Do centrioles contain ribosomes?

Bioscience Reports 3, 1155-1162 (1983) 1155 Printed in Great Britain

Do c e n t r i o l e s c o n t a i n r ib o so mes?

Rein 3. KALLENBACH

Department of Zoology, University of California, Berkeley, California 94720, U.S.A.

(Received 7 November 1983)

C e n t r i o l e s induced via a number of pa r thenogene t i c agents regular ly reveal the presence of one or more granules within thei r cen t ra l cores . Though not a new discovery, these cen t r io la r granules have ca re fu l ly been r e -eva lua ted here . Considering various c r i t e r i a , it is p r o p o s e d t h a t t h e s e g r a n u l e s a r e i n t r a - c e n t r i o l a r r ibosomes. More specif ical ly , they are more compar - able to 705 r ibosomes than to 805 cy tor ibosomes . The d a t a s u g g e s t t h a t w i th in the l u m en of cent r io les , c e r t a in cen t r io la r proteins are synthes ized on r ibosomes tha t may be uniquely cen t r io la r .

Cent r io les are typical components of many eukaryo t i c cells and are involved in a va r ie ty of cel lular ac t iv i t ies . While many aspects of c e n t r i o l e s s t i l l r e m a i n u n c l e a r , one th ing tha t has been f i rmly e s t a b l i s h e d is t h a t c e n t r i o l e s r ep roduce themselves (27,28,43,48) , t hough t he f i n e r d e t a i l s of th i s un ique repl ica t ion are, as yet , unknown . Various hypotheses have been advanced in r ecen t years regarding the reproduc t ive cont inui ty of cen t r io les (11,29,30,38,43,45). T h e s e h y p o t h e s e s involve a rep l ica t ion at the molecular level and genera l ly are supported both by ear l ie r repor t s of cen t r io la r DNA and by m o r e r e c e n t f i nd ings of c e n t r i o l a r RNA, for which t h e r e is cons iderable co r robora t ive ev idence (10,36,47) .

S t r u c t u r a l d e s c r i p t i o n s of r eso lvab le cel lular cons t i tuen t s have g e n e r a l l y p r e c e d e d d e t a i l e d f u n c t i o n a l a n a l y s e s i nvo lv ing o ther approaches . As such, cy top lasmic and organel lar components still exis t for which the re is not as ye t any ascr ibed funct ion. This is ce r t a in ly t rue for various cen t r io la r f ea tu res , most of which were descr ibed soon a f t e r e l ec t ron microscopy became a biological research tool. In fac t , many of the ear ly studies still guide us with r e spec t to many of the morphological deta i ls of both cen t r io les and basal bodies (1 ,5 ,42,43,49) . Whereas the p resence of the ca r twhee l , a readi ly apparen t and of ten r e g u l a r f e a t u r e of cent r io les , genera l ly has rece ived most of the a t t e n t i o n of t he i n t r a - c e n t r i o l a r components , a va r i e ty of o ther s t ruc tures occur within the lumen of cen t r io les (10,47) . These include r e l a t i v e l y sma l l d e n s e granules, ev idence of which is revea led in numerous u l t r a s t ruc tu ra l studies, both old and new, though the subject is now rare ly speci f ica l ly addressed. This repor t reexamines these g r a n u l e s in l i gh t of the se l f -dupl ica t ive na ture of cent r io les . In addit ion to a number of o ther considera t ions , the in t e rp re t a t ion of the

01983 The Biochemical Society

1156 KALLENBACH

data supports the existence of RNA in centrioles. Specifically, the intra-centriolar granules appear to be centriolar ribosomes, implying that at least two sources of RNA may be present in centrioles.

Mater ia l s and Methods

Mature sea-urchin eggs will readily produce numerous 'de novo' (17) c e n t r i o l e s fo l lowing exposures to any one of a number of p a r t h e n o g e n e t i c agents (18,20,24). Some of these parthenogenet ic procedures were used in this study and details on experimental design and e l e c t r o n m i c r o s c o p y have a l r eady been reported (16,19,21). Basically, eggs of Strongylocentrotus purpuratus were subjected to two different treatments to induce centriologenesis. One treatment was simply a continuous hypertonic-seawater exposure (21), whereas the other was a two-step approach involving urea and D20 (19). Eggs Of Lytechinus pictus were t reated wi th two separate two-step procedures (19), Eggs were exposed to NH3-seawater followed by D20-seawater ; a l ternat ively, eggs were treated with NH3-seawater fol lowed by hyper ton ic-seawater contain ing polyethylene glycol. Add i t iona l l y , between the 20th and 60th minute following normal fert i l ization, eggs of L. p i c t u s were exposed to seawater containing 55% D20 (30,31); following the parthenogenetic treatment, eggs were cultured in seawater.

