JOURNAL

OF STRUCTURAL

BIOLOGY

Centriole

109,

1-12 (19%)

Modification

V. B. BYSTREVSKAYA, Institute

of Experimental

Cardiology, Received

in Human Aortic

V. V. LICHKUN, Cardiology December

Research 24, 1991,

The structure of centrioles in endothelial cells of embryonic (22-24 weeks old) and definitive (2, 14-17, and 3040 years) human aorta in situ and also in aortic endothelial cells dividing in organ and cell cultures (donor age 30-40 years) was studied. It was found that in the endothelial cells from definitive aorta the lengths of mother centrioles vary from 0.5 to 2 pm, whereas the length of daughter centrioles remains constant (0.4-0.5 pm). The distal part of the cylinder of long mother centrioles consists of microtubule doublets. In aorta of donors 30-40 years old in multinucleated cells and in one of 30 singlenucleated cells analyzed, C-shaped long centrioles were seen. These centrioles exhibit a doublet organization along all their length. Mitotic cells in organ and cell culture had a nonequal structure of spindle poles: at one pole, the long mother centriole was seen, while at the other a mother centriole of standard size was found. In such cells of organ culture long centrioles make contact with the remnant of primary cilia until the end of anaphase. In cell culture mitotic cells are also observed containing C-shaped centrioles. In these cells the number of mother centrioles is odd and their number is not equal to the number of daughter centrioles. The possible mechanism for transformation of endothelial centrioles and its role in the control of cell-cycle progression are discussed. i 1992 Academic Press, Inc.

From a number of electron microscopic studies of the centriolar machinery it is clear that the structure of microtubular skeleton of centriole is the species-specific feature which can change in germ-line cells, but is preserved extremely stable in somatic tissue (Wheatley, 1982). For example, in embryonic and definitive tissues of mammals the centrioles are found generally consisting of nine triplets of microtubules having lengths of 0.5-0.6 km. There is only one type of somatic cells (pinealocytes) known in which the structure of centrioles changes during differentiation (Lin, 1970). The increase of centriolar length followed by the disintegration of the centriolar cylinder into separate triplets of microtubules takes place in rat pineal cells at the postnatal stage.

A. V. KRUSHINSKY, Center, and

3rd

in revised

Endothelial

Cherepkovskaya form

April

V. N.

AND

str. 15A, 23,

Cells

SMIRNOV 121552

Moscow,

Russia

1992

The structure of centrioles begins to change when the mitotic activity in pineal body drops from the neonatal maximum to the trace level. Lin (1970) pointed out that the centriolar transformation may be considered as a degeneration of centrioles that have resigned from their functions in mitosis and in ciliogenesis when pinealocytes lose their ability for renewal. The centrioles longer than 0.6 pm and centrioles with a defective cylinder were recently found in many endothelial cells of atherosclerotic human aorta (Bystrevskaya et al., 1988). A question arises of whether this picture means that the population of cells which are responsible for cell replacement in endothelium is considerably reduced with age or the progression of atherosclerosis. The data available today are insufficient to judge whether Lin’s hypothesis is valid for endothelial cells. Some insight into this issue might be gained by comparing the organization of centrioles in dividing endothelial cells with that in cells being irreversibly arrested in a quiescent state. Since the most likely representative of irreversibly Go-arrested cell subpopulation in the endothelium are multinucleated endothelial cells, we have undertaken a comparative study of the organization of centrioles in (1) multinucleated and mononucleated endothelial cells in atherosclerotic human aorta, (2) mononucleated endothelial cells of embryonic human aorta (22-24 weeks) and in the postnatal period (2, 14, and 17 years), and (3) aortic endothelial cells that are able to divide in organ and cell culture. MATERIAl3

AN11 METHODS

The organization of the centriolar apparatus in mononucleated and multinucleated endothelial cells taken from the descendent part of the aortic arch of three males aged 2,38, and 40 years and three females aged 14, 17, and 35 years was studied. For organ and cell cultures the segments of the thoracic aorta from two males and two females aged 3C40 years were used. In all cases the aorta was taken at autopsy within 1.5 hr after death caused by a brain trauma. The aorta of two 22- to 24-week-old human embryos (intrauterine death) was also studied. To estimate the approximate frequency of multinucleated endothelial cells in various zones of the aortic arch and the number of nuclei per cell, light microscopy of en face preparations of the endothelium from grossly normal or lesioned zones (gelatinous

Ali

(‘opynght I-lgbts of

i

1047-8477,‘92 $5.00 1992 by rlcademic Press, Inc

reproduction

in any form

reserved.

