redistribution of golgi stacks and other organelles during mitosis

Redistribution of Golgi Stacks and OtherOrganelles during Mitosis and Cytokinesis in Plant Cells1[w]

Andreas Nebenfuhr*, Jennifer A. Frohlick, and L. Andrew Staehelin

Department of Molecular, Cellular and Developmental Biology, University of Colorado,Boulder, Colorado 80309

We have followed the redistribution of Golgi stacks during mitosis and cytokinesis in living tobacco BY-2 suspension culturecells by means of a green fluorescent protein-tagged soybean a-1,2 mannosidase, and correlated the findings to cytoskeletalrearrangements and to the redistribution of endoplasmic reticulum, mitochondria, and plastids. In preparation for celldivision, when the general streaming of Golgi stacks stops, about one-third of the peripheral Golgi stacks redistributes tothe perinuclear cytoplasm, the phragmosome, thereby reversing the ratio of interior to cortical Golgi from 2:3 to 3:2. Duringmetaphase, approximately 20% of all Golgi stacks aggregate in the immediate vicinity of the mitotic spindle and a similarnumber becomes concentrated in an equatorial region under the plasma membrane. This latter localization, the “Golgi belt,”accurately predicts the future site of cell division, and thus forms a novel marker for this region after the disassembly of thepreprophase band. During telophase and cytokinesis, many Golgi stacks redistribute around the phragmoplast where thecell plate is formed. At the end of cytokinesis, the daughter cells have very similar Golgi stack densities. The sites ofpreferential Golgi stack localization are specific for this organelle and largely exclude mitochondria and plastids, althoughsome mitochondria can approach the phragmoplast. This segregation of organelles is first observed in metaphase andpersists until completion of cytokinesis. Maintenance of the distinct localizations does not depend on intact actin filamentsor microtubules, although the mitotic spindle appears to play a major role in organizing the organelle distribution patterns.The redistribution of Golgi stacks during mitosis and cytokinesis is consistent with the hypothesis that Golgi stacks arerepositioned to ensure equal partitioning between daughter cells as well as rapid cell plate assembly.

Cell division requires the duplication of all criticalcell components, their distribution into different do-mains within the cytoplasm, and the separation ofthese domains into two independent daughter cells.These events have been characterized most thor-oughly for the genetic material, where duplicationoccurs during S phase of the cell cycle, followed bydistribution of the resulting chromatids in mitosis,and the subsequent formation of two daughter cellsby cytokinesis. The membranous organelles of eu-karyotic cells can be expected to follow a similarsequence of events, the specific manifestation ofwhich should be determined by the structural orga-nization of these organelles and their functional re-quirements. In recent years the inheritance of theGolgi apparatus in cultured mammalian cells hasreceived considerable interest (Lowe et al., 1998;Roth, 1999). In this case the single perinuclear Golgicomplex breaks down at the onset of mitosis andreforms after cytokinesis. The distribution of Golgimembranes over a larger area of the cell is expectedto aid in the even distribution of this organelle intothe daughter cells (Warren and Wickner, 1996). Al-though the mechanics of this partitioning are stilldebated (Shima et al., 1998; Roth, 1999; Zaal et al.,

1999), there is general agreement that during thisstage of the cell cycle, protein export from the endo-plasmic reticulum (ER) is blocked and the secretorysystem is shut down (Warren, 1993; Farmaki et al.,1999). In contrast to the centralized Golgi of mamma-lian cells, the Golgi apparatus of plants cells consistsof many independent stacks that continue to producesecretory products during all steps of mitosis andcytokinesis (Andreeva et al., 1998; Dupree and Sher-rier, 1998). Thus the mode of Golgi partitioning inplant cells can be expected to differ from that inmammalian cells, reflecting both the different struc-tural properties and the different functional require-ments of plant cells.

Cytokinesis in terrestrial plants occurs by cell plateformation. This process entails the delivery of Golgi-derived vesicles carrying cell wall components to theplane of cell division and the subsequent fusion ofthese vesicles within this plane (Staehelin and Hep-ler, 1996; Heese et al., 1998). After formation of anearly tubulo-vesicular network at the center of thecell, the initially labile cell plate consolidates into atubular network and eventually a fenestrated sheet(Samuels et al., 1995). The cell plate grows outwardfrom the center of the cell to the parental plasmamembrane with which it will fuse, thus completingcell division. Formation and growth of the cell plateis dependent upon the phragmoplast, which is re-quired for proper targeting of Golgi-derived vesiclesto the cell plate. The phragmoplast is a complexassembly of microtubules (MTs), microfilaments

1 This work was supported by the National Institutes of Health(grant no. GM18639 to L.A.S.).

[w] Indicates Web-only data.* Corresponding author; e-mail andreas.nebenfuehr@colorado.

edu; fax 303– 492–7744.

Plant Physiology, September 2000, Vol. 124, pp. 135–151, www.plantphysiol.org © 2000 American Society of Plant Physiologists 135

(MFs), and ER elements, that assemble in two oppos-ing sets perpendicular to the plane of the future cellplate during ana- and telophase (Staehelin and Hep-ler, 1996). As the cell plate matures in the central partof the cell, the phragmoplast disassembles in thisregion and new elements are added on its outside.This process leads to a steady expansion of thephragmoplast, and concomitantly, to a continuousretargeting of Golgi-derived vesicles to the growingedge of the cell plate. Once the cell plate reaches andfuses with the plasma membrane the phragmoplastdisappears. This event not only marks the separationof the two daughter cells, but also initiates a range ofbiochemical modifications that transform the callose-rich, flexible cell plate into a cellulose-rich, stiff pri-mary cell wall. In highly vacuolated cells these com-mon mechanisms of cytokinesis are preceded by twoadditional events. First, the nucleus migrates to acentral position in the cell defining the future divi-sion site. Subsequently, a cytoplasmic bridge, thephragmosome, is formed at this position in whichmitosis and cytokinesis will occur (Lloyd, 1991).

The heavy dependence of cell plate formation onactive Golgi stacks explains why plant cells, unlikemammalian cells, do not disassemble their secretionmachinery during cell division. This dependence alsoraises the question as to whether there exists a specialspatial relationship between the Golgi apparatus andthe cytokinetic machinery. It has long been knownthat Golgi stacks appear in the vicinity of the phrag-moplast. In fact this spatial proximity, together withthe similarity in Golgi vesicle and cell plate staining,was the first indication that Golgi products may beinvolved in cell plate formation (Whaley and Mollen-hauer, 1963). However, unlike the distribution of ERduring mitosis and cytokinesis (Hepler, 1980, 1982),the relationship of Golgi stacks to the phragmoplasthas received little attention. Only recently has astudy attempted a rigorous assessment of Golgi stackdistribution during cytokinesis. Using a double-staining approach, it was found that Golgi stacksapproach the maturing cell plate more closely thanmitochondria or plastids (Kawazu et al., 1995). But,since organelles were detected in chemically fixedand sectioned material, it was impossible to followdynamic changes in living cells, or to visualize thethree-dimensional distribution in an entire cell. Fewstudies have addressed the distribution of other or-ganelles during cell division in terrestrial plants(Schopfer and Hepler, 1991), although proper segre-gation of organelles clearly is important for cellviability.

We have previously described the creation of an invivo Golgi marker by expression of a fusion betweena resident cis-Golgi protein (soybean a-1,2 mannosi-dase [GmMan1]) and green fluorescent protein (GFP)in tobacco BY-2 suspension culture cells (Nebenfuhret al., 1999). Using this tool we demonstrated thatGolgi stacks can display characteristic translational

movement (cytoplasmic streaming) during inter-phase, which occurs along actin filaments. This stop-and-go movement is highly specific for individualstacks and presumably tightly regulated. The appar-ent regulation of Golgi stack movement suggests thattheir distribution in plant cells is not random, andthat Golgi stacks can be recruited to specific locationswhere they perform secretory functions (Nebenfuhret al., 1999). Cell plate formation is one examplewhere Golgi products are needed at a specific loca-tion and may therefore serve as model for the postu-lated recruitment of Golgi stacks.

