The Golgi Apparatus during Mitosis in Human MelanomaCells in Vitro1
Gerd G. Maul and B. R. Brinkley
Department of Pathology, Temple University Health Sciences Center, School of Medicine, Philadelphia, Pennsylvania ¡9140 [G. G. M.], andDepartment of Biology, M. D. Anderson Hospital and Tumor Institute at Houston, Houston, Texas 77025 fB. R. B.J
SUMMARY
The ultrastructural changes of the Golgi apparatus ofhuman melanoma cells in vitro were investigated duringmitosis. In interphase, the Golgi apparatus consists of numerous interconnected dictyosomes, each of which is composed of4 cisternae, as a rule. Some of these cisternae have fenestra-tions along their lateral edges. During prophase, the membranous tubular connections between the dictyosomes breakdown. Also, no fenestration of the cisternae can be observed.The number and size of cisternae are reduced. The frequencyof Golgi elements found reached a minimum during anaphase,but some tubular and vesicular membrane elements werepresent at the lateral edges of single cisternae which were considered remnants of dictyosomes. Reconstruction of dictyosomes starts at the time of nuclear envelope reformation inearly telophase. The increase in number of cisternae appears tobe due to apposition of smooth endoplasmic reticulum. Thepersistence of Golgi elements throughout mitosis was established. The presence of coated vesicles suggested that thecisternae are functional during mitosis. No relationship between premelanosomes and Golgi elements was found duringmitosis.
INTRODUCTION
The importance of the Golgi apparatus in pigment formation of the melanocyte was recognized early (8, 13). Thephysical proximity of premelanosomes to the Golgi apparatuslead to the hypothesis that these organdÃes were directly involved in melanogenesis (1, 25). Evidence was presented for acontinuity of premelanosomes with SER2 (17) and possibly
with the dictyosomes of the Golgi apparatus (14). It has alsobeen suggested that proteins are formed on the ribosomes,transferred into the RER, channeled through the Golgi apparatus to be concentrated, and packaged for transport within(9) or out of (2, 11, 12, 26, 27) the cell.
In order to study the role of the Golgi apparatus in mel-anosome formation, it seemed important to determine thetime during the cell cycle when the Golgi apparatus is inactiveas far as melanogenesis is concerned. The reports on the pres-
'This research was supported by Grants CA 11654 and GM 15887
from the NIH.3The abbreviations used are: SER, smooth endoplasmic reticulum;
RER, rough endoplasmic reticulum; ER, endoplasmic reticulum.Received January 8, 1970; accepted May 15, 1970.
enee of the Golgi apparatus during mitosis vary. Golgi elements were reported to be absent in several tumor cells duringmitosis (5). For HeLa cells, histochemical and fine structuralevidence was presented that the Golgi apparatus disappearsduring metaphase and reappears rapidly in telophase (19-21).In hepatic cells of the rat, however, it was found that typicaldictyosomes persist during mitosis (6). Dictyosomes were alsopresent in metaphase and anaphase in spermatocytes of theEuropean corn borer and the giant amoeba (23). In transplant-able hepatomas of rats, the Golgi apparatus did not break intosmall vesicular clusters but retained its laminar and vesicularcomponents throughout different phases of the mitotic cycle(4).
This investigation was undertaken to determine whether ornot the Golgi apparatus was present during mitosis and tofollow any structural changes from its interphase morphology.Particular emphasis was placed on the association of persistingGolgi elements and premelanosomes.
MATERIALS AND METHODS
A cloned cell line of human melanotic melanoma (LeCa19-4, formerly H-19) was propagated (22) in Falcon plastic Tflasks, fixed in situ at room temperature for 1 hr with 3%phosphate-buffered glutaraldehyde (24), and postfixed in 1%OsO4 buffered in a similar manner. The cell monolayer wasprestained in 2% aqueous uranyl acetate for 30 min, dehydrated quickly in a graded series of ethanol up to 90%, followed by hydroxypropyl methacrylate, and flat embedded (3)in Epon 812 (11). This method allowed light microscopicselection and photography of individual cells, followed by thinsectioning of the same cell with a diamond knife on a LKBultratome III. All sections were stained with uranyl acetateand lead citrate (18). Serial sections were picked up on slottedgrids (LKB No. 4829A-l 3) coated with 0.5% collodion in amylacetate. A Hitachi HU-11C and a Siemens 101 electron microscope were used at 75 and 80 kV, respectively. At least 5 cellsof each mitotic stage were scanned, including the centriolearea. One hundred to 200 sections were scanned per cell.
