JOURNAL

OF STRUCTURAL

BIOLOGY

108,

107-128 (1992)

Centrosome Organization Their Sensitivity MICHEL PAINTRAND, Centre

de G6nktique

and Centriole Architecture: to Divalent Cations

MOHAMMED MOUDJOU, MolPculawe

du CNRS,

HERVB DELACROIX, AND MICHEL BORNENS’

2 Avenue

de la Terrasse,

91198

Gif-sur-Yvette,

France

ReceivedOctober 17, 1991, and in revised form November 12. 1991

portant organelle has emerged as the role of the microtubule array in several cellular activities is increasingly being appreciated. A role of the centrosome in the organization, polarity, and division of the animal cells has been proposed since the early days of cell biology and we know today that such a role, if it exists, is linked to the properties of the centrosome to nucleate and to anchor microtubules. Yet the nature, and sometimes the existence, of the centrosome is still the subject of debates more than a century after its discovery (see Bornens, 1992). The main reason is that the definition of the centrosome has been largely morphological until now and with very poor resolution, a criterion which is dependent on the cell system under study among divergent species. A general definition should involve criteria holding for any sort of centrosome, or centrosome equivalent, despite divergent morphology. Such a definition could be tentatively formulated as follows: a centrosome is a central body, tightly associated with the nucleus, which duplicates once during the cell cycle and which acts as a microtubule organizing center (Bornens, 1992). These four criteria appear as the minimal requirement to identify a centrosome. In animal cells, the old description of the cent,rosome as a polar corpuscule containing two centriales (Boveri, 1901) corresponds to the most common situation. This description is completed in modern times by that of the arrangement of the centrioles as nine triplets of microtubules, and usually by mentioning “the electron-dense, ill-defined osmiophilic pericentriolar material which is the site of microtubule nucleation” (i.e., Stearns et al., 1991). Indeed, the numerous publications on the morphology of the centrosome have not yet led to a comprehensive and accepted description, although thick serial sectioning and high voltage microscopy represented a significant step toward a better understanding of centrosome organization (Rieder and Borisy, 1982). Three-dimensional reconstructions from serial sectioning have been rare, however, not only due to the

The centrosome plays a major role in the spatial organization of the microtubular network and has a controlled cycle of duplication, the two duplicated centrosomes functioning as mitotic poles during subsequent cell division. However, a comprehensive description of the overall organization of the centrosome in animal cells is lacking. In order to integrate the various pieces contributing to the centrosome structure and to optimize the quality of the data, we have undertaken an extensive ultrastructural study of centrosomes isolated from human lymphoblasts, which involved 6) orientation of centrosomes by sedimentation before embedding and sectioning, (ii) ultrathin serial sectioning, (iii) digitalization of micrographs to obtain quantitative data, and finally, (iv) comparison between two methods of isolation, which differ by the presence or absence of EDTA. Using this strategy, we have unambiguously described the pericentriolar organization of two distinct sets of appendages (distal and subdistal) about the so-called parental centriole. New structures have been also observed in association with the microtubule sets in this study: (i) external columns, which are dense structures localized at the basis of the subdistal appendages and (ii) internal columns, which are made of globular subunits integrated in a more luminal and probably helical structure. We have also obsewed that removal of divalent cations by the EDTA during the isolation procedure could affect the centrosomal structure at different levels (subdistal appendages, internal and external columns, pericentriolar matrix), including a significant variation in centriole diameter. A *theme of the overall organization of the centrosome from animal cells and of its modulation by divalent cations can be drawn from this study. Our data gives a view of the centrosome as an organelle displaying a complex and possibly dynamic structural organization. p 1992 Academic Press. Inc. INTKODUCTION The centrosome is still largely terra incognita in cell biology. Recent interest for this potentially im-

’ To whom correspondenceshould be addressed. 107

All

1047.8477’92 $3.00 CopyrIght i 1992 by Academic Press, Inc rights of reproductmn m any form reserved.

108

PAINTRAND

difficulties inherent to the technique itself but also because these studies could only be realized on in situ centrosomes, where random orientation of the centrioles is expected. Advances in the isolation of centrosomes has allowed us to overcome this problem: isolated centrosomes could be oriented by sedimentation on a coverslip before processing them for electron microscopy (Bornens et al., 1987; Komesli et al., 1989). Using this approach, we have undertaken an extensive ultrastructural study by serial sectioning of centrosomes isolated from human lymphoblasts and by quantitative analysis of the serial micrographs. Moreover, we carried out this study on centrosomes isolated either in the presence or in the absence of divalent cation chelators, as a role of divalent cations, and particularly of Ca2+ on the structure of isolated centrosomes has been observed in our laboratory, as has the presence of centrosomal calcium-binding proteins (Moudjou et al., 1991). The structural analysis presented here has led us to reinterpret previous observations from the literature on centrosomes in situ, and from ours on isolated centrosomes, and to make several new observations which will provide the bases for a three-dimensional model of the isolated centrosome. The main conclusion from such a structural study is twofold: (i) the different parts of the centrosome and of the centrioles are organized in a quite precise and constant manner from one centrosome to the other despite the high complexity of the structure; and yet, (ii) a significant flexibility is possible at several levels in the centrosome organization, judging from the isolates realized in the presence or the absence of EDTA. MATERIALS The

Isolation

Centrosomes were isolated (KE37 cell line) according to described previously (Bornens ilar to method I except that acid) has been omitted in all Chemical

AND METHODS

of Centrosomes

Extractions

from human lymphoblastoma cells two procedures: Method I has been et al., 1987); and method II is simEDTA (ethylenediaminetetraacetic steps.

of Isolated

Centrosomes

Isolated centrosomes were extracted with 1 M urea, 1% phosphotungstic acid (PTA), or 0.5 M KI (potassium iodide) and processed for the electron microscopy as described by Klotz et al. (1990) and Moudjou et al. (1991). Transmission

Electron

Microscopy

of Isolated

Centrosomes

Centrosomes have been sedimented on coverslips as described previously (Bornens et al., 1987; Komesli et al., 1989) except that the centrosome fraction was first mixed with latex beads (0.75 pm in diameter) at a ratio of one bead per two centrosomes. The distribution of latex beads among centrosomes on the coverslip facilitates the identification of an individual centrosome in serial sections. After sedimentation, centrosomes were fixed in two steps: Prefixation with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.3, during 40 min at room temperature, to prevent any

ET AL. possible disassembly of microtubular structures; and postfixation with 0.5% osmium tetroxide in the same buffer during 10 min at 0-4”C, to minimize alteration of microfilaments (Szamier et al., 1978) potentially present within the centrosome structure. Between pre- and postfixation, centrosomes were treated with 0.5% tannic acid in cacodylate buffer during 20 min to better distinguish the centriolar microtubules from the pericentriolar material. Centrosomes were embedded by inversing Epon capsules on the coverslip. After polymerization, Epon was detached from the coverslip by thermal shock with liquid nitrogen. Sections performed on an ultramicrotome Reichert Ultracut were collected one by one on grids with slot, contrasted with 4.9% uranyl acetate in water during 30 to 60 min and with 0.2% lead citrate during 2 min (Venable and Coggeshall, 1965), and observed on an electron microscope Philips EM 201. Eleven series of 3 to 5 sections have been made for centrosomes isolated by method II (chosen as the reference method) and 8 series of 3 to 5 sections for those isolated by method I. The theoretical thickness was 1000 A, the interference color being pale yellow, but was probably less from the number of sections necessary to section the whole length of a centriole (which can be known with accuracy from longitudinal sections). Because the centrosome is an organelle with a complex three-dimensional organization, we had to choose sections perpendicular to the axis of one of the two centrioles to obtain the maximum structural information. More than five sections perpendicular to the longitudinal axis cover the total length of a centriole. Sections were first micrographed at x 7000 magnification. Centriolar transverse sections that were optimally oriented (perpendicular to the coverslip) were identified and localized with respect to the pattern of latex beads. They were further micrographed at magnification x 45 000. Most often, the centriole in a given centrosome which sedimented perpendicular to the coverslip was the centriole possessing appendages at the distal end (called parental or mother centriole by convention, the method proposed by Rieder and Borisy (1982) to distinguish the parental centrosome from the daughter one holds only for replicating centrioles), the distal end normally being on the coverslip. Quantitative

