the addition of CD and stained for Tyr and Glu MTs as described above. Microtubule Regrowth Experiments

Wounds were made in confluent monolayers of NRK cells on glass coverslips and cells at the wound edge were allowed to develop oriented Glu MTs as described above. At 90 minutes after the wounding, nocodazole was added to a final concentration of 10 pM in order to completely depolymerize microtubules . This concentration of nocodazole also blocked further cell migration and prevented the cells at the wound edge from coming into contact during the course of the experiment. After a two-hour incubation in nocodazole, the medium was switched to fresh DMEM containing 10% calf serum after two washes with the same medium. The cells were fixed in methanol at different intervals after nocodazole removal and stained for Tyr and Glu MTs as described above. RESULTS Differential Distribution of Glu MTs in Cells at the Wound Edge and Inside the Monolayer

It has been previously shown that stable Glu MTs are selectively localized in the leading edge of NIH 3T3 cells at the margin of an experimental wound [Gundersen

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Fig. 1. Distribution of Tyr and Glu MTs in NRK cells at the wound edge. A confluent monolayer of NRK cells was wounded and incubated for two hours as described in the text. Cells were then fixed and double immunofluorescently stained for Tyr tubulin (a), and Glu tubulin (b). Bar, 20 pm.

and Bulinski, 19881. The distribution of Glu MTs is such that they are oriented toward the wound, which is the direction of movement of the cells. We have observed a similar pattern of Glu MTs in wounded monolayers of NRK fibroblasts (see Fig. l ) , and also other fibroblast cell lines (Swiss 3T3, Balb/c 3T3, and Rat-6 fibroblasts; data not shown). Of the cell lines tested, NRK cells yielded particularly uniform monolayers that were free of overlap, and therefore, we chose to use them for the present study. The high density and oriented nature of Glu MTs at the leading edge is characteristic of the cells at the wound margin since cells inside the monolayer rarely contain such Glu MTs (see Fig. 1). Instead, the cells inside the monolayer, which are in contact with surrounding cells, have a small number of Glu MTs that are usually coiled around the nucleus. The shape of Glu MTs in these cells is generally a curled one, which is readily distinguishable from the extended Glu MTs found in cells at the wound edge. A small percentage (-5%) of cells at the wound edge and inside the monolayer do not contain immunologically detectable Glu MTs. These presumably represent cells about to undergo mitosis, since they frequently contain separated centrosomes (see Fig. 1 for an example), and we have found that the breakdown of interphase Glu MTs begins prior to the onset of mitosis (unpublished observation). Oriented Glu MTs Are Not Present in Contacting Cells That Become Surrounded The different distributions of Glu MTs in cells at the wound edge and within the interior of the monolayer

suggested that cell-cell contact may trigger a change in the oriented array of Glu MTs present in migrating cells. To test this idea, we allowed migrating NRK cells from the two sides of the wound to come into contact in order to mimic the process of a monolayer formation in a controlled manner, and determined when and how the Glu MT distribution switched from “the wound edge” pattern to ‘‘the monolayer’ ’ pattern. Time-lapse recordings were made of the contacting cells to provide a record of events before, during, and after the cell-cell contact. From the time-lapse recordings, we could determine the time of initial cell-cell contact, and the time at which contacting cells became completely surrounded by other cells. Following fixation and immunofluorescence staining, cells that had been recorded were relocated and their distributions of Tyr and Glu MTs were examined. An example of such an experiment is shown in Figure 2, which shows DIC images from the recording and illustrates the behavior of cells during contact, and in Figure 3, which shows immunofluorescence images of the fixed contacting cells. At the time of initial contact (0 min in Fig. 2), the ruffling of the leading edges of the two contacting cells is locally inhibited. This response is characteristic of contacting cells during contact inhibition of motility [e.g., Trinkaus et al., 19711 and provided us with a convenient visual marker which confirmed that a productive contact had been established. The inhibition of ruffling becomes more extensive as the contact between the cells becomes more extensive [19 min in Fig. 21, Although the forward translocation of the cell’s leading edge was completely stopped when an extensive con-