2

BYSTREVSKAYA

elevations, lipid spots, and plaques) of the vessel was carried out. En face preparations stained with Bemer hematoxylin and silver were made in collodion (Silkworth and Stehbens, 1975). The ultrastructural study of centriolar organization in endothelial cells with different numbers of nuclei was carried out using en face endothelial preparations made in gelatin. Aortic segments were fixed with 2.5% glutaraldehyde in 0.1 M PBS for 40 min. Then the samples were immersed in PBS and 2 x 3 mm specimens were excised from grossly normal or lesioned areas (gelatinous elevations, lipid spots, and plaques). The specimens were gently blotted with filter paper and applied with their endothelial surfaces to gelatin-covered glass slides heated to 37°C (slides were covered with 20% gelatin 3 hr prior to their use). Within l-2 min the endothelium-free segment of the aorta was removed from the glass and en face endothelium preparations were rinsed with PBS, fixed with 1% 0~0, in PBS, dehydrated, and embedded in Epon 812. Using phase-contract microscopy mononucleated or multinucleated cells were selected and ultrathin sections were made. In the aorta of donors aged 30-40 years the ultrastructure of the following cells was analyzed: 30 mononucleated cells; 6 binucleated cells; 4 trinucleated cells; multinucleated cells with one compact nuclei cluster containing 4 nuclei (4 cells), 5 nuclei (4 cells), 6 nuclei (1 cell), 9 nuclei (1 cell), and 11 nuclei (1 cell). In addition, 2 giant multinucleated cells having approximately 70 nuclei distributed over several nuclear clusters were analyzed. In the aortas from 14- and 17-year-old donors, and also in the aorta from a 2-year-old donor 30 mononucleated cells were investigated. An electron microscopic study of the endothelium from human embryonic aorta was carried out on sections prepared as described earlier (Bystrevskaya et al., 1988). In this case, the ultrastructure of 30 cells was studied. To prepare organ cultures, the segment of aorta without the adventitia and the outer part of the media was washed off blood with medium 199 and incubated at 37°C for 1.5 hr in medium 199 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 kg/ml streptomycin, 0.1 pg/ml hydrocortisone (GIBCO), and 5 pg/ml insulin (from bovine pancreas, Fluka). The pieces, about 4 x 6 mm with their long axis oriented along the blood flow, were then cut and from two short edges of each piece part of the endothelium was removed with a scalpel. Each piece was placed in 3 ml 199 medium supplemented with fetal calf serum (25%), L-glutamine, antibiotics (in concentrations mentioned above), and sodium bicarbonate (0.3%), and cultured for 5 days in an atmosphere of 95% air:5% COz. The culture medium was changed daily. Morphological analysis of the endothelium in organ culture was carried out each day using a modification of the method described by Rogers and Karnovsky (1988). The explants were fixed with 2.5% glutaraldehyde in cacodilate buffer and stained with silver to visualize the cellular borders (Zand et al., 1982). The preparation was next placed in a drop of propidium iodide (0.05 pg/ml) and analyzed by fluorescent microscopy. The first mitotic cells (located at the surface of the explants) were found on the 4th day along the margins of the wound inside and outside of the endothelial layer. For electron microscopic investigation the explant was fixed for 5 days with 2.5% glutaraldehyde in 0.1 M PBS. Then, the thin inner layer of the intima was separated, fixed with OsO,, dehydrated, and impregnated with Epon 812 by routine procedure. The impregnated samples were placed with the endothelium down on smooth glass slips, covered with a flat rubber ribbon, and the glass plate was placed on top. Using special clamps complete flattening of the intima samples was achieved. After polymerization the outer layer of the intima was sawed off, so that the endothelium and two to three layers of subendothelial cells were left on the surface of the glass. Then the samples were reembedded into Epon 812. Mitotic cells for electron microscopic examination were selected in preparations by phase contrast microscopy, and ultrathin sections were cut. The cells analyzed were

ET AL. in prophase (4 cells), prometaphase (6 cells), anaphase (6 cells), and telophase (5 cells). Primary cultures of endothelial cells were made as described earlier (Antonov et al., 1986). Cells were seeded at a density of 3-4 x lo2 cells/mm’. After 5 days in culture the cells were fixed with 2.5% glutaraldehyde and 1% 0~0, (both in 0.1 M PBS), dehydrated, and embedded in Epon 812 according to a standard procedure. The cells in prophase (4 cells), prometaphase (10 cells), anaphase (6 cells), telophase (10 cells), and also 2 sister cells in early G, period were analyzed. All endothelial cells assayed (both in interphase and in mitosis) were studied using serial ultrathin sections of the same thickness (about 100 nm). Sections were cut with an LKB-5 ultratome using a diamond knife, stained with uranyl acetate and lead citrate, and examined in a JEM-100CX electron microscope. To determine the length of centrioles sectioned obliquely, the projection of the cylinder-forming microtubules was measured in one of the intermediate sections (Albrecht-Buehler and Bushnell, 1979); the angle of slope (a) was determined assuming the section thickness to be 0.1 pm (Mullins and Wette, 1966). The total length of the centriole projection (1) was calculated as the sum of microtubule projections in all sections where the centriole was seen. The centriole length was determined from the formula licosa. The centrioles were considered to be different in length if the length difference was longer than 0.1 pm. RESULTS