Study

of the EM

Pictures

The quantitative analysis of the serial sections of individual centrosomes involved several steps: 1. Identification of individual sections. The level corresponding to any centriolar tranverse section in a given series was estimated with respect to two reference series of five sections (presented on Fig. 3 under Results). In this way, each micrograph is identified within a series by a pseudocoordinate from 1 at the distal end to 5’ at the proximal one. 2. Digitalization of micrographs. Data were collected by digitalizing the pictures from the negative films obtained at magnification x 45 000, with an Optronics PlOOO photoscan system at a raster step of 50 pm. Each pixel on the digitalized picture corresponds to 1.11 nm. 3. Definition of the coordinates. Each digitalized picture was then visualized on a high-definition screen (1280 x 1024) on a Vax-station and the coordinates of the center of each of the nine microtubules A and of the nine microtubules B were recorded interactively (with a cursor). 4. Calculations. From these data, several dimensions have been calculated: -The location of the center C of each centriolar section, by searching for the center of the tightest circle passing through the nine centers of the tubule A; -the mean length of the nine corresponding radii which gives the value of the average diameter of the section, together with the corresponding standard deviation which gives an indication of the excentricity of the section and therefore of its possible deformation;

ISOLATED

HUMAN

-the angle between two adjacent radii (average and its fluctuations; -the angle between the doublets and the tangent cle (plus standard deviation). Values were obtained by lation lI12 - the angle between vector CA and vector

value

CENTROSOMES

AND

109

CA”

40”)

to the cirthe calcuAB.

RESULTS Methodology

An example of low magnification serial micrographs used to select individual centrosomes (isolated by method II) is shown on Fig. 1. It illustrates how optimally oriented centrioles could be selected and how the distribution of the latex beads enabled us to unambiguously recognize a given centrosome from one section to the next. It also demonstrates the purity of the centrosome preparations used in this work. The

Overall

Organization Isolation

C When desired, the ninefold symmetry of transverse centriole sections was used to specifically reinforce the contrast of the elements obeying the symmetry: nine images have been recomputerized from one section, each of the nine images corresponding to the original image translated (recentered about the point C) and rotated by nx2Il/9 (0 G n G 8). The final image is then obtained by mutually averaging the nine images. It represents the average image of a 2H19 angular sector of the corresponding centriole section.

a FIG. 1. identification

of Centrosomes Procedure

Depends

upon

A representative section of individual centrosomes isolated according to method I and to method II is presented on Fig. 2. Several differences can be noted. First, the intercentriolar link, which in method I appears as a narrow and long bundle of thin filaments, is spread in method II as a complex network surrounding the two centrioles, which are accordingly closer to each other. The network in method II appears to possess a rather definite boundary (see also below). Denser aggregates can be observed within this network which, altogether, is

b Low magnification of individual

of three serial sections centrioles. Bars, 0.5 Wm.

la-6

the

of centrosomes

isolated

by method

II.

Latex

beads

allow

unambiguous

PAINTRAND

ET AL.

Method II

FIG. 2. Overall organization of centrosomes isolated by method I (A) and method II (B). Centrioles are associated by a narrow and long bundle of thin filaments in method I, which sometimes seems to possess transversal stripes (small arrows). Centrioles are immersed in a complex network in method II which shows a definite boundary and in which denser aggregates (asterisk) are reminiscent of in situ pericentriolar satellites. Note the difference in the aspect of subdistal (large black arrows) and the distal appendages (open arrows) on the mother centriole in both methods. Note also that the centriole diameter is slightly different in each method. A latex bead is visible in the lower left corner in B. Bars, 0.2 urn.

more reminiscent of the “ill-defined osmiophilic pericentriolar material” described in situ than is the link observed in method I. Second, two sets of appendages, that we call subdistal and distal appendages, are associated with the mother centriole; they do not present the same aspect in both methods, particularly the subdistal ones. Whereas the latter look fuzzy and thick and are transversally oriented with respect to the centriole axis in method I, they are quite narrow in method II and are obliquely oriented toward the distal end. Third, centrioles themselves appear slightly narrower in method II than in method I, whereas their length is similar in both methods. An important issue when dealing with isolated organelles is whether or not the isolation procedure has led to structural or functional modifications. Both methods lead to centrosomes which are able to perform under controlled conditions two of the basic functions normally associated with centrosomes, i.e., the nucleation of microtubules and the establishment of a functional spindle during the parthe-

nogenic test (Tournier et al., 1989). As for the structural features, there are several reasons to consider method II as providing centrosomes more native than method I. (i) The absence of EDTA from the isolation procedure is more likely to conserve the native association of divalent cations with the centrosomal structures. The effect of divalent cations such as Mg2+ or Ca2+ on subcellular structures has been substantiated in the past, for ribosomes or chromatin for example, where they are very important. In the case of isolated nuclei from which divalent cations dissociate easily, the presence of millimolar divalent cations in the isolation medium is necessary to provide nuclei with internal structures looking like those observed ilt situ (Olins and Olins, 1972). (ii) The axial symmetry of the centrioles is better. (iii) Several features of the centriolar structure, which will be detailed below, are more reminiscent of what was observed in situ during an extensive survey of numerous sections of KE37 cells. For these reasons, method II has been taken as the reference method: extensive serial sectioning en-

ISOLATED

HUMAN

CENTROSOMES

compassing the whole length of the mother centriole, as described under Material and Methods, has been performed on these centrosomes to establish the detailed organization. Individual or serial sections realized on centrosomes isolated by method I were then observed to study the structural modifications brought about by the presence of EDTA during the isolation procedure. Serial

Sectioning Isolated

along the Parental-Centriole by Method II Demonstrates Proximo-Distal Architecture

of Centrosomes a Graduated

Two independent series are shown on Fig. 3. They are not exactly in register, showing that the first section may not be exactly at the same level above the coverslip. Therefore, one could distinguish at least 10 levels in the different series that we performed. Each series is numbered from the distal to the proximal end; the coordinate numbers in Fig. 3 are used throughout the paper to identify any section. From the distal to the proximal end, several features can be observed which are indicated in Table I. They will be further detailed in the following section where a systematic comparison between methods I and II is done for each level. One may distinguish the features which concern the centriole itself from those which belong to the pericentriolar material. In order to make the description clearer, we have also shown the average images of an ideal 40” angular sector recalculated from the complete sections (Figs. 3A’ and 3B’). Organization

of the Centriole

The centriolar cylinder displays nine sets of microtubule triplets at the proximal end, with a connection between tubules A and C of adjacent triplets, and nine doublets at the distal end apparently unconnected. Intermediary situations exist in sections 2, 2’, and 3, where incomplete tubules C are present. Observed from the distal end, the vector from tubule A to tubule B in each triplet is oriented clockwise. This orientation indicates whether from one section to the next one gets closer to one end or the other. Quantitative analysis of several series or individual sections has allowed us to obtain the mean dimensions of the centriole. The diameter of the circle defined by the centers of the nine tubules A varies from about 0.145 km at both ends to 0.155 pm in the middle, thus indicating that the centriole is slightly swollen (Fig. 4A). Whether this feature is native or promoted during sedimentation on the coverslip is difficult to decide. Longitudinal sections are not sufficient to decide either, as we have no way to be sure that such sections are strictly parallel to the centriole axis. The angle between the triplets and the tangent to the circle passing through the tubules A (see Mate-

