VARIATIONS IN THE DISTRIBUTION AND MIGRATION OF … · final centriole duplex location, their migration patterns and th,e timing of the different events. Breakdown of cytoplasmic

J. Cell Sci, 43, 177-194 (1980)Printed in Great Britain © Company of Biologists Limited 1980

VARIATIONS IN THE DISTRIBUTION AND

MIGRATION OF CENTRIOLE DUPLEXES IN

MITOTIC PtK2 CELLS STUDIED BY

IMMUNOFLUORESCENCE MICROSCOPY

JANE E. AUBIN*, MARY OSBORN AND KLAUS WEBERMax Planck Institute for Biophysical Chemistry, D-3400 Gottingen,Federal Republic of Germany

SUMMARYThe localization and migration of centriole duplexes have been studied in PtK2 cells by indirect

immunofluorescence microscopy using specific tubulin antibodies. The study demonstrated theusefulness of the immunofluorescence technique to quantitate studies of centriole migration andconcomitant events such as cytoplasmic microtubule breakdown in large populations of cells.Centriole duplex locations in normal and Colcemid-treated interphase populations have beencompared with duplex locations in prophase cells. A higher percentage of duplexes were foundclose to the nucleus in prophase than in interphase cells, but approximately 5 % of the duplexesremained in the cytoplasm far removed from the nucleus in prophase and throughout thecourse of duplex separation. Duplex separation occurred along a wide variety of paths andduplexes did not have to be closely juxtaposed to the nuclear envelope for separation to occur.Some duplexes separated in the cytoplasm with no detectable nuclear attachment, with spindlesforming far to the side of the condensing chromosomes. The timing of duplex separation didnot always coincide either with chromosome condensation or with nuclear membrane break-down, and in a small percentage of the cells separation occurred as late as prometaphase. Thesedata suggest that normal spindle formation can occur despite the large variability in initial andfinal centriole duplex location, their migration patterns, and the timing of the different events.Breakdown of cytoplasmic microtubules began in prophase and progressed until prometaphase;the last cytoplasmic microtubules disappeared soon after the loss of the nuclear membrane.

INTRODUCTION

How the formation of the mitotic apparatus is controlled at the molecular level isan intriguing and still open question. One aspect of the formation and the laterfunctioning of the mitotic spindle involves the role of the centrioles. Historically thecentrioles have been considered as directly involved in spindle organization andfunction (Stubblefield & Brinkley, 1967). A variety of information suggests howeverthat the centrioles themselves may not be necessary (Szollosi, Calcarco & Donahue,1972; Berns & Richardson, 1977; Pickett-Heaps, 1969, 1975; Dietz, 1966), but that

• Present address: Medical Research Council Group in Periodontal Physiology, Universityof Toronto, 4384 Medical Sciences Building, Toronto, Ontario, Canada.

Correspondence to: Dr J. E. Aubin, Medical Research Council Group in Periodontal Physi-ology, University of Toronto, 4384 Medical Sciences Building, Toronto, Ontario M5S 1A8,.Canada.

178 J.E.Aubin, M. Osborn and K. Weber

intact pericentriolar material may have an active role in spindle formation and function(Berns, Rattner, Brenner & Meredith, 1977; Robbins & Gonatas, 1964; Robbins &Jentzsch, 1968; Robbins, Jentzsch & Micali, 1968). Evidence from drug studiessupporting centriole involvement (for discussion, see Berns et al. 1977) and thepresence in vitro of microtubules arising both from centriole ends and from peri-centriolar material (Gould & Borisy, 1977; Telzer & Rosenbaum, 1979) furthercomplicates the question.

Whether or not the centrioles or the pericentriolar material or both are directlyinvolved, knowledge of the intracellular location of the centrioles with their accompany-ing pericentriolar material would facilitate the understanding of spindle formation.It is especially important to document the migration of the centrioles and the micro-tubules associated with them, as well as the behaviour of other cytoplasmic micro-tubules during this period. The timing of these migrations to form spindle polesversus the timing of other mitotic events, e.g. chromosome condensation, is also ofinterest.

