Forced Expression of a Dominant-NegativeChimeric Tropomyosin Causes Abnormal

Motile Behavior During Cell Division

Kit Wong, Deborah Wessels, Sonja L. Krob, Amanda R. Matveia,Jenny Li-Chun Lin, David R. Soll, and Jim Jung-Ching Lin*

Department of Biological Sciences, University of Iowa, Iowa City

Forced expression of the chimeric human fibroblast tropomyosin 5/3 (hTM5/3) inCHO cell was previously shown to affect cytokinesis [Warren et al., 1995:J. CellBiol. 129:697–708]. To further investigate the phenotypic consequences ofmisexpression, we have compared mitotic spindle organization and dynamic 2Dand 3D shape changes during mitosis in normal cells and in a hTM5/3misexpressing (mutant) cell line. Immunofluorescence microscopy of wild typeand mutant cells stained with monoclonal anti-tubulin antibody revealed that theoverall structures of mitotic spindles were not significantly different. However, theaxis of the mitotic spindle in mutant cells was more frequently misaligned with thelong axis of the cell than that of wild type cells. To assess behavioral differencesduring mitosis, wild type and mutant cells were reconstructed in 2D and 3D andmotion analyzed with the computer-assisted 2D and 3D Dynamic Image AnalysisSystems (2D-DIAS, 3D-DIAS). Mutant cells abnormally formed large numbers ofblebs during the later stages of mitosis and took longer to proceed from the start ofanaphase to the start of cytokinesis. Furthermore, each mutant cell undergoingmitosis exhibited greater shape complexity than wild type cells, and in every caselifted one of the two evolving daughter cells off the substratum and abnormallytwisted. These results demonstrate that misexpression of hTM5/3 in CHO cellsleads to morphological instability during mitosis. Misexpression of hTM5/3interferes with normal tropomyosin function, suggesting in turn that tropomyosinplays a role through its interaction with actin microfilaments in the regulation ofthe contractile ring, in the localized suppression of blebbing, in the maintenance ofpolarity and spatial symmetry during cytokinesis, and in cell spreading aftercytokinesis is complete. Cell Motil. Cytoskeleton 45:121–132, 2000.r 2000 Wiley-Liss, Inc.

Key words: cytokinesis; blebbing; misalignment of mitotic spindles; computer-assisted 2D and 3D dynamicimage analysis

INTRODUCTION

Human tropomyosins are encoded by four geneslocated on different chromosomes [Eyre et al., 1995;Hunt et al., 1995; Laing et al., 1995; Wilton et al., 1995,1996]. Through the use of multiple promoters andalternatively spliced exons, tissue-specific and cell type-specific tropomyosin isoforms can be generated fromthese four genes [see references in Lees-Miller andHelfman 1991; Pittenger et al., 1994; Lin et al., 1997]. At

K.W. and D.W. contributed equally to this work.

Contract grant sponsor: National Institutes of Health; Contract grantnumbers: HD18577, DK47673; Contract grant sponsor: W. M. KeckFoundation.

*Correspondence to: Dr. Jim Jung-Ching Lin, Department of Biologi-cal Sciences, University of Iowa, 138 Biology Building, Iowa City, IA52242-1324. E-mail: Jim-Lin@Uiowa.Edu

Received 22 September 1999; accepted 18 November 1999

Cell Motility and the Cytoskeleton 45:121–132 (2000)

r 2000 Wiley-Liss, Inc.

least eight distinct tropomyosin isoforms (hTM1, hTM2,hTM3, smooth slow, hTM5a, hTM5b, hTM5, and hTM4)have been detected in a human fibroblast cell line [Novyet al., 1993a]. In addition, an isoform (called TC22)identical to hTM5 except the last coding exon (IX) has

been cloned from a colon cancer cell line and found to beexpressed in human fibroblasts. Figure 1A depicts 4different genes encoding human tropomyosins and sum-marizes a structural comparison among these fibroblasttropomyosin isoforms and their respective striated muscle