RESULTS
In interphase, the Golgj apparatus of human melanoma cellsin vitro consists of an enormously developed membranesystem. As judged from the reconstruction of serial sections,
most dictyosomes are interconnected by tubular SER. Fig. 1 isan example of 4 dictyosomes connected by tubular SER. Incross-sections, most dictyosomes showed 4 flat cisternae.Some of these were fenestrated at the lateral edges (largearrow). In cross-sections, these fenestrations as well as thetubular SER connecting the dictyosomes appear as vesicles. Acloser inspection of favorable sections, however, reveals thatthe "vesicles" (arrows) are cross-sections through the bridges
between fenestrations (Fig. 2) or through tubular membraneelements. RER was usually found at the convex side of thedictyosomes.
Fig. 3 shows the Golgi region in a cell at late prophase orprometaphase. The nuclear envelope has not yet broken downcompletely, as several nuclear pores can still be observed. TheGolgi apparatus has decreased in size. Many serial sections atdifferent levels of the cell were required to locate parallelstacks of cisternae, as seen in Fig. 3. Coated vesicles werealways present at their vicinity. In fact, they were used tolocalize the dictysomes by checking the same area of the cellseveral sections apart. In many cases, the number of parallelcisternae in the dictyosomes decreased from 4 to 1 or 2.
At metaphase, one seldom finds parallel cisternae of thetype characteristic of dictyosomes. If several cisternae arefound, they are usually randomly oriented (Fig. 4). However, adictyosome with 3 cisternae was found in 2 different cells. Thecisternae with the spacing characteristic for the innermostcisternae in dictyosomes are surrounded by ER. The ER formsa network of anastomosing tubules of varying diameter andflatness. Ribosomes are attached to part of this membranesystem. A fine fibrillar material is contained within the ER.
Many fine membranous tubules appear in oblique or cross-sections as "Golgj-like vesicles.' These can be seen as forked
(Fig. 4b, arrow), oval (Fig. 4, a, c, i, and /, arrow), androunded vesicles which are often found in subsequent sections,indicating a depth which is several times their diameter (Fig.4, b to /, vertical arrows). Free vesicles are demonstrated inFig. 4, d and e (arrows pointing right). Remaining dictyosomalcisternae were found in the centriole region, as well as in thearea of the equatorial plate in metaphase.
During anaphase, remaining dictyosomal cisternae wereseldom found. Of 5 cells investigated, the cisternae were onlyfound in 1 cell (Figs. 5 and 6). In later anaphase, it could beobserved that the number of tubular and vesicular elementsaround individual cisternae remained greatly reduced (Figs. 7and 8). In early telophase (Figs. 9 and 10), remaining dictyosomal cisternae were infrequently seen in the centriole area,but in 1 case 2 parallel cisternae were found. A remainingdictyosomal cisterna with accompanying vesicles and tubuleswas found in the cleavage furrow (Fig. 11). At this stage,reconsitution of the nuclear membrane had begun, as judgedby several small areas of membrane attachments to the chromosome and the presence of occasional nuclear pores.
Stacks of 4 cisternae were first encountered in telophase atthe time of advanced nuclear membrane reconstitution (Fig.12). However, as soon as nuclear envelope formation is completed (Fig. 13), one can find several dictyosomes within 1section, most of them having at least 4 cisternae. Most dictyosomes observed at this stage were found in close apposition tothe nuclear membrane. By comparing Figs. 9 and 13, it was
evident that nuclear envelope formation, as well as dictyosomeaccumulation, occurs at the onset of cytokinesis. At late telophase (Figs. 14 and 15) when cytokinesis is completed, butdecondensation of the chromosomes has just begun, manydictyosomes can be found in the centriole region and a few inother areas of the daughter cells. The number of cisternaevaries widely. The greatest number found was 8. There wereless vesicular and tubular membrane elements at the lateraledge of the dictyosomes than in metaphase. Coated vesicles arepresent, but not as many as in prophase or later in early Glcells. It seems as if SER becomes deposited parallel to thecisternae of the dictyosomes (Fig. 15, d2, arrows). The apposition of small cisternae can often be seen in dictyosomes withmore than 4 cisternae (Fig. 15, dl). Images such as that shownat the right in Fig. 15 (d2) suggest that some of the dictyosomes are again in membranous continuity at this stage ofmitosis. There is also some suggestion of fenestration (parallelarrows).
As soon as the chromosomes have decondensed, while atthe same stage of cell division as judged from the light microscope images, the Golgi apparatus is fully reformed, includingfenestrated cisternae (see Fig. 10 in Ref. 15). At this stage,melanogenesis is resumed. No structural involvement of theremaining cisternae from dictyosomes with premelanosomescould be detected during mitosis.