Light

Microscopy

of en Face Preparations

In human aortas of 30- to 40-year-old donors multinucleated cells were found in grossly normal and injured areas (gelatinous elevations, lipid spots, and plaques), which confirms the results of earlier investigations (Cotton and Wartman, 1961). The unexpected finding was that on the surface of plaques no cells were depicted having five or more nuclei as commonly seen in all other zones of aortas. Usually all nuclei in multinucleated cells are picked together thus forming one compact nuclear cluster (Figs. 1A and 1B). The number of nuclei in these cells do not exceed 20. In gelatinous elevations, cells are also found containing several (2-10) compact nuclear clusters (Fig. 1C). The number of nuclei in these cells may be as high as 70. The absence of plasma membranes separating nuclear clusters was confirmed by the results of an electron microscopic study of the en face endothelial preparations fixed with glutaraldehyde. In the aorta of young donors aged 2, 14, and 17 years, binucleated cells are sometimes depicted; cells containing more than two nuclei are not found. In en face preparations of endothelium fixed with glutaraldehyde and OsO,, osmiophylic granules are present (Fig. 1D) in all aortas of donors aged 14-40 years. This electron microscopic study demonstrates that these granules are typical lipid inclusions in both single-nucleated as well as multinucleated cells (Fig. 2A). The Structure

of Centrioles

In multinucleated cluster all centrioles

in Multinucleated

Cells

cells containing one nuclear form one (2-6 nuclei) or two (9

ENDOTHELIAL

FIG. 1. En face preparations of endothelium and gelatinous elevations (Cl of human aortic multinucleated cells. The broken line indicates phase-contrast microscopy). Magnifications: A,

CENTRIOLE

TRANSFORMATION

(A-C, collodion covering; D, gelatin covering) overlying a grossly normal area (A, B, D) arches from donors aged 30-40 years. Several nuclei clusters (NC) are seen in some the borders of multinucleated cells (A-C, stained with silver and hematoxylin; D, x 90; B-D, x 550.

and 11 nuclei) centriolar groups localized between nuclei (Fig. 2A). These groups of centrioles will subsequently be called as “centriolar complexes.” In giant multinucleated cells having approximately 70 nuclei distributed over several clusters, centriolar complexes are found in the center of each nuclear cluster (Figs. 3A and 3B). In the clusters containing a large number of nuclei (up to 501, several centriolar complexes are usually present (Figs. 3A and 30. Homogenous electron-dense picnotic nuclei are seen in these large nuclei clusters (Fig. 3C). Sometimes single-nucleated cells are seen, which are surrounded by one multinucleated cell (Figs. 3A, 3C, and 3D:. in bi- and trinucleated cells the number of mother centrioles (having satellites and appendages) is equal to the number of the daughter centrioles (no satellites and appendages) and equal to the number of nuclei. In the majority of cells having 4-11 nuclei, no correspondence is found between the number of mother and daughter centrioles as well as between the number of mother centrioles and the number of nuclei. The number of mother centrioles may be more (4-6 nuclei) or less (9 and 11 nuclei) by l-2 than the number of nuclei per cell. The difference in the number of mother and daughter centrioles varies from 1 to 4 in different cells; this is due to either

a larger number of mother centrioles or a larger number of daughter centrioles. No centrioles were seen at the periphery of cells. All multinucleated cells analyzed are characterized by a large variation in the length of the cylinder of the mother centrioles and by a constant length of the daughter centrioles (0.4-0.5 km, Fig. 2B). The length of the mother centrioles varies from 0.5 to 2 p.m (Figs. 2C and 2D). The minimal difference in the length of centrioles varies from 0.1 to 0.5 km in different multinucleated cells. The length of the mother centrioles in binucleated cells may differ by 0.1-l pm. In each of the analyzed cells with 3-11 nuclei the centrioles of similar length are repeated not more than two times. Analysis of serial cross sections of long mother centrioles demonstrated that the structure of their cylinder is different at the proximal and distal end. In the proximal region the cylinder of the centriole consists of microtubule triplets having the usual structure (Fig. 2E). Around the centriole in this region the halo of fine fibrillar material (width 0.2 FrnJ is seen. The length of this satellite-free region may vary from 0.3 to 0.5 p.m starting from the proximal end of the centriole. The other part of the centriole (satellite-bearing) consists of microtubule doublets (Figs. 2F-2H). The B-microtubules of each doublet