Detailed studies of centriole location and migration have been limited to light- andelectron-microscopic analyses. While the former technique has the advantage ofallowing one to follow centriole migration in a living cell and to relate it to otherphenomena such as chromosome behaviour, the usefulness of the approach is limitedby the difficulty of distinguishing the centriole duplexes from cellular vesicles andgranules that appear similar to them in the light microscope. Certain staining pro-cedures have somewhat enhanced centriolar visibility for some studies in the lightmicroscope (Stubblefield & Brinkley, 1967; Brinkley, Stubblefield & Tsu, 1967;Wilson, 1934), however, routinely only a small number of cells has been available foranalysis. Furthermore, any information on the accompanying microtubules is belowthe resolution of the light microscope. Use of the electron microscope, either alone orin combination with light microscopy, has allowed investigation in greater detail ofcentriolar ultrastructure and simultaneously the state of the nuclear membrane,chromosomes and microtubules. However, the latter technique also has been limitedto relatively small numbers of cells and often it has been difficult even from serialsections to get a good detailed overview of the entire cell. Even given the limitationsof the 2 techniques, several detailed studies have been attempted and considerable dataaccumulated (Stubblefield & Brinkley, 1967; Szollosi et al. 1972; Berns & Richardson,1977; Pickett-Heaps, 1969, 1975; Dietz, 1966; Berns et al. 1977; Robbins & Gonatas,1964; Robbins & Jentzsch, 1969; Robbins et al. 1968; Brinkley et al. 1967; Wilson,1934; Roos, 1973; Rattner & Berns, 1976a, b; Zeligs & Wollman, 1979). Manyunanswered questions as to the generality of some observations: e.g. the variability oflocation of the 2 centriole duplexes in early prophase (Roos, 1973; Rattner & Berns,1976a) have not been approached. Further, several important questions have givenapparently contradictory answers, possibly due to the small sample sizes under study.

Immunofluorescence microscopy has been used to study microtubular profiles inboth interphase and mitotic cells (see for example Weber et al. 1975 a, b; Fuller,Brinkley & Boughter, 1975). The advantages of immunofluorescence microscopy tostudy centriole locations in large numbers of cells has been suggested in studies

Centriolar duplexes in PtK2 cells 179

employing both tubulin antibody (Osborn & Weber, 1976) and some nonimmunerabbit sera (Connolly & Kalnins, 1979). Here we report the use of specific tubulinantibodies which we have used to quantitate for the first time the percentages of cells inlarge populations showing particular locations of centriole duplexes in interphase andduring early mitotic events: i.e. from early prophase through metaphase. Centrioleduplexes at various stages of separation were analysed. Besides the frequently de-scribed migration of duplexes around the nucleus, centriole duplexes having noapparent association with the nucleus migrated to form presumptive spindle poleswith the establishment of prometaphase spindles. Cytoplasmic microtubules havebeen monitored at the same time to ascertain the timing of various stages of centriolemovement with the appearance of astral microtubules and the disappearance of cyto-plasmic microtubules; the relation of both these events to nuclear membrane break-down and chromosome condensation has been determined. To increase further theavailable sample size of mitotic cells, cells were sometimes synchronized in 5-phaseby double thymidine block (Rao & Johnson, 1970) and their subsequent progressthrough mitosis analysed. Several general conclusions are presented demonstratingand quantitating the extreme variability in locations of centrioles in prophase, theirnumerous paths of migrations, and their different final locations giving rise to pro-metaphase spindle formation.

MATERIALS AND METHODS

Cell culture

The established epithelial cell line, PtK2, was grown and maintained in culture as described(Aubin, Weber & Osborn, 1979; \Veb9ter, Osborn & Weber, 1978). In preparation for immuno-fluorescence, cells were seeded on to glass coverslips and allowed to grow for at least 24—36 h.When synchrony was used, cells were grown on glass coverslips and treated with a doublethymidine block. 24-36 h after seeding at a density of io6 cells per 100-mm diameter Petri dish,medium was removed, and medium prewarmed to 37 °C containing 2 mM thymidine was added.After 24 h, the medium was removed and replaced with fresh medium without thymidine. Aftera further 24 h, cells were exposed a second time to medium containing thymidine again for aperiod of 24 h. Then the medium with thymidine was replaced with fresh medium and thepopulation monitored with time for mitotic index (for further details see Aubin, Osborn,Franke & Weber, 1980). The synchronization process itself did not affect the observations oncentrioles since untreated cultures gave identical results. In some experiments, cells on cover-slips were treated with Colcemid (5 /*g/ml) for 4 h and then allowed to recover for 30 min inColcemid-free medium.