In this study we use the Golgi-localized fluorescenttag to quantitatively describe the three-dimensionaldistribution of Golgi stacks in living BY-2 cells dur-ing mitosis and cytokinesis. In addition we use anER-targeted GFP construct and the fluorescent Mito-Tracker dye, which labels mitochondria and plastids,to localize those organelles during this part of the cellcycle. By comparing the distribution of these or-ganelles we demonstrate that BY-2 cells can sort theirorganelles during mitosis and assign them to specificpositions within the phragmosome. Finally we dem-onstrate that cytoskeletal elements play only a lim-ited role in maintaining the specific Golgi stacklocalization.

RESULTS

Golgi Stacks Accumulate near Spindle Poles and in anEquatorial Belt during Metaphase

Attempts at synchronizing the GmMan1-GFP-expressing cells by aphidicholin treatment (Nagata etal., 1982) were met with limited success (mitotic in-dex approximately 15%), which may be due to theslightly lower growth rate of the transgenic linewhen compared with untransformed BY-2-cells (datanot shown). We did not try to increase the mitoticindex by an additional propyzamide treatment (Sam-uels et al., 1998) since the disruption of MTs can beexpected to alter the cellular organization at the onsetof mitosis. The results presented here therefore werederived from unsynchronized cultures that were notexposed to any drugs, unless indicated. Observationswere conducted 2 to 4 d after transfer of the cells tofresh medium, when the number of dividing cells wasrelatively high (mitotic index approximately 6%).

During interphase, Golgi stacks appeared to berandomly distributed throughout the cortical and pe-rinuclear cytoplasm (Fig. 1A; Nebenfuhr et al., 1999).The distribution of Golgi stacks in different parts ofthe cytoplasm was analyzed in three-dimensional im-age stacks of three interphase cells using an auto-mated peak-finding algorithm to identify stacks fol-lowed by manual corrections. Identification of Golgistacks in the cells was complicated by the dynamicnature of stack localization and limitations of theoptical equipment. We have therefore analyzed onlythe upper one-half of each cell where the Golgi stacks

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136 Plant Physiol. Vol. 124, 2000

were more easily identifiable. The numbers thereforerepresent approximations that illustrate the patternof Golgi stack distribution within some unknownmargin of error. In this way between 600 and 1,200Golgi stacks were detected in the cells. The inter-phase cells analyzed had approximately two-thirdsof their Golgi stacks in the cortical cytoplasm. Only

about one-tenth of all stacks were found in the pe-rinuclear cytoplasm with the remainder (about one-fifth) being localized to the transvacuolar strands(Table I).

To test whether this distribution of Golgi stacks istruly random we have correlated the number ofGolgi stacks in different regions of the cells with theavailable volume of cytoplasm. For this purpose, cy-toplasm was defined as the area covered by the com-bined fluorescence of our GFP-marker, which stainsthe Golgi and to some extent, the ER (Nebenfuhr etal., 1999), and the MitoTracker dye, which stainsmitochondria and plastids (see below). An additionalbackground subtraction largely eliminated the dif-fuse ER-like fluorescence. This approach excludescytoplasmic domains that do not contain these largerorganelles, such as very thin layers of the corticalcytoplasm, the nucleoplasm, or the mitotic spindle.The absolute values of cytoplasmic volume obtainedin this way therefore should be considered approxi-mations and be interpreted with caution. However,the relative distribution of cytoplasm within cells canbe expected to average out these uncertainties andthus constitute a relatively robust measure. The over-all distribution of cytoplasm in the three interphasecells analyzed closely resembles the distribution ofGolgi stacks (Table I). When the numbers of Golgistacks found in the different regions of these cellswere divided by the approximate cytoplasmic vol-umes in the respective regions, no statistically signif-icant difference could be found between cortical andinterior, or between perinuclear and transvacuolarcytoplasm (Fig. 2, black bars). Thus the distributionof Golgi stacks in interphase cells reflects the distri-bution of cytoplasm and appears to be random.

As the cells prepared for mitosis, the nucleus mi-grated to a central position in the cell. At this stage,large amounts of cytoplasm accumulated in a perinu-clear location, forming a so-called “phragmosome”(Lloyd, 1991). The nucleus was usually displaced toone side, almost touching the plasma membrane. Onthe opposing side, the phragmosome was connectedto the adjacent cortical cytoplasm by one or a fewthick cytoplasmic strands. Most other transvacuolarstrands of cytoplasm, on the other hand, disap-peared. During early mitotic stages, most Golgi

Figure 1. Stereo images showing the Golgi stack distribution ina living tobacco BY-2 cells in interphase (A) and metaphase (B).A, Interphase cell. Projections of 30 individual deconvolved epiflu-orescence images taken at 1-mm intervals. N, Nucleus; w, cell wall.B, Metaphase cell. Projections of a three-dimensional reconstructionderived from 60 individual deconvolved epifluorescence imagestaken at 0.5-mm intervals. Arrows denote the location of the spindlepoles. The dashed line traces an equatorial accumulation of Golgistacks, the “Golgi belt”. Animated three-dimensional reconstructionsof these cells can be viewed at http://mcdb.colorado.edu/znebenfue/cytokinesis.

Table I. Distribution of Golgi stacks in cortical and internal cytoplasm during interphase and metaphaseGolgi stack distributions were derived from direct counting of stacks and manual outlining of relevant regions. Cytoplasmic volume was

defined as the area covered by the combined GFP and MitoTracker fluorescence, i.e. it represents the part of the cytosol accessible to largerorganelles. Values are arithmetic means 6 SE, n 5 3.

Interphase Cells Metaphase Cells

Location Golgi stacks Cytoplasm Location Golgi stacks Cytoplasm

% %

Cortical cytoplasm 70 6 4 62 6 4 Cortical cytoplasm 54 6 4 44 6 4Interior cytoplasm 30 6 4 38 6 4 Interior cytoplasm 46 6 4 56 6 4Perinuclear 9 6 4 12 6 5 Spindle region 19 6 3 13 6 3Transvacuolar 21 6 3 26 6 3 Transvacuolar 27 6 6 42 6 6

Plant Golgi Stacks during Mitosis and Cytokinesis

Plant Physiol. Vol. 124, 2000 137

stacks accumulated in the phragmosome region,leaving the cortical cytoplasm depleted of Golgistacks. The redistribution of Golgi stacks into thephragmosome did not involve the rapid stop-and-gomovements described for interphase cells (Neben-fuhr et al., 1999), which could not be observed duringany of the mitotic and cytokinetic events. When thecells reached metaphase, the Golgi distribution withinthe phragmosome underwent further changes. In par-ticular, a large number of Golgi stacks accumulatedaround the spindle MTs and the spindle poles (Figs.1B, arrows and 3A). The number of stacks tightlyassociated with the mitotic spindle was quantified inthree-dimensional image stacks of three cells in meta-phase. Thus about 20% of all stacks in the cells ana-lyzed were localized in immediate proximity to themetaphase spindle (Table I). This number may be anunderestimate since the selected spindle area waschosen conservatively to ensure that cortical stacks(in the Golgi belt) and those in other parts of thephragmosome would not be included. A slightlylarger number of stacks was found in the rest ofthe interior cytoplasm, leaving approximately 55%(64%, se) of all stacks in the cortical cytoplasm.

