The role of the flagellar transition region: inferences from the analysis of a Chlamydomonas mutant with defective transition region structures

JONATHAN W. JARVIK and JOSEPH P. SUHAN

Department of Biological Sciences, Carnegie Mellon Uniuersity,4400 Fifth Auenue, Pittsburgh, PA 15213, USA

Summary

Thin-section electron microscopy of the Chlamy- autotomy the process by which doublet micro- domonas reinhardtii mutant vfl-2 revealed striking tubules are severed and flagella are released from defects in the transition region between basal body the cell. It has been claimed that autotomy is caused and flagelhrm. In place of the highly organized by contraction of the centrin-containing stellate transition cylinders and stellate fibers characteristic fibers, resulting in the mechanical severing of the of wild type, variable quantities of poorly organized doublet microtubules and a concomitant reduction of electron-dense material were present. In many cases the diameter of the axoneme adjacent to the abscis- the transition region was penetrated by central pair sion point. Our observations do not support this microtubules that passed from the axoneme into the claim in that vfl-2 cells, which lack organized stellate basal body. On the basis of these observations we fibers, display effective autotomy unaccompanied by propose that an important function of the structures detectable narrowing of the axoneme. present in the normal transition region is to physi- cally exclude the central pair microtubules from the basal body. Key words: autotomy, flagella, Chlamydomonas, transition The transition region is the site of flagellar region, microtubules, centrin.