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tact with another cell was established [e.g., 19 min in Fig. 21, the nucleus continued moving forward in many of the sequences we observed. This is particularly evident for the nucleus in the contacting cell on the right in Figure 2 (compare 19 rnin and 35 min). This continuing movement of the nucleus is interesting, since it suggests that the forward movement of the nucleus is not directly dependent on the forward protrusion of the leading edge. For the experiment shown in Figure 2, the two cells in the middle became completely surrounded by other cells at 19 rnin for the cell on the left and at 25 min for the cell on the right. From this point until the cells were fixed at 37 min, neither cell exhibited ruffling or lamellar protrusions on any of its surfaces. Thus, the cell on the left and the cell on the right had been completely surrounded and had stayed immotile for 18 min and 12 min, respectively, at the time they were fixed. The distributions of Tyr and Glu MTs in the surrounded cells are shown enlarged in Figure 3, a and b, respectively. The two surrounded cells have a distribution of Glu MTs similar to that observed in cells in the interior of the monolayer (see Fig. lb). The Glu MTs in these cells are in a knot around the nucleus rather than extending toward the leading edge, or what had been the leading edge. These cells had been in contact for 37 rnin and they had been surrounded for an even shorter period of time (12 min and 18 min), suggesting that the signals that lead to the juxtanuclear distribution of Glu MTs in cells within the monolayer are closely related to the actual cell-cell contact during monolayer formation. This hypothesis was tested more thoroughly in the experiments described below. Alteration of Oriented Glu MTs in Migrating Cells Occurs Rapidly Once the Cells Become Surrounded To determine whether cell-cell contact induced an alteration in the distribution of Glu MTs, it would be ideal to observe the Glu MTs in real time as two cells collide with each other. However, real time observation

Fig. 2. Time-lapse sequence of NRK cells as they become surrounded by virtue of contact with other cells. Selected frames from a time-lapse recording using DIC optics are presented. Elapsed time (in min) is indicated at the upper left corner of each panel. The first panel (- 16 min) shows the polarized appearance of motile cells at the edges of a wound before the initiation of contact. At 0 min, two cells in the middle made initial contact. At 19 min, the cell at the left was completely surrounded by other cells. At 25 min, the cell at the right was completely surrounded by other cells. At 35 min, the recording was stopped, and cells were fixed in methanol (at 37 min). Immunofluorescent images of the cells in the boxed area in the last panel are shown at higher magnification in Figure 3. Bar, 20 pm.

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Fig. 3. Immunofluorescence of the surrounded NRK cells shown in Figure 2 . NRK cells shown in Figure 2 were fixed and double stained for Tyr tubulin (a) and Glu tubulin (b) as described in the text. The cell on the left and that on the right had been surrounded for 18 min and 12 min, respectively, at the time of fixation. Bar, 20 p,m.

of Glu MT dynamics is not currently possible, since Glu MTs are generated from pre-existing Tyr MTs by the enzymatic detyrosination of Tyr tubulin [Gundersen et al., 19871. Nonetheless, we were able to conduct a statistical analysis of changes in the Glu MT distribution induced by cell-cell contact, since the distribution of Glu MTs in the cells at the wound edge prior to contact was quite uniform (see Fig. lb). Depending on the experiment, between 50 and 70% of cells at the wound edge

had straight Glu MTs oriented toward the leading edge, 10-20% had juxtanuclear Glu MTs similar to those in cells within the monolayer, 15-3596 contained Glu MTs characterized as intermediate between these groups, and 0-5% had no detectable Glu MTs. These percentages are based on determinations from over 10 separate experiments, in which over 500 individual cells were scored for each experiment. In most of the cells of the intermediate category, the majority of the Glu MTs were oriented toward the leading edge but did not extend all the way to the margin of the cell, and therefore these cells were not included in the oriented category. Cells with no detectable Glu MTs may be in the late G2 stage of the cell cycle (see above). Because the distribution of oriented Glu MTs at the leading edge was the most unambiguous and also the most dissimilar from that in cells within the monolayer, we were particularly interested in determining whether the percentage of cells containing this array was altered as a consequence of cell-cell contact. We evaluated the distribution of Glu MTs in 139 individual cells that had been fixed at various intervals after they had become surrounded by cells migrating from the other side of the wound. As shown in Figure 4,the percentage of cells with an oriented array of Glu MTs extending to the leading edge did not change significantly from that in noncontacting cells during the first 10 min after the cells became completely surrounded. However, over the next time interval (1 1-30 min) and for the remainder of the time course, the percentage of cells displaying such an oriented array of Glu MTs dropped to only 10-15% of the entire population, approaching the level (3%) exhibited by nonmotile cells within the monolayer. Thus, when cells become surrounded by virtue of cell-cell contact, they rapidly lose the Glu MT array most characteristic of motile cells. While the percentage of cells with extended Glu MTs oriented toward the leading edge decreased after the cells became surrounded, the percentage with curly perinuclear Glu MTs steadily increased (Fig. 4). This increase in the population of cells with perinuclear Glu MTs paralleled the loss of cells with oriented Glu MTs. The percentage of cells with intermediate Glu MTs (oriented toward, but not extending to, the leading edge) did not change significantly during the first 30 min after the cells had become surrounded, and at later times decreased to a level below that prior to contact. Little change was noted in the percentage of cells with no immunodetectable Glu MTs over the entire time course. Taken together, these data suggest that the extended and oriented array of Glu MTs in migrating cells is converted to the juxtanuclear array within 30 min after the cells have become surrounded. Because Glu MTs have been shown to be long-lived MTs [Webster et al., 19871, this

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100

0 51-70min

071-ll0min 0 Monolayer

n

Oriented Glu MTs

Intermediate Perinuclear Glu MTs Glu MTs

Fig. 4. Distribution of Glu MTs in cell populations that had been surrounded for varying lengths of time. A total of 139 cells, which had become surrounded by other cells, were identified from recorded timelapse sequences. The sequences were analyzed to determine the time at which the cells became completely surrounded. The distribution of Glu MTs was then scored and the results plotted as a function of time after the cell became completely surrounded by other cells. For comparison, the distributions of Glu MTs in cells prior to contact (“wound

rapid alteration in the Glu MT distribution has interesting implications for the way that the stable MT array is regulated in cells (see Discussion).