AND

CA’+

111

rials and Methods) varied continuously from 45” at the proximal end to 20” at the distal one (Fig. 5A). The lumen of the centriole appears empty at the proximal end on one-third of the total length. We never observed any cartwheel structure at the proximal end such as that observed during the assembly of basal bodies which preceeds ciliogenesis (Kalnins and Porter, 1969). However, in the absence of serial sectioning of a replicating centrosome, we cannot eliminate the possibility of such structure in human centrosomes. There exists however an internal organization in the centrioles which might be continuous from one end to the other and which, in its distal part, is described for the first time. At the proximal end, in agreement with numerous reports in the past (Wheatley, 19821, a small spur is protruding from each tubule A toward the center. On the distal end, a sort of ring has been reported in the past. We observed that this ring which is internally adjacent to the centriolar wall is actually rather organized, somehow like a ratchet wheel: it appears as nine thick domains, that we call internal columns, touching each doublet A-B with the same clockwise orientation, and of nine thin connections between adjacent internal columns having a slightly different orientation (they would represent the concave edges of the notches in the internal ratchet wheel). These internal columns often display three subglobular domains, a feature which is clearly reinforced in the average images (Fig. 3B’, levels 2’ and 3’). From the serial sectioning, the proximal spurs and the internal columns may be continuous one with another. Such an organization could ensure the cohesion of the centriole structure. The internal ratchet wheel composed of the nine internal columns on the distal end might also belong to a more complex organization: there is a material filling in the lumen of the centriole at this level, except in the more central part where an apparently empty axial annulus can be observed. This material is not easily defined but sometimes shows dense curved and elongated structures going from the wall of the annulus to the peripheral internal columns where they abut (see Fig. 3B, level 3’; see also Figs. 6A and 6B, levels 3-3’1, with the same clockwise orientation as that of the internal columns or of the microtubules sets. They are reinforced by averaging in some sections (Fig. 3B’, level 2’) but not in others, except at the periphery, close to the globular domains of the internal columns (Fig. 3B’, level 3’). These structures seem to be organized as the blades of an iris diaphragm. Together with the nine internal columns where they apparently abut, these could represent a single structural system, a possibility which is suggested by the concerted modifications of both elements in Method I (see below). This unique structural system could possess an helical

112

PAINTRAND

ET AL.

-7

Proximal

3

2 _(:-

Distal

ISOLATED

HUMAN

CENTROSOMES

AND

113

CA2’

TABLE I Structural Features Identified in Each Sectional Level along the Mother Centriole of Centrosomes Isolated According to Method II (see Fig. 3) Section

level

Distal 1

Centriole Centriole wall Triplets Incomplete C tubule Doublets A-C linkage Centriole internal structure

1’

+

2’

+/-

+

3

Spur on A tubule Column internal to A (and B) tubule Connections between columns Centriole-associated structures Distal appendages Subdistal appendages Column external to A and B tubule

+

t + +

organization, as suggested in Fig. 6B, where an authentic sagittal section of daughter centriole (levels 3-3’1, as judged by the two flanking sections which are tangential, is observed (see also Fig. 11A). of the Pericentriolar

Material

Centrioles are surrounded by, or immersed within, a thin fibrillar material associated with them, that we name centrosome matrix rather than pericentriolar matrix (Baron and Salisbury, 1988) in order not to define the centrosome simply with respect to centrioles. In the case of the mother centriole, there are two additional sets of appendages, that we name subdistal and distal appendages due to the level of their attachment to the centriole. They are different from the so-called pericentriolar satellites which correspond to dense parts of the matrix (Rieder and Borisy, 1982; Baron and Salisbury, 1988; see Fig. 2). The subdistal appendages. The number, thickness, and distribution of the subdistal appendages can apparently vary significantly from one centrosome to the other in the same preparation, whereas the length is quite constant (about 0.14 pm), the tips of all the appendages defining an almost perfect circle with a diameter of about 0.400 pm. However, there is rather compelling evidence that the basic organization of these appendages is in

Proximal 5

3’

4

4’

+

+

+

+

+/-

+

+

-+

+/-

+

i

5’

+:-

/+

+

Distal plate

Organization

2

+ t

i+

+ +

+ t

t +

+

i +

+ +i-

fact conserving a g-fold symmetry (see section 2 in Fig. 3A and the corresponding averaging in Fig. 3A’), with their base enlarging and interacting with two adjacent microtubule sets, and that these appendages are capable of significant lateral flexibility and of interaction with adjacent appendages (see Fig. 6, levels 2-2’; Fig. 9B, right section). The tips and the edges of these appendages are dense, whereas the internal part is less compact, a situation which is compatible with these arms being tilted (as observed on Fig. 2) and hollow. The latter possibility is supported by transversal sectioning of the appendages observed in grazing sections with respect to the centriole wall (see below, Fig. 10). The averaging of section 2 in Fig. 3A suggests that the subdistal appendages could be made of filaments. The interaction of the appendages with the centriole wall is complex, producing an external dense thickening along the microtubule sets from section 1’ to 3 that we call external columns (compare in Fig. 3B’ the average image of level 2’ to that of level 4’). Grazing sections are necessary to observe how the appendages interact with the centriole wall (see below, Fig. 10). This interaction is also significantly modified in centrosomes obtained by method I and this will be detailed below together with the substructures present in these appendages. The distal appendages. In contrast to the sub-

FIG. 3. Serial sectioning along the parental centriole of centrosomes isolated by method II. Series A and B are staggered, allowing the identification of 10 levels, numbered from 1 to 5’ from the distal to the proximal end. Average images for the whole series A and B are shown side by side in A’ and B’ and are used to point to the different substructures: spur, small black arrowhead; internal column, large black arrowhead; external column, thin arrow; incomplete C tubule, black and white arrowhead; subdistal appendage, fat arrow; distal appendage, open arrow. Note also the annulus in levels 24. The sections 3, 4, and 5 in series A are slightly compressed. The corresponding average images are less precise than those from series B. In particular, the internal columns in levels 3 and 4 do not show the three subglobular domains which are visible in some of the internal columns in the original section (level 4) and which are clearly reinforced in series B. Bars, 0.2 pm.

114

PAINTRAND

ET AL.

__

“”

A-

Method

A-

II

Method

II

50 60ii

Q :*

0

5

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q

. q

q

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z ?! P

40-

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30-

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2 x

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level 60

B-

B- Method

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50.

60

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z 2 co 70 2 0 e

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40-

r 0,

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q l

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4

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5

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level

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1

level

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.