Antibodies

The preparation of rabbit antibodies against porcine brain tubulin has been described as hasthe isolation of specific tubulin antibodies by passage over Sepharose 4 B coupled with covalentlybound tubulin (Weber, Wehland & Herzog, 1976). Fluorescein-labelled goat-anti-rabbit IgGs.(Miles-Yeda, Israel) were used as the second antibody. The low level of non-specific bindingof these IgGs was eliminated by preabsorption of these antibodies on methanol-fixed PtKzcells (see Aubin et al. 1979). Specific tubulin antibodies were used at 0-05 mg/ml; fluorescein-labelled goat-anti-rabbit IgGs were used at approximately 0-5 mg/ml.

180 J. E. Aubin, M. Osborn and K. Weber

Indirect immunofluorescence

PtK2 cells on glass coverslips were immersed in —10 °C methanol for s min, followed by abrief wash in phosphate-buffered saline (PBS). Staining with antibodies and washing wereperformed as described (Aubin et al. 1979; Webster et al. 1978). Coverslips were mounted inMoviol4-88.

Cells displaying centriole duplexes in various locations were counted by scanning coverslipsand focussing through the cells. Centriole duplexes in some locations (e.g. above the nucleus)were difficult to visualize simultaneously with cytoplasmic microtubules. It was necessary tofocus up to the duplex, then to focus down to the cytoplasmic array of microtubules to determinetheir orientation and appearance. Such problems with depth of field sometimes limited thephotography as is evident in several of the photographs shown here. Often we have focussed onand exposed for the centriole duplexes resulting in loss of information in the photographic printsince: (a) cytoplasmic microtubules may be out of focus, and (b) cytoplasmic microtubules maybe underexposed compared to the bright centriolar duplexes. At the microscope, determinationof locations was normally possible and to determine the percentages of cells, 300-5030 cells werecounted per coverslip. To increase the sampling size of mitotic cells, we frequently used cellssynchronized in .S-phase and then allowed to proceed through mitosis (Aubin et al. 1980).

The designation 'close beside' the nucleus refers to duplexes lying less than o-5 mm from thenuclear membrane (on 3 5-mm film with 160 x magnification) or less than ~ 3 /tm from themembrane. Duplexes located further away could be seen to be removed from the nuclearmembrane and located in the cytoplasm. Centriolar duplexes could be found in the cytoplasmat any distance from the nucleus; some were measured up to approximately 25 fim from thenuclear membrane.

Nomenclature

Prophase was taken to be that period of the mitotic cycle from earliest detectable chromosomecondensation up to breakdown of the nuclear envelope. Prometaphase was taken as beginningafter loss of the nuclear envelope.

RESULTS

Interphase

In interphase cells stained with tubulin antibody, the centriole duplexes can oftennot be visualized against the extensive background of microtubules. However, in anormal population of PtK2 cells we were able to distinguish in about 20—30 % of thecells the centriole duplex as a bright dot of fluorescence, sometimes with a hollow core,which served as a focal point for many cytoplasmic microtubules. A correspondingphase-dense black dot could be seen only occasionally. Intracellular locations of theduplexes in several hundred cells were determined relative to the substrate (coverslips)to which the cells were attached. In normally growing cells, the centriole duplexeswere found at variable locations: (a) in direct juxtaposition to the nuclear membrane,i.e. close beside the nucleus. This was the most common location (~ 56 %); (b)' on topof or below the nucleus (22%); and (c) noticeably removed from the nucleus andoften far out in the cytoplasm (22 %) (Fig. 1 A). In category (6), when it was possibleto distinguish the orientation, the majority of the centriole duplexes were on top ofthe nucleus relative to the substrate; for convenience we shall continue to refer to themhere as 'on top*. To try to determine how representative of the whole populationthese percentages were, a similar quantitation was attempted after treatment of the