Further quantitative analysis of metaphase cellsdemonstrated that the distribution of Golgi stacksdid not match the distribution of cytoplasm as well asin the interphase cells (Table I). In particular, thephragmosome outside the spindle region accountedfor 42% 6 6% of the cytoplasm, but it contained only27% 6 6% of all Golgi stacks. In contrast, the imme-

diate vicinity of the metaphase spindle representedonly 13% 6 3% of the total cytoplasm, but contained19% 6 3% of the Golgi stacks (Table I). This differ-ence is readily apparent when the Golgi stack densi-ties are plotted for the different cellular regions (Fig.2, gray bars). The perispindle region of the interiorcytoplasm had a more than 2-fold higher density ofGolgi stacks than the rest of the phragmosome andtransvacuolar strands (90 6 14 pL21 versus 37 6 6pL21). We expect this difference, although as suchstatistically not significant (paired t test, P . 0.05),due to the small sample size, to persist when a morerobust method of assessing cytoplasmic volume isdeveloped. In contrast, the slightly higher Golgi stackdensity in the cortical cytoplasm may disappear withbetter measurements of cytoplasmic volume. Ourdata, despite their inherent uncertainties, thereforesuggest that the distribution of Golgi stacks duringmetaphase is not random.

Most metaphase cells also accumulated Golgistacks in a narrow, band-like region of cortical cyto-plasm around the edges of the metaphase plate,which we have termed the “Golgi belt” (dashed linein Fig. 1B). This Golgi belt region appears to corre-spond to the site of the former preprophase band(PPB) of MTs since it coincides with the future site ofcell division (see below). We have quantified theaccumulation of Golgi stacks within the Golgi belt bycounting the number of stacks in a cortical layer ofcytoplasm in the same three cells at metaphase. Forthese experiments, the Golgi belt area was definedvisually and individual stacks were identified man-ually. Only stacks immediately underlying theplasma membrane were analyzed to avoid complica-tions due to uneven thickness in the cortical cyto-plasm. The Golgi belt covered between 12% and 20%of the cell surface and the average density of stackswithin the belt region was about 3-fold higher than inthe rest of the cortical cytoplasm. In two cases wewere able to determine the overall stack density inthe cortical cytoplasm for two adjacent sister cells inmetaphase and interphase, respectively. In both pairsof cells, the metaphase sister had a lower density ofGolgi stacks in the cortical cytoplasm than the inter-phase sister (75% and 80% of the interphase sister,respectively). This difference was more pronouncedin the peripheral cortex (i.e. the cortex excluding theGolgi belt in metaphase and the perinuclear cortex ininterphase) where the Golgi stack density dropped toabout 60% of the interphase value during metaphase,suggesting that about one-third of the cortical Golgistacks in the mitotic cells had been relocalized to thephragmosome.

Although we attempted to eliminate variations instack distribution due to changes in the thickness ofthe cortical cytoplasm, we cannot exclude the pos-sibility that in some regions the space availablebetween the plasma membrane and the tonoplast

Figure 2. Golgi stack density in different regions of the cytoplasm ininterphase (black bars) and metaphase cells (gray bars). Total num-bers of Golgi stacks were determined with an automated peak-finding algorithm. Cytoplasmic volume in manually delineated re-gions of the cells was defined as the area covered by the combinedGFP and MitoTracker fluorescence, i.e. the part of the cytosol acces-sible to larger organelles. Golgi stack density is given as number ofstacks per picoliter of cytoplasm. Error bars represent the SE (n 5 3).Note the much higher density of Golgi stacks in the immediatevicinity of the spindle in metaphase cells.

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was too small to accommodate larger organellessuch as Golgi stacks. We have therefore analyzedthe cortical distribution of mitochondria, which usu-ally are only slightly smaller than Golgi stacks inBY-2 cells, in the same cells. Mitochondria werestained with the MitoTracker dye that partitions pref-erentially into membranes with high membrane po-tential (Haugland, 1996). In plants these are mitochon-dria and plastids, which can be easily distinguishedbased on their different sizes. Again, only organellesimmediately underlying the plasma membrane wereconsidered. Although mitochondria also show a slightaccumulation in the region of the Golgi belt, this pref-erential localization was much smaller that that foundfor Golgi stacks (Table II). We conclude that the ap-parent accumulation of Golgi stacks in the Golgi belt isnot an artifact of differential cytoplasmic accumula-tion, but that it reflects the specific concentration ofthis organelle around the cell equator.

Table II. Density of Golgi stacks and mitochondria in the corticalcytoplasm during metaphase

Individual stacks or mitochondria immediately underneath theplasma membrane were marked manually. Different regions of thecortical cytoplasm were identified by eye and outlined. Values arearithmetic means 6 SE (n 5 3) and given in mm22.

LocationOrganelle Density

Golgi stacks Mitochondria

mm22

Overall 0.09 6 0.01 0.13 6 0.02Belt region 0.17 6 0.02 0.16 6 0.03Peripheral cortex 0.06 6 0.01 0.11 6 0.02Concentration in belt over periphery 2.9 6 0.3 1.5 6 0.4

Figure 3. Comparison of GmMan1-GFP (A) andGFP-hdel (B) fluorescence in metaphase cells.On the left are fluorescence images and on theright corresponding differential interferencecontrast (DIC) image of the same cells. A, BY-2cell expressing GmMan1-GFP. Note the pres-ence of hazy, string-like fluorescence in the mi-totic spindle in addition to the punctate appear-ance of Golgi stacks. B, BY-2 cell expressingER-targeted GFP (GFP-hdel). A similar hazy flu-orescence pattern is seen in the spindle.



Diffuse Fluorescence during Mitosis May Result fromPartial ER Localization of the cis-Golgi Marker

During interphase, the GFP fluorescence was largelyrestricted to Golgi stacks, although a fraction of thefusion proteins could be detected in the ER (Neben-fuhr et al., 1999). When the cells entered mitosis, thefluorescence appeared to become more diffuse (com-pare Fig. 1, A and B) and also spread to areas thatexcluded Golgi stacks such as the spindle region (Fig.3A). The latter localization indicates the presence ofthe GmMan1-GFP fusion protein in the ER. This in-terpretation is supported by observations of cellsexpressing GFP in the lumen of the ER, whichshowed a similar distribution of fluorescence (Fig.3B). In particular, the ER marker was redistributedinto the phragmosome, leaving only small amountsof ER in the cortical cytoplasm and transvacuolarstrands (compare Fig. 3, B and 4, central cell). Withinthe phragmosome, the highest ER density was foundnear the spindle poles at metaphase (Fig. 3B), andlater in the region of the phragmoplast (Fig. 4, outercells), thus confirming earlier reports in the literaturefor other cell types (Hepler, 1980, 1982; Schopfer andHepler, 1991). The apparent increase in ER-like fluo-rescence in dividing cells expressing GmMan1-GFPraises the possibility that our Golgi marker partiallyrelocalizes into the ER during mitosis and cytokine-sis. To address this question we have attempted toquantify the relative level of diffuse ER-like versuspunctate Golgi fluorescence in optical sectionsthrough the center of the cell. However, the intermin-gling of the two organelles made it impossible toreliably quantify the contributions of the two or-ganelles independently.

Golgi Stacks Are in Close Proximity to thePhragmoplast during Cytokinesis

As the cells progressed into ana- and telophase,individual stacks appeared in the region between thedaughter nuclei (Fig. 5, A and B). After phragmoplastformation, Golgi stacks were excluded from the re-gion of high cytoskeletal density (Fig. 5, C and D).Only diffuse, ER-like fluorescence was visible in thephragmoplast, whereas Golgi stacks became en-riched in the surrounding cytoplasm (Fig. 5, C andD). In many cells, the daughter nuclei flattened outand approached the new cell plate while the phrag-moplast was growing centrifugally. During the out-ward growth of the phragmoplast, Golgi stacks werecontinuously associated with the periphery of thecytoskeletal elements. In addition, stacks could befound in close proximity to the more mature cellplate in the center of the cell (Fig. 5D).