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141

50 -

zS 2

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54 1

2

3 section

4

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level

FIG. 4. Variation of the mother centriole radius defined by the center of the nine tubules A. In A (method II) the five different symbols correspond to five series. In B (method I), squares correspond to four individual sections. The three curves correspond to three series. In C, method I (small squares) and method II (large squares) are compared. The section levels have been estimated as described under Materials and Methods.

distal ones, the nine distal appendages display a remarkably constant and radially symmetrical organization (section 1 in Fig. 3A). They appear as rigid sticks protruding at an angle from the microtubule sets like the blades of a turbine and display a swollen tip. Observed from the distal end, they are ori-

FIG. 5. Distal-proximal variation of the angle between the microtubule triplets and the tangent of the circle passing through tubule A. Note that the variation is monotonous from one end to the other and similar in both methods. Symbols correspond to those in the legend for Fig. 4.

ented base to tip counterclockwise, opposite to the orientation of the microtubule triplets. In longitudinal sections (see Fig. 2), they appear obliquely oriented toward the distal end, lying in the median direction between the subdistal appendages and the centriole wall. Averaging the section 1 in Fig. 3A indicates that the base of the distal appendage could be associated with the edge of tubule B (Fig. 3A’).

ISOLATED

HUMAN

CENTROSOMES

AND

CA2 +

115

3 -3’ ‘. ‘.

.I ‘..,

:‘.

.

%“- 2’ ‘. ,..’ 4. : 1 i .v 3 1

n FIG. 6. Serial sectioning demonstrating the mutual relationship between the two rentrioles in centrosomes isolated by method II. In both series, the association between both centrioles is realized by a three-dimensional system of apparently very thin filaments between proximal sides of each centriole. Specialized structures (arrows in A, levels 5-5’) might be involved in the attachment of the centrosome matrix of the mother centriole. The sagittal section of daughter centriole (shown in B, levels 3-3’) demonstrates a helical organization of dense internal structures (arrowheads). Sections have been labeled by the coordinate number of the mother centriole. Note in levels P ~2’ how adjacent subdistal appendages can laterally associate (see also right section in Fig. 9Bl Bars, 0.2 pm.

From the observation of complete serial sectioning through the length of the mother centriole, we can conclude that the centriolar organization and that of the associated material are highly complex at all levels. The most striking feature is that all the ele-

ments present a conspicuous proxim+-distal zation. Serial sectioning The centrosome matrix. strating the mutual relationship between centrioles in a centrosome is shown on Fig.

organidemonthe two 6. Sec-

116

PAINTRAND

ET AL.

tions have been labeled by the coordinate number of the mother centriole. One can see that the association is realized by a three-dimensional system of apparently very thin filaments between proximal end of each centriole. The possibility that specialized structures are involved in the attachment of this material at the proximal end of the mother centriole is sometime suggested (Fig. 6A, arrows at the levels 5-5’). It was also suggested by averaging section 5’ in Fig. 3B.

observed on an individual section (levels 4-4’) is shown after averaging, alongside a corresponding section from method II in Fig. 8. Another feature concerning the centriole wall observed in method I must be noted here: often in transverse sections, half of the triplets on one side were not cut transverse whereas the other side was. This was true also of centrioles in method II but to a considerably lesser extent. This question will be addressed below.

Centrosomes

The Radial Symmetry of Centriole Structures is Blurred

Isolated

in the Presence Modified at Several

of EDTA Levels

(Method

I) Are

Serial sectioning of a mother centriole from centrosomes isolated by method I is shown in Fig. 7, as well as the averaging of each section. The levels in method I have been estimated with respect to the more constant features: the presence of luminal material, the doublet-triplet transition, the presence of both kinds of appendages. Several observations can be made. The Centriole

Diameter

Increases Significantly

A most striking difference between the centrosomes isolated with method I and method II is the centriole diameter which can vary by more than 20% (Fig. 4). Centrosome preparation made according to method I was used to measure the diameter of centrioles from individual and serial sections (Fig. 4B). There is some variation, the mean diameter being larger in the individual sections than in serial ones. Moreover, in the latter case, there was variation along the length of the centriole in two out of the three series: the diameter increased from a value, at the proximal end, not significantly greater than the diameter measured in method II (see Fig. 4C) to large values at the distal end. The third series was more constant in diameter along the length of the centriole and, in fact, had a value close to that of method II. By comparison, the values obtained for the centriole diameter in method II are more constant for a given coordinate number (Fig. 4A). The significance of the variations observed in method I will be addressed below. The same sections were used to measure the angle of the sets of microtubules with respect to the tangent to the circle passing through the center of tubules A (Fig. 5). For any given coordinate number, the angle is quite similar in methods I and II, indicating that the variations in diameter take place through transversal sliding of adjacent triplets with respect to each other, like the parts of an iris diaphragm. Apparently, the increase in centriole diameter occurs at the proximal end through a significant extension of A-C linkages. The maximum extension

Internal

Comparison of the different levels in the proximal end of centrioles observed in methods I and II (Figs. 3 and 7) leads to the following observations: (i) spurs associated with the internal edge of tubules A at levels 4-5 are quite visible in method I, as if they were elongated or have changed their orientation with respect to tubule A (see also Fig. 8); (ii) the internal columns adjacent to the microtubule sets from level 2 to level 4 in method II are not visible in method I. Instead, one observes a more or less continuous ring and the lumen is more fuzzy than in method II. Averaging the images suggests sometimes the possibility that one out of the three globular domains observed in method II, could maintain its association with the internal edge of tubule A (see Fig. 8 and Discussion); (iii) the axial annulus observed in method II from levels 2 to 4 is not present in method I. Instead, one observes an axial denser area which could sometimes look like a hub embedded in a fuzzy material (Fig. 8 and 9; see also Bornens et al., 1987; Komesli et al., 1989). This illdefined material contrasts with the linear structures radially organized which are sometimes observed in method II. Another feature is obvious in all sections: the part of centrosomal matrix, which is closely associated with the proximal part of each centriole, looks like a thick rim around the centriole in method I (Fig. 7) whereas it appears more dispersed in method II (Fig. 3A and 3B). The Base of the Subdistal Appendages from the Centriole Wall

Dissociates

The subdistal appendages appear thicker in method I (Fig. 9A1, as if swollen such that averaging the images often leads to a continuous ring (Fig. 7A’, level 2, and Fig. 8). Substructures are clearly visible: each individual appendage is apparently made of numerous radially assembled fibrils (long black arrowheads in Fig. 9A) which are laterally associated in specific places at the tip of the appendage resulting in transversal dark lines (small arrows in Fig. 9A). This lateral attachment appears as

ISOLATED

of the mother 1;IG. 7. Serial sectioning ( orresponding average Images are shown

HUMAN

centriole from in A’. Compare

CENTROSOMES

AND

a centrosome isolated by method with Fig. 3. Bars, 0.2 +m.

a narrower zone at the tip of the appendages in method II (small arrows in Fig. 9B). The tips define an almost perfect circle with a mean diameter of 0.484 pm to be compared to a mean value of 0.400

117

CA’+

I iAi.

The

level

5 io missing

isee

Fig.

81.