Centnolar duplexes in PtK2 cells 181

Fig. i. Immunofluorescence micrographs of interphase and mitotic PtK2 cells fixedwith methanol and labelled with rabbit antibody against tubulin and fluorescein-label-led goat-anti-rabbit IgGs. A, areas of 2 cells are shown. A cytoplasmic edge of an inter-phase cell in the left of the photograph contains an interphase centriole duplex (arrow)with microtubules radiating from it. The cell on the right is in prophase. Increasedsize and intensity of the centriolar region (double arrows) is evident. The enlargement(c) shows the centriolar region of the same cell exposed to indicate the substructurevisible in the centriolar region. Note that both the interphase and prophase centriolarduplexes shown here are in the cytoplasm far from the nucleus, B, interphase PtK2cells treated with Colcemid for 4 h, and then allowed to recover for 30 min withoutdrug. The position of the fluorescent dot - which corresponds to the centriolar region- can be distinguished in almost all cells. In A and in some subsequent micrographs,cytoplasmic microtubules are not always clearly visible since: (i) the microscope isfocussed on the centriolar region leaving many of the microtubules out of focus; and(ii) in order to print the photograph such that the prophase centriolar area does notappear overpoweringly bright, the exposure times do not also allow clear visualizationof the majority of the cytoplasmic microtubules although these are clearly visible inthe microscope. Magnification 1 A, c, x 1080; B, x 240.


PtK2 cells with Colcemid and recovery in Colcemid-free medium for 30 min. Underthese conditions the cytoplasmic microtubules which are destroyed by the drug treat-ment have only just started to regrow and the centriolar position is clearly visible as abright dot or ring of fluorescence (cf. Osborn & Weber, 1976). After Colcemid treat-ment fields of cells were chosen in which the positions of the centriole duplexes couldbe distinguished in > 95 % of the cells (e.g. Fig. 1 B). The relative percentages of cellswith centrioles in various locations were: (a) 60% with centriole duplexes closebeside the nucleus; (b) centrioles on top (or below) the nucleus (19 %); and (c) centriolesnoticeably removed from the nucleus (21 %).

Prophase

The earliest indication of prophase detectable in immunofluorescence in cellsstained with tubulin antibodies was a striking increase in the size and intensity of thebright fluorescent spot marking the centriolar region (Figs. 1, 2). At this very earlytime, the general display of cytoplasmic microtubules appeared unchanged, with nobreakdown or decrease in abundance of the tubules yet detectable. Occasionally 2 veryclose black dots, presumably the 2 centriole duplexes, could be distinguished in phasecontrast, although while they remained very close together, their fluorescent imagesappeared fused probably because of the wealth of microtubules radiating from them(e.g. Fig. 5A, B, p. 188). Chromosome condensation had always begun already by thisstage. The bright fluorescent dots could be seen in about 95 % of the early prophasecells identified by observing chromosome condensation either in phase-contrastmicroscopy or using Hoechst stain (Hilwig & Gropp, 1972). In the remaining prophasecells, chromosome condensation was seen to have begun without coincidence of theincrease in fluorescence staining of the centriolar regions (see below).

Although it has been suggested or implied that the centriole duplexes migrate to thenuclear envelope prior to their separation in prophase (see e.g. Rattner & Berns, 1976 a,b), we have noted that in PtK2 cells this does not always occur. When prophase cellswith centriole duplexes at various locations were counted, we found: 72 % withcentriole duplexes closely juxtaposed to the nuclear membrane, 22 % with centrioleduplexes on top of the nucleus, and 6 % with centriole duplexes still far removed fromthe nucleus in the cytoplasm (e.g. Figs. 1, 2). Comparing these numbers with thosefrom interphase cells would suggest that some centriole duplexes migrated closer tothe nucleus prior to prophase, but that prophase development of centrioles could occurin the cytoplasm as judged by the high percentage of cells that displayed prophasecentrioles located there.