In many cells the nucleus assumed an asymmetri-cal position prior to mitosis, which often resulted inthe cell plate reaching one parental wall long beforethe other. Careful observation of Nomarski images,

however, revealed that in such instances a phragmo-plast was present on both sides of the cell immedi-ately prior to fusion of the cell plate with the plasmamembrane (Fig. 5D and data not shown). This sug-gests that the cell plate fuses with the plasma mem-brane nearly simultaneously all around the cell. Latestage phragmoplasts in BY-2 cells typically had awidth of approximately 10 mm when viewed withNomarski optics (Fig. 5D). The entire process fromana-/telophase to completion of the cell plate wasaccomplished within 90 min. Time-lapse video analy-sis of individual cells maintained in a perfusion cham-ber revealed that Golgi stacks continuously “wiggled”and shifted positions within the phragmosome while

Figure 4. Distribution of ER in interphase and cytokinetic in GFP-hdel expressing BY-2 cells. A, Fluorescence image showing thedistribution of the GFP-hdel marker. B, DIC image of the same groupof cells. The left cell is in telophase/early cytokinesis with a promi-nent phragmoplast between the daughter nuclei. The right cell is ina later stage of cytokinesis with a phragmoplast near to top edge ofthe cell. The central cell is in interphase. Arrows point at the formingcell plates within the phragmoplasts. The strong GFP-hdel fluores-cence within the phragmoplasts reveals the abundance of ER in thisregion. Note the change in ER distribution between interphase andcytokinesis. The strong labeling of cortical cytoplasm and transvacu-olar strands during interphase is nearly absent in dividing cells.

Nebenfuhr et al.


maintaining their general spatial relationship to thephragmoplast (compare time-lapse video sequence athttp://mcdb.colorado.edu/znebenfue/cytokinesis).

It has been shown for mammalian cells that thedistribution of mitotic Golgi fragments into daughtercells is very close to the predicted 1:1 ratio (Shima etal., 1997, 1998). We have determined the distributionof Golgi stacks in pairs of daughter cells to testwhether BY-2 cells with their much larger number ofindividual stacks also partition their Golgi apparatusevenly. Analysis of three pairs of cells immediatelybefore or after completion of the cell plate revealedthat the daughter cells did not receive identical num-bers of Golgi stacks (ratio 1:1.16 6 0.10). However, itwas evident that the two cells usually were of differ-ent size, with the larger of the two receiving morestacks. We have therefore determined the approxi-mate volume of cytoplasm in the two daughter cellsas either the area covered by the combined fluores-cence of GmMan1-GFP and MitoTracker (see above)or as the total cell volume minus that of the manuallydelineated vacuole in three-dimensional DIC imagestacks. The difference in normalized Golgi stack den-sities between pairs of cells was found to be only 9%(67%). Thus, the Golgi stacks in dividing BY-2 cellsappear to be approximately evenly distributed intothe cytoplasm of the resulting daughter cells.

Cell Plates Grow toward Surface RegionsDefined by the Golgi Belt

As described earlier, Golgi stacks accumulated inan equatorial belt underlying the plasma membraneduring metaphase (Fig. 1B). We have examined thespatial relationship of this Golgi belt with other land-marks and events during mitosis and cytokinesis togain some insight regarding its possible function.The early phragmoplast is assembled in the regionof the mitotic spindle during ana- and telophase,and the pole MTs are thought to provide the initialguidance (Lambert et al., 1991). The phragmoplast

Figure 5. Time-lapse observation of Golgi stack distribution in adividing BY-2 cell. Fluorescence (left) and DIC images (right) weretaken in close succession at various time points during mitosis andcytokinesis. A, Metaphase. Arrow denotes plane of metaphase plateof chromosomes. Bracket indicates the position of the “Golgi belt” inthe cortical cytoplasm. B, Telophase. The daughter nuclei (N) havemoved out to the spindle poles and Golgi stacks are seen entering theinternuclear region (arrows). C, Early cytokinesis. The phragmoplast(angle brackets) has formed between the daughter nuclei. Golgistacks are excluded from the phragmoplast but accumulate in itsvicinity. D, Late cytokinesis. The phragmoplast (angle brackets) hasgrown centrifugally and has almost reached the parental plasmamembrane. Golgi stacks, but not ER, continue to be excluded from thephragmoplast but are present in close proximity. Note the presence ofGolgi stacks close to the more mature cell plate in the center of thecell. A time-lapse video sequence of this cytokinesis can be viewed athttp://mcdb.colorado.edu/znebenfue/cytokinesis.



thus maintains the same orientation as the mitoticspindle, and formation of the initial (internuclear)cell plate occurs in the same plane as the metaphaseplate of chromosomes (compare Fig. 5, A and C). Wehave observed eight cells in which the metaphaseplate was oriented at an angle relative to the Golgibelt (Fig. 6A). In these cases the early cell plate wasalso created in the same plane as the metaphasechromosomes (Fig. 6B). However, when the phrag-moplast left the interzone between the daughter nu-clei, it changed direction leading to a kink in theimmature cell plate (Fig. 6C). The reorientation of thephragmoplast always occurred toward the area ofGolgi stack accumulation identified in metaphase,and cell plate fusion with the parental plasma mem-brane was always in the region of the Golgi belt

(brackets in Fig. 6). Thus the Golgi belt appears toaccurately predict the future site of cell division afterthe disassembly of the PPB of MTs (Mineyuki, 1999).

Regions of Golgi Stack Accumulation ExcludeMitochondria and Plastids

We have shown that the distribution of Golgistacks during metaphase is not random and does notfollow the distribution of cytoplasm (Fig. 2; Table I).The observed accumulation of stacks in the Golgi beltis specific for this organelle and is not mimicked bymitochondria (Table II). It is unfortunate that wewere not able to perform a similar analysis in the cellinterior since the high density of mitochondriawithin the phragmosome together with limitations inour optical equipment precluded us from reliableidentification of individual organelles in this region.Nevertheless we were able to correlate the Golgistack localization to the distribution of mitochondriaand plastids in living cells. In interphase cells, the redfluorescence of the MitoTracker dye and the greenfluorescence of the GFP tag was intermingled andshowed no particular arrangement (Fig. 7A, left cell).In contrast, during metaphase, the two sets of or-ganelles marked by the two labels partitioned intocomplementary cellular regions (Figs. 7A, right celland 8A). In particular, whereas the Golgi-specific andER-like green fluorescence were seen preferentiallyaround the spindle poles, in the Golgi belt, and in thespindle, the red fluorescence showed that mitochon-dria and plastids accumulated outside the spindleand in the cortical cytoplasm outside the Golgi belt(Figs. 7A, right cell and 8A). Analysis of the fluores-cence intensities in a 5-mm-wide strip that stretchedacross the phragmosome and the spindle poles re-vealed that the green GFP fluorescence markingGolgi stacks and ER peaked several mm (range of 1–.5mm) closer to the spindle than the mitochondria andplastid-specific fluorescence (Fig. 7C).