Re su l t s

Over 400 s e p a r a t e EM pro f i l e s of c e n t r i o l e s were carefully examined for any evidence of small granules. ~ Only a small percentage of thin sections provided relatively clear images of discrete intra- centriolar granules (Fig. la) , though many sections through centrioles revealed the presence of probable granules which likely would have become more fully apparent in an adjacent thin section. While no information is as yet available with respect to their frequency of appearance, the granules were observed in de novo centrioles induced via a variety of parthenogenetic procedures and were readily detected in eggs of either species. Many sections through centrioles picked up only a single granule (Fig. lb), but various images clearly displayed the presence of more than one granule. The distribution of multiple granules tended to be quite variable within centriolar lumens. These granules sometimes appeared in rather spread-out arrangements between the cartwheel and the distal end plate (Fig. lc) , whereas other profiles provided examples of intra-centriolar granules in close association with each other (Fig. ld). Other images exhibited views of granules seemingly positioned in a circular configuration (Fig. le). This type of configuration was also noted in a case where five distinct granules could be seen (Fig. 1[); this was the largest number of granules that could clearly be identified within a single centriote.

The micrographs in Fig. [ allow a close comparison to be made between the in t ra -cen t r io la r granules and cytoplasmic ribosomes immediately surrounding the centrioles. Careful comparisons reveal not only a d i s t i nc t s i m i l a r i t y , if not identicalness, between the pos i t ive ly s t a ined images of granules and ribosomes, but also an unmistakable similarity in the variable morphologies of both types of

DO CENTRIOLE5 CONTAIN RIBOSOMES? 1157

par t ic les . The major i ty of granules exhibi ted an el l ipt ical shape and these el l iptoids were measured for thei r lengths and widths with a re t i cu la ted 10x magnif ie r . These measu remen t s were averaged to give a size of 23.7 x 16.1 nm Ior granules of L. p i c t u s cen t r io les and 23.6 x 16.8 nm in S. # u r p u r a t u s . These values were compared with similarly de te rmined dimensions of both cy tosomal and mi tochondr ia l r ibosomes in the eggs of both species (Table I ) .

Disc u ss ion

I n t r a - c e n t r i o l a r granules have rece ived l i t t le a t t en t i on since the ear ly descr ip t ive studies on cent r io les . This, no doubt, is because the smal l g r a n u l e s a r e no t c o n s i s t e n t l y d e t e c t e d in thin sect ions of cen t r io les and the i r p resence or absence has never been co r r e l a t ed to any p a r t i c u l a r f u n c t i o n a l phase of the cen t r i o l e (47) . In many sect ions of cen t r io les , smaIl granules are not l ikely to be present ; this cou ld be because of the obvious size d i f f e r en ce be tween the two bodies, or because the granules, at some points during the cen t r io l e cycle , may f l u c t u a t e in number or disappear en t i re ly . In any case, inves t igators have observed the granules o f ten enough to conclude tha t they fall within a size range of 20 to 30 nm (8) . This is basical ly conf i rmed in the da ta p resen ted here , as these granules were re la t ive ly f r e q u e n t l y d e t e c t e d in eggs containing numerous pa r thenogene t i ca l ly induced cent r io les .

Table I. Lengths and widths of measured centriolar granules and mitochondrial and cytosomal ribosomes

in parthenogenetically stimulated eggs of two species of sea urchin

Only distinct and clearly defined granules were measured from a large number of EM micrographs. The indicated lengths and widths represent averages for the total number of granules or ribosomes measured (numbers in parentheses). Averages are given to account for various possible experimental errors which could be reflected in the actual sizes of particles on different micrographs with various primary and secondary magnifications. The sizes of all particles must be considered preliminary, as more exact dimensions can more accurately be determined from high-resolution negatively-stained images of isolated granules or ribosomes. Though effective procedures exist for the isolation of cytasters from eggs, it has been very difficult, so far, to get pure preparations of centrioles, from which ribosomes could then easily be obtained on density gradients.

Sizes of Granules or Ribosomes (nm)

Centriolar Mitochondrial Cytosomal

Lytechinus 23.7 x 16.1 25.4 x 17.4 27.0 x 17.5 pictus (58) (20) (I00)

Strongylo- 23.6 x 16.8 25.4 x 16.4 29.3 x 18.5 centrotus (7) (20) (50) purpuratus