The separation of the 2 duplexes to form spindle poles proceeded by a number ofroutes as would have been expected from their variable cytoplasmic locations observedin early prophase (see above). It was evident that even amongst those duplexes foundclose to the nuclear envelope, there was variation in their paths of migration. Forexample, centrioles could move progressively further apart while remaining in closeproximity to the nuclear envelope, until finally reaching opposite sides of the nucleus(Fig. 3). The microtubules associated with this class of duplexes seemed to radiatefrom both duplexes and often curved around the nucleus. Alternatively, the duplexes


Fig. 2. Phase and fluorescence micrographs of PtK2 cells indicating the variation inlocations of centriolar duplexes prior to their separation, A, centriolar duplexes closeto the nuclear membrane. Cytoplasmic microtubules can be seen to have begun tobreak down in a large area of the cytoplasm, B, centriolar duplexes on top of thenucleus. C, a prometaphase cell with late duplex separation. Note the absence of nuclearmembrane and cytoplasmic microtubules as well as the well-progressed chromosomecondensation. In these figures the duplexes are visible as one fluorescent spot fromwhich extensive astral microtubules radiate. Magnification, x 720.


Fig. 3. Phase and fluorescence micrographs showing the increased separation of thecentriole duplexes as they migrate around the nucleus. A, the centriole duplexes inearly prophase are visible as one fluorescent spot. B, early duplex separation. Astralmicrotubules are evident, as is the loss of cytoplasmic microtubules, especially on theside of the nucleus opposite from the migrating centriole duplexes. C, further separa-tion of duplexes showing the angle of astral microtubules from the two duplexes, whichare distinguished as two separate fluorescent spots. The remaining cytoplasmic micro-tubules are largely out of focus. D, E, duplexes are well-separated and loss ofcytoplasmic microtubules is clear. In E the astral microtubules from the two duplexesare clearly at an angle to each other. F, here the centriolar duplexes are at oppositesides of the nucleus. The nuclear envelope is still present and although astral micro-tubules are evident, no pole-to-pole microtubules are visible. G, shortly after loss ofthe nuclear membrane, pole-to-pole microtubules are present. In H, the prometaphase

Centrtolar duplexes in PtK2 cells

H

spindle and in I the metaphase spindle indicate the further development of the micro-tubules in the form of chromosome-pole and pole-to-pole microtubules with relativelyfewer astral microtubules. Magnification, x 720.

13 CEL 43

i86 J. E. Aubin, M. Osborn and K. Weber

Fig. 4. Phase and fluorescence micrographs of centriole duplexes in prophase. A—c, aseries of micrographs showing phase (A) and fluorescence (B, C) of centriolar duplexeson top of the nucleus. In B the microscope has been focussed on the separatingduplexes, with some astral microtubules in focus also, but the majority of the cyto-plasmic microtubules out of focus. When the cytoplasmic microtubules which remainare put in focus (c), the separating duplexes appear as one diffuse fluorescent spot.Often one duplex appears to migrate over the top of the nucleus while the other issituated close beside the nucleus (D-G). Magnification, x 720.


could be removed a short distance from the nucleus (arbitrarily defined here as ^ 3 /tm),and could apparently move apart on a straight line path at an oblique angle to thenuclear envelope. Often one duplex was found close to the nucleus while the other wasfound in the cytoplasm at various angles to the nucleus. It seemed that probably nearlyall angles of the centrioles relative to the nucleus were possible. The second type ofmovement involved those duplexes found, relative to the substrate, above the nucleus.Duplexes on top of the nucleus appeared to migrate across the top to final positions atopposite sides of the nuclear membrane (Fig. 4A-C). Occasionally, one duplex wasfound close beside the nuclear envelope and the other apparently migrated across thetop (Fig. 4D-G). The third, and perhaps most intriguing path, involved those centrioleduplexes located far away (> 3 jura) from the nucleus in the cytoplasm. Numerousexamples of such duplexes were found in which the duplexes moved apart to apparentlynormal distances, but without any detectable association with the nucleus (e.g. Fig. 5).