No Glu MTs

edge” cells) and in cells that had been surrounded throughout the recording (“monolayer” cells) are shown. Categories used for the scoring are defined in the text. The number of cells examined in each group was as follows: 22 cells for 1-10 min, 29 cells for 11-30 min, 23 cells for 31-50 min, 33 cells for 51-70 min, 32 cells for 71-110 min. For “wound edge” cells and “monolayer” cells, 200 and 322 cells, respectively, were scored in the dishes that were used for timelapse recording and fixed at two to three hours after the wounding.

The DIC images in Figure 5 show an example of the sequence of events that occurs as two cells come into contact with each other. Prior to contact, the leading edges of both cells are actively ruffling (Fig. 5 , -10 Altered Distribution of Glu MTs Induced by min). As the initial contact is made (0 min) and expanded Cell-Cell Contact is a Localized Event at later times (3-15 rnin), ruffling ceases in the region of The responses of cells during contact inhibition of contact. In most of the early contacts that we recorded, locomotion are generally thought to be localized to the ruffling and lamellar protrusions were observed intermitregion of cell-cell contact [Abercrombie, 19701. For ex- tently at the free cell edges adjacent to contact sites. In ample, the most characteristic response of contact inhi- the example shown in Figure 5 , active ruffling is particbition of locomotion, the paralysis of ruffling of the lead- ularly evident in the lower portion of each contacting cell ing edge, is restricted to the area of contact between the at 3 rnin (arrowheads) and in the top portion of each cell colliding cells [Trinkaus et al., 19711. We were inter- at 9 rnin (arrowheads). The last panel in Figure 5 shows ested in determining whether the alteration in the ori- the cells just before they were fixed for immunofluoresented array of Glu MTs is a localized event similar to the cence (at 17 rnin after contact). The distributions of Tyr contact inhibition of ruffling. Accordingly, we examined and Glu MTs in the contacting cells (boxed area in Fig. earlier stages of cell-cell contact when the colliding cells 5, 15 min) are shown enlarged in Figure 6, a and b, had not yet become completely surrounded. Time-lapse respectively. While Tyr MTs are located throughout the recordings were made as described above to monitor the cytoplasm of the contacting cells, Glu MTs are conspiccontact between two cells from opposite sides of the uously absent from the regions of the cytoplasm subjawound. A cell was considered to have made contact (0 cent to the contact site. Instead, Glu MTs are found min) when active ruffling at the leading edge ceased due around the nucleus and extending to one of the nonconto encroachment of another cell. Cells were fixed at in- tacting and ruffling edges of each cell (arrows in Fig. tervals after the contact, and the distribution of Tyr and 6b). The other noncontacting margins do not show Glu Glu MTs was determined by immunofluorescence stain- MTs extending all the way to the ruffling edges (arrowheads, Fig. 6b). ing.

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In all, we determined the distributions of Tyr and Glu MTs in 66 cells that had established localized contacts on portions of their leading edges with the duration of contact ranging from 10 to 30 min. Because of differences in the width of the leading edge and the orientation of the contacting cells, the extent of contact varied substantially within this group (see additional examples, Fig. 7). Nonetheless, we consistently found that oriented Glu MTs were absent in the immediate vicinity of the contact site, and were still present and oriented toward the free cell margins in noncontacting areas (see arrows, Fig. 7b and d). Among the 66 cells we examined, only 14% contained an oriented array of Glu MTs extending to the contact site. Since 50-70% of the cells at the wound margin contain such an oriented array of Glu MTs before contact (see Fig. 4), this shows that even a localized contact at the leading edge can cause a rapid alteration in the Glu MT array. However, in contrast to cells that became surrounded, cells with a localized contact apparently do not lose their entire array of oriented Glu MTs . Further analysis of the Glu MTs in cells with localized contacts supports the idea that the immediate effect of cell-cell contact on Glu MTs is localized. Cells that had made a limited contact for 10-30 min, and still had a part of their original leading edge free and ruffling at the time of fixation, frequently contained Glu MTs extending to the ruffling edge (47% of cells examined, n = 53). In most of the remaining cases (40% of total), Glu MTs were oriented in the direction of the ruffling edge, but did not extend all the way to the cell margin (as with the intermediate Glu MTs in Fig. 4). These percentages are similar to those in cells before contact (see Fig. 4, wound edge cells), suggesting that the localized contact does not affect the bulk of the oriented Glu MTs. These data, combined with the observation that Glu MTs are lost from the area subjacent to the contact site, show that the early (10-30 min) effects of contact in the ori-