Frn measured in method II: the difference is due in part to the tilt of the appendages on the centriole wall in method II (see Fig. 2) and to their close association with the centriole wall in that method,

118

PAINTRAND

FIG. 8. Summary of the major differences, outlined by averaging images, in the mother centriole structure observed in method II (left) and method I (right). Top row corresponds to levels 4’ and 5. Note the extension of A-C link and the lengthening of the internal spur (see also level 4 in Fig. 7A’). Middle row corresponds to levels 3 to 4. Note the variation in the aspect of the internal column. Only one globular domain (arrowhead) maintains its association with microtubule sets in method I; the others dissociate and give the aspect of a continuous ring. Note the empty annulus in method II and the presence of axial material in method I. Bottom row corresponds to level 2. The outer columns dissociate from the centriole wall in method I demonstrating the doublet structure of the centriole blades at this level. Note also the disorganization of the subdistal appendages in method I. Internal columns and the annulus are modified as in the middle row. Bars, 0.2 pm.

rather than a difference in their length. A clear dissociation of the base of the subdistal appendages from the centriole wall is often observed in method I (Fig. 9A) and never in method II (Fig. 9B; compare also the average images of sections at level 2 in Figs. 3A and 7). This feature, independent of its functional signification, facilitates the observation of the

ET AL.

bases of the subdistal appendages. Dense masses, likely to correspond to the external columns observed in method II, are apparently participating in the bases of two adjacent appendages (white arrowheads in Fig. 9A) and face microtubule incomplete triplets (black and white arrowheads in Fig. 9A) or microtubule doublets. These dense masses are apparently tightly associated with the outer edge of the microtubule sets in method II and sometimes can be confused with incomplete tubule C in this method (see for example the average image of level 2 in Fig. 3A’), as they may be kinked (Fig. 9A, middle section in the bottom row, facing the doublet at 3 o’clock). They are apparently in continuity with the interrupted tubule C. That these masses are common to two adjacent subdistal appendages in both methods I and II is supported by the aspect of transverse sections in the plan of the subdistal appendages when the centriolar structure has been extracted by chaotropic agents or by phosphotungstic acid: the masses are still visible and seem to hold together the subdistal appendages after extraction of centrosomes isolated either with method I (Figs. 9C and 9D) or with method II (Fig. 9E). In the latter case the system of connected internal columns is still present (small black arrowheads in Fig. 9E). To better understand how the bases of the subdistal appendages associate with the centriole wall, we looked for grazing sections of centrioles isolated by method II, such as those presented on Fig. 10. Figure 10B (section CX)shows how complex this association is: it involves a longitudinal column of about 0.12 pm, probably along a microtubule set, which is in continuity at its proximal end with a tranversal segment (upward arrows in Fig. lOB, CX) that appears to further curve back toward the distal end and terminates tangential to the next longitudinal column. The longitudinal columns are possibly interacting at their distal end through an additional globular structure (downward arrows inFig. lOB, CY). However, these quite distal structures could also correspond to the bases of the distal appendages: from the averaging of transverse sections at level 1’ shown in Fig. 3B’, the bases of subdistal appendages could be globular and interacting with the extreme end of tubule B. The complex configuration of the attachment of subdistal appendages with the centriole wall is indeed compatible with the possibility, already suggested by transverse sections, that each subdistal appendage interacts with at least two adjacent microtubule sets. Figure 10A confirms that the subdistal appendages are rather hollow and apparently made of substructures. The Distal

Appendages

Are More Radial

The comparison of sections at level 1 in both methods shows that the distal appendages are thicker in

ISOLATED

HUMAN

CENTROSOMES

method I than in method II and that they are less tilted with respect to the centriole wall in method I (compare Figs. 3A and 3A’ with Figs. 7A and 7A’, level 1): the increase in centriole diameter is apparently accompanied by the unwinding of the distal appendages. As was observed for all the other levels, the part of centrosomal matrix, which is closely associated with the centriole, is mostly collapsed on the distal appendages and on the centriole in method I, whereas it is dispersed in method II. Ca*+

Can

Reduce

the Distal Time-Dependent

Diameter of Centrioles Manner

in a

We have attempted to monitor the effect of adding Ca2+ to isolated centrosomes in a time-dependent fashion. Centrosomes isolated with method II (with cations natively associated) were further incubated with 5 n-J4 CaCl, during 10 or 30 min. They were then processed for EM as usual. The results are presented on Fig. 11. For a short binding time, the centriole diameter at the distal end (Fig. 11A) and at the proximal end (not shown) were not significantly different (0.145 pm on average in three determinations on the distal end) from the value observed in method II (Fig. 4A), indicating either that Ca2+ could not produce any further changes in the structure of centrioles isolated according to method II or would require longer time to do it. Note, however, in Fig. 11A that the intercentriolar distance has been significantly shortened by Ca2+, as already reported (Moudjou et al., submitted for publication), often bringing the two centrioles in an orthogonal configuration. Note also that the edges of the subdistal appendages appear highly contrasted. When incubation in Ca2’ was protracted for 30 min, a dramatic decrease in the centriole diameter was observed (Fig. 11B) at the distal end (diameter about 0.125 km) and to a lesser extent at the proximal end (0.140 pm). The distal diameter was so reduced that the centriole symmetry was broken (white arrows in Fig. 11B). That such a treatment leads to further binding of Ca”’ to centrosomal structures is suggested by the inverse contrast of the microtubule triplets often observed in this experiment (Fig. 11B). The subdistal appendages appear very contrasted and the internal centriolar structure is also thickened, the internal columns being almost adjacent. We conclude therefore that structural changes induced by Ca2+ in the centrosome, and particularly in the centrioles, correspond to a slow process and that there exists a proximo-distal variation in the susceptibility of the centriole structure to Ca2’. One possibility is indeed that the subdistal appendages participate in the constriction of the distal centriole, but it could as well be promoted by the contraction of the internal system.

AND

Do Microtubule

119

CA” + Triplets

Possess

a Pitch

along

the Centrioles?

Several models of centrioles have proposed that microtubule triplets should display a pitch in order to take into account the fact that in transverse sections half of the triplets on one edge were often not cut transverse when those on the other one were (Anderson, 1972; Albrecht-Buehler, 1990). We also frequently observed this situation with centrosomes isolated by method I (see Fig. 7). By contrast centrosomes isolated by method II displayed most often a remarkable radial symmetry, although a close observation of even the most symmetrical ones demonstrated that the triplets on one edge were not completely identical to those on the other one (see for example the serial sections in Fig. 3B, where one side all along the centriole is slightly less sharp than the other). We therefore looked carefully at longitudinal sections to assess any pitch and never obtained any such evidence (see Fig. lOB, l3; and Fig. 11A). Figure 12 shows three tangential sections of centrioles, two from centrosomes isolated according to method I observed either classically (left) or in vitrous water (right; processed and photographed by D. Chretien and R. H. Wade in CENG, Grenoble) and one in situ in a KE37 cell observed after cryofixation in helium vapor and cryosubstitution in osmic acetone (middle). In all cases, most of the triplets were obviously parallel and longitudinal, and more perfectly so when in situ, with optimal fixation (Fig. 12, middle). However, in all three cases, one triplet, and possibly more if they were superimposed, was apparently not parallel to the others (indicated by a thin arrow in each case). This suggests that, rather than a general pitch of all triplets, there may exist some flexibility in the centriole structure concerning the position of one or several adjacent triplets among the others. The

Centrosome

Matrix:

A Unique

Filament

System?