DISCUSSION
An active involvement of the Golgi apparatus in melanogenesis was assumed because of the physical proximity of thisorganelle with premelanosomes of all stages (1). It has beenshown that some melanosomes are in membranous continuitywith SER and possibly with Golgi elements (14). In order tounderstand fully the role of the Golgi apparatus in melanogenesis, structural changes as well as the functional activitymust be investigated at all stages of the cell cycle.
Several investigations have been concerned with ultrastructural changes in the Golgi apparatus during mitosis in differentcell systems. The Golgi apparatus was reported to be completely absent in several dividing tumor cells (5). In HeLa cells,the Golgi apparatus disappeared during metaphase and ana-phase and reappeared during telophase (19). However, inhepatic cells (6) as well as in transplantable hepatomas of therat (4), it was found that the Golgi apparatus did not completely dissappear, but retained its laminar components andcould be recognized throughout mitosis. Dictyosomes werealso found in metaphase and anaphase in spermatocytes of theEuropean corn borer and the giant amoeba (23). None of theseinvestigations, however, relate the structure of the Golgi apparatus in interphase cells to the one in mitosis.
In interphase, the Golgi apparatus of the human melanomacells in vitro consists of many dictyosomes which are connected by a tubular network of SER (see also Ref. 16). Theconvex side of single dictyosomes is often part of the RER.Some of the cisternae are fenestrated at the lateral edges.Often, the pores are arranged hexagonally and sometimes correspond to the pores of adjacent cisternae (15).
During mitosis profound changes occur in the Golgi apparatus. All fenestrated cisternae disappear during prophase.
The Golgi apparatus breaks down into single dictyosomeswhich become much smaller than in interphase. The numberof cisternae is decreased. By anaphase, only a single cisternawith accompanying tubules and vesicles exist. Parallel stacks ofcisternae are conspicuously absent. For the reasons givenearlier, most membrane components at the lateral edges ofsingle cisternae were considered to be tubules. It is often asdifficult to demonstrate a free vesicle as it is to demonstratemembranous continuity. Membranes in face on views appearonly slightly gray against background. As the diameter of mosttubules is smaller than the section thickness, it is difficult tofollow an obliquely sectioned tubule from 1 section to thenext, because the densest part of the immage will not appeardirectly above the underlying image of the same tubule.
In anaphase and early telophase, hardly any remaining dic-tyosomal cisternae could be found. Negative findings (19-21)
may partly be due to the inherent problems of sampling. InFig. 20 of Ref. 19, a cluster of vesicles suggests that in ana-
phase He La cells have dictyosomes similar to the one presented here. The fact that 1 dictyosomal cisterna could befound at late anaphase in 1 of 5 cells investigated proves thatthey are present at this stage, but in very small numbers. Itdoes not prove, however, whether the dictyosomes are newlyformed in later telophase or whether they are formed aroundthe persisting dictyosomal cisternae. The small numbers ofsingle dictyosomal cisterna in anaphase and the high numberof dictyosomes in late telophase let it seem likely that dictyosomes are formed anew and not only by enlarging remainingdictyosomal cisternae.
The dictyosomes enlarge and appear to "proliferate"
rapidly during the time which is characterized by the reformation of the nuclear envelope. Morphologically, it appears as ifthis enlargement and increase in number of dictyosomal cisternae comes about by apposition of preexisting membranesrather than by membrane formation de novo. However, thelatter cannot be ruled out.
The function of the remaining dictyosomal cisternae duringmitosis remains obscure. Coated vesicles were observed in allmitotic stages, but in very small numbers. They are consideredto be primary lysosomes (7). Whether or not coated vesiclesare left over from interphase or formed anew during mitosiscannot be answered by the present study. The activity of dictyosomes during cell plate formation has been demonstrated inplant cells (28). In melanoma cells, a typical dictyosomalcisterna could be found at the equatorial plate of metaphasecells and at the cleavage furrow in the telophase cells. At thatstage they were found in physical proximity to electron-dense,membrane-bound granules, presumably lysosomes. Because oftheir proximity to the cleavage furrow, one might speculate ontheir involvement in cytokinesis, but, at the terminal stage oftelophase, dictyosomal cisternae can no longer be observedaround the midbody.