During cytokinesis, this differential distribution oforganelles was largely maintained (Fig. 8, B and C).Again, diffuse ER-like fluorescence was detected inthe region of the phragmoplast, whereas the strongerand more particulate staining of the Golgi stacks wasevident in the perinuclear cytoplasm, also betweenthe daughter nuclei and the phragmoplast, as well asin the Golgi belt (Fig. 8B). Plastids were not presentin these regions, whereas the smaller mitochondriacould approach the phragmoplast more closely (Fig.8, B and C). During late cytokinesis, a few mitochon-dria were evident in the region between the nucleiand the phragmoplast/cell plate, whereas the largerplastids were completely excluded from this region,usually remaining on the far side of the daughternuclei (Fig. 8C).

Figure 6. Time-lapse series demonstrating that the Golgi belt pre-dicts the site of cell plate fusion with the plasma membrane. Fluo-rescence images (left) and DIC images (right) of a BY-2 cell with aslanted metaphase plate, taken at metaphase (A), telophase/earlycytokinesis (B), and late cytokinesis (C). Note that the early cell plateappears in the same plane as the metaphase plate (arrows). Later theleft end of the phragmoplast curves upwards toward an area of Golgistack accumulation, the Golgi belt (bracket).

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Golgi Stack Redistribution Commences in Metaphase

The results described so far indicate that plant cellssegregate their organelles into distinct domains priorto metaphase, i.e. during early mitotic stages. Toidentify the precise time at which this segregationfirst becomes apparent, we have identified prophasecells by their lack of a nucleolus when viewed withNomarski optics. Unfortunately, only a small num-ber of cells could be identified in this way. At thisstage, no Golgi stack accumulation near the prospec-tive spindle poles was evident, and mitochondriaand plastids were intermingled among the stacks(data not shown). However, all of these cells eitherreverted back to interphase during the observationperiod or failed to form a tight metaphase plate. Wesuspect that these effects were caused by the disrup-tive stress of observing the cells with high light in-tensities. To circumvent this problem we have fixedcontrol BY-2 cells with 1% (v/v) glutaraldehyde anddetermined the arrangement of Golgi stacks in the

context of mitotic stage-defining MTs visualized byimmunofluorescence (Fig. 9).

The dispersed distribution of Golgi stacks that istypical of interphase cells (Fig. 1A) was retained inpreprophase before the breakdown of the nuclearenvelope (Fig. 9A). During prometaphase, when pe-rinuclear MTs start to form a spindle, some cellsdisplayed an increased number of Golgi stacks adja-cent to the narrow PPB (Fig. 9B); however, not allcells at this stage exhibited this accumulation in theGolgi belt. The accumulation of Golgi stacks aroundthe spindle poles described above (Figs. 1B and 7)was not evident in cells fixed during the early stagesof spindle development (Fig. 9B). On the other hand,cells with fully formed mitotic spindles and a meta-phase plate of chromosomes clearly displayed anaggregation of Golgi stacks near the spindle poles(Fig. 9C). In general, the distribution of Golgi stacksin cells fixed during or after metaphase was essen-tially the same as that found in living cells. Forexample, only a few Golgi stacks were found be-tween the chromosomes and the forming phragmo-plast in telophase (Fig. 9D). This number of internu-clear Golgi stacks increased as the phragmoplastbegan to grow outwards (Fig. 9, E and F). In addition,Golgi stacks were found to accumulate near thephragmoplast during cytokinesis (Fig. 9, E and F).Thus it appears that the specific distribution of Golgistacks observed in metaphase cells arises after break-down of the PPB during the late stages of spindleformation. It should be noted, however, that the ex-tensive processing of the samples for immunostain-ing may have led to some subtle rearrangements oforganelles and cytoskeletal elements, making it moredifficult to correlate precisely the living and the fixedcell images.

Disruption of MTs Does Not Lead to aMixing of Organelles

The specific localization of Golgi stacks and theremoval of mitochondria and plastids from regionsof Golgi stack accumulation was found to occur dur-ing the early stages of metaphase as the nuclearenvelope breaks down and the spindle forms (Fig. 9,B and C). It is tempting to speculate that this redis-tribution of organelles is brought about by break-down of the cortical MTs and formation of the mitoticspindle. To test this hypothesis we have treated cellsundergoing mitosis with the MT-disrupting drug,propyzamide (6 mm). Analysis of living cells main-tained in a perfusion chamber showed that althoughthe drug produced the expected cell cycle arrest, theoverall distribution of the Golgi stacks changed verylittle. In particular, the breakdown of the metaphasespindle allowed larger organelles to approach thechromosomes more closely (Fig. 10, compare A andB). However, the general segregation of Golgi stackscloser to the chromosomes and mitochondria and

Figure 7. Comparison of fluorescence distribution for Golgistacks/ER and mitochondria/plastids in adjacent interphase andmetaphase cells. A, Merged fluorescence image showing Golgistacks and ER in green and mitochondria and plastids (labeled withMitoTracker) in red. The stars denote the positions of the spindlepoles. The brackets mark the region of the Golgi belt. B, DIC imageof the same cells in interphase (left, note nucleolus) and metaphase(right, note metaphase plate), respectively.



plastids in the periphery of the phragmosome waslargely maintained.

It should be noted that under these conditions thespindle usually did not disappear completely (Fig.10B), which presumably reflects the insensitivity ofkinetochore fibers to MT-disrupting drugs (Binarovaet al., 1998). If, on the other hand, the cells weretreated with propyzamide under continuous agita-tion, the spindle collapsed completely and large or-ganelles were directly opposed to the condensedchromosomes (Fig. 10C). We speculate that the fewkinetochore MTs left after propyzamide treatmentare not sufficient to resist the strong physical forcesin tumbling cells. Under these conditions, the segre-gation of Golgi stacks, mitochondria, and plastidswas partially lost although still recognizable (arrow-heads in Fig. 10C), suggesting that MTs are not ab-solutely required for maintenance of organelle segre-gation. At the same time, however, this resultdemonstrates that the physical strains imposed ontumbling cells in agitated cultures can affect the cell

architecture in the absence of stabilizing cytoskeletalelements.

Golgi Stacks Do Not Appear to Require Actin MFs forMaintaining Their Specific Localization

We have shown previously that Golgi stacks cantravel along actin filaments in interphase BY-2 cells(Nebenfuhr et al., 1999). This raises the possibilitythat Golgi stack distribution during mitosis and cy-tokinesis also depends on actin filaments. We havetherefore examined the spatial relationship betweenGolgi stacks and actin filaments in fixed cells, as wellas the effect of actin-disrupting drugs on Golgi dis-tribution in dividing BY-2 cells. Control cells werefixed and stained for actin with an anti-pea actinmonoclonal antibody (Andersland et al., 1994). Inmetaphase cells, thick actin filaments were foundthroughout the cortical cytoplasm, in transvacuolarstrands, and surrounding the mitotic spindle (Fig.

Figure 8. Comparison of Golgi stack and ER (green) distribution with that of mitochondria and plastids (red) at various pointsduring mitosis and cytokinesis. Each row shows from left to right green fluorescence channel, red fluorescence channel,merged fluorescence images, and DIC images. A, Metaphase; B, early cytokinesis; and C, late cytokinesis (same cell as inB). Brackets delineate the Golgi belt at metaphase. Arrows point at the growing ends of the cell plate. Note that in this casethe late cell plate (C) curves upward toward regions that were rich in Golgi stacks earlier (B). N, Nucleus; p, plastid; m,mitochondrion.