The matrix which extends to the intercentriolar link and contains the satellites, subdistal, and distal appendages, all represent the elements of the pericentriolar material. As shown in this work and elsewhere (Moudjou et al., 1991 and submitted for publication) on isolated centrosomes and as suggested by several reports on the centrosome in situ (see i.e., Baron and Salisbury, 19881, Ca2+ is likely to modulate in a rather dramatic fashion the structure and extension of the centrosomal matrix. As shown elsewhere, the centrosomal matrix is only slightly modified by mild treatments with chaotropic agents, whereas the triplets of microtubules are totally extracted and the tubulin solubilized (Klotz et al., 1990). The possibility that all the pericentriolarassociated structures belong to a unique filament system is suggested by such experiments in which the general organization of the centrosome is main-

120

PAINTRAND

ET AL.

”

“.

(0 1M

Urea

00 .SM

KI

a

P

v

B

A FIG. 10. Two grazing serial sections of mother black arrows) are hollow and apparently made of appendages with the centriole wall (black arrows of the subdistal appendages. Open arrows point to along the centriole. Note that the mother centriole

centrioles isolated by method II. Series A shows that the subdistal appendages (large substructures. Series B shows how complex is the association of the bases of subdistal in B, o, and left large black arrow in B, Bl. Small black arrows point to the tip structure the distal appendages. The parallel lines in B, 3 are to show the absence of obvious pitch in series B is duplicating. Bars, 0.2 km.

PIG. 9. Comparison of level 2 of mother centrioles in method I (A) and method II (Bl. The subdistal appendages are swollen in method I, displaying numerous radially assembled fibrils (long black arrowheads in A). These fibrils are laterally associated in specific places at the tip of the appendage resulting in transversal dark lines (small arrows in A and Bl. A clear dissociation of the base of the subdistal appendage (white arrowheads in A) from the centriole wall is often observed in method I and never in method II (compare also average images of level 2 at bottom row in Fig. 8). The bases of subdistal appendages correspond to the external columns observed in method II (white arrowheads in Bi, where they are tightly associated with the outer edge of the microtubule sets and sometimes can be confused with incomplete tubule C in this method as they may have a kinked shape (A, middle section in the bottom row, facing the doublet at 3 o’clock). These bases apparently serve two adjacent appendages, and face microtubule incomplete triplets (black and white arrowheads in A, top row) or microtubule doublets. This possibility is supported by the aspect of sections where the centriolar structure has been extracted by chaotropic agents (C and El or by phosphotungstic acid (B): the external columns (white arrowheads in C-E) are still visible and seem to hold together the subdistal appendages after extraction of centrosomes isolated either with method I (C and D) or with method II (El. In the latter case the system of connected internal columns (small black arrowheads in B and E) is still present. Note that in urea extracted centrosomes (Cl, radial fibrils are visible (large black arrowheads), and are tightly associated with the tip of each appendage (see also Di. Bars, 0.2 pm. 121

PAINTRAND

ET AL.

III+Ca++

FIG. 11 Effect of adding Ca2 + to centrosomes isolated by method II. Centrosomes were incubated with 5 m&f CaCl, during 10 min (A) or 30 min (B, II + Ca2+ ; the left centrosome in B is the control). Note in A that the transverse section of mother centriole corresponding to level 2 is not significantly different from control (compare with Fig. 9B). By contrast the corresponding sections in B show a dramatic decrease in the centriole diameter as much as to break centriole symmetry (white arrow in B). The proximal diameter is also reduced but to a lesser extent (left section in B, II + Ca2+). The contrast is reversed in this condition. Note in A that the intercentriolar distance has been significantly shortened by Ca2+, bringing the two centrioles in an orthogonal configuration (see Discussion). Large arrowheads in A show the interruption of tubule C on daughter centrioles. The small arrows point to internal helically organized structures. The parallel lines show that there is no obvious pitch along daughter centrioles. Bars, 0.2 pm.

tained (Fig. 13; see also Figs. 9C-9E). Such an approach should allow us to gain more insight into centrosomal morphogenesis. It is difficult to observe how the matrix attaches to the centriole wall. However, averaging images often suggests that, indeed, the matrix attaches in respecting the g-fold symmetry of the centriole wall (see Fig. 6; see also Komesli et al., 1989). What appears clearly from the present work is that the ma-

trix attachment to the centriole wall is such that when divalent cations are chelated during the isolation procedure, the matrix collapses in a rim of constant thickness around the centriole. There is also some evidence that extended thin, rigid transverse structures (or a disc) could be distributed around the proximal end of centrioles (at least the parental one) according to a g-fold radial symmetry and could participate in the association of the inter-

ISOLATED

HUMAN

CENTROSOMES

FIG. 12. Tangential sections of centrioles. Centrosomes isolated according to method I are observed either classically (left) or in vitrous water (right; processed and photographed by D. Chretien and R. H. Wade in CENG, Grenoble). The middle one has been observed in situ after cryofixation in helium vapor and cryosubstitution in osmic acetone. In all cases, most of the triplets were obviously parallel and longitudinal (indicated by black lines). In all three cases, however, one triplet was apparently not parallel to the others (indicated by a thin arrow in each case). White arrow in the right centriole indicates the level of interruption of C tubules. Bars, 0.2 km.

centriolar matrix to the centrioles (see Fig. 3B, level 5’ after averaging; see also Fig. 6, levels 5-5’). The precise relationship between the centrioles and the centrosome matrix is one of the riddles in the understanding of the centrosome organization. DISCUSSION

Understanding the molecular bases of centrosome structure and functions is an important issue in cell

B FIG. 13. The centrosome matrix as observed after centrosome extraction with urea (A) or PTA (B). The overall organization maintained and the subdistal appendages can be recognized fhlack arrows). Bars. 0.2 wrn

is

AND

123

CA”+

biology. This organelle plays a major role in the spatial organization of the microtubular network. Moreover, it has a controlled cycle of duplication, the two duplicated centrosomes functioning as mitotic poles during subsequent cell division. Correct coupling between centrosome duplication cycle and the mitotic clock could be critical for cell cycle control (Bailly et al., 1989 and submitted for publication; Maniotis and Schliwa, 1991). However, a comprehensive description of the overall organization of the centrosome in animal cells is lacking. The possibility of isolating centrosomes represents a decisive step in the study of their structure, organization, and biochemical composition (see Bornens, 1992). The present work is aimed at integrating the various pieces contributing to the centrosome structure. The Overall

Centrosome

Organization

Two centrioles paired through a very complex three-dimensional network of ultrathin filaments appear as the minimum requirement to define a centrosome in human cells. Rather than talking of pericentriolar matrix or pericentriolar material to define what is not centrioles within the centrosome, we propose to use “centrosome matrix” as encompassing all parts to which centrioles are associated. Questions about this matrix are many given that we are ignorant of almost everything except that it is the seat of the microtubule nucleation activity: they concern the spatial extension of the matrix within the cell, the structural bases, and the chemical nature of its various parts, its interaction and relationship with both centrioles and with other cellular components (Golgi apparatus, nuclear membrane, microfilaments and intermediate filaments), its function and indeed its mode of duplication (is it a true duplicative process or a simple subseqeunt redistribution on duplicating centrioles?). We have documented in this work that the centrosome matrix proper is a 3-D domain with a definite outer boundary, which associates along the proximal end of both centrioles, along more than half the length of the centriole wall, up to the insertion of the subdistal appendages isee Fig. 14~. Deciphering the precise relationship between the centrioles and the centrosome matrix is indeed an important issue for the understanding of the duplication mechanism of the centriole, as we know that the reproductive capacity of centrosomes depends upon the presence of centrioles (Sluder and Rieder, 1985; Sluder et al., 1989). We have also documented that, as reported elsewhere (Moudjou et al., submitted for publication), the centrosome matrix is susceptible to divalent cations, and particularly to calcium. The overall shape of the centrosome matrix is quite different depending on the presence of EDTA in the isolation me-