Fig. 1. Human tropomyosin isoform diversity and comparison ofcoding exons among hTM3, hTM5 and chimeric hTM5/3 isoform.A:All known human tropomyosin isoforms are encoded by four differentgenes (a, b, g, andd). The isoforms are grouped according to theirrespective genes and compared with their respective striated muscleisoforms. The different type of rectangular colored-boxes indicatesexons with different sequences from that of their respective muscleisoforms. The numbers above or below each exon indicate the aminoacid residues encoded by that exon. For comparison purposes, we referto these exons as region I–IX to distinguish them from the exonnumbers given in the genomic organization. It is known that humanfibroblast can express hTM1, hTM2, hTM3, smooth slow, hTM5a,

hTM5b, hTM5, and hTM4 [Novy et al., 1993a; Lin et al., 1997].Isoform TC22 was recently cloned from a colon cancer cell line andcontained 247 amino acid residues. Skeletal/smooth fast and skeletal/smooth slow are fast-and slow-, respectively, migrating tropomyosinbands in SDS-PAGE gel of skeletal/smooth muscle. FT, fast-twitch;ST, slow-twitch. B: Exon differences among hTM3, hTM5, andchimeric hTM5/3. Using a PCR technique known as splicing byoverlap extension, a chimeric hTM5/3 was generated from hTM5 andhTM3 as described previously [Novy et al., 1993b]. As a result,hTM5/3 contains the N-terminus of hTM5 and the C-terminusof hTM3.

122 Wong et al.

isoforms. Some of these isoforms have been expressed asrecombinant proteins and characterized in vitro. Resultsdemonstrated that different isoforms differ in a varietyof properties including binding to actin filaments, promot-ing in vitro movement of actin filaments on myosin-coated surfaces, and stimulating actin-activated myosinATPase activity [Novy et al., 1993b]. Furthermore, thedifferential expression of tropomyosin isoforms in nor-mal and transformed cells [Hendricks and Weintraub1981; Matsumura et al., 1983; Tanaka et al., 1993;Cooper et al., 1985; Leavitt et al., 1986; Lin et al., 1984;1985; Novy et al., 1993a; Bhattacharya et al., 1988;Prasad et al., 1993; Takenaga and Masuda 1994] andthe differential localization of tropomyosin isoformsin nonmuscle cells [Lin et al., 1988, Hannan et al.,1995; Schevzov et al., 1997] imply that different tropo-myosin isoforms have distinct intracellular functions.Tropomyosin isoforms may be involved in stabilizingactin filaments, cell shape, intracellular granule move-ment and cytokinesis [see references in Lin et al.,1997].

The genetic analysis of the cell-cycle defectivemutant cdc8 inS. pombeprovided the first evidencethat yeast tropomyosin may be required for actin re-distribution and the formation of the contractile ringduring cytokinesis [Balasubramanian et al., 1992]. Wepreviously generated a human fibroblast chimeric tro-pomyosin, hTM5/3 (Fig. 1B), which exhibited abnor-mally strong binding to actin filaments and the loss ofKCl-dependent binding to actin filaments [Novy et al.,1993b], and which was capable of replacing preboundtropomyosins from actin filaments [Warren et al., 1995].Forced expression of this dominant-negative tropomyo-sin isoform in CHO cells resulted in a high incidenceof multinuclear cells, indicating a defect in cytokinesis[Warren et al., 1995]. Furthermore, we have shown thatthe tropomyosin isoform 4, which is recognized by themonoclonal antibody LC24, is preferentially localized inthe contractile ring of dividing CHO cells [Lin et al.,1997]. These results suggest that tropomyosin plays arole in the regulation of cell division. To understandthis role, we have analyzed the structure and orientationof the mitotic spindle, the timing of mitosis and thedynamic 2D and 3D morphology of cells during mitosisin hTM5/3 nonexpressing (wild type) and misexpress-ing (mutant) cell lines. We have found that expression ofthe dominant-negative hTM5/3 isoform leads to in-creased misalignment of the mitotic spindle along thelong axis in a significant proportion of dividing cells,excessive blebbing of dividing cells, twisting of themitosing cell, detachment of one of the two evolvingdaughter cells from the substratum, and longer times toproceed from the start of anaphase to the start ofcytokinesis.