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11A). This latter distribution near the spindle matchesthat of Golgi stacks (Fig. 11B). However, only a fewGolgi stacks could be clearly localized to actin fila-

ments within the phragmosome (Fig. 11D). Disrup-tion of actin filaments with either 20 mm cytochalasinD or 1 mm latrunculin A did not lead to a redistribu-tion of Golgi stacks in mitotic living cells (data notshown). Similarly, the segregation of Golgi stacks(and ER) from mitochondria and plastids was notaffected in cells treated with the actin-filament dis-rupting drugs. However, the treated cells becamearrested in their given stage of mitosis or cytokinesis,

Figure 9. Golgi stack distribution relative to MTs. MTs (red) weredetected in fixed BY-2 cells by indirect immunofluorescence. DNA(blue) was marked with DAPI, and Golgi stacks are green. A, Pre-prophase. Note the condensing chromosomes and the broad PPB ofMTs (brackets). No particular arrangement of Golgi stacks can bediscerned. B, Pro-metaphase. Chromosomes are fully condensed.The spindle is forming and the PPB (brackets) has almost disap-peared. Golgi stacks show some accumulation near spindle polesand PPB/Golgi belt. C, Metaphase. Spindle is fully formed and chro-mosomes are arranged in metaphase plate. Golgi stacks are ac-cumulated at spindle poles; the Golgi belt is not visible in this cell.D, Telophase. Chromosomes have moved to spindle poles andphragmoplast is starting to form in the interchromosomal region. FewGolgi stacks can be found between the MTs. E, Early cytokinesis. Thephragmoplast has mostly disappeared from the center, allowing a fewGolgi stacks into the internuclear region. F, Late cytokinesis. Thephragmoplast has almost reached the plasma membrane. Golgistacks are present close to the phragmoplast as well as surroundingthe daughter nuclei.

Figure 10. Effect of MT-disrupting drugs on organelle distribution.A and B, Individual BY-2 cell in metaphase before (A) and after a15-min treatment with 10 mM propyzamide (B). The cell was treatedin a perfusion chamber on the microscope stage. Arrow denotes theplane of the metaphase plate of chromosomes. Note the separation ofGolgi and ER (green) and mitochondria and plastids (red) that ismaintained even after the spindle has partially collapsed. C, BY-2cell at metaphase that has been treated with 10 mM propyzamide for30 min under constant agitation. The spindle has completely col-lapsed and the organelles have closely approached the chromosomes(arrow). Note that green fluorescent Golgi stacks are clustered nearthe center of the metaphase plate (arrowheads).



making it impossible to observe the effects of thedrugs on the redistribution of organelles. Thus actinfilaments do not appear to be necessary for maintain-ing the specific distribution of Golgi stacks in divid-ing cells.

DISCUSSION

Golgi stacks in plant cells are the sites of de novosynthesis of cell wall matrix polysaccharides, in ad-dition to their role in sorting and modification ofproteins and membrane lipids (Driouich and Staehe-lin, 1997; Andreeva et al., 1998; Dupree and Sherrier,1998). Based on our analysis of the tightly regulatedGolgi stack movement in interphase cells, we havespeculated that the stacks can be recruited to loca-tions where their products are needed (Nebenfuhr etal., 1999). The forming cell plate in dividing cellsconstitutes such a region of localized requirement forGolgi products and may therefore serve as a modelwith which to test the predictions of our hypothesis.In addition to playing a central role in cytokinesis,the Golgi stacks (together with the other organelles)should be distributed roughly equally among the

daughter cells to ensure optimal cell viability. In thisstudy we have addressed both of these points bydetermining the distribution of Golgi stacks in living,dividing plant cells and comparing their distributionwith that of mitochondria and plastids. Our resultsindicate that during mitosis and cytokinesis Golgistacks are indeed sorted to specific areas of the cyto-plasm that appear to be related to their sites of action.

Plant Cells Sort Golgi Stacks, Mitochondria, andPlastids into Distinct Cytoplasmic Domains duringCell Division

We have used a Golgi-targeted fusion of GFP tosoybean mannosidase to follow the redistribution ofGolgi stacks during mitosis and cytokinesis in livingtobacco BY-2 suspension culture cells. Throughout allstages of cell division, we observed an accumulationof Golgi stacks in the phragmosome region (e.g. Figs.1B and 5). In particular we found that at metaphase,about 40% of the Golgi stacks accumulate in imme-diate vicinity of the spindle and in an equatorial beltunderlying the plasma membrane, whereas inter-phase cells contain only 25% of their Golgi stacks in

Figure 11. Golgi stack distribution relative to actin filaments in a metaphase cell. Actin filaments (red) were detected in fixedBY-2 cells by indirect immunofluorescence. A, Actin filaments (Cy3 channel). B, Golgi and ER distribution (fluoresceinisothiocyanate channel). C, Chromosomes (DAPI channel). D, Merged fluorescence image showing actin (red) Golgi and ER(green) and chromosomes (blue). Thick actin filaments are found surrounding the phragmosome, in transvacuolar strands,and throughout the cortical cytoplasm. Thinner actin filaments are found throughout the phragmosome and inside themitotic spindle. The Golgi stack distribution only partially matches that of the actin filaments.

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the equivalent perinuclear and equatorial regions.Golgi stacks therefore are recruited into the phrag-mosome during early mitotic stages. It is not cur-rently known whether this recruitment involves ac-tive movement of Golgi stacks along actin filamentsor is the result of passive stack movement caused bythe major reorganization of cellular architecture.Within the phragmosome, the stacks assume pre-ferred positions, namely at the spindle poles and in acortical belt that surrounds the mitotic machinery.This Golgi belt appears to predict the plane of cellplate fusion with the plasma membrane (Fig. 6) andthus constitutes a novel marker of the future divisionsite after the disassembly of the PPB of MTs(Mineyuki, 1999). The Golgi belt may correspond to areticulate band of cytoplasm underlying the plasmamembrane in mitotic sycamore maple callus cells(Roberts and Northcote, 1970).

By comparing the distribution of Golgi stacks withthat of mitochondria and plastids labeled with theMitoTracker dye we were further able to show thatthese organelles are largely localized to separate andcomplementary domains within the phragmosome.The regions of highest Golgi stack density at meta-phase, the spindle poles and the Golgi belt, are com-pletely devoid of plastids and contain only few mi-tochondria (Figs. 7 and 8). During cytokinesis, Golgistacks are found in close proximity to, but not within,the phragmoplast (Figs. 5C and 9, E and F). Plastidsare also absent from this region of Golgi stack accu-mulation, whereas some mitochondria can be foundnear the phragmoplast (Fig. 8, B and C). In the laterstages of cytokinesis when the ring-shaped phragmo-plast expands centrifugally, Golgi stacks and somemitochondria can approach the more mature parts ofthe cell plate, whereas plastids are still excluded fromthe region between the daughter nuclei and the cellplate (Fig. 8C).

To our knowledge, the striking, cytokinesis-relatedsegregation of organelles in living plant cells docu-mented in this study has not been described before.In the past, the distribution of organelles during mi-tosis and cytokinesis has received only little attentioncompared with the extensive literature on cytoskel-etal rearrangements. For example, an accumulationof ER membranes near spindle poles was describedfor mitotic barley cells based on osmium tetroxide/potassium ferricyanide staining and observation inthe electron microscope (Hepler, 1980). Similarly, ERwas found to be closely associated with the phrag-moplast and the forming cell plate (Hepler, 1982).More recently, the exclusion of plastids from thephragmoplast was described based on DIC images(Schopfer and Hepler, 1991). One study tried to com-pare the distribution of several organelles at the sametime by employing a double-staining approach thatallowed the simultaneous identification of Golgistacks, mitochondria, and plastids in thin sections ofembedded material under the fluorescence light mi-

croscope (Kawazu et al., 1995). In this case it wasfound that Golgi stacks approach the maturing cellplate more closely than mitochondria and plastids(Kawazu et al., 1995). However, the segregation oforganelles during metaphase described here was notrecognized in the earlier study, presumably due tothe thinness of the sections (0.5 mm), which did notallow an appreciation of the three-dimensional ar-rangement of organelles over larger volumes.