FIG. 2. Centriolar complex of multinucleated human aortic endothelial cells. (A) Nuclei and centrioles in adjacent cells containing three and five nuclei; large arrows indicate the border between these cells. (B) Two daughter centrioles. (C, D) Subsequent longitudinal sections through a long mother centriole; the border between the proximal and distal portions of the cylinder is seen (arrows). (E-H) Four cross sections through a long mother centriole; the proximal portion of the cylinder (E) consists of microtubule triplets, while the distal portion (F-H) consists of microtubule doublets; arrowheads indicate projections on a B-microtubule of doublets. (I-L) C-shaped centrioles in cross (L) and oblique (I, K) sections. (M) Weibel-Palade bodies (arrows). N, nucleus; LI, lipid inclusions; C, centriole; Cc, C-shaped centriole; IF, interconnecting fibers between centrioles; F, pericentriolar electron-dense material; S, satellite; A, appendages; Cil, cilium; Mt, microtubules. Magnifications: A, x 3400; B-D, x 25 000; E-H, L, x 65 000; I, K, x 17 000; M, x 60 000.

ENDOTHELIAL

CENTRIOLE

TRANSFORMATION

multinucleated endothelial cell containing five nuclei clusters in gelatinous elevations of human aorta. iA .) Phase F ‘I(;. 3. Giant con tras ;t microscopy. B-D) Ultrastructure of the areas limited by a broken line in A. Arrows (A-D) indicate single-nucleated end othelial by a multinucleated cell, and the asterisks indicate the location of centriolar complexes. (E) Centriolar c omplex, cell s SInrounded associated with microtubules of centriole cylinder is indicated by arrowheads. PN, picnotic nuclei; other symbols elec :troi n-dense material are the same as described in the legends for Figs. 1 and 2. Magnifications: A, X 550; B, X 2080; C, x 1500; D, x 2500; E, x 50 000.

6

BYSTREVSKAYA

yield a projection resembling an open C-microtubule (Fig. 2H). Rounded electron-dense masses (diameter is about 20 nm) joined to the doublets is observed in cross-centriole sections (Figs. 2H and 3E). In oblique sections of long centrioles this material is seen as a continuous uniform dense structure extending along the cylinder-forming microtubules (Fig. 3E). The centrioles, as long as 1.5 pm, form a primary cilia having a common organization; dense material extended along the centriolar cylinder terminates at the appendage level (Fig. 3E). The doublet organization of the long centrioles is not an artifact resulting from the en face preparation technique, since centrioles of the same structure are found in the endothelial sections after standard in situ fixation and embedding (Bystrevskaya et al., 1988). The specific feature of the centriolar apparatus of all analyzed cells having four or more nuclei is the presence of C-shaped centrioles (Figs. 21-2L and 3E). The length of C-shaped centrioles varies from 0.9 to 1.4 pm. In one centriolar complex there may be from 1 to 3 centrioles having a defective cylinder. In cross sections of C-shaped centrioles it is seen that they have a doublet organization (7-8 doublets) all along their length (Fig. 2L). The B-microtubules yield the projections of the configuration described above. Microtubule doublets are associated with electron-dense material (Fig. 2L). C-shaped centrioles reveal appendages and satellites (Figs. 21 and 2K). Sometimes the appendages of these centrioles make contact with the plasma membrane or an intracellular membrane vesicle, but no primary cilium was found to be associated with a C-shaped centriole. All multinucleated cells analyzed had WeibelPalade bodies (Fig. 2M). The Structure

of Centrioles

in Mononucleated

Cells

In all endothelial cells analyzed from embryonic aortas, the aortas of 2- to 17-year-old donors, and in the vast majority of endothelial cells from aortas of 30- to 40-year-old donors, two centrioles (mother and daughter) were found. In the endothelial cells of embryonic aortas the length of the mother centrioles was 0.5-0.6 Frn (Fig. 4). In some cells of an aorta from a 2-year-old donor, longitudinally sectioned mother centrioles with a length of 0.8 pm were found. The length of the centrioles in the aorta of donors 14 and 17 years old varied from 0.5 to 1.1 pm. The length distribution of the mother centrioles from aortic endothelial cells of donors 30-40 years old revealed the same appearance as in multinucleated cells (Fig. 4). The analysis of centrioles on cross sections shows that the structure of the long mother centrioles is similar in the aorta of donors in all age groups studied: the cylinder at 0.3-0.5 pm from the proximal end has a triplet structure that continues as a doublet with satellites and appendages at its