During mitosis, no structural evidence was found whichwould suggest a direct relationship between the remainingdictyosomal cisternae and the process of melanogenesis.Despite the fact that several premelanosomes were found inmitotic stages of the melanoma cell in vitro, it is concludedthat the remaining dictyosomal cisternae which persist duringmitosis are not involved in melanogenesis. A structural account
of the melanoma cell in vitro during Gj, S, and G2 phase willbe given in a subsequent paper.
In careful studies where serial sections have been examined,the persistence of Golgi elements throughout mitosis seems tobe the rule rather than the exception. This is especially true ofcancer cells, possibly because more of them were thoroughlyinvestigated (4). The functional status of the Golgi elements inmitosis should be more thoroughly established in order toevaluate the significance of the present observation in regardto cancer cells.
ACKNOWLEDGMENTS
Thanks are extended to Dr. M. Romsdahl for supplying the melanoma cells.
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Fig. 1. Oblique view of 4 dictyosomes interconnected by tubular SER. Long arrow, fenestrated cisterna. The secreting side consists of a networkof anastomosing tubules (lower left). Coated vesicles (cV) are numerous and often attached to membrane tubules of the dictyosomes. X 38,000.
Fig. 2. Section through the lateral edge of a dictyosome. Arrows, cross-sections of the bridges between fenestrations. They appear in lessfavorable sections as small vesicles. RER is in direct apposition to the dictyosome. Ribosomes were attached to this membrane, as seen in aconsecutive serial section (see also Ref. 15, Figs. 1 and 9). X 137,000.
Fig. 3. Late prophase. The nuclear membrane has not completely broken down. Chromatin is still attached to membranes, mostly aroundpersisting pores (arrows). The Golgi apparatus has decreased in size. Fenestrated cisternae are no longer to be seen. The average number of cisternaeper dictyosome is 2. Only very seldom are 4 cisternae found. X 82,000.
Fig. 4. Ten consecutive sections through remaining dictyosomal cisternae in metaphase. Vesicular and tubular elements surround singlecisternae. SER is present as an anastomosing network. RER also comes in close vicinity of the tubular elements of the cisterna (h to j). Arrowspointing right in d and e, single vesicles. Branching membrane tubules can be seen in b (arrow). In c, the arrow (upper right) points to an area whichis interpreted as tangentially sectioned membrane to illustrate the difficulty in following membranous continuity. See same area in a and b. X75,000.
Fig. 5. Obliquely sectioned anaphase; e, ceninole. Boxed area is magnified in Fig. 6. X 4,000. inset, light micrograph of this anaphase. X 1,000.Fig. 6. Single cisternae with tubular and vesicular elements in the centriólearea. This micrograph was taken 5 sections apart from the boxed area
shown in Fig. 5. Arrow, coated vesicle. X 21,000.Fig. 7. Late anaphase cytokinesis has begun. A chromosome break has occurred. Boxed area is magnified in Fig. 8. X 4800. a, light micrograph
of this anaphase. X 1200.Fig. 8. Single cisterna as found in late anaphase. X 33,000.Fig. 9. Early telophase. Nuclear membrane reformation has begun, c, centriole. Boxed areas are magnified in Fig. 10 to 12. X 5400.Fig. 10. Dictyosomal cisterna found at the cleavage furrow, m, melanosome; L, lysosomes (?). X 52,000.Fig. 11. Only dictyosome found with 2 parallel cisternae after prophase and before late telophase. Hardly any tubular or vesicular membrane
elements are present, nm, nuclear membrane; cv, coated vesicle. X 70,000.Fig. 12. Telophase. Nuclear membrane reconstitution is well advanced, but not completed. At this time, the first stacks of 4 cisternae can be
found. On the light microscope level, no difference can be noted until early telophase as in Fig. 9. Arrows, dictyosomes. X 32,000.Fig. 13. Telophase. The nuclear membrane has been completed, but cytokinesis has not markedly advanced. Several dictyosomes with 4
cisternae can be noticed close to the nuclear envelope. A few dictyosomes were also found in the cleavage furrow and in other areas (arrows). X23,000.
Fig. 14. Late telophase. Cytokinesis is complete. The chromosomes start to decondense. Many melanosomes are found in the centriole area, butno early premelanosomes. mb, midbody. Boxed area is magnified in Fig. 16. X 6000. Inset, light micrograph of late telophase as shown in Fig. 15.X 2000.
Fig. 15. Dictyosomes in centriole area of late telophase are numerous and have cisternae from 2 to 8. Vesicular and tubular elements are not yetdominant. Some indication of a fenestrated cisterna was found (arrows). No early premelanosomes are present, m, melanosomes;d, dictyosomes;/,lipid droplet. X 64,000.