Fig. 5 . Time-lapse sequence of the initial localized contact between NRK cells in an experimental wound. Cell-cell contact in a wounded monolayer was recorded as in Figure 2, but cells were fixed before they became surrounded by other cells. Shown here is a sequence of DIC images of migrating cells as they make contact. Elapsed time is indicated at the upper left corner of each panel in min. The first panel (- 10 min) shows the wound edge cells before the initiation of contact. At 0 min, two cells in the middle made an initial contact. At 3 min, both cells continue the ruffling on their lower side (indicated by arrowheads). At 9 min, contact is more extensive and local inhibition of ruffling is apparent. Both cells exhibit ruffling edges on the top side of the contact site (arrowheads). At 15 min, the recording was termnated, and cells were fixed at 17 min. Immunofluorescent images of the cells in the boxed area in the last panel are shown at higher magnification in Figure 6. Bar, 20 pm.

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tact on the Glu MT array, we found in a small number of cases (14%, n = 172) that there was an array of cytoplasm just behind the contact site that contained a substantially lower level of Tyr MTs. These areas were about the size of the nucleus, and in contrast to immediately adjacent areas, contained an easily enumerated number of MTs. Examples of such areas can be seen clearly in two of the four contacting cells shown in Figure 7a and c. Although the apparent density of MTs was very low in these areas, we have not observed a case in which MTs were completely absent from an equally sized region behind the contact site. Such large regions of low MT density were only rarely detected in the leading edge of noncontacting cells ( l % , n = 261; statistically different from the contacting cells at a 95% confidence level). While the significance of these relatively large regions of lower MT density is unclear at this time, it is possible that they reflect an effect of cell-cell contact on most of the MTs near the contact site (see Discussion). Inhibition of Forward Locomotion by CD Does Not Effect a Rapid Alteration of Glu MT Arrays

Fig. 6. Immunofluorescence of the NRK cells with localized contacts shown in Figure 5 . Immunofluorescent images of Tyr MTs (a) and Glu MTs (b) in cells shown in Figure 5 . The arrows indicate noncontacting ruffling edges that still contain oriented Glu MTs. Arrowheads show noncontacting, ruffling edges that do not contain oriented Glu MTs. Bar, 20 pm.

ented array of Glu MTs are restricted to the vicinity of the contact site. In addition to the effect of the initial cell-cell con-

The depletion of oriented Glu MTs after collision of two cells may result from transmembrane signals triggered by the interaction of the two cell surfaces. Alternatively, the alteration of Glu MT arrays may result indirectly from the cessation of forward locomotion that is caused by cell-cell collision. To test the latter possibility, we blocked forward locomotion of motile NRK cells at the edge of a wound with CD, and determined the effect on the oriented array of Glu MTs. At high (> 1-2 pM) concentrations of CD, fibroblasts will undergo a dramatic shape change in which most of the cytoplasm contracts around the nucleus, leaving extended processes at points where the cell is still attached to the substratum [ “arborization”; Goldman and Knipe, 1973; Yahara et al., 19821. However, at lower concentrations (< 1-2 pM), arborization does not occur, yet forward protrusion and ruffling of the leading edge are still inhibited [Goldman and Knipe, 1973; Yahara et al., 19821. Because of this extreme sensitivity of the leading edge activities to CD, we reasoned that we could inhibit forward locomotion of the cells at concentrations of CD that would not cause a dramatic alteration in cell shape. This was an important consideration, since we wished to examine the distribution of Glu MTs in cells rendered immotile, but without such a dramatic shape change that it would be difficult to relate the distributions to normal untreated cells. To find the lowest concentration of CD that would inhibit forward locomotion of NRK cells, we treated wounded monolayers of NRK cells with 0.1, 0,2, 0.5, and 1.0 pM CD while recording their motility by time-

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Fig. 7. Immunofluorescence of locally contacting NRK cells in an experimental wound. Motile NRK cells were recorded and then fixed for immunofluorescence after they had established contacts on portions of their leading edges, as described in Figures 5 and 6. Shown are the immunofluorescent images of Tyr MTs (a,c) and Glu MTs (b,d) in two such cell pairs with localized contacts. a and b: Cells were fixed 15 minutes after the initial contact. The cell on the top

shows a small number of Glu MTs oriented toward the ruffling edge (indicated by an arrow), while, in both cells on top and bottom, Glu MTs are absent at the immediate vicinity of the contact region. c and d: Cells were fixed 20 minutes after the initial contact. Glu MTs are absent in the immediate vicinity of the contact area, but are seen clustered toward the noncontacting ruffling region (indicated by an arrow) in the cell on the top. Bars, 10 pm.