lntroduction membrane from the plasma membrane (Musgrave et al. 1986; Kaneshiro, 1989), partitioning the flagellar interior The specialtzed region between the eucaryotic cilium or from the cytosol (Besharse and Horst, 1990), and auto- flagellum and its basal body is known as the transition tomy, or flagellar shedding (Bluh, L97L). In addition, the region or transition zone. The transition region can be said transition region may play a major role in the control of to begin where the C-tubules of the basal body end (i.e. flagellar assembly (Oentler, 1987). where triplet microtubules give way to doublet micro- The biflagellate green alga Chlamydomonas reinhardtii tubules) and to end where the central pair microtubules provides an excellent model system for studying organelle begin (Pitelka, L974), or, in a variation on this definition, structure and function using genetic, molecular and to end where the axoneme first assumes its standard ultrastructural approaches (Harris, 1989). As a means of ultrastructure (Gibbons, 1961). Most transition regions studying basal body structure and locahzation, we have are 200-400 nm in length, although longer examples found it useful to analyze a class of mutants that, instead exist. of being biflagellate, have a variable number of flagella The transition region can be conveniently divided into per cell. These mutants are designated ufl, for uariable an inner zone and an outer zone, with the doublet flageIIa number. ufl mutants are typically defective in microtubules forming the boundary between the two. The basal body placement andf or segregation at cell division, ultrastructure of the outer zone shows great evolutionary and, in a number of cases, their functional defects have conservation, with all transition regions possessing been shown to be associated with particular ultrastruc- Y-shaped elements linking the doublets to the membrane tural abnormalities (Wright et al. 1983; Hoops et al. 1984; as well as two or more rings of distinctive membrane- Adams et al. 1985; Wright et al. 1985; Wright et al. L989). associated particles known collectively as the ciliary Previously published results from our laboratory necklace (Ringo, L967; Gilula and Satir, L972; Rohlich, (Wright et al. 1989) showed that the mutant ufl-2 has L975). In contrast, inner zorae structures vary greatly defects with respect to the quantity and locahzation of the among organisms, and, accordingly, they go by a variety of protein centrin (also known as caltractin) - a small acidic names such as axosomes, transverse septa or basal plates. calcium-binding protein related to calmodulin. In wild In Chlamydomonas the transition region inner zotte type, centrin is found in the contractile nucleus-basal contains two prominent transition cylinders and a set of body connectors that link the basal bodies to the nucleus, stellate fibers that link the cylinders to the doublet in the distal striated fibers between the members of each microtubules. basal body pair, and in the inner zone of the transition The transition region is responsible for a number of region (Wright et al. 1985; Huang et a\.1988; Melkontan et critical functions, including partitioning the flagellar al. 1988; Salisbury et al. 1988; Sanders and Salisbury, Journal of Cell Science 99,73L-740 (1991) Printed in Great Britain @ The Company of Biologists Limited 1991 73r 1989; Wright et al. 1989). In this report we describe the the standard laboratory strain 137c and five other natural ultrastructure of the ufl-2 basal body region. Not only are isolates (see Materials and methods) were observed by NBBCs and distal striated fibers absent, but dramatic thin-section transmission electron microscopy. The tran- defects are present in the transition region - defects whose sition regions in all strains appeared identical and showed nature has interesting implications with regard to normal the constellation of structural features typical of C. transition region function. reinhardtii (Ringo, L967; Cavalier-Smith, L974). These features (Fig. 1) include: (1) two prominent electron-dense transition cylinders, one distal and one proximal; (2) a set Materials and methods of stellate fi,bers linking the transition cylinders to the surrounding outer doublet microtubules. In longitudinal Strains and culture conditions sections the stellate fibers appear as projections between Wild-type C. reinhardtii were either 137c mt+ or the 137c the transition cylinders and the outer doublet micro- derivitive NO mt+ obtained from the Chlamydomonas Culture tubules, and in transverse sections they are seen as a set of Collection, Duke University, Durham, NC. Cell wall-defective fibers that appear to originate at the A-tubules of the outer strains cw15 mt+ and cw92 mt+ (Hyams and Davies, 1972) and doublets and to contact the transition cylinders tangen- the Chtamydomona.s natural isolates C. smithll (Hoshaw and Ettl, tially; collectively the stellate fibers produce the image of a 1966) and S1D2 (Gross et al. 1983) were obtained from the same (3) specialized segment of the flagellar (Kuchka nine-pointed star; a source. The mutant ufl-2 was isolated in our laboratory membrane that maintains a constant distance of about as were the C. reinhardtii natural isolates 6, and Jarvik, L982), is to (Spanier, Graham and Jarvik, unpublished). Cells 50 nm from the axoneme and refractory solubilization 224 and 356 (Goodenough, were grown under constant light in M-medium (Medium I of with nonionic detergents 1983; Kamaya and Sager and Granick, 1953) supplemented with 0.LVo yeast extract, Witman, 1984). We refer to this as the transitional O.IVo proteose peptone and 0.01 vr sodium acetate. membrane; @) a set of Y-shaped membrane-microtubule connectors, apparent in transverse sections, connecting CelI ghosts the outer doublet microtubules to the transitional mem- (see (5) Cell ghosts were prepared by pelletin g 40 ml samples of log phase brane Fig. 1C and D); a set of ,fuzzy V-shaped cells at approx. l06cellsml-t for Smin at 2500revsmin-' in a annular connections projecting from the transitional Beckman TJ6 centrifuge, resuspending in 8 ml nucleus buffer membrane towards and between the doublet microtubules without calcium (NB minus Ca2*: 25mM KCl, 10 mvr MgC12, (Fig. lE-G). We suspect that in transverse sections the 3.7 mm EGTA, 0.67 mM Tris-HCl, pH 7.3), repelleting as before, annular connections are represented by the clouds of and resuspending in NB minus Ca2* plus LVo Nonidet P-40 electron density that surround the doublet microtubules (NP-40). After extraction for 30 min at room temperature, the cell and extend outwards to the transitional membrane (e.g. microscopy in L 7o glutaraldehyde in ghosts were fixed for electron Fig. 1C); (6) a clear zone of reduced electron density about NB minus Ca2*. 100 nm deep between the proximal transition cylinder and the internal matrix of the basal body; (7) a transition N ucleo - ellar apparatus e s flag cylinder fiber extending from the proximal transition Nucleo-flagellar apparatuses (Wright et al. 1985) were prepared cylinder through the clear zotte and into the internal by a procedure identical to that described above, except that the matrix of the basal body (e.g. Fig. 1F). In our micrographs, t}r,e or cw92 mutation. cells carried cw75 the central pair microtubules typically end above the distal transition cylinder (Fig. lE and F), but it is not at Deflagellated cells all uncommon for them to reach right to it (Fig. 1G). Note prepared by centrifuging 20 ml samples Deflagellated cells were also the prominent transitional fibers associated with the of log-phase cells at appio*. 106 cells ml-t fot 3 min at 2500 revs microtubules of the basal body just below the a Beckman TJ6 centrifuge; resuspending in lml M triplet min-t in region (e.g. Fig. 1B). These serve to link the medium; adding 1ml of 10 mM dibucaine, 5 7o stcrose; incubating transition for 30s; and fixing for electron microscopy by adding 2ml of 2Vo basal body to the plasma membrane; despite their name, glutaraldehyde in M medium. they are not strictly part of the transition region. vfl-2 has abnormal transition regions that are often Electron microscopy penetrated by central pair microtubules Log-phase cells were harvested by centrifugation (5 min at 2500revsmin-1 in a Beckman TJ6 centrifuge) and resuspended Thin-section transmission electron microscopy of cells and tn 27o glutaraldehyde in growth medium or NB minus Ca2* as cell lysates of ufl-2 revealed grossly abnormal transition appropriate. After 30 to 60 min they were washed several times in regions, with no normal case seen in many hundreds of fresh medium with 2Vo glutaraldehyde, and the pellets were transition regions examined. Examples are shown in postfixed for 30 min. with L Vo osmium tetroxide and rinsed twice Fig.2. Electron-dense material reminiscent of the tran- with water. Samples were en bloc stained in 1 Vo ura;nyl acetate for sition cylinders was typically observed inside the tran- 30 min, dehydrated in a graded ethanol series, rinsed twice in sition region, but its abundance and morphology were infiltrated with resin (Polybed 812). propylene oxide, and highly variable and it was never organi zed into a true InfiItration was facilitated by use of a vacuum oven (50'C, 10psi;) connec- and the resin was polymenzed at the same setting fot L2-24h. cylinder as in wild type. Membrane-microtubule Silver to gold sections were cut using a diamond knife on a Sorvall tors were present, aS were transitional membranes MT-28 microtome. Sections were stained with L Vo aqueous uranyl maintaining a standard distance from the axoneme. acetate and Reynold's lead citrate and examined using a Philips Stellate fibers were not observed nor were annular 300 electron microscope at 60 kV. connections. A clear zorte was sometimes present but transition cylinder fibers were absent. Finally, in about one third of the cases, the central pair microtubules were Results seen to penetrate into or through the transition region, and it was not at all unusual to observe them extending The wild-type transition region ":f, A number of wild-type strains of C. reinhardtii, including :H: $ *.'1i J I Sxit rf#:.' ffi"j' ;i i 3 :HilHilf