ET AL. donor

I

aoe 35 and 38 vrs. 3 mhinucleated &Is (6. 9 and 11 nuclei)

ucleated

22-24-week-old 1 CENTRIOLE FIG. 4. Distribution lengths in endothelial aorta. Each diagram

1.l

cells

embryos 1.7

LENGTH

2

(Cr)

of mother centrioles according to cylinder cells of embryonic and definitive human represents 30 centrioles.

distal end. A C-shaped mother centriole was found in only one of the mononucleated cells (30 years). In this particular cell 4 centrioles were found, including one C-shaped mother centriole and 3 daughter centrioles. The length of the daughter centrioles in all mononucleated cells is 0.4-0.5 km. All cells analyzed had Weibel-Palade bodies. Light

Microscopy

Characteristics

of Mitotic

Cells

In aortic organ culture, the cells located on the surface of the explant revealed only standard bipolar mitotic figures. Normally, the spindle long axis appears oriented parallel to the longer dimension of the explant. In primary cultures of aortic endothelium, cells at the early stages of mitosis could be differentiated by their chromosome morphology: some prometaphase and metaphase cells were characterized by the appearance of twofold thicker chromosomes with a more clearly pronounced double-stranded structure. From time to time, rounded cells with randomly located chromosomes were seen. The electron microscopic study revealed a tripolar mitotic spindle in the latter cells. Meanwhile, all cells that could be identified unambiguously as anaphase and telophase cells by light microscopy, exhibited a standard bipolar mitotic configuration. Only pairs of mononucleated sister cells were found at early G, period. Centriolar

Apparatus

of Mitotic

Cells

The study of the ultrastructure of mitotic cells in organ and cell culture demonstrated that the vast majority of cells contained an asymmetric mitotic apparatus: the length of the mother centrioles in the

ENDOTHELIAL

CENTRIOLE

two spindle poles was not the same. Mother centrioles differing in size were found in cells at all stages of mitosis (prophase, prometaphase, anaphase, and telophase). In these cells one of the diplosomes contained a mother centriole 0.5-0.6 pm in length (Figs. 5E and 5D), whereas the length of the mother centriole in the second diplosome varied from 0.8 to 1.2 pm (Figs. 5B, 5F, 5H, 5T-5V). The length of the daughter centrioles was always 0.4-0.5 km (Figs. 5G, 51-5N). The daughter centriole in each diplosome is located perpendicularly to the mother centriole at one of its ends (proximal) (Figs. 5A-5C, 5S--5U). At the opposite (distal) end of the mother centriole appendages were present (Fig. 5B, 5D, 5S5V). In prophase and prometaphase the appendages were seen only on the long centriole, whereas in anaphase and telophase they were found on both the long and short mother centriole (Figs. 5B and 5D). At all stages of mitosis analyzed the short mother centriole was surrounded evenly with a fibrillar halo; in contrast, the fibrillar material associated with the long mother centriole was concentrated at the proximal portion of the cylinder (Fig. 5H). It is not clear whether the structure of the microtubule skeleton of the long centriole in the proximal and distal portions of the cylinder differs during mitosis, since all these centrioles are seen in longitudinal (prophase, prometaphase, and anaphase) or close-tolongitudinal sections (telophase). The analysis of the serial cross sections of the centrioles having a standard length (mother and daughter) demonstrates that all of them consist of microtubule triplets. Some features related to the organization of the long centrioles and of their microenvironment differed in organ and cell culture. In organ culture the long centriole makes contact with an invagination of the plasma membrane (prophase and prometaphase) or with a highly flattened membrane cisterna of complicated configuration (anaphase). The invagination of the plasma membrane looks like the shaft of primary cilia. However, there are no microtubules found within this structure (Figs. 50 and 5Q). The invagination of the plasma membrane makes contact with the ends of microtubules that represent a continuation of the centriolar cylinder outside its amorphous bush (Figs. 5P-5R). The length of these microtubules varies from 0.1 to 0.2 Frn in different cells. In anaphase, a closed membrane cisterna flattened along most of its length such that the distance between the two membranes is approximately equal to the width of the membrane itself (Figs. 5S-5W) is located near the distal end of the long centriole. The intermembrane distance increases in the close vicinity of the centriole where vesicular-like broadenings are seen (Figs. 5S-5VJ. The peculiar feature of these centrioles lies in the fact that within their cylinder at a distance of about 0.2 pm from the distal end the