lapse video microscopy. We found that the cells were MTs (data not shown). In contrast, the effect of cell-cell still partially motile at 0.1 pM CD, but at 0.2 pM, the contact on the proportion of cells with oriented Glu MTs cells exhibited almost no motility. As shown in Figure (data taken from Fig. 4 and plotted as a dashed line in 8a, the inhibition of forward locomotion of NRK cells Fig. 9) indicates that cell-cell contact leads to a more with 0.2 p M CD was virtually instantaneous and was rapid and complete change in the oriented Glu MTs than maintained for at least 2 hr. Analogous to cell contact- does simply blocking cell locomotion. This suggests that induced inhibition of locomotion, the movement of nu- the loss of oriented Glu MTs after cell-cell collision is clei continued in the presence of 0.2 p M CD. It is also not directly related to the resulting inhibition of forward worth noting that at this concentration of CD, the NRK locomotion, but is due to other events triggered by cellcells did not undergo a dramatic change in cell shape, cell contact. whereas arborization was observed within minutes with 1.0 pM CD. Establishment of Newly Formed Glu MTs Is We then examined the effect of inhibiting locomo- Dependent on Cell-Cell Contact tion with 0.2 p M CD on the oriented array of Glu MTs in cells at the wound margin. Cells at the edge of a The results described above suggest that cell-cell wound that had been treated with 0.2 p M CD for 30 min contact, as a result of cell collision, triggers the loss of still exhibited oriented Glu MTs (Fig. 8c) that were vir- Glu MTs from the leading edge of migrating cells. This tually indistinguishable from those in cells incubated loss of Glu MTs could be induced by either: (1) depolywithout CD (Fig. 8b). A comparison of the percentage of merization of pre-existing Glu MTs and formation of cells exhibiting oriented Glu MTs (as defined in Fig. 4) new Glu MTs at a new location, (2) relocation of preshowed that inhibiting locomotion had only a small ef- existing Glu MTs, or (3) a combination of both. As an fect on the population of cells with oriented Glu MTs initial step in determining the mechanism of the Glu MT when compared with motile control cells (Fig. 9). At redistribution in contacting cells, we have determined the later time points (90 and 120 min), the CD-treated cells pattern and kinetics of Glu MT formation in wounded still contained oriented Glu MTs, although some of the monolayers of NRK cells. cells appeared to have a reduced number of oriented Glu Monolayers of NRK cells were wounded, cells at

Glu Microtubules in Contacting Cells

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Fig. 8. Effect of inhibiting locomotion with CD on the oriented array of Glu MTs. In (a) is shown a series of micrographs taken from a time lapse recording of NRK cells moving into a wound. Times on the right are given in min. At 0 min, CD was added to a final concentration of 0.2 pM, and the recording was continued for another 120 min. The white line on each of the panels represents the position of the leading

edges of the cells at the time of CD addition. The figure was constructed by overlaying a trace of the position of the cells at t = 0 on each of the images. Bar, 30 pm. The immunofluorescence images in (b) and (c) show the distributions of Glu MTs in (b) control cells 30 min after adding medium without CD and in (c) 0.2 pM CD-treated cells 30 rnin after adding medium with CD. Bar, 20 pm.

the wound edge were allowed to generate oriented Glu MTs, and then MTs were completely depolymerized by nocodazole treatment. Following removal of nocodazole, MTs were allowed to grow back for different intervals before fixation and staining. If the state of cellcell contact is a contributing factor in establishing the distribution of Glu MTs, then the newly formed Glu MTs would be distributed within the cell in a manner similar to that before the drug treatment: i.e., Glu MTs would be oriented toward the leading edge in cells at the wound margin, and coiled around the nucleus in cells within the monolayer. Figure 10 shows the distribution of newly formed Tyr and Glu MTs at 10,30, and 60 rnin after the removal of nocodazole. After 10 min, the distribution of newly formed Tyr MTs (Fig. 10a) was already indistinguishable from that in untreated cells (see Fig. la). On the other hand, the formation of Glu MTs was considerably slower than that of Tyr MTs. Glu MTs first became detectable at 20-30 rnin after the drug release (see Fig. 10d for 30 min) and 50-60 rnin were required before the array of Glu MTs resembled that in untreated cells (see

Fig. 10f for 60 rnin). These results are similar to those obtained with other cell lines [e.g., Gundersen et al., 19871 and are consistent with the idea that Glu MTs in NRK cells are formed by the post-polymerization detyrosination of Tyr MTs. In these experiments, we consistently observed a slightly longer delay (5-10 min) in the reappearance of Glu MTs, but not Tyr MTs, in the cells within the monolayer when compared with that in the cells at the wound edge (data not shown). We do not know whether this apparent delay represents actual differences in the rates of Glu MTs formation or whether it is because of the relative ease of detecting Glu MTs in the cells at the wound edge. Significantly, the newly formed patterns of Glu MTs are similar to those in cells that had never been treated with nocodazole. Cells at the wound edge generated Glu MTs oriented toward the free leading edge, whereas cells inside the monolayer formed mostly Glu MTs coiled around the nucleus (Fig. lOf). Thus, the generation of new Glu MT arrays in these cells appears to be tightly coupled to the state of cell-cell contact.