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Fig. 1. Wild-type transition region. (A) Consecutive transverse serial sections. (B-D)Transverse sections at three consecutive levels. Note dot in center of transition cylinder in C that probably represents the transition cylinder fiber, and the ring inside the transition cylinder in D that probably represents the end of a central pair microtubule. (E-G) Longitudinal sections. A,B,D, strain 137c. C,E, strain 6. F,G, strain 356. ac, annular connection; cpnt /, central pair microtubules; cz, clear zone; dtc, distal transition cylinder; mmc, membrane-microtubule connector; ptc, proximal transition cylinder; sf, stellate fiber; tcf, transition cylinder fiber; tf, transitional fiber; tm, transitional membrane. Bar, 200nm. short microtubules of unknown significance inside the ufl- morphology of the transition region changes during 2 transition region. Examples can be seen in Fig. 28 and C autotohy, with a distinct reduction in the diameter of the (see also Fig. 3D). axoneme just below the point of severing, accompanied by a narrowing of the open end of the distal transition cylinder and an apparent contraction of the stellate fibers Flagellar autotomy in vfl-2 (Lewin and Lee, 1985; Sanders and Salisbury, 1989). Many treatments are known to indu ce Chlamydomonas to Wild-type and ufl-2 cells were treated with the deflagel- shed its flagella by an active calcium-dependent process lating agent dibucaine for 30 s and then fixed and prepared known as flagellar autotomy (Lewin et al. 1982; Huber et for electron microscopy (Fig. 3 ). Both strains were ef- aI. 1986). This process involves the severing of the outer ficiently deflagellated by the procedure. Although the doublet microtubules and a pinching-off of the flagellar outer doublet microtubules of ufl-2 were severed, there membrane just distal to the transition region, so that the was little or no narrowitrg of the axoneme below the basal body remains with the cell and the flagellum is abscission point as in wild type. In a number of cases released into the medium. It has been shown that the central pair microtubules were observed to pass out of the

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Fig. 2. Defective transition region in ufl-2. (A-C) Three sets of consecutive transverse serial sections. (D,E) Longitudinal sections, one showing the penetration of the transition region by central pair microtubules. (F ) Transverse section at the level of the basal body (note triplet microtubules) showing central pair microtubules. nunc, membrane-microtubule connector; tf, transitional fiber' tnt, transitional membrane; cpnt /, central pair microtubules. Bars, 200 nm; all micrographs except F are at the same scale.