In all these cases, astral microtubules could be seen radiating from the migratingcentriolar duplexes, with microtubules from each overlapping frequently. Often thecentrioles and the astral microtubules were at angles to each other, i.e. there was not astraight line between the centrioles (e.g. Fig. 3 E). Although occasional microtubulescould be found there, normally microtubules could not be detected which ran directlybetween the separating duplexes, that is beginning on one and ending on the other.Astral microtubules grew noticeably in length as the migration process progressed.A prometaphase spindle began to form when centriole duplexes reached some distanceapart, whether they were at opposite sides of the nucleus (Fig. 3), with one beside thenucleus and one in the cytoplasm (Fig. 4), or with both in the cytoplasm (Fig. 5).It has not been possible to determine this distance exactly, since varying degrees offlatness exist in the mitotic PtK.2 cells used and the precise orientation of the substrateto the axis joining the centriole duplexes cannot be determined in the fluorescencemicroscope. Fluorescent dots such as are seen in Figs. 4B and c are occasionallyobserved; their significance is not known, although they have been observed in asecond cell line (Marchisio, Osborn & Weber, 1978; Fig. 11).

The onset of breakdown of cytoplasmic microtubules occurred sometime afterchromosome condensation began. It could be detected always in cells where centrioleduplex migration had begun. Thus, in cells where 2 spots of fluorescence could bedistinguished indicating that the centrioles had begun to separate, some disappearanceof microtubules could be seen. Breakdown of microtubules began apparently from theperiphery of the cell in one particular area and then moved in the direction of thenucleus. Although microtubular breakdown could be followed in many cells, nopreferential location of this early breakdown could be determined. Although often thebreakdown appeared to progress more rapidly on the side of the nucleus oppositefrom where the centrioles were migrating, this was not always so. Occasionally, growthof astral microtubules somewhat obscured the appearance of cytoplasmic micro-tubules and the determination of the breakdown process. Focussing up and downthrough the cell normally helped to determine what kinds of microtubules were present,and in particular, to identify those originating from the migrating centrioles.

13-2

J. E. Aubin, M. Osborn and K. Weber


PrometaphaseThe transition from prophase to prometaphase is generally regarded as the time

when the nuclear membrane disintegrates (Roos, 1973). In those cases where centriolesmoved around the nuclear membrane, they appeared to reach opposite sides beforenuclear membrane breakdown (Fig. 3F). Similarly, when centriole duplexes migratedthrough the cytoplasm, they appeared to reach distances of separation equivalent tothose reached by centrioles proximal to the nucleus before nuclear membrane dis-integration occurred (Fig. 5). It is possible that some localized areas of membranebreakdown might have been present and yet been undetected in the phase microscope,but in almost all cases looked at in the phase microscope, a phase-dense line could beobserved at these times. A nucleolus and recognizable state of chromosome condensa-tion and packing were also evident. Immediately after nuclear membrane breakdown,when the nucleolus was also gone and chromosomes were further condensed, pole-to-pole microtubules were clearly visible for the first time in the corresponding fluorescentimages (Fig. 3 G). At this point, a few cytoplasmic microtubules still persisted. Thelast cytoplasmic microtubules disappeared shortly after this as the prometaphasespindle developed. Well before metaphase, no microtubules outside the arrays ofastral, pole-to-pole, and chromosome-to-pole microtubules were visible (Fig. 3 H).

Apparently the centriole duplexes which migrated through the cytoplasm awayfrom the nucleus could form spindle poles. In Fig. 5, a prometaphase spindle far tothe side of the chromosome mass is shown. Such a configuration of prometaphaseoccurred with approximately the same frequency (8 %) as the early prophase centrioleduplexes in the cytoplasm removed from the nucleus (6 %).