MATERIALS AND METHODS

Cell Culture

hTM5/3 non-expressing (C68) and misexpressing(C70) CHO cell lines have been previously described[Warren et al., 1995]. The two lines were maintained inDMEM supplemented with 10% FCS and G418 (500µg/ml, GIBCO BRL, Gaithersburg, MD) in a humidifiedincubator at 37°C and 5% CO2.

Immunofluorescence Staining and Microscopy

Cells grown on glass coverslips were fixed, perme-abilized, and prepared for immunofluorescence micros-copy as described [Warren and Lin, 1993]. A monoclonalantibody DM1B againstb-tubulin [Blose et al., 1984]was used to visualize the mitotic spindles of stablytransfected CHO cells. The secondary antibody wasfluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Sigma, St. Louis, MO). Micrographs weretaken with a Zeiss phase-contrast/epifluorescence photo-microscope III.

2D Analysis of Cell Dynamics During Mitosis

Dividing cells in monolayers 80% confluent weregrown on glass coverslips. For analysis, the coverslip wastransferred to a perfusion chamber [Berg and Block,1984] and cells video-recorded through a 633 objectiveon a Zeiss phase-contrast photomicroscope III equippedwith Argus-10 Image processor (Hamamatsu PhotonicSystems Corp., Oak Brook, IL) and 70 series camera(DAGE-MTI Inc., Michigan City, IN). The microscopestage was pre-warmed to 37°C with an air-curtain. Therecorded images were then digitized at a rate of one frameper second on to the hard disc of a PowerComputingPowerTower Pro 225 computer (Apple Computer, Inc.,Cupertino, CA) equipped with a framegrabber board(Data translation, Inc., Marlboro, MA) and 2D-DIASsoftware [Soll, 1995; Soll and Voss, 1998]. Cell perim-eters were traced manually as previously described [Soll,1995; Warren et al., 1996; Soll and Voss, 1998]. TheDIAS software program generated both cell centroid andcell perimeter tracks. The parameters’ ‘‘instantaneousvelocity’’ and ‘‘directional change’’ were automaticallycompiled from the centroid paths. The parameters’ ‘‘area,’’‘‘maximum length,’’ ‘‘perimeter,’’ ‘‘ roundness,’’ ‘‘radiallength,’’ ‘‘radial deviation,’’ ‘‘concavity,’’ and ‘‘convex-ity’’ were computed from the cell perimeters. Detaileddescriptions of these parameters have been presentedelsewhere [Soll, 1995; Soll and Voss, 1998]. In brief, theinstantaneous velocity of a cell in frame n was computedby drawing a line from the cell centroid in frame n-1 tothe cell centroid in frame n11, and dividing that distanceby 2Dt, whereDt is the time interval between analyzedframes. In this analysis,Dt was 2 second. Directionalchange was calculated as the absolute value of the

Roles of Tropomyosin in Mitotic Cell Motility 123

difference in the direction of centroid translocation be-tween consecutive frames. If the directional change wasgreater than 180°, it is subtracted from 360° providingvalue between 0 and 180°. Instantaneous velocity anddirectional change were averaged over the period ofanalysis of each cell, and the mean of the averagescomputed for a number (N) of cells. The parameter‘‘area,’’ ‘‘perimeter,’’ and ‘‘maximum length’’ are self-evident. Radial length was computed as the averagedistance from boundary pixels of the cell perimeter to thecentroid. Radial deviation was computed by the formula100 3 SD/Radial Length, where SD is the standarddeviation of radial lengths. Roundness was computed bythe formula 1003 4 p (area / perimeter2). A roundnessvalue of 100 is a perfect circle and a roundness value of 0is a straight line. Convexity and concavity were computedby drawing line segments connecting the vertices of thefinal cell shape, and then measuring the angles of turning.Convexity was computed as the absolute value of the sumof positive turn angles in degrees, and concavity wascomputed as the absolute value of the sum of negativeturn angles in degrees. A cell with n projections will havea convexity of n3 180 and a concavity of n3 180–360.Again, these morphology parameters were averaged overthe period of analysis for each cell and the mean of theaverage computed for a number (N) of cells.