Nagasaki et al.

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601

‘f -I O

20

0 4 ”

0

I

30

”

I

”

60

1

90

”

’

120

Time (rnin)

Fig. 9. Quantification of the effect of inhibiting forward locomotion with 0.2 pM CD on the oriented array of Glu. Wounded monolayers were treated with or without 0.2 pM CD for different intervals and then fixed and stained for Glu and Tyr MTs. The percentage of wound edge cells containing an oriented array of Glu MTs (as defined for Fig. 4) was then determined. The results shown were obtained from three separate experiments in which over 200 cells were scored for each time point. (Error bars are S.E.M.) The dashed line (labeled “cell-cell contact”) was redrawn from the data in Figure 4 for the category “oriented Glu MTs,” using the end time within each bar as the “time” for plotting in this figure.

Orientation of the Microtubule Organizing Center Is Not Rapidly Altered by Cell-Cell Contact

In many types of motile cells, such as neutrophils [Malech et al., 19771, endothelial cells [Gotlieb et al., 19811 and fibroblasts [Kupfer et al., 1982; Gundersen and Bulinski, 19881, the MTOC is positioned so that it comes to lie between the nucleus and the leading edge. We found that the reorientation of the MTOC in motile NRK cells at the wound edge was similar to that reported by Kupfer et al. [1982]. At 2-3 hr after the wounding, approximately 60% of the NRK cells contained the MTOC at the front of the nucleus, or between the nucleus and the leading edge. In the rest of the cells, the MTOC was located on either side of the nucleus or behind the nucleus, relative to the direction of cell migration. When we examined the position of the MTOC in cells whose movement had been recorded in time-lapse sequences, we found that cell-cell contact did not induce a rapid change in the orientation of the MTOC (data not shown). DISCUSSION

In this study using NRK fibroblasts, as well as in a previous study using 3T3 fibroblasts [Gundersen and Bulinski, 19881, we have found that the distribution of Glu MTs in motile cells at the edge of a wound is distinct from that in nonmotile cells within the monolayer. The

results presented here show further that the cytoplasmic distribution of Glu MTs in motile NRK cells changes rapidly after the leading edge makes contact with another cell. When the contact is restricted to a portion of the leading edge, the oriented array of Glu MTs characteristic of migrating cells is no longer found in the vicinity of the contact area (Figs. 5-7). When the entire leading edge is in contact with other cells, i.e., when the cell is surrounded, Glu MTs are primarily found coiled around the nucleus and do not extend to the cell edges (Figs. 2-4). These changes in the Glu MT distribution suggest that cell-cell contact results in the generation of cytoplasmic signals that effect an alteration in the array of Glu MTs. These putative signals must be triggered shortly after cell-cell contact since we observed localized alterations in the Glu MT arrays in some cells as early as 10-15 min after contact had been initiated, and a more complete transformation of the Glu MT array by 11-30 min after cells had become surrounded (Fig. 4). Presumably, the localized depletion of Glu MTs triggered by a localized contact is integrated over areas in which cells have contacts so that when the cells become surrounded, they lose their entire array of extended Glu MTs. These surrounded cells must also become incapable of generating a new array of extended Glu MTs, since we did not observe the reappearance of such an array in cells for over one hour after they had become surrounded (see Fig. 4). This idea is also supported by the inability of cells within the monolayer to generate an extended array of Glu MTs in regrowth experiments (Fig. lo). These observations suggest that signals triggered by cell-cell contacts can be sustained as long as the state of cell-cell contact is maintained. Further experiments will be necessary to identify the nature of the signals that trigger the alteration in the Glu MT array (see below). Significantly, the intracellular signals that effect an alteration in the oriented array of Glu MTs do not appear to be directly related to the inhibition of locomotion that occurs during cell-cell collision. When we blocked forward translocation of the cells at the wound edge with a low concentration of CD, we did not find as rapid or as extensive an effect on the oriented Glu MTs as we had observed in the contacting cells. This suggests that immobilization per se is not sufficient to cause the loss of extended Glu MTs from the leading lamellae of contacting cells, and that additional intracellular signals are elicited by the actual interaction of the two contacting cell surfaces. This interpretation is consistent with previous observations that showed that oriented Glu MTs could form in cells at the wound edge prior to the onset of locomotion [Gundersen and Bulinski, 19881. Although we inhibited locomotion of cells with CD, an agent known to interfere with actin assembly