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Fig. 3. Transition region in deflagellated wild-type and ufl-2 cells. rA-C) Wild-type (strain NO* t. Note distal narrowing of (D,E) axoneme and transition cylinder. Two sets of consecutive serial sections of t,fl-2.Note absence of narrowing at distal end and penetration by central pair microtubules. Bar, 200 nm.

Role of flagellar transition region 7Bs that link the members of each wild-type basal body pair together (Fig. 5). Observations of numerous ufl-2 basal bodies in a variety of preparations - including whole cells, cell ghosts, and free nucleo-flagellar apparatuses - never revealed a complete or partial distal striated fiber, even in the not-infrequent cases in which the basal bodies were found in pairs. ufl-2 is thus more defective with respect to the distal striated fiber than the previously described mutants ufl-l and ufl-7, in which many basal bodies carry partially assembled distal striated fibers (Wright et al. 1983; Adams et al. 1985).

Discussion

vfl-2 and the protein centrin The ultrastructural results reported here demonstrate that ufl-2 is defective for all cellular structures known to contain the protein centrin: the nucleus-basal body connectors (NBBCs), the distal striated fibers, and the stellate fibers. These observations are fully consistent with our previously reported light-microscopic results 4 showing that centrin immunostaining in ufl-2 was absent in the region of the NBBC and unusually weak at or near the basal bodies (Wright et al. 1989). The ufl-2 phenotype is (strain Fig. 4. Wild-type cell ghost 137c) showing unique; we have examined approximately 20 other ufl nucleus-basal body connector. nbbc, nucleus-basal body mutants by electron microscopy and none has shown this connectot; n, remnant of nucleus. Bar, 500 nm. phenotype. Whether the ufl-2 mutation is in the structural gene for centrin or in a gene involved in regulating centrin severed axoneme and through the tip of the flagellar gene expression or centrin function is unknown. stump. TWo sets of consecutive serial sections showing this The ufl-2 transition region is defective with respect to are included in Fig. 3. The appearance of the electron- four distinct structures: the stellate fibers, the transition dense amorphous material in the ufl-2 transition regions cylinders, the annular connections, and the transition did not seem to change as a consequence of deflagellation, cylinder fibers. Since ufl-2 has a general defect with although, because of the variability from case to case, w€ respect to centrin, and since evidence exists to show that cannot be certain that there was no change at all. centrin is localized in the stellate fibers (Melkonian et al. In both wild type and ufl-z the point of severing 1988; Sanders and Salisbury, 1989), we think it likely that corresponded closely to the most cell-distal end of the the mutant's primary transition region defect is in the transitional membrane. Thus the pre-deflagellation stellate fibers, with the defects in the other three lengths from the base of the basal body to the end of the components arising as secondary consequences. transitional membrane were 582+47 nm (n-10) for wild type and 603 + 33 nm (n - 10) for ufl-2, and the post- The transition region as a physical barrier to the deflagellation lengths from the base of the basal body to penetration of the basal body by the central pair the end of the flagellar stump were 556 +24nm (n-8) for microtubules wild type and 553+33 nm (n-22) for ufl-2. As documented in this paper, the mutant ufl-2 is grossly defective in the transition region and it frequently suffers vfl,-2 cells lack nucleus-basal body connectors a penetration of the central pair microtubules into the We knew from previous analysis that ufl-2 cells are basal body. On the basis of these facts, 8s well as other defective for the centrin-containing fibers that link the considerations discussed below, we propose that an basal bodies to the nucleus (Wright et al. 1989). Electron important function of the structures in the inner zone of microscopy extended this result to the ultrastructural the normal transition region is to act as a physical barrier level. To make visualization of the NBBC easier, we to prevent the central pair microtubules from entering the examined cell ghosts that had been extracted with the basal body. nonionic detergent NP-40 prior to fixation (Kimaya and The immediate barrier to central pair penetration Witman, 1984); extractions were performed in calcium- appears to be the distal transition cylinder