7

TRANSFORMATION

electron dense lamina is present (Figs. 5T and 5U). In addition, two rows of appendages are seen: one is at the level of the electron dense lamina, and the other is at the level of the vesicle-like broadening of the membrane cisterna (Figs. 5S-5V). It should be noted that in all these cells the appendages of the mother centriole are seen less clearly compared to those found in telophase and interphase cells. In telophase cells one row of appendages is located at the distal end of the long centriole. In contrast to organ culture, in all mitotic cells analyzed in cell culture the mother centriole did not make contact with either the plasma membrane or a membrane vesicle (Fig. 5H). One row of appendages is seen at the distal end of the long centriole in both anaphase and telophase. In organ and cell culture some mitotic cells were found in which the mother centrioles in two diplosomes had the same size (0.5-0.6 km). Mitotic cells in cell culture yielded considerably less Weibel-Palade bodies compared to interphase cells (Antonov et al., 1986). By thorough analysis of all serial sections only single Weibel-Palade bodies could be found in prophase and prometaphase cells. In organ culture single Weibel-Palade bodies were found only in one cell at early prophase (in one of its poles the long centriole was located). Mitotic

Cells with C-Shaped

Centriole

Dividing cells with a C-shaped mother centriole were found only in cell culture. The C-shaped centriole is found in all assayed prometaphase cells with thickened chromosomes, in a rounded cell with randomly located chromosomes (tripolar anaphase with asynchronous disunion of chromosomes), and in one of two sister postmitotic cells (early Gi period). The total number of mother centrioles in these cells varied: some cells contained two mother centrioles (prometaphase), and others contained three (prometaphase, anaphase, sister cells in early G, period). The length of the C-shaped centrioles varied from 1 to 1.5 Frn (Fig. 6). The structure of their wall was not, known since in all cases these centrioles were sectioned obliquely. In contrast to the long cylindrical centrioles (see above), the C-shaped centrioles (1) have no appendages either in prometaphase or in anaphase cells; (2) along all their length they are surrounded by a fibrillar halo, where the microtubules of the spindle end; and (3) they do not have one, but two daughter centrioles. The daughter centrioles are located at the opposite ends of the C-shaped centriole (Figs. 6A-K). One of them is located perpendicularly to the C-shaped centriole at a distance of approximately 0.1 Frn (Figs. 6B, 6N, and 60). The distance between another end of a mother centriole and the second daughter centriole varied

FIG. 5. Centriole organization in cells that had proliferated at the wound edges of an endothelial layer in an aorta explant (A-F, G-W), and in primary cell culture of aortic endothelium (G-N). (A-F) Cell in telophase: mother centriole of length 0.5 nm (D) is located at one pole of the spindle (E), and at the other pole (F) a mother centriole of length about 0.8 pm is found (A-C, subsequent sections). (G-N) Cell in telophase: mother centriole at one mitotic pole is seen in six cross sections (G); mother centriole of length 1 pm is found at the other pole (H); daughter centrioles at both poles (G, I-N) are 0.4-0.5 pm long (I-N, subsequent sections). (O-R) Cell in prophase: microtubules, which are a continuation of the centriole cylinder, make contact with an invagination of the plasma membrane (P-R, subsequent sections). (S-W) Cell in anaphase: a long mother centriole makes contact with a flattened membrane cisterna that forms a vesicle-like broadening (arrowheads) in the close proximity of the centriole (S-V, subsequent sections). The small arrows indicate centriolar appendages. MC, mother centriole; DC, daughter centriole; B, centriolar bush; MI, invagination of the plasma membrane; MB, membrane cisterna; P, spindle pole; other symbols are the same as described in the legend for Fig. 3. Magnifications: A-D, P-R, and S-V, X 34 000; G-N, x 20 000; E and F, x 4000; 0, x 4700; W, X 3800.

FIG. 6. C-shaped centrioles in cycling cells of a primary culture of human aorta endothelium (5 days after plating). (A-E) One of the mitotic centers in a prometaphase cell: daughter centrioles of 0.4 pm length (A, E) are located at the opposite ends of the C-shaped centriole (A-D, subsequent sections). (F-K) Four sections through the mitotic center of a prometaphase cell; two daughter centrioles (F, Ii are located at the opposite ends of a C-shaped centriole (G, H). At higher magnification it is seen (J, K, subsequent sections) that some microtubules continuously go along the middle part of the long centriole. (L-O) Cell in prometaphase: mother centriole of 0.5 km length (L, M) is located at one pole of the spindle, and a long C-shaped mother centriole (N and 0, subsequent sections) is found at another pole. (P-R) Centrioles of one of the two sister postmitotic cells: a long C-shaped centriole (P) and two centrioles of standard size are located in the perinuclear region (asterisk); a diplosome of standard structure i&j is seen at midbody (R). Small arrows indicate daughter centrioles. Mb, midbody: other symbols are the same as described in t,he legend for Fig. 5. Magnifications: A-E, x 25 000; F-I, x 7500; J-K, 30 000: L, r 4000; M-O. ~17 500: P, Q, * 30 000; R. L. 2000.