3D Reconstruction and Motion Analysisof Living Cells

For 3D reconstruction, cells were positioned on thestage of a Zeiss Axioplan 2 microscope equipped with

DIC optics and a 63 Planapo objective. The coarse focusknob of the microscope was connected to a computer-regulated microstepper motor programmed to move thefocus from the substratum through 20 µm in the z-axis in2 seconds. Images were acquired with a cooled CCDcamera (Optronics Inc., Galito, CA] and recorded ontothree-quarter-inch videotape. Thirty optical sections wereacquired in 2 seconds at 0.67-µm increments. Thisprocedure was repeated every 4 seconds. The opticalsections were digitized with a Media 100QX framegrab-ber board (Media 100 Inc., Marlboro, MA) in a Power-Computing PowerTower Pro 225 computer (Apple Com-puter Inc.). The optical sections were then automaticallyoutlined, stacked and converted to a faceted image with3D-DIAS software as previously described [Soll, 1995;Soll and Voss, 1998; Wessels et al., 1998). Cellular blebswere identified and color coded by methods developedfor the 3D reconstruction of pseudopods [Wessels et al.,1998].

RESULTS

Misexpression of Chimeric Mutant hTM5/3 Resultsin Increased Blebbing and MorphologicalDistortion During Cytokinesis

The majority of cells of the transformed, nonexpress-ing cell line C68 divided and spreaded in a fashion similarto that of non-transfected CHO cells (Fig. 2A–D),demonstrating that transfection per se does not affect cellmorphology or behavior during mitosis. In the case of

Fig. 2. Phase-contrast micrographs of dividing cells from chimerichTM5/3-nonexpressing C68 (A–D) and misexpressing C70 (E–H)lines. A,E: cells at the start of anaphase;B,F: cells at the start ofcytokinesis;C,G: cells at 50% of cytokinesis;D,H: cells at approxi-

mate 5 min. after reaching 50% cytokinesis. At the end of videorecord-ing for 2D-DIAS analysis, the majority of C68 cells successfully divideand begin to spread, whereas more than half of mutant C70 cellsexamined have extensive blebs covering their surface.

124 Wong et al.

C70 cells, which misexpress the chimeric mutant tropo-myosin hTM5/3 at about 3 times the level of endo-genous TM4 [Warren et al., 1995], several behavioralaspects of mitosis were aberrant. First, the general shapeof the emerging C70 daughter cells midway throughcytokinesis (50% cytokinesis, defined as 50% reductionof the size of the cleavage furrow) was not as roundas that of C68 cells, and this is evident in a comparison ofthe representative C68 and C70 cells midway throughcytokinesis in Figure 2C and 2G, respectively. Second,the cleavage furrows of C70 cells were not always asdistinct and symmetrical. Third, C70 cells formed moreblebs over their surface during cytokinesis than C68cells, and in some cases underwent extensive distortionand blebbing late in cytokinesis, as is evident in Figure2H.

During cytokinesis, it has been demonstrated thatnormal cells undergo blebbing [Porter et al., 1973; Lasterand Mackenzie, 1996], but not to the extent of C70 cells.To demonstrate this point, a quantitative comparison wasperformed between control C68 and test C70 cells. In thiscase, excessive blebbing was defined as blebs distributedat any one time over half the length of a dividing cell.After chromosome segregation and 50% cytokinesis,56% of C70 cells exhibited excessive blebbing, comparedto only 15% of C68 cells (Table I). To demonstrate thispoint visually, a representative C68 and a representativeC70 cell, both approximately halfway through cytokine-sis, were reconstructed as faceted images in whichprotrusions from the main cell contour were color-codedred (Fig. 3). It is clear from the top views in Figure 3 thatwhile the C68 cell formed a few small blebs close to thedeveloping furrow throughout the 112-second period ofanalysis (Fig. 3A), the C70 cell formed far more exten-sive blebs along its entire length (Fig. 3B). Therefore,both the size and distribution of blebs differed markedlybetween the representative C68 and C70 cells. Thisdifference was also observed between four additional C68and four additional C70 cells analyzed in the samefashion.