A further interesting prometaphase type was observed. Fig. 6 summarizes thisbehaviour. In a small percentage of cells in prometaphase (3 %), the chromosomeswere seen after nuclear membrane breakdown and at a late stage of chromosomecondensation to be grouped around a single fluorescent spot. Cytoplasmic micro-tubules were absent as expected for this stage of nuclear events. The observation of asingle spot did not appear to be due merely to viewing the spindle end on, lookingdown along the pole axis, since focussing through the cell did not reveal the otherpole. Rather it seemed that in those few cells, the centriole duplexes had not yetmigrated apart. That they were however capable of doing so and forming spindlepoles a short time later is shown by Fig. 6. In these micrographs, it is evident thatcentriole duplexes could be found among the condensed chromosomes at variousstages of separation.

Fig. 5. Phase and fluorescence micrographs of PtK2 cells in prophase and later stagesof mitosis with centriole duplexes in the cytoplasm. In phase (A), 2 duplexes are visiblein the cytoplasm, while only one fluorescent spot is visible in fluorescence (B). AS theduplexes separate, 2 fluorescent dots become distinguishable, corresponding to the 2phase-dense dots (see c). Centriole duplexes separate to considerable distances in anormal fashion (D, E) and a prometaphase spindle eventually forms (E) which is off-centre relative to the chromosome mass. Pole-to-chromosome microtubules are visible(E). Progression to the metaphase spindle seems to occur with positioning of thechromosomes to the metaphase plate (F). Magnification, x 720.


6 A

Fig. 6. Phase and fluorescence micrographs of cells with late separation of centrioleduplexes occurring in prometaphase. Magnification, x 720.


DISCUSSION

The migration of centriole duplexes and its temporal relationship to other micro-tubular events has been investigated by immunofluorescence microscopy in interphaseand mitotic PtK.2 cells with antibodies to tubulin. By allowing visualization of a largenumber of cells both in normal cultures and in synchronized populations, an un-ambiguous determination of the location of centrioles and their spatial orientation tothe nucleus has been obtained. Thus the technique has been useful in defining and inquantitating a number of possible paths of centriolar migration and early spindledevelopment. It has been possible also to determine the overall state of the cytoplasmicmicrotubules relative to centriole movement and the progression of prophase. Thisstudy therefore extends earlier studies of mitotic cells by immunofluorescence usingtubulin antibodies in which only isolated photographs of cells in different mitoticstages have been presented (Fuller et al. 1975; Weber et al. 1975a; Brinkley, Fuller &Highfield, 1976).

Centrioles could be seen in about 25 % of interphase cells without any drug treat-ment as dots of fluorescence with a sometimes extensive array of microtubules radiatingfrom the dots. In essentially all prophase cells (95 %), the centrioles were clearlyvisible as very bright fluorescent spots, such that prophase cells could easily bedistinguished in fluorescence. The staining intensity of the centriolar duplexes inprophase cells was much increased over that observed in interphase cells and in theprophase cells the centriolar regions were obvious foci for increased numbers ofmicrotubules. These observations support previous conclusions obtained from electron-microscopic observations which suggest an increase during mitosis in the abundanceof pericentriolar material and microtubules radiating from it (Robbins & Gonatas,i964;Robbins&Jentzsch, 1969; Robbins et al. i968;Roos, 1973; Telzer&Rosenbaum,1979; Snyder & Mclntosh, 1975; Rattner & Phillips, 1973).

The majority of previous data has suggested that each duplex migrates as a singleunit through interphase cells and ends as a duplicated unit close to the nucleus priorto separation to form the spindle poles (e.g. Roos, 1973; Rattner & Berns, 1976a).We have verified the observation that the centriolar duplex may be located in a widevariety of places in the interphase cell and quantitated these locations. That at leastsome duplexes must migrate from their interphase positions closer to the nucleus isindicated by the higher percentage of cells with centrioles removed from the nucleusin interphase versus prophase cells. However, a considerable fraction (6%) of thePtK2 cells observed in this study revealed centriole duplexes which remained in thecytoplasm far removed from the nucleus and which separated normally as prophaseproceeded. Thus, it seems that a fixed spatial orientation of centriole duplexes to thenucleus is not required for duplex migration and ultimately for spindle formation.Further, amongst those centriole duplexes located close to the nucleus, either besideor on top, much variability was observed in their paths of migration. The straight linejoining pole-to-pole was observed at all possible angles to the substrate and nucleus.Variation in possible paths of migration has been seen in light-microscope examinationof prophase cells (Rattner & Berns, 1976a). Interpolating from the variety of positions