Maintenance of Organelle Segregation Is Only PartiallyDependent on Intact Cytoskeletal Elements

The segregation of Golgi stacks, mitochondria, andplastids into distinct domains demonstrates thatplant cells are able to specifically control the posi-tioning of these organelles during mitosis, but notnecessarily during interphase (Fig. 7). We testedwhether MFs or MTs are required for the setup andmaintenance of the observed segregation by selec-tively disrupting either of the cytoskeletal elementsby specific drugs. However, the treated cells did notprogress through mitosis or cytokinesis, thus pre-cluding us from observing any drug effects on or-ganelle rearrangements. In a similar manner, wewere not able to follow cells as they created the initialsegregation of organelles. Thus we can only assessthe role of the cytoskeleton in maintaining the previ-ously setup organelle distributions. Both MF- andMT-disrupting drugs had only limited effect on or-ganelle segregation in cells maintained in a perfusionchamber (Fig. 10B and data not shown), suggestingthat these cytoskeletal arrays play only a minor rolein keeping the organelles segregated. A completecollapse of the spindle by propyzamide treatmentduring continuous agitation resulted in a more pro-found effect on organelle distribution, although clus-ters of Golgi stacks were still maintained (Fig. 10C).This finding indicates that the spindle plays a centralrole in organizing the cytoplasm during metaphase.At the same time, the persisting Golgi clusters sug-gest that the stacks may be bound to other cellularstructures, allowing them to maintain their associa-tion even after removal of the major organizingprinciple.

It is well known that the tight packing of spindleMTs excludes all larger organelles and thereforeleads to an accumulation of these organelles in thesurrounding phragmosome. However, the strikingsegregation of Golgi stacks and mitochondria plusplastids goes beyond this simple steric exclusion andappears to be at least partially dependent on thepresence of an intact mitotic spindle. The role of thespindle MTs in organizing this segregation of or-ganelles can be either direct or indirect. A direct rolecould be envisioned as a physical interaction be-tween a Golgi-associated MT-binding protein and thespindle MTs, which results in the specific localizationof Golgi stacks. The indirect model would postulate



that the spindle serves as an anchor for a separatesignal that can recruit Golgi stacks into this area. Ourdata do not allow us to distinguish between thesepossibilities.

A central implication of the observed sorting ofGolgi stacks is that the organelles and/or their targetregions in the phragmosome are distinguished byspecific molecules that facilitate the sorting process.A possible candidate molecule for the sorting mightbe myosin. Indeed it has been shown for pollen tubesthat different types of myosin are associated withorganelles of different sizes (Miller et al., 1995). Wehave previously demonstrated that Golgi stacks em-ploy myosin motors for movements along MFs dur-ing interphase, although such rapid movements werenot visible during mitosis and cytokinesis (seeMineyuki et al., 1984). The presence of actin filamentsnear Golgi stacks in dividing cells (Fig. 11 and datanot shown) is also consistent with an acto-myosinbased localization process. The more limited distri-bution of Golgi stacks compared with that of MFsmay be caused by specifically localized “stop sig-nals” (Nebenfuhr et al., 1999) that prevent the Golgistacks from leaving these areas. A possible candidatefor such a stop signal is calcium, since elevated levelsof this ion are known to inhibit myosin motors inplants (Shimmen and Yokota, 1994; Yokota et al.,1999). A likely source of calcium is the ER at thespindle poles, which contains high concentrations ofcalcium (Wolniak et al., 1980). In this context thepossible role of the spindle MTs may be the localiza-tion of ER to the spindle poles (Fig. 3B; Hepler, 1980).However, other areas of Golgi stack accumulation(Golgi belt, phragmoplast) lack such conspicuous cal-cium stores (Wolniak et al., 1980). Clearly more re-search is necessary to clarify the mechanisms of Golgistack recruitment and organelle segregation.

The Mitotic Spindle Plays Only a Minor Role in theInheritance of Golgi Stacks

Although Golgi stacks play an important secretoryrole during cytokinesis in plant cells, they also haveto be evenly partitioned to ensure that both daughtercells receive a functioning secretory system. Theproblem of Golgi inheritance has attracted much at-tention in mammalian cell culture cells where theGolgi apparatus is organized in to a single perinu-clear complex, the Golgi ribbon. During mitosis, theGolgi ribbon breaks apart into smaller fragments,which are subsequently partitioned into the daughtercells (for review, see Roth, 1999). Association of asubset of these Golgi fragments with the mitotic spin-dle is thought to ensure near equal distribution ofGolgi membranes (Shima et al., 1997, 1998).

In this study we describe a similar association ofplant Golgi stacks with the mitotic spindle (Figs. 2, 7,and 8A), with almost identical numbers of stackspresent at both poles (50% 6 1%). Nevertheless, the

numbers of stacks differed considerably between sis-ter cells and appeared to depend on their relative cellsizes. Normalization of Golgi stack numbers to theapproximate cytoplasmic volumes of the two cellsaccordingly showed smaller deviations in Golgi stackdensities. Thus it appears that the random cytoplas-mic distribution Golgi stacks at metaphase likelyplays a more important role in Golgi stack inheri-tance than the ordered arrangement of stacks aroundthe spindle. This conclusion is further supported bythe small proportion of stacks in the immediate vi-cinity of the spindle (Table I).

It has been reported that a doubling of the numberof Golgi stacks occurs during metaphase (Garcia-Herdugo et al., 1988). We have tried to document thisevent in living BY-2 cells transformed with our Golgimarker. However, we were not able to identify anyspecific time period during which the number ofGolgi stacks showed such a dramatic increase (datanot shown). This failure to observe Golgi doublingmay have resulted from the large number of stackspresent in BY-2 cells (often more than 1,000) and thedynamic distribution of these stacks, which made iteffectively impossible to account for all stackspresent in a cell. Alternatively, the doubling of Golginumbers in BY-2 suspension cultures may not occurwithin such a narrow time window as documentedfor root-tip cells (Garcia-Herdugo et al., 1988). Ourobservation that larger interphase cells contain moreGolgi stacks (not shown) is also consistent with thisinterpretation.

Golgi Stacks Accumulate at Specific Sites to EnsureRapid Formation of the Cell Plate

The observed segregation of Golgi stacks, mito-chondria, and plastids also raises the questionwhether these organelles perform specific functionsat their respective positions, or whether specific lo-cations serve just as “parking” sites during mitosisand cytokinesis. In other words, are any of theseorganelles recruited to specific places within thephragmosome to satisfy a functional need at thislocation? For Golgi stacks near the phragmoplast,this possibility is most likely true. The role of Golgi-derived vesicles in cell plate formation has been doc-umented extensively (Staehelin and Hepler, 1996;Heese et al., 1998) and an accumulation of Golgistacks near the growing edge of the cell plate makesgood biological sense. This observation thereforelends support to the concept that Golgi stacks can berecruited to places where their products are needed(Nebenfuhr et al., 1999).

A similar argument can be made for the Golgistacks that accumulate near the spindle poles or inthe Golgi belt. For example, the stacks in the Golgibelt may be involved in preparing the plasma mem-brane and cell wall of the future division site for theanticipated fusion of the cell plate (Mineyuki and

Nebenfuhr et al.


Gunning, 1990). In a similar manner, the Golgi stacksaccumulating near the spindle poles may already beinvolved in the production of vesicles destined forthe cell plate. Such a population of “prefabricated”cell plate components could help explain the remark-able rate of initial cell plate growth (20 mm in diam-eter within a few minutes of cell plate initiation)despite the limited number of Golgi stacks in theregion between the reforming nuclei and the phrag-moplast (Figs. 5 and 9D; see Gunning, 1982). Exper-iments with the drug brefeldin A, which inhibitssecretion and leads to a breakdown of the Golgiapparatus have demonstrated that the early cell plateis formed from material that has left the Golgi inmetaphase, long before cell plate formation starts(Yasuhara et al., 1995). These results therefore sup-port the idea that Golgi stacks at the spindle poles arealready actively producing cell plate material.