Glu Microtubules in Contacting Cells

Fig. 10. Distributions of newly formed Tyr and Glu MTs in a wounded monolayer of NRK cells after removal of nocodazole. A confluent monolayer of NRK cells was wounded and treated with nocodazole as described in the text. After complete depolymerization of microtubules, the cells were transferred to nocodazole-free me-

57

dium. Cells were fixed for double immunofluorescence at intervals after the removal of nocodazole. Shown here are immunofluorescence of Tyr MTs (a, c, e) and corresponding Glu MTs (b, d, 0 in cells fixed at 10 min (a, b), 30 min (c, d) and 60 min (e, f) after nocodazole was removed. Wounds are on the right of each image. Bar, 20 bm.

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[reviewed in Cooper, 19871, it is important to point out that our study with CD does not rule out the possibility that microfilaments are involved in the formation, maintenance and/or turnover of the oriented Glu MTs. Different microfilament arrays within cells exhibit different sensitivities to cytochalasins with the microfilament network in the lamellapodium being one of the most sensitive [Goldman and Knipe, 1973; Yahara et al., 19821. At the concentration of CD used in our experiments (0.2 FM), we observed inhibition of lamellapodium protrusion and leading edge ruffling without a major change in the shape of the cells. Therefore, it is reasonable to assume that the effects of 0.2 pM CD on NRK cells were restricted to highly sensitive arrays of microfilaments. This leaves open the possibility that other microfilament arrays are important for the formation and/or turnover of Glu MTs. We are extending our studies with cytochalasins to examine this question in more detail. Given the enhanced stability of Glu MTs in vivo [reviewed in Bulinski and Gundersen, 19911, the contact-induced alteration in the oriented array of Glu MTs has interesting implications for the regulation of the array of stable MTs in cells. Although our study necessarily relied upon a statistical analysis of the Glu MT distributions before and after cell contact, our data suggest that the Glu MTs in contacting motile cells exhibit dynamic behavior previously unrecognized for stable MTs in vivo. In addition to the decrease in the percentage of cells displaying an oriented array of Glu MTs in response to contact, there was a parallel increase in the population of cells with juxtanuclear Glu MTs. The simplest interpretation of these data is that the oriented array of Glu MTs is converted to the juxtanuclear array. This would imply that cell-cell contact induces either the coordinated depolymerization of the oriented Glu MTs followed by formation of new Glu MTs around the nucleus, the gross relocation of existing Glu MTs away from the contact site, or a combination of these two activities. Although lateral movements of MTs in interphase cells have been observed directly [Sammak and Borisy, 1988; Cassimeris et a]. , 19881, the movements characterized in these studies were limited in nature and seemed to occur randomly, unlike the coordinated relocation that may be happening in the contacting cells. If relocation were the primary mechanism responsible for the loss of Glu MTs from the leading edge area, the presence of the juxtanuclear Glu MTs could be explained by the mass movement of Glu MTs toward the vicinity of the nucleus. Alternatively, a localized depolymerization of Glu MTs triggered by cell-cell contact could also account for the loss of Glu MTs from the leading edge. If depolymerization were the predominant mechanism for loss of Glu MTs after contact, then the remaining juxtanuclear array of Glu MTs may represent partially depolymerized MTs. It

is also possible that the juxtanuclear array may have been present all along, and that only after depolymerization of the oriented array is it revealed as a distinct array. Judging from the results of the nocodazole/regrowth experiments, it is unlikely that the Glu MTs in the juxtanuclear array arise de novo after cell-cell contact. Following release from nocodazole treatment, it took almost one hour for NRK cells to regenerate a juxtanuclear array of Glu MTs (Fig. 10); yet in contacting cells, an increase in the number of cells with a juxtanuclear array had already occurred by 30 min (Fig. 4). Pulse-chase experiments with microinjected tagged tubulin will be necessary to distinguish between alternative mechanisms of GIu MTs remodeling based on relocation and/or depolymerization. In this study, we have focused on the effects of cell-cell contact on the polarized array of Glu MTs. However, it is worth noting that Tyr MTs, which reflect the total MT population, may also be affected by cellcell contact. In 14% of the cells with localized contacts, Tyr MTs were significantly reduced in a relatively large area behind the contact site. The low percentage of contacting cells exhibiting such regions with reduced MTs suggests that cell-cell contact either does not consistently result in an alteration of the total MT array or that it has a consistent effect, but one that is only transient. A transient effect would be difficult to capture in fixed cell preparations, since we would expect rapid regrowth of the dynamic Tyr MTs into the areas of low MT density. Although further experiments will be necessary to establish the effect of contact on Tyr MTs, these observations raise the possibility that the mechanism responsible for the remodeling of the Glu MTs upon contact (relocation and/or depolymerization) may also involve the entire MT array. The selective stabilization of the dynamic array of MTs in proliferating cells has been hypothesized to play an important role in the generation of cellular asymmetry [Kirschner and Mitchison, 1986; Bulinski and Gundersen, 19911. Cellular asymmetry in turn is responsible for important biological functions such as morphogenesis of the early embryo and subsequent development of complex tissues and organs. It has been well documented that MT stabilization occurs during a number of morphogenetic events: during the polarization of fibroblasts as they migrate into an in vitro wound [Gundersen and Bulinski, 19881, during the alignment of myoblasts prior to fusion to generate multinucleated myotubes [Gundersen et al., 19891, during neurite outgrowth [Baas and Black, 19901, and during formation of a polarized epithelium [Pepperkok et al., 19901. The results presented here and also by Pepperkok et al. [1990] show that cell-cell interactions can directly influence the stability of MTs. Interestingly, cell-cell interactions leading to the formation of an epithelium have an effect on MT stability opposite of