Misexpression of Chimeric Mutant hTM5/3Destabilizes Mitotic Spindle AlignmentDuring Anaphase

Warren et al. [1995] reported an increased incidenceof multinuclearity in C70 cells. We attributed this to

unsuccessful cytokinesis and suspected that the phenom-enon might be a consequence of a defective mitoticapparatus. Therefore, we immunofluorescently stainedcells of the C68 and C70 lines with antibody againstb-tubulin to check the integrity and orientation of themitotic spindles. When CHO cells enter cell division,they become less adhesive to the substratum and roundup. During anaphase, the mitotic spindle functions toseparate the pairs of sister chromatids as the cell begins toelongate. During this period, the axis of the mitoticspindle parallels the long cell axis. Mitotic spindles thatformed at an angle greater than 30° relative to the longaxis of the cell were considered to be ‘‘misaligned.’’Figure 4 shows immunofluorescence micrographs of C68and C70 cells stained with DM1B antibody againsttubulin. Mitotic spindles in representative cells of strainsC68 and C70 at prophase (data not shown), metaphase(data not shown), anaphase (Fig. 4A,B), and telophase(Fig. 4C,D) were intact and exhibited similar structure.However, while 30% of C70 cells (N5 208) containedmisaligned mitotic spindles (example in Fig. 4A,B ofC70), only 12% of C68 cells (N5 188) containedmisaligned mitotic spindles, a 2.5-fold difference that wassignificant, with aP value,0.005. The observed increasein the proportion of misaligned mitotic spindles in C70cells might account for the increase in the average timefor C70 cells to proceed from the start of anaphase to thestart of cytokinesis. The interval for C68 and C70 cellswas 2776 38 and 2976 101 seconds, respectively (N510 in both cases). The difference was just significant, withaP value of 0.045.

Misexpression of Chimeric Mutant hTM5/3 Affectsthe Motile Behavior of Cells During Division:A 2D Analysis

To assess the effects of hTM5/3 misexpression onthe dynamics of mitosis, C68 and C70 cells undergoingmitosis were motion-analyzed with the 2D-DIAS soft-ware program [Soll, 1995; Soll and Voss, 1998]. Motilityparameters were computed from the dynamics of the cellcentroid, and dynamic morphology parameters from thechanging contour of the cell perimeter. The size param-eters included mean area, mean maximum length, meanperimeter, and mean radial length. All size parameterswere greater for C70 cells over the period between thestart of anaphase and 50% cytokinesis (measured as thetime required for 50% reduction of the size of thecleavage furrow) (Table II). Although in no case was thedifference statistically significant, the fact that all param-eters were greater for C70 cells suggested a trend. Thisresult could be obtained if C70 cells were on average lessround, and this was suggested by the mean roundness andmean radial deviation parameters. The mean roundnessparameter was lower for C70 cells, indicating that they

TABLE I. Cell Morphology in C68 and C70 After Cytokinesis *

Divided and spread(%)

Excessive blebbing(%) Total

C68 23 (85) 4 (15) 27C70 14 (44) 18 (56) 32

*Excessive blebbing is defined as blebbing over more than half of thecell length.

Roles of Tropomyosin in Mitotic Cell Motility 125

Figure 3.

are less round than C68 cells. The mean radial deviationparameter was larger for C70 cells, again suggesting thatthey were less round, or that their shape was morecomplex (Table II). These differences held for cellsduring the period between anaphase and the start ofcytokinesis and during the period between the start ofcytokinesis and 50% cytokinesis (data not shown). Com-puting mean convexity and mean concavity directlyassessed the difference in the complexity of the cellperimeter. The former was 30% higher and the latter 66%higher in C70 cells, and the increases were significant(P 5 0.023 and 0.021, respectively) (Table II). Thesedifferences again held for cells during the period between

anaphase and the start of cytokinesis and the periodbetween the start of cytokinesis and 50% cytokinesis(data not shown).