noted here in hundreds of cells, we have also found that centrioles migrate around orover the top (relative to the substrate) of the nucleus. One centriole duplex was oftenfound in the cytoplasm, whereas the other was proximal to the nucleus. In all of thesecases, however, the centrioles seemed to move apart equally well reaching oppositesides of the nucleus or some distance apart in the cytoplasm before nuclear envelopebreakdown. Frequently astral microtubules from the migrating duplexes appeared tobe at angles to each other rather than along the straight line joining the poles. Thiscould be seen in cells even after the breakdown of the nuclear membrane. This hasalso been observed in electron-microscopic studies (Roos, 1973; Rattner & Berns,1976J) and may indicate that some final realignment of centriole duplexes can occurbefore formation of the actual spindle axis.

The timing of centriole migration relative to other events, for example the degree ofchromosome condensation, was variable. The majority of cells (~9S%), showingearly prophase chromosome condensation, also displayed the increase in intensity ofcentriolar duplexes stained with tubulin antibody. A few prophase cells (~ 5 %) inwhich chromosome condensation had already begun, did not display this increase instaining in the duplex region. A comparable number of cells displayed duplexesseparated by only short distances when chromosome condensation was well progressed.The most extreme case occurred in a small percentage of cells in which the duplexesappeared not to migrate until after nuclear membrane breakdown. For this class ofcells, duplexes separated normally through the region of the prometaphase chromo-somes. This occurred in the absence of cytoplasmic microtubules which seemed tohave disappeared with the usual timing and were completely gone after nuclearmembrane breakdown (see below).

Only after nuclear membrane breakdown did clearly identifiable pole-to-polemicrotubules appear. The loss of the nuclear membrane with accompanying loss ofthe nucleolus and further chromosome condensation coincided with the completionof depolymerization of cytoplasmic microtubules. Duplex separation is thus not alwayscoupled to the trigger for a cell's entry into mitosis as evidenced by chromosomecondensation and ultimately in loss of the nuclear envelope. Breakdown of cytoplasmicmicrotubules may be tied to some extent to these events. However, maturation ofpericentriolar material as evidenced by the increase in staining intensity, and centrioleseparation are more variably timed events. Thus, what the trigger is for prophase asdefined by the onset of chromosome condensation and the trigger for and mechanismof duplex separation remain intriguing questions.

This report verifies a number of light- and electron-microscopic observations.More importantly, the study has extended the understanding of the variability inboth the locations of centriole duplexes in prophase and their paths of migration. Apreliminary study of centriole movement in mouse 3T3 cells has shown that thepercentages of cells displaying certain types of centriole behaviour is cell-line depen-dent. For example, a higher percentage of 3T3 cells displayed centrioles separatingafter nuclear membrane breakdown than in PtK2 cells; no 3T3 cells were observedwith duplexes separating in the cytoplasm. Such a rapid technique for visualizationboth of the centrioles and of their milieu against the depolymerizing cytoplasmic


microtubules is excellent for quantitation and for the general definition of centriolemigration. If these processes can be studied at the light- and electron-microscopiclevels using the same mitotic cells, understanding of the molecular events whichaccompany mitosis, as well as their sequence, should be advanced.

J.E.A. was a Postdoctoral Fellow of the Medical Research Council of Canada.

REFERENCES

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AUBIN, J. E., WEBER, K. & OSBORN, M. (1979). Analysis of actin and microfilament-associatedproteins in the mitotic spindle and cleavage furrow of PtK2 cells by immunofiuorescencemicroscopy: a critical note. Expl Cell Res. (In Press.)

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{Received 22 November 1979)

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VARIATIONS IN THE DISTRIBUTION AND MIGRATION OF … · final centriole duplex location, their migration patterns and th,e timing of the different events. Breakdown of cytoplasmic