1158 KALLENBACH

DO CENTRIOLES CONTAIN RIBOSOMES? 1159

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1160 KALLENBACH

Not only do the intra-centr iolar granules stain in a manner similar to that of ribosomes) but they also take on the general shape of these RNP particles. Different views of granules compare favorably to the various images of cytoribosomes as they appear in EM; fur thermore , the granular dimensions undisputably fall within the reported size range of ribosomes (33). Compared to the egg's cytoplasmic ribosomes, the size of intra-centr iolar granules is distinctly smaller, as is the size of the egg's mitochondrial ribosomes. This reveals that the granular d imensions a re more c lose ly r e l a t e d to those of mitochondrial, chloroplast, and prokaryotic ribosomes (2,3,25,26,33). On the basis of the above cri ter ia , it is tenta t ively proposed that the granules, in fact , a re r ibosomes ; but they appear to be more like the smaller 70S p r o k a r y o t i c = t y p e r ibosomes than the l a rge r 80S eukaryot ic=type ribosomes. The granules' apparent confinement to centr iolar cores suggests that they may be uniquely centriolar in nature; no 70S-like ribosomes are found in the cytosol of eukaryotes. If the existence of intra=centriolar ribosomes can be confirmed, then centrioles will join the ranks of mitochondria and plastids as organelles with their own set of prokaryot ic- type ribosomes.

The presence of centriolar ribosomes implies the presence of both r ibosomal and messenger RNA, both of which could represent the source o f RNA de tec ted in centrioles (7)i1,12)39/~2). This RNA is seemingly involved in the process of centriolar duplication (32)) an observation that is also revealed in experiments involving antibiotics. Not only is centriolar replication known to be inhibited by actinomycin D (4,51) and cycloheximide (37), but it is also suppressed by the action of chloramphenicol (CAP) (6,44). All of these @rugs selec- tively inhibit protein synthesis. But what is of particular interest here is that CAP specifically interferes with translation on2y on the smaller ribosomes from prokaryotes and not on the larger eukaryotic cy to- ribosomes; also, CAP inhibits protein synthesis on both mitochondrial and plastid ribosomes (15,23,35,40), whereas cycloheximide does not prevent organellar ribosome activit ies. On the basis of these facts, the da ta he re imply the exis tence of 70S or 70S-like ribosomes involved in normal centriolar duplication.

This c o n t e n t i o n is cor robora ted by the finding that CAP also inhibits the formation of cytasters (46), a process which, along with the f o r m a t i o n of m i t o t i c a s te r s ) also r equ i r e s c e n t r i o l a r RNA (14)34)41)52). In this case, CAP's e f fec t s on cytas ter formation were presumed to be due to a suppression of de novo centr iole formation (46) , and recent studies have shown that cy tas ter formation does depend, in fact , s tr ict ly on the activation and subsequent maturat ion of latent centrioles (18-20). Thus, any inhibition in the development of newly induced centrioles also inhibits the formation of cytas ters , since the two are complementary structures which develop in unison (20). Inasmuch as procentrioles are already clearly associated with recognizable cytastral structures, CAP's known ef fec t s on both protein synthesis and aster formation suggest that centriolar ribosomes may be engaged in the synthesis of certain unique centriolar proteins needed for p r o c e n t r i o l e f o r m a t i o n . Conceivably , this could involve the synthesis of proteins for the construction of special s t ructures such as the cartwheel, one of the first centriolar components to appear during centriologenesis (47). Since other protein inhibitors also suppress the

DO CENTRIOLES CONTAIN RIBOSOMES? 1161

deve lopment of centrioles (4,37,51), i t appears that the complete formation of a centriole into a ful ly mature organelle is a semi- autonomous event involving information from both the nucleus and the parent centriole. In the case of sea-urchin eggs this information, in mult iple copies, must already be present in the cytoplasm of mature ova, because neither the nucleus (9,13,22,50) nor centrioles, known to be absent in these eggs (16), need to be present for both the induction and formation of new centrioles.

R e f e r e n e e s

1. Anderson R (1972) J . C e l l B i o l . 54 , 246-265 . 2. B i e l k a H (1982) The E u k a r y o t i c Ribosome, S p r i n g e r - v e r l a g , New

York. 3. Con RA (1977) Prog. Biophys. Mol. Biol. 32, 103-231. 4. DeFoor PH & Stubblefield E (1974) Expt. Cell Res. 85,

136-142. 5. de Harven E (1968) in: Ultrastructure in Biological Systems,

vol 3, The Nucleus (Dalton AJ & Haguenau F, eds), pp 197-227, Academic Press, New York.

6. Deutch AH & Shumway LK (1973) Protoplasma 76, 387-395. 7. Dippell RV (1968) Proc. Natl. Acad. Sci. U.S.A. 60, 461-468. 8. DuPraw EJ (1970) DNA and Chromosomes, Holt, Rinehart &

Winston, New York. 9. Fry HJ (1932) Biol. Bull. 63, 149-186.

i0. Fulton C (1971) in: Origin and Continuity of Cell Organelles, vol 2 (Reihart J & Ursprung H, eds), pp 170-221, Springer-verlag, New York.