The increased complexity of the shape of C70 cellsundergoing cytokinesis suggested that they were moredynamic and protrusive than C68 cells. In Figure 5A andB, time sequences are presented of the perimeter of arepresentative C68 and C70 cell, respectively, duringcytokinesis. The two cell types differed in several ways.First, the general shape of the C68 cell was rounder.Second, the C70 cell formed more complex protrusions.Third, the symmetry of the emerging daughter cells wasfar less in the C70 cell. Finally, the cleavage furrow wasfar more complex in the C70 cells. The increase in shapedynamics displayed by the C70 cells translated into amore expansive centroid track. In Figure 5C and D, thecentroid tracks of the representative C68 and C70 cells inFigure 5A and B are expanded to the same extent.It is clear that the distance intervals between centroidsare far greater in the C70 track. The differences inperimeter and centroid tracks exhibited by the C68 andC70 cell in Figure 5A and B were representative of 8additional C68 and 7 additional C70 cells analyzed in thesame fashion.

The difference in centroid tracks is clearly reflectedin the mean instantaneous velocity of the centroids of 9C68 and 8 C70 cells monitored in each case from theonset of anaphase through 50% cytokinesis. While the

Fig. 3. Time series of 3D reconstructions of representative chimerichTM5/3-nonexpressing C68 (A) and misexpressing C70 (B) cellspresented at 16-sec intervals. Dividing cells grown on coverslips wereobserved under a Zeiss Axioplan 2 microscope equipped with DICoptics. To obtain optical sections, the plane of focus was automaticallyraised in 0.67 µm increments at 0.067 sec intervals using a microstep-per motor regulated by a Macintosh-based operating program. Section-ing of a cell was complete in 2 sec and repeated every 4 sec. Theseoptical section images were continuously videorecorded onto 3/4-inchtape. Perimeters of the in-focus portion of each section were manuallydigitized into the 3D-DIAS data file. The digitized perimeters of thein-focus portions of the optical sections were filled and stacked tocreate a pseudo 3D reconstruction. Both C68 and C70 reconstructionswere then slanted 60° relative to the grid plane. The distance betweenhorizontal lines in the grid plane is 10 µm. The red color areas representsurface blebs.

Fig. 4. Immunofluorescence microscopy on C68 and C70 cells from astable hTM5/3-nonexpressing and overexpressing, respectively, lineswith DM1B antibody againstb-tubulin. A,C: Phase-contrast micro-graphs;B,D: fluorescence micrographs. During anaphase (A, B), theaxis of the mitotic spindle from most C68 cells parallels the long axis ofthe cell or forms an angel less than 30° with the cell long axis. Incontrast, the axis of the mitotic spindle in many anaphase C70 cells (A,B) forms an angle greater than 30° with the cell long axis. At telophase

(C, D), the chromosomes of both C68 and C70 cells decondense, thenuclei reform, and the cleavage furrows deepen. Microtubules arefound in asters and radiate out and absence in the intercellular bridges,midbodies. At this stage, one of the evolving daughter C70 cells liftedfrom the substrata and twisted to either the left or the right relative tothe other daughter cell. Thus, the midbody was not easily detected inC70 cells by immunofluorescence microscopy with the anti-tublinantibody. Bar5 10 µm.

Roles of Tropomyosin in Mitotic Cell Motility 127

mean instantaneous velocity of C68 cells was 4.46 1.1mm per min, the mean instantaneous velocity of C70 cellswas 6.36 0.9 mm per min, a difference of 43% (TableIII). The difference was significant, with aP value of0.001. In contrast to mean instantaneous velocity, meandirectional change was extremely high for both C68 andC70 cells, and statistically indistinguishable (Table III),reflecting randomness of movement for both cell types[Soll, 1995].

Misexpression of Chimeric Mutant hTM5/3 AffectsAdherence to the Substratum of One of the TwoEmerging Daughter Cells During Cytokinesis:A 3D Analysis

Computer-assisted three-dimensional reconstruc-tions of the surface of living cells undergoing mitosiswhen viewed from on top revealed an abnormal increasein blebbing of C70 cells along the long cell axis (Fig. 3B).These reconstructions, when viewed from the side (Fig.6), revealed two additional abnormal aspects of C70 cellbehavior. First, one of the two emerging daughter cellslifted off the substratum (Fig. 6B). This abnormal eventoccurred in five out of five C70 cells reconstructed in 3D,but in zero of five C68 cells reconstructed in 3D. Second,the daughter cell that lifted off the substratum twisted tothe left or right of the cell adhering to the substratum (Fig.6B). This twisted phenomenon was also detected in fixedand stained cells (Fig. 4C and D; C70). This abnormalcontortion occurred in five out of five 3D C70 cellsreconstructed in 3D and zero out of five 3D C68 cellsreconstructed in 3D.