Glu Microtubules in Contacting Cells

that reported here, suggesting that cell-cell contact elicits a cell type-dependent effect on the MT array. It is interesting to note that the MT array is influenced by interactions with neighboring cells, because it shows that external environmental factors can affect the specification of the MT array. Since the distribution of MTs directly determines the arrangement of a number of organelles in the cell, MTs may be important mediators in the coordination of cellular activities. While MTs are clearly necessary for the locomotion of fibroblasts [Vasiliev et al., 1970; Goldman, 19711 and endothelial cells [Gotlieb et al., 19831, the role of stable Glu MTs in cell motility is less certain. The orientation of Glu MTs in the direction of migration [Gundersen and Bulinski, 19881 and the maintenance of Glu MTs at ruffling edges but not at cell-cell contact sites (this study) are consistent with the idea that Glu MTs are important in determining or maintaining the direction of motility [see Discussion of Gundersen and Bulinski, 19881. It is worth pointing out that in both this and the previous study, even though the percentage of wound edge cells exhibiting an oriented array of Glu MTs was high, it was not 100%.It is not clear at this time whether this reflects the lack of an absolute requirement of these stabilized MTs €or locomotion, or whether stabilized MTs are required only part of the time for locomotion. The influence of cell-cell contact on the distribution of Glu MTs has one immediate and practical implication. Although contact inhibition of locomotion has been recognized as one of the fundamental properties of cultured cells [Abercrombie, 1970; Heaysman, 19781, molecular and biochemical analyses have been hampered by lack of appropriate markers which can be used, for example, to purify molecules involved in the event. Since the distribution of Glu MTs can be determined easily, the contact-induced loss of Glu MTs can be employed as a marker for contact inhibition of locomotion. We have obtained preliminary results that plasma membranes prepared from NRK cells can cause a similar change in the distribution of Glu MTs when added to motile NRK cells at the wound edge. Thus, the use of Glu MTs as a marker for cell-cell contact should prove useful in obtaining molecular and biochemical information about events which comprise contact inhibition of locomotion. ACKNOWLEDGMENTS

The authors thank Dr. J. Kilmartin for YL 1/2 antibody, Dr. R. Assoian for NRK cells, Dr. F. Maxfield (Columbia University, Department of Pathology) and Dr. M. Gershon (Columbia University, Department of Anatomy and Cell Biology) for the use of the SIT camera, Dr. J.C. Bulinski (Columbia University, Depart-

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ment of Anatomy and Cell Biology) for the use of Nikon Optiphot, and I. Kim and M. Brewer for excellent technical assistance. This work was supported by grants from American Cancer Society (ACS-CD-398) and National Institute of Health (GM-42026) to G.G.G., and a Damon run yon-Walter Winchell Cancer Research Fund Fellowship (DRG-1099) to T.N.

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Schulze, E., Asai, D.J., Bulinski, J.C., and Kirschner, M. (1987): Posttranslational modification and microtubule stability. J. Cell Biol . 105:2 167-21 77. Trinkaus, J.P., Betchaku, T., and Krulikowsh, L.S. (1971): Local inhibition of ruffling during contact inhibition of cell movement. Exp. Cell Res. 64:291-300. Vasiliev, J.M., Gelfand, I.M., Domnina, L., Ivanova, O.Y., Komm, S.G., and Olshevskaja, L.V. (1970): Effect of colcemid on the locomotory behavior of fibroblasts. J. Embryol. Exp. Morph. 241625-640. Webster, D.R., Gundersen, G.G., Bulinski, J.C., and Borisy, G.G. (1987): Differential turnover of tyrosinated and detyrosinated microtubules. Proc. Natl. Acad. Sci. U.S.A. 84:9040-9044. Yahara, I., Harada, F., Sekita, S . , Yoshihira, K., and Natori, S . (1982): Correlation between effects of 24 different cytochalasins on cellular structures and cellular events and those on actin in vitro. J. Cell Biol. 92:69-78.