II. Hartman H (1975) J. Theoret. Biol. 51, 501-509. 12. Hartman H, Puma JP & Gurney T Jr (1974) J. Cell Sci. 16,

241-259. 13. Harvey EB (1938) Biol. Bull. 75, 170-188. 14. Heideman SR, Sander G & Kirschner MW (1977) Cell IO, 337-350. 15. Jimenez A (1976) Trends Biochem. Sci. I, 28-30. 16. Kallenbach RJ & Mazia D (1982) Europ. J. Cell Biol. 28,

68-76. 17. Kallenbach RJ (1982) Biosci. Rep. 2, 959-966. 18. Kallenbach RJ (1982) Cell Biol. Intern. Rep. 6, 1025-1031. 19. Kallenbach RJ (1983) Europ. J. Cell Biol. 30, 159-166. 20. Kallenbach RJ & Mazia D (in preparation). 21. Kallenbach RJ, Paweletz N & Finze E (1983) Cell Calcium 4,

13-26. 22. Kato KH & Sugiyama M (1971) Devel. Growth Differ. 13,

359-366. 23. Kroan AM (1969) in: Handbook of Molecular Cytology (Lima de

Faria A, ed), North-Holland, Amsterdam. 24. Kuriyama R & Borisy GG (1983) J. Cell Sci. 61, 175-189. 25. Kurland C (1977) Ann. Rev. Biochem. 46, 173-198. 26. Lake JA (1981) Sci. Am. 245, 84-97. 27. Loewy AG & Siekevitz P (1969) Cell Structure and Function,

Holt, Rinehart & Winston~ New York.

1162 K A L L E N B A C H

28. Lwoff A (1950) Problems of Morphogenesis in Ciliates: The Kinetosome in Development, Reproduction and Evolution, Wiley & Sons, New York.

29. Mazia D (1961) in: The Cell (Brachet J & Mirsky AE, eds), pp 77-412, Academic Press, New York.

30. Mazia D (1977) in: Mitosis Facts and Questions (Little E, Paweletz N, Petzelt C, Ponstingl H, Schroeter D & Zimmermann H-P, eds), pp 196-213, Springer-verlag, Heidelberg.

31. Mazia D (1978) in: Cell Reproduction (Dirksen ER, Prescott DM & Fox CF, eds), pp 1-14, Academic Press, New York.

32. McGill M, Highfield DP, Monohan TM & Brinkley BR (1976) J. Ultrastr. Res. 57, 43-53.

33. Nomura M, Tissieres A & Lengyel L, eds (1974) Ribosomes, Cold Spring Harbor Laboratories, New York.

34. Pepper D (1979)Nucleation and Polarity of Microtubules, Amer. Soc. Cell Biol., Toronto, Canada.

35. Pestka S (1971) Ann. Rev. Microbiol. 25, 487-562. 36. Peterson ST & Berns MW (1980) Intern. Rev. Cytol. 64, 81-106. 37. Phillips SG & Rattner JB (1976) J. Cell Biol. 70, 9-19. 38. Pickett-Heaps JD (1971) Cytobios 3, 205-214. 39. Rieder CL (1979) J. Cell Sci. 80, 1-9. 40. Sisler HD & Siegel MR (1967) in: Antibiotics Mechanism of

Action, vol 1 (Gottlieb D & Shaw PD, eds), pp 283-307, Springer-verlag, Berlin.

41. Snyder JA & McIntosh JR (1976) J. Cell Biol. 70, 368a. 42. Stubblefield E & Brinkley BR (1967) in: Symposium of the

International Society for Cell Biology, vol 6, Formation and Fate of Cell Organelles (Warren KB, ed), pp 175-218, Academic Press, New York.

43. Went HA (1966) Protoplasmatologia 6, 1-109. 44. Went HA (1977) Expt. Cell Res. 108, 63-73. 45. Went HA (1977) J. Theor. Biol. 68, 95-100. 46. Went HA (1978) in: Mitosis Facts and Questions (Little E,

Paweletz N, Petzelt C, Ponstingl H, Schroeter D & Zimmermann H-P, eds), pp 571-580, Springer-verlag, Heidelberg.

47. Wheatley DN (1982) The Centriole; A Central Enigma of Cell Biology, Elsevier Biomedical Press, Amsterdam.

48. Wilson EB (1925) The Cell in Development and Inheritance, Macmillan, New York.

49. Wolfe J (1972) in: Advances in Cell and Molecular Biology, vol 2 (Dupraw EJ, ed), pp 151-192, Academic Press, New York.

50. Yatsu N (1905) J. Expt. Zool. 2, 287-312. 51. Younger KB~ Banerjee S, Kelleher JK, Winston M & Margulis L

(1972) J. Cell Sci. II, 621-637. 52. Zackroff R, Rosenfeld A & Weisenberg R (1976) J. Supramol.

Struct. 5, 577-589.