DISCUSSION

We previously demonstrated that in vitro the chi-meric tropomyosin hTM5/3 has increased actin bindingactivity and actin-activated HMM ATPase activity, whencompared to either endogenous hTM5 or endogenoushTM3 [Novy et al., 1993b]. Furthermore, we previouslydemonstrated that this chimeric tropomyosin replacesprebound endogenous tropomyosins from actin filaments[Warren et al., 1995]. Unlike endogenous tropomyosins,the binding of chimeric hTM5/3 tropomyosin to actinfilaments is not regulated by KCl concentration [Novy etal., 1993b]. From these results, one would predict thathTM5/3 generates more force than endogenous tropomyo-sin and may not be regulated in a normal fashion. Severalof the behavioral characteristics of the hTM5/3 overexpres-sion mutant C70 support this prediction. First, we foundthat C70 cells undergoing mitosis lift one of the twoemerging daughter cells off the substratum, then undergoa twist to the left or right. This bizarre behavior occurredin every mitosing C70 cell reconstructed in 3D. In normalcells, the contractile ring participates in symmetric con-traction at the center of the cell. Both daughter cells of thecontrol strain C68 remained adhered to the substratumand the longest distance through the cell remained astraight line through mitosis. Therefore, contraction aroundthe cell center appeared symmetric in normal cells. Thecontortion that occurred during cytokinesis in C70 cellsappeared to result from an extreme asymmetric flex at thedivision furrow, which is consistent with the predictionthat hTM5/3 generates too much tension. This result inturn, supports the conclusion that tropomyosin plays arole either in the force-generating mechanism of ringcontraction or in ring stabilization.

Second, we found that overexpression of hTM5/3affects the behavior of post-mitotic cells in a fashionconsistent with a role in cortical tension. While 85% ofC68 cells spread immediately after cytokinesis, only 44%of C70 cells spread. This result suggests that overexpres-sion results in the abnormal maintenance of increasedcortical tension, which involves the actin-myosin cytoskel-eton [Pasternak et al., 1989].

Third, we found that C70 cells have a propensity forspindle misalignment. While approximately 12% of C68cells contained spindles aligned perpendicular to the longaxis of the dividing cell, 30% of C70 cells exhibitedmisalignment. In anaphase, the spindle apparatus ofbudding yeast is always oriented with one spindle pole ator through the neck between the mother cell and growingbud [Palmer et al., 1992]. Using mutants and cell cycleinhibitors, it has been demonstrated that astral microtu-bules, dynein and actin filaments play a role in orientation[Palmer et al., 1992; Li et al., 1993; Yeh et al., 1995]. Adelay in the cell cycle occurs in a dynein mutant before

TABLE II. Computer-Assisted Measurements of C68 and C70Cell Shape During the Intervals Between the Start of Anaphaseand 50% Cytokinesis*

Numberof cells

Meanarea

(µm2)

Meanmaximum

length (µm)

Meanperimeter

(µm)

Meanroundness

(%)

C68 9 4456 139 286 5 806 15 886 6C70 8 5826 216 326 5 956 17 826 9% diff 131 114 118 27P value NS NS NS NS

Numberof cells

Meanradiallength(µm)

Meanradial

deviation(%)

Meanconvexity(degree)

Meanconcavity(degree)

C68 9 126 2 12.46 3.4 6666 164 3066 164C70 8 146 2 13.56 3.9 8646 158 5086 159% diff 117 19 130 166P value NS NS 0.023 0.021

*% diff: percent difference of C70 to C68; NS: not significant,P value. 0.05.