124

PAINTRAND METHOD

I

ET AL. METHOD Ij

II

Microtubule

Doublet

Distal Spur

FIG. 14. treatment a question

Tentative interpretation in uitro. The nomenclature mark between parentheses

Appendage

of centrosome organization and centriole architecture observed in methods I and II and after used in the text is indicated on this figure. Ca” and EDTA treatments which are indicated indicate experiments which have been monitored by immunofluorescence only.

dium. When isolated centrosomes are further incubated in Ca2 + , the centrosome matrix is apparently able to contract, a process which brings the two centrioles close to each other, often according to an orthogonal configuration (see Fig. 11A). This latter feature could be indeed artifactually favored by the sedimentation of centrosomes on the coverslip before fixation. However, examples of specific basal body reorientation (up to 180”) mediated by Ca2+modulated contractile proteins are known in unicellular algae (Hyams and Borisy, 1978; McFadden et al., 1987). Indeed in these cases, it happens through specialized connecting structures and for micromolar calcium concentrations. The centrosome matrix is apparently less organized, a feature which could allow more flexibility (and could explain that regulatory elements of the calcium effect have been lost); but if matrix filaments are anchored on each centriole according to the revolution symmetry of the latter’s, one may predict that shortening of the matrix filaments would act on the orientation of the centrioles (see Fig. 14) and bring them back into the very same mutual position they had when the association was established, i.e., during the orthogonal budding of the procentriole. As a matter of fact, the intercentriolar organization appears to be critical for centrosome duplication (Tournier et al., 1991a,b). There are other reasons to pursue the comparison with the flagellar apparatus of unicellular algae. We have recently identified a major 62/64 kDa Ca2+-

Ca2+ with

binding protein in the matrix of human centrosome which is immunologically related to centrin and is involved in microtubule nucleation (Moudjou et al., 1991). More work is necessary to biochemically characterize the filaments which constitute the centrosome matrix. The possibility that it contains y-tubulin (Oakley et al., 1990) has been recently substantiated (Zheng et al., 1991; Stearns et al., 1991). The Polarity

in Centrosome

Organization

A remarkable feature of the centrosome in animal cells is indeed the presence, as a rule, of two centriole copies. The significance of this rule is unknown and one could think of several possibilities. But it is clear that both centrioles are not equivalent in the centrosome: only one possesses appendages. This supposes, incidentally, that the complete centrosome cycle encompasses at least two cell cycles (Rieder and Borisy, 1982; see also the different situations for the reproduction of the flagellar apparatus in unicellular algae; Heimann et al., 1989a,b). Two sets of appendages have been described in this work, which are distally associated with one of the two centrioles only. Bessis et al. (1958) were the first to observe two rings of appendages (called “spherules”) around the centriole, although neither their configuration nor their position were correctly interpreted in these early days of electron microscopy. The subdistal appendages were designated as “pericentriolar satellites” by Bernhard and de Har-

ISOLATED

HUMAN

CENTROSOMES

ven (19561 and by many subsequent authors. With random sectioning, the tips of these appendages could appear as separated from the centriole. There exist in KE37 cells within the pericentriolar material in situ several dense aggregates (see, i.e., Gosti et al., 1986) which, having no direct connection with the centrioles, can be called “satellites,” in agreement with Rieder and Borisy (1982) and Baron and Salisbury (1988). We have shown in this work that the individualization of these satellites could depend upon the presence of divalent cations. Correspondence between the nomenclature used in the present work and that used in other reports is summarized in Tables II and III. The significance of the subdistal appendages is apparently to anchor microtubules at their tips, as judged by transmission EM of KE37 cells after cryofixation and cryosubstitution (not shown; see also De Brabander et al., 1982). Each appendage is apparently made of many longitudinal subfilaments which laterally interact in a rather rigid and hollow structure which is tilted with respect to the centriole wall. There is a basic set of nine appendages in centrosomes isolated from KE37 cells, a situation which might be different in other cell types (see Komesli et al., 1989). Each appendage seems to contact two adjacent centriole doublets in such a way that the nine appendages appear often as making a continuous and very complex necklace close to the centriole wall, whereas the tips are capable of important lateral and longitudinal flexibility. More work will be necessary to really decipher the interaction of these appendages with the centriole wall. The radially symmetrical distribution of the subdistal appendages close to the centriole wall, and the great flexibility which is apparently possible in the orientation of the tip, would be compatible with a role in the orientation of microtubules. Chelation of divalent cations with EDTA disturbs significantly the subfilament arrangement of subdistal appendages, leading them to lose their tilted orientation with respect to the centriole wall. Interestingly, the base of each subdistal appendage (actually common to two adjacent appendages1 dissociates in a distinct manner tram t,he centriole wall in these conditions. This observation suggests the possibility that the interac-

AND

125

CA2 +

TABLE III Comparison of the Present Data and Features Described by Anderson (1972) on Basal Body of Vertebrate Ciliated Cells ~~ ~ __ ~~ _____ Parental centriole (This work) ~~ ~__ ~ KE37 cells In vitro Distal Proximal Nine distal appendages Nine subdistal appendages

Basal (Anderson,

body 1972)

Rhesus monkey In situ Apical Basal Nine alar sheets One basal foot ~~~ __ ~~ --_~

oviduct

tion between subdistal appendages and the centriole wall might be dynamic. Distal appendages, like the subdistal ones, are observed exclusively in association with the mother centriole. They correspond to the transition fibers or the alar sheets of previous work. They have been often confused with the subdistal appendages (Szollosi, 1964; Zeligs and Wollman, 1979; Dustin, 1978). They were mostly described in cells undergoing ciliogenesis where they participate in the association of the basal body to plasma membrane (Anderson, 1972) but also in cells which possess a primary cilium where they play the same role (AlbrechtBuehler and Bushnell, 1980; Baron and Salisbury, 1988). They have been observed also in cells deprived of any kind of cilium such as in rat salivary glands or in rat liver or in human leukocytes (O’Hara, 1970). The latter observations suggested that these structures were part of the centrosome. Our data establish that this is the case. Their functional significance is unknown. They insert very distally to the centriole wall and their bases apparently do not dissociate from the centriole in the presence of EDTA, as do those of the subdistal appendages. By analogy with their role in the association of kinetosomes to the subcortical domain of the plasma membrane, one could propose a role in the anchoring of the centrosome within the cytoplasm to the actin system (see, i.e., Kleve and Clark, 1980). The Polarity

of the Centriole

Architecture

There is a definite proximo-distal differentiation along the centriole cylinders. The proximal part dis

TABLE II Correspondence between the Nomenclature Cell This work Vorobjev-Chentsov, Rieder-Borisy, Schweitzer-Brown, Baron-Salisbury,

1982 1982 1984 1988

KE37 PE PtK2 Lymphocyte (murine) PtK2

Used in This Work

and That

Used in Previous

Reports

CTR Isolated In situ In situ In situ

Distal appendage Appendage

Subdistal Satellite Appendage Condensed

(resting In situ

Alar

appendage

Basal

foot

appendage

satellite cell)

Satellite Satellite Condensed (stimulated Satellite

satellite cell)