Is the Transport between ER and Golgi Altered duringMitosis and Cytokinesis?

During our observations of cells undergoing mito-sis or cytokinesis we noticed that the green fluores-cent label, which during interphase largely is con-fined to Golgi stacks (Nebenfuhr et al., 1999), alsoappeared as a hazy staining particularly in the mi-totic spindle and the phragmoplast. Comparison ofthis pattern with cells expressing an ER-targeted GFP(GFP-hdel) suggests that this diffuse fluorescencerepresents the ER (Fig. 3). The apparent increase inGmMan1-GFP fluorescence in the ER during mitosiscould result from one of several changes in the en-domembrane system. For example, the ER of cellsundergoing mitosis and cytokinesis redistributesdramatically from the cortical cytoplasm into thephragmosome, leading to a concentration of this or-ganelle in the spindle and the phragmoplast (Figs. 3Band 4; see Hepler, 1980, 1982; Schopfer and Hepler,1991). Thus the small amount of GmMan1-GFP,which is present in the ER throughout the cell cyclemay be sufficient to appear as hazy fluorescence inregions of high ER density. This interpretation doesnot invoke any changes in membrane transport andrelies simply on the redistribution of ER togetherwith a relatively small number of (at steady state)ER-localized GmMan1-GFP molecules.

Alternatively, the apparent increase in ER fluores-cence could result from a reduction of anterogradeER-to-Golgi transport of newly synthesized protein,or from an increased retrograde Golgi-to-ER trans-port of already existing protein, or both. A reducedexport from the ER would be consistent with earlierreports suggesting a change in membrane fluxthrough the secretory system (e.g. Mollenhauer andMollenhauer, 1978). We have tried to quantify therelative contribution of Golgi and ER to total cellfluorescence at different stages of the cell cycle. It isunfortunate, though, that the values derived from

this analysis are inherently unreliable since we werenot able to unequivocally distinguish between thesignals from the two organelles due to the intermin-gling of ER and Golgi stacks in large parts of the cells.We are therefore not able to address the relativecontribution of this potential change in ER-Golgitransport to the observed fluorescence pattern.

CONCLUSIONS

We have determined the distribution of severalorganelles, namely Golgi stacks, ER, mitochondria,and plastids, in living, dividing plant cells. We showfor the first time that during metaphase these or-ganelles are sorted to distinct cytoplasmic regions.This segregation of organelles is largely maintainedthroughout cytokinesis. The accumulation of Golgistacks near the spindle poles and in the Golgi belt,the future site of cell division, suggests specific rolesfor these stacks during cell plate formation and istherefore consistent with our model predicting Golgistack recruitment to sites where their products areneeded (Nebenfuhr et al., 1999).

MATERIALS AND METHODS

Cell Lines and Culture Conditions

The transgenic BY-2 cell line expressing a fusion of Gm-Man1 and GFP (GmMan1-GFP) has been described previ-ously (Nebenfuhr et al., 1999). BY-2 cells expressing GFPwith an ER-retention signal (GFP-hdel) were kindly pro-vided by M. Ziegler and K. Danna (University of Colorado,Boulder). Cells were grown in a modified Linsmaier andSkoog medium (Nagata et al., 1982) with constant shaking(120 rpm) at 27°C in the dark. Cells were subculturedweekly into fresh medium at a dilution of 1:50. Cells wereharvested by low-speed centrifugation (500g for 2 min) 2 to4 d after subculturing and immediately used for experi-ments. Treatment of the cells with anticytoskeletal drugswas performed as described (Nebenfuhr et al., 1999).

Microscopy

Mitochondria and plastids were fluorescently labeledwith 50 nm MitoTracker Red CMXRos (Molecular Probes,Eugene, OR) for at least 15 min in the dark prior to obser-vation. Cells were observed on an epifluorescence micro-scope (DM-RXA, Leica, Wetzlar, Germany) with standardfluorescein isothiocyanate and Cy3 filter sets. Extendedobservations of living cells were carried out in a perfusionchamber with a continuous supply of fresh medium at aflow rate of 0.5 mL/min. No additional MitoTracker dyewas added during observations in the perfusion chamber.Images were captured with a Sensicam (Cooke Corp.,Tonawanda, NY) and SlideBook software (Intelligent Imag-ing Innovations, Denver, CO) on a Macintosh computer(Apple Computer, Cupertino CA). Image processing oc-curred in SlideBook (deconvolution), NIH Image (three-dimensional reconstructions, quantifications; program avail-



able at http://rsb.info.nih.gov/nih-image/Default.html),and Photoshop (final image assembly; Adobe Systems, SanJose, CA). Stack distributions were quantified in NIH Im-age. Positions of individual stacks were either marked byhand or automatically using a peak-finding algorithm fol-lowed by manual corrections. Regions of interest (entirecell, Golgi belt, perinuclear cortex, spindle region, and cellinterior) were selected manually and the number of stackswithin them determined using the “Analyze Particles”command. Duplicate stacks that were marked in adjacentslices of three-dimensional images were subtracted from thetotal. Cytoplasmic volume was estimated by measuring thearea of combined GFP and MitoTracker fluorescence afterbackground subtraction (which largely removes the ER-likefluorescence) in a three-dimensional-image series. All valuesgiven in the text are arithmetic means 6 se.

Immunofluorescence

BY-2 cells 2 to 4 d after subculturing were fixed with 1%(v/v) glutaraldehyde in culture medium for 15 min. Aftertwo washes in phosphate-buffered saline (PBS), the cellswere transferred to 4 to 7 mL of PBS containing 0.5% (w/v)NaBH4 to reduce autofluorescence of the fixative. After thisovernight incubation, cells were again washed in PBS andtheir cell walls were partially digested by treatment with1% (v/v) cellulase (Worthington, Freehold, NJ) and 1%(v/v) pectinase (Fluka, Buchs, Switzerland) for 30 min.Following two additional washes in PBS, the cells wereallowed to settle onto a multiwell microscope slide coatedwith 0.1% (w/v) polyethyleneimine. Membranes were per-meabilized by a 15-min treatment with 1% (w/v) IGEPAL(Sigma, St. Louis). To detect MTs, a mouse monoclonalantibody against Drosophila melanogaster a-tubulin wasused (1:30; M. Fuller, Stanford University, Stanford, CA).Actin MFs were detected with a mouse monoclonal anti-body against pea actin (undiluted; Andersland et al., 1994).The secondary antibody for both antitubulin and anti-actinantibodies was anti-mouse Alexa594-IgG (1:200; MolecularProbes). Primary and secondary antibody incubationslasted for at least 1 h each and were followed by at leastthree 5-min washes in PBS. The first wash after the second-ary antibody application included 0.1 mg/mL 49,6-diamino-phenylindole (Molecular Probes) to stain DNA.After the final wash the cells were either mounted in 50%(w/v) glycerol in PBS or viewed directly.

ACKNOWLEDGMENTS

We thank M. Ziegler and K. Danna (University of Colo-rado, Boulder) for the BY-2 cell line expressing ER-targetedGFP, M. Fuller (Stanford University) for the anti-tubulinantibody, and R. Cyr (Pennsylvania State University, Uni-versity Park) for the anti-actin antibody. We also thankM. Winey for use of the microscope and M. Otegui forcritical comments on the manuscript.

Received April 24, 2000; accepted June 13, 2000.

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redistribution of golgi stacks and other organelles during mitosis

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