10

BYSTREVSKAYA

in various cells, but did not exceed the width of three sections. Other mother centrioles (one or two) in each cell had an usual size (0.5-0.6 pm) (Figs. 6L and 6M) and formed diplosomes of standard structure with their daughter centrioles. The length of all daughter centrioles in each cell was 0.4-0.5 p,m (Figs. 6A, 6E, and 60). In prometaphase cells with seven centrioles (3 + 2 + 2) the spindle exhibited a clear bipolar configuration on serial sections, and the C-like mother centriole was always found at one pole (Figs. 6G60). At the other pole one diplosome of standard structure was seen (Figs. 6L and 6M). The second diplosome was located on the side of the spindle, and the number of microtubules around this diplosome was significantly less compared to their number around diplosomes located at the poles. In a rounded cell in anaphase diplosomes of standard structure were found at two poles of the tripolar spindle, and the C-like centriole with two daughter centrioles was seen at the third pole. In this cell some chromosomes were seen near the poles, and the others (with disunion of sister chromatids at the kinetochore regions) at the equator of the spindle. At early G, period in one of the sister cells two centrioles of standard size oriented perpendicular to each other were found. In the second sister cell one out of five centrioles found was C-shaped (Fig. 6P). Three centrioles (the C-shaped centriole and two centrioles with a length of 0.5-0.6 pm) were located in the perinuclear region of these cells (Fig. 6R), whereas the diplosome of standard structure was found near the midbody (Figs. 6Q and 6R). DISCUSSION

The characteristic feature of the endothelial cells from postnatal human aorta is the variability of the length of their mother centrioles. Based on the analysis of cross and longitudinal sections of centrioles longer than 0.6 pm, one can conclude that this variability stems from the variation in the length of the cylinder segment consisting of microtubular doublets. The existence of a variable doublet region and the triplet part having relatively constant length cannot be interpreted as “triplet basal body with a doublet ciliary process of a denuded nature coming from it” (Wheatley, 1982). First of all, the pericentriolar satellites are localized on the doublet portion of the cylinder, and the appendages are seen at its distal end. Second, inside the cylinder doublet segment electron-dense material is seen, so-called amorphous bush (Vorobjev and Chentsov, 19821, which is characteristic of centrioles and is absent in cilia axonemes. Finally, the long centrioles are capable to form primary cilia having the usual organization. Mother centrioles longer than 0.6 p.m are

ET AL.

found in most endothelial cells of donors aged from 2 to 40 years; however, no cells containing long daughter centrioles were found. Apparently, the increase in the length of the endothelial centriole over 0.6 pm begins only after the formation of its own procentriole takes place and the centriole becomes capable to function as a microtubule-organizing center (MTOC) in mitosis and in interphase (Zeligs and Wollman, 1979; Rieder and Borisy, 1982; Vorobjev and Chentsov, 1982). Some of the long endothelial centrioles have a C-like shape and consist of microtubule doublets along their whole length. Since C-shaped daughter centrioles are not found, it is reasonable to assume that in endothelial cells the mother centrioles that are longer than 0.9 p.rn may undergo a transformation involving the disruption of the C-microtubules at the proximal region of the centriole and the breakdown of its cylinder upon disassembly of some doublets. The possibility of structural modification of the centriolar skeleton of somatic cells in the process of their differentiation was first found by Lin (Lin, 1970) who studied pinealocytes (see Introduction). The results of our investigation demonstrate that in contrast to pinealocytes in endothelial cells (1) only mother centrioles undergo transformation; (2) the increase in the length of the centriolar cylinder is accompanied by changes of its internal structure; and (3) apparently, the disruption of the cylinder does not proceed beyond the disappearance of one to two microtubule doublets. The analysis of cycling cells reveals that the presence of long centrioles and even the presence of centrioles with their cylinder disrupted does not rule out the possibility for endothelial cells to enter mitosis under certain conditions of stimulation. The perculiarity of the mitotic apparatus of endothelial cells lies in the fact that the structure of its poles is asymmetric as only one mother centriole is transformed. Quite unexpected was the finding that the organization of the mitotic pole with a long centriole is not the same in cells that undergo division in organ or in cell culture. In contrast to cell culture, in organ culture the long mother centriole until anaphase makes contact with membrane structures resembling the shaft of primary cilia. It is known that in proliferating cells the primary cilia is normally destroyed before the onset of mitosis (Wheatley, 1982). If the resorption of cilia occurs at the early stages of mitosis, its membrane is the first to be destroyed (early prometaphase), and only then the axonemal microtubules are disassembled (metaphase) (Rieder et al., 1979). In prophase and prometaphase cells from aortic explants, microtubules with the length approximately 0.1 km extend beyond the amorphous bush of the centriole and their ends make contact with the membrane. In anaphase cells, irrespective of the total length of the