126

PAINTRAND

plays the typical nine triplets of microtubules and shows a tangential A-C link between adjacent triplets. The distal part displays nine unlinked doublets of microtubules. A continuous variation of this orientation with respect to the tangent to the circle passing through the tubules A, from 45” at the proximal end to 20” at the distal one, represents for each doublet/triplet a continuous and important twist. The way in which tubule C is interrupted involves apparently a short distance on which it is incomplete. It is at this level that the insertion of the subdistal appendages initiates and spreads toward the distal end. We have not been able to document in such a reliable way the structure of tubule C in daughter centrioles where there are no associated appendages, but it is also apparently interrupted (see Fig. 11A). One could therefore see that the centriole cylinder is made of two parts, a proximal one which is more like the classical organization of the basal body with nine opened triplets and a distal one which is more reminiscent of the organization of an axoneme with nine doublets oriented in a more tangenitial manner. It is noteworthy that it is at this end that, in certain cell types, primary cilium would grow (Wheatley, 1982). The centriole would already possess the duality basal body/cilium in its organization. We have observed an unexpected susceptibility of the centriole wall organization to the presence of divalent cations: the diameter can increase significantly (by 20%) in the presence of EDTA, by the mutual transversal sliding of the microtubule blades, like the parts of an iris diaphragm. Apparently this EDTA-dependent change in centriole diameter involves the extension of the A-C linkages at the proximal end and the dilation of the internal structures at the distal one. In contrast to what is often assumed, centriole architecture is therefore not invariant and is particularly susceptible to the presence of divalent cations. A somewhat similar effect of divalent cations on the structure of axonemes has been reported in the past, however (Warner, 1978; Zanetti et al., 1979): millimolar concentrations of divalent cations produce a 15% reduction in the diameter of isolated axonemes, due to uniform dynein cross-bridging to all B tubules. Extraction of dynein arms prior to the addition of divalent cations suppresses the effect. The centriole organization differs markedly from that of axonemes and there is nothing apparently like outer or inner dynein arms either in the microtubule triplet-containing proximal part (the A-C link is more reminiscent of a nexin link) or in the microtubule doublet-containing distal part. It is therefore elsewhere that one must look for the effect of divalent cations on the centriole structure.

ET

AL.

Internal structures in the centriolebasal body of animal cells have always been ill-defined. From the present work, it appears that the major reason for this could be that internal organization of animal centrioles is highly susceptible to external conditions and particularly to divalent cations. Various connections between microtubule sets inside the centriolebasal body have been reported (protuberances, triplet bases, ring, and clots in Vorobjev and Chentsov, 1980, 1982; connecting sheets in Anderson, 1972). Our analysis of serial sections has led to the possibility of a single internal structure which changes along the centriole. This internal structure is apparently quite complex and more work will be necessary to elucidate its 3-D organization. Several features must be noted and require correct elucidation: (i) the internal structure apparently involves a helical distribution of the internal columns (see Figs. 6B and llA), a quite intriguing feature; (ii) the nine internal columns appear often to comprise subunits, usually three, which appear sometimes, at least for two of them, as the globular head of an elongated tail extending in a curved manner from the axial annulus; (iii) precise and apparently “empty” annulus coincides precisely with the axis of the centriole on the distal end. However, this apparently precise internal organization is almost completely blurred when centrosomes are isolated in the presence of EDTA, the annulus being replaced by a more or less axial accumulation of some material and the nine columns vanishing. Actually, one part of the internal columns (one globular domain out of the three) might persist (see arrowhead in the average section in Fig. 8). This part looks rather similar to the spur observed on the proximal end. We cannot therefore exclude, from the available data, that the spur on the proximal end might correspond to a small internal column running longitudinally all along tubule A. A tentative interpretation of the structural changes brought about by divalent cations in centriole architecture might be proposed if one assumes mechanisms similar to those observed in axonemes (Warner, 1978; Zanetti et al., 1979). We can see the internal structure of centrioles as made of identical subunits possessing a globular domain close to the internal edge of microtubules and a more extended domain pointing radially toward the axial area of centrioles. When the globular domains internally interact with the microtubule sets to constitute the nine internal columns (method II), the extended domains would be pulled centrifugally in such a way as to create an empty axial annulus. In the presence of EDTA (method I), the subunits would dissociate from the microtubule sets, the internal columns vanishing in an ill-defined ring-like structure, and the annulus, filled by extended domains of the sub-

ISOLATED

HUMAN

CENTROSOMES

units looking more like a hub (Bornens et al., 1987; Komesli et al., 1989). It is critical to establish the existence of individual subunits in the internal structure and to know the chemical nature of these subunits. Their shape and size, if the globular domain and the elongated part do belong to the same molecule, would be compatible with those of dyneins. We are trying to chemically dissect the centrosome and have obtained compelling evidence for the presence of at least two different dyneins in purified preparations that we are currently trying to localize in the centrosome (Moudjou and Bornens, manuscript in preparation). Indeed, the functional significance of a centriole configuration in which the internal structure would correspond to an assembly of dynein molecules would be of interest as it would strongly suggest that centrioles are capable of active movement, currently a mere hypothesis (Bornens, 1979). A real parallel between the effect of divalent cations on axonemal and on centriolar structures would thus exist, although the dynein molecules would be differently distributed. Except that the centriole configuration observed in method II (divalent cations present) is clearly-from an extensive survey of numerous sections of KE37 cells--the configuration observed in situ, whereas the extensive crossbridging induced by divalent cations in isolated axonemes does not correspond to the in situ configuration. The possibility that a luminal structure is able to contract and reduce the diameter of the basal body1 axoneme transition has also been substantiated in Chlamydomonas (Sanders and Salisbury, 1989). In this case, dynein was not apparently involved. We have interpreted the differential aspect of the triplets on one edge of a transverse section with respect to the other (which is more prominent in method I than in method II) as indicating that there is some flexibility in the position of some adjacent triplets with respect to the others rather than reflecting a general pitch, as often proposed in the past [see for example Anderson, 1972; Albrecht-Buehler, 1990). This tentative conclusion adds to the need of experimentally addressing the possibility of an a~Live movement in centrioles. In conclusion, the present report advances our description of the highly complex centrosome structure. The significance of this complexity remains: why are there two centrioles, for example, or why are there two different sets of appendages on only one centriole? Where are microtubules nucleated and is their anchorage to the centrosome a separate process? What principles govern centrosome assembly during the duplication process? Progress toward this latter goal can be expected from the development of an in vitro assay in which centriole orthog-

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onal budding can be initiated (Tournier et al., 1991b). However, the centriole duplication process may be only one part of the centrosome duplication. How do cell cycle-dependent structural changes of the centrosome take place? Our structural data reinforces the notion that the different parts of the centrosome are built from subunits, a general principle in biological structures. A biochemical study of isolated centrosomes and a molecular characterization of their major components will be necessary to identify and to list the different subunits, such that a function might be assigned to the different parts of the centrosome described in this work. We thank Dr. J. Lepault for his help in processing digitalized pictures, Dr. C. Klotz for her help in early experiments with urea on isolated centrosomes, Drs. D. Chretien, R. H. Wade, and D. Job for allowing us to observe KE37 centrosomes in vitrous water, Drs. S. Brown and G. Keryer for critical reading of the manuscript, and I. Gaspard and D. Meur for art work. M.M. has received a fellowship from Association francaise contre les Myopathies (AFM). This work was supported by CNRS, by Grant 6762 from ARC (Association pour la recherche sur le cancer), and by a grant from AFM to M.B. REFERENCES Albrecht-Buehler,

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