Cell Biology International Reports, Vol. 4, No. 11, November 1980 1037

THE ULTRASTRUCTURE OF CENTRIOLE IN MAMMALIAN TISSUE CULTUHE CELLS

1.B. Vorobjev and Yu.S. Chentsov

A.N. Belozersky Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University, Moscow,

JJSSR

Abstract Structural polarity of centriole has been shown by analyzing serial sections of centrioles in the tissue culture cells of mouse, man, pig and Chinese hamster. Its major component is nine microtubule trip- lets. The inclination of the triplets towards the ra- dius at the proximal end of the centriole is smaller than at the distal one. The internal tubule of the triplet has a smaller diameter than the middle and ex- ternal ones; The triplets are bound by links of various nature all over their length. In the middle part, in the centriole lumen there is an amorphous hub; in the distal part, a thin fibre that is helically wound. In the proximal part, there are bases along the triplets, and handles stretch from the internal tubules. In the middle and distal parts, there are accumulations of an electron dense substance along the middle tubules. At the distal end, the centriole lumen is filled with an amorphous substance, whereas the proximal end is free from it. Prom outside, appendages are attached to the triplets at the distal end. The centriole structure is identical in all the cell types studied, except for those of Chinese hamster.

Introduction The first description of centriole ultra- structure goes back to the middle fitties (de Harven, Berhard, 1956), but not much has been added to it since then centriole is known to be a polar structure with proximal and distal ends (Bernhard, de Harven, 1958). On replication, a daughter centriole forms at the pro- ximal end; a cilium can grow from distal end. The ques- tion of how this functional polarity of centriole is associated with its structure remains unsolved. There is only one paper in the literature that is concerned with the three-dimentional reconstitution of centriole in mammal cells (Stubblefield, Bmkley, 1967); however its authors did not use complete series of sections

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1038 Cell Biology International Reports, Vol. 4, No. 11, November 1980

Abbreviations used: A,B,C - inner, middle and outer tri- plet microtubules; ap - appendages; tb - triplet bases; con - connectives (A-C links); h - hub; f - fibre; rl - ring of links in the middle part. Fig. l-5. Cross sections of centrioles in different cell

lines: a - proximal end, b - middle part, - distal end.

gar represents 0.1 pm,

Fig. 1. L-strain fibroblasts

Fig. 2. Rotation images of section viewed on fig. 1.

Cell Biology international Reports, Vol. 4, No. 11, November 1980 1039

for analysis of the centriole structure, and the model suggested by them is to a considerable extent hypo- thetic.

In this connection, the purpose of this work was to study and compare centriole ultrastructure in tis- sue culture cells of various species of mammals basing on reconstitution of the three dimentional structure by serial sections.

Materials and Methods Use was made of tissue culture 11 f (leucemic cells line J-41), mouse (line

Tani iriigy embryonic fibriblasts), pig (embryonic kidney epithelium, line PE) and Chinese hamster (epi- thelial cells, line BIId, clone 23'7~). They were grown on cover slips in penicillin flasks, fixed, dehydrated, contrasted and embedded in Epon-812 mixture using con- ventional techniques (Zatsepina et al., 1377). Serial sections were mounted on formvar-coated grids and stained with lead citrate according to Reinolds. The preparations were examined and photographed in Hitachi HU-11B and HU-12 and Jeol JEM-100B electron microsco- pes. Series of sections that are perpendicular to the centriol axis were taken for further analysis. Rota- tional photos were prepared by the method of Markham et al. (1963) on an inclined table, which allowed one to compensate for deformations of the centriole ari- sing on sectioning. When describing the details of the ultrastructure, we used, where possible, the terms from the work by Stubblefield and Brinkley (1967).

Results In the cells of all the lines studied by us, mcentrioles have a similar structure, the only exception being the cells of Chinese hamster (the dif- ferences will be described below). The first thing to be noticed when the series of sections perpendicular to the centriole axis are analyzed, is a striking lon- gitudinal heterogeneity of its structure (Figs l-5). Thus, we can distinguish the proximal and distal ends of the centriole not only b y the presence of additional structures (daughter centriole, cilia), but also by its structure. In Figs l-9 the sections (a,b,c) are numerated from the proximal to distal end.

Morphologically, the major component of the cent- riole are nine triplets of microtubules which stretch from one end of the centriole to the other, thus for- ming a centriolar cylinder. The distribution of the triplets all over the length of the cylinder is not uniform; their inclination towards the radius in the proximal part is 50-55O, reaches 6%70°.

and in the distal part it The internal (A) tubule of each trip-

let has a complete wall all over the len th, whereas the external (C) and sometimes middle (B 7 tubule may

1040 Cell Biology International Reports, Vol. 4, No. 11, November 1980

Fig. 3. Human J-41 cells

Pig. 4. Primary murine fibroblasts

Fig. 5. Chinese hamster cells BIId, clone 237~.