128 Wong et al.

cytokinesis [Li et al., 1993; Yeh et al., 1995; Muhua et al.,1998], suggesting a new checkpoint for correcting defectsin mitotic spindle orientation. Muhua et al., [1998] haverecently provided evidence that EB1, a tumor suppressorgene, may be involved in this checkpoint. It, therefore,may be no coincidence that C70 cells exhibit a dramaticincrease in spindle misalignment, as demonstrated here,and an average increase in the period of cytokinesis, aspreviously demonstrated [Warren et al., 1995]. Throughits interaction with actin filaments, tropomyosin may playa role in positioning the mitotic spindle, and the efficiencyof this process is diminished in the hTM5/3 overexpres-sion mutant C70.

Fourth, we found that C70 cells extend moreextensive blebs than C68 cells, and these blebs aredistributed across the cell surface of C70 cells, rather thanlocalized at the cleavage furrow, as in the case of C68cells. Blebbing has been demonstrated to be a normalcharacteristic of cultured cells both during interphase andduring mitosis [Porter et al., 1973; Cunningham, 1995;Laster and Mackenzie 1996], although its function isunknown. The mechanism underlying blebbing is be-

lieved to be actin-based, and this suggestion is reinforcedby a number of observations. Microinjection of unregu-lated myosin light chain kinase causes blebbing individing normal rat kidney cells [Fishkind et al., 1992]. Inaddition, prolonged, extensive blebbing occurs in spread-ing cells of melanoma lines deficient in the actin cross-linking protein, ABP-280, and this defect is rescued byreexpression of ABP-280 through transfection [Cunning-ham et al., 1992]. Using scanning electron microscopy,Laster and Mackenzie [1996] observed that during mito-sis in fibroblasts, a period of microvilli formation wasfollowed by the formation of blebs containing actinfilaments.

We have found that overexpression of hTM5/3affects the distribution as well as extent of blebs. ControlC68 cells also formed blebs localized to a central regionencompassing the cleavage furrow. In contrast, dividingC70 cells formed larger protrusions that were distributedacross their long axis throughout mitosis. Unlike mela-noma cells lacking ABP-280, however, C70 cells did notbleb abnormally during interphase. The abnormal forma-tion of blebs in C70 cells would not seem to be directlyrelated to the contortion phenomenon that affects symme-try and contact with the substratum, suggesting thatoverexpression of hTM5/3 interferes with two differenttropomyosin-dependent events. The actin-myosin cyto-skeleton has been implicated in the suppression of lateralpseudopod formation in the posterior half of crawlingamoebae [Wessels et al., 1988, 1990; Spudich, 1989], andour results suggest that tropomyosin plays a similar rolein suppressing blebbing in the regions of a mitosing celldistal to the cleavage furrow.

In conclusion, our results suggest that throughinteraction with actin filaments, tropomyosins play a rolein the regulation of the contractile ring, in the localizedsuppression of blebbing, in the maintenance of polarityand spatial symmetry of the emerging daughter cellsduring cytokinesis, and in cell spreading after cytokinesisis complete.

Fig. 5. Stacked perimeter (A,B) and centroid tracks (C,D) generatedby 2D-DIAS of a representative cell each from C68 (A and C) and C70(B and D) lines during division. The interval time between perimeter orcentroid images was 20 sec. Scale bar for stack images is 8.5 µm. Scalebar for centroid track5 1.7 µm.

TABLE III. Computer-Assisted Measurements of MotilityParameters of C68 and C70 Cells During the Intervals Betweenthe Start of Anaphase and 50% Cytokinesis*

Numberof cells

Meaninstantaneous

velocity(µm/min)

Meandirectional

change(degree/2 sec)

C68 9 4.46 1.1 826 3C70 8 6.36 0.9 806 5% Diff 143 22P value 0.001 NS

*% diff: percent difference of C70 to C68; NS: not significant,P value. 0.05.

Roles of Tropomyosin in Mitotic Cell Motility 129

Fig. 6. Faceted 3D reconstructions from the side view (0°) of the C68 (A) and C70 (B) cells presented in Figure 3.

ACKNOWLEDGMENTS

We thank Dr. Stephen Blose for the monoclonalantibody againstb-tubulin. We thank Rebecca Reiter forcritical reading of the manuscript.

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