ENDOTHELIAL

CENTRIOLE

centriole, the size of its distal segment that is limited by two rows of appendages stays constant (about 0.2 pm). Such a morphological picture suggests that the length of endothelial centrioles increases by the same value equal to 0.2 km each time during mitosis, provided the resorption of the cilia at early mitosis stages has occurred and the contact between the centrioles and the membrane is preserved until the end of anaphase. This interpretation assumes that the increase in the length of the endothelial centrioles over 0.6 pm may take place only when the centriole acquires the ability to undergo ciliogenesis. It is known that de nouo formed centrioles should participate in two mitotic divisions before they acquire the ability to form primary cilia (Vorobjev and Chentsov, 1982). The proposed mechanism of the centriolar lengthening allows for a satisfactory explanation of the observed large variations in the length of the doublet mother centrioles, whereas the length of the daughter centrioles remains constant an observation that undoubtedly requires more direct evidence. This suggestion also offers an explanation for why in endothelial cells of embryonic aorta, in which the cilia resorption occurs in interphase (Bystrevskaya et al., 1988), only standard length centrioles are found. Additionally, in cell culture, where no cilia remnant is found in mitotic cells, we failed to detect mononucleated cells with long centrioles even after the second passage (data not presented). Interestingly, the mitotic cells with long cylindric centrioles are seen both in organ and cell cultures, whereas the cells with a C-shaped centriole are found only in cell culture. The point is worth emphasizing that in the mitotic cells having a C-shaped centriole, the number of centrioles (usually seven) is greater than that which is generally found in diploid cells during mitosis. In addition, abnormal reproduction of the centrioles is likely to occur since such cells reveal an odd number of mother centrioles and the discrepancy between the number of mother and daughter centrioles is also observed. In this context we must inquire whether the cells with a C-shaped centriole are diploid or tetraploid and what is the dynamic of their cell-cycle progression. As the present study demonstrates, these cells are characterized by the appearance of twofold thicker chromosomes with a more clearly pronounced doublestranded organization. This morphology of chromosomes at early stages of mitosis may result from either premature disunion of the chromatids or the doubling of their number (diplochromosomes). Undoubtedly, the assumption that only the cells had undergone endoreduplication of their chromosomes under cultural conditions (Gatti and Olivieri, 1976) reveal a C-shaped centriole during mitosis, looks very attractive. Further studies are needed to clarify

TRANSFORMATION

11

whether the appearance of C-shaped centrioles is related to the polyploidization of mononucleated endothelial cells. As the analysis of the structure of multinucleated cells demonstrated, the variability of the length of the mother centrioles in these cells is the same as in mononucleated cells. The simplest explanation of this observation is that even multinucleated cells with a small number of nuclei are formed by cellular fusion. The specific feature of multinucleated endothelial cells is the presence of C-shaped centrioles seen very rarely in mononucleated cells. In addition, in multinucleated cells having about 70 nuclei, C-shaped centrioles in each of the investigated centriolar complexes are found with approximately the same frequency as in the centriolar complexes of cells having 9-11 nuclei. One may conclude that either C-shaped centrioles are always formed in multinucleated cells or for mononucleated cells having a C-shaped centriole cell fusion is inevitable and obligatory. Thus, endothelial cells from human aorta are an unique example of somatic cells in which only the centriole functioning as a MTOC is transformed. This transformation occurs in two steps: (1) the lengthening of the cylinder and (2) its destruction (C-shaped centriole). Apparently, the mechanism of centriole lengthening is related to the changes in the cycle of formation and resorption of endothelial cilia in aorta after birth. A long centriole in the cell functions the same as a mother centriole with a normal structure: it serves as the basal body for cilia and is involved in the organization of the pole of the mitotic spindle. The second step in the transformation rules out the involvement of the centriole in ciliogenesis. Cells with a C-shaped centriole are characterized by an excessive (compared to diploid cells) number of centrioles at the discrepancy between the number of mother and daughter centrioles. On the basis of the existing concept on the function of primary cilia, it can be proposed that absence of ciliogenesis makes the retardation in the restriction point of the cell cycle, where both centriole and DNA synthesis cycles can be reversibly arrested {Tucker et al., 1979; Tachi, 1984), quite unlikely for the cell with a newly formed C-shaped centriole. From this point of view it seems reasonable to suggest that the transformation of centrioles in the slowly turning over population of endothelial cells is an integral part of growth-regulating events switching the cell to the program of polyploidization after a certain number of divisions (accompanied by the centriole lengthening). Thus, one may expect that a future study of the regulatory mechanism of cellcycle progression in aortic endothelium in organ and cell culture will elucidate the role of the primary cilia in the growth control of endothelial cells.

12

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