Cell Biology international Reports, Vol. 4, No. 11, November 1980 1041

have an incomplete wall in the distal part of the cent- riolar cylinder. The diameter of the triplet microtubu- les is not the same; it equals 22 nm in tubule A and 25 nm in tubules B and C. All over the length of the cent- riolar cylinder the triplets are bound by various con- nectives. In the proximal part of the centriole these connectives stretch from tubule A of one triplet towards tubule C of the adjacent triplet (A-C-links). Protube- rances resembling arms of a cilium axoneme stretch from tubules A into the cylinder lumen. Electron dense struc- tures of diffuse contours, the so-called triplet bases, stretch from the arms towards the A-C-links. The bases localize asymmetrically with respect to triplets and they can hardly be revealed in rotational photos. In the middle and distal parts of the centriolar cylinder there are no rmas of A-tubules and bases. Inside the centrio- lar cylinder there is a solid ring of links attached to tubules A and B of each triplet. In the middle and distal part of the centriolar cylinder, at the junction of tubules A and B there are small electron dense clots seen as columns along the triplets.

In the proximal part, there are no electron dense structures in the lumen of the centriolar cylinder. In its middle part there is an amorphous hub. It has a 100-120 nm external diameter and a 40 nm internal. The hub can attach itself by short knobs to the ring of links. The number of these knobs varies and is always less than nine in one section. In the distal part, there are accumulations of amorphous material near the hub, with a 5 nm filament surrounding them in the form of an incomplete circle. The filament is never attached to the triplets. We always observed it only in one sec- tion of the series.

Outside the triplets of microtubules are surroun- ded all over the length of the centriolar cylinder with a rim of amorphous or fine fibrillar material, i.e. ma- trix. The matrix is most distinctly seen in the proximal part of the centriole. In the distal part, the triplets have appendages, i.e. knobs attached to them from out- side. They stretch perpendicular to the longitudinal axis of the triplet and are inclined at an angle of 45" towards the longitudinal axis of the centriolar cylinder. The appendages are never seen within one section; as a rule, two sections are needed for that. They do not lo- calize quite symmetrically with respect to the triplets ana can be hardly intensified in rotational photos (Fig 2~). Since all nine appendages are hardly ever seen equally well (in one section), we believe that they do not localize at the same level.

Ei general scheme of the structure and sections of centriole is shown in figure 16. For convenience, the

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a

Cell Biology International Reports, Vol. 4, No. 11, November 1980

Pig. 6, Model of the centriole from a mammalian cell, a- three-dimentional reconstruction, b-d - - croa section8 of proximal;-kLddle and dietal parts of the centriole.

Cell Biology International Reports, Vol. 4, No. 11, November 1980 1043

appendages and bases of triplets are arranged in the figures symmetrically.

In the cells of Chinese hamster (Fig. 5), centrio- les differ from the above structure in the following way. They are devoid of appendages, the inclination angle of the triplets to the radius is constant all over the length of the centriole and equals 5%60°. Tubules B and C have a complete wall all over the length. In other respects, the structure of centrioles in Chinese hamster cells is identical to that described above.

Discussion The centriole ultrastructure offered by us ff

B&E?iy from the model suggested by Stubblefield and

(Stubblefield, Brinkley 1967). The main diffe- rence is that the centriole struiture ie heterogeneous all over its length. For example, in Stubblefield and Brinkley's model, bases stretch along the triplets all over the length of the centriole, whereas according to out data, they localize only in the proximal part of the cylinder. The ring of links, situated in the middle and distal parts of the centriole, was described pre- viously for a cell culture of line L-929 (Rattner, Phil- lips, 1973); however, it has been mentioned in none of the descriptions of the centriole structures but ours. Moreover, in the work of McGill et al. (McGill et al., 1976), various centriole sections (containing and de- void of the ring of links) illustrate changes in the structure of centriole caused by various treatments.

The question of the functional role of all the structural components of the centriole remains open at present. Moreover, the present work shows.that the two structures to which Stubblefield avid Brinkley had atta- ched great functional importance (Stubblefield, Brink- ley, 1967; Stubblefield, 1968), the helix, situated in the lumen of the centriolar cylinder, and the membrane bound vesicle in the middle of the centriole, are not indispensable components of centriole and, we believe, they cannot be responsible for such universal features of the centriole as replication and spindle formation.

The absence of appendages in centrioles of Chinese hamster cells (line Blld, clone 237c), allows the sug- gestion to be made that appendages are not the necessa- ry component of the centriole. It could be regarded as an atavistic sign indicating that centrioles and basal bodies in mammal cells form in the same way.

References

Bernhard 'J., de Harven E. (1960) L-ultrastructure du centriole et d'auteres elements de l'appareil achromatique. In: "4-th Intern. Congr. Electron

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De Harven E., Bernhard W. (1956) Etude an microscope electronique de l'ultrastructure du centriole chez les vertebrates, Z. Zellforsch., 45, 375-398.

Markham E., Prey S., Hills G. (1963) Methods for the enhancement of image detail and accentuation of structure in electron microscopy. Virology, 20, 88-102.

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Rattner J.B., Phillips S.G. (1973) Independence of cent- riole formation and DNA synthesis. J. Cell Biology, 57, No. 2, 359-372.

Stubblefield E. (1968) Centriole replication in a mam- malian cell. In: "The proliferation and spread of neoplastic cells", Baltimore, 1968, 175-189.

Stubblefield E., Brinkley B.R. (1967) Architecture and function of the mammalian centriole. In: "Formation and fate of cell organelles", N.Y., 6, 175-218,

Zatsepina O.V., Polyakov V.Yu., Chentsov Yu.S. (1977) Some structural aspects of the fate of the nuclear envelope during mitosis. Cytobiologie, 16, 130-144.

Received: 14th July 1980 Accepted: 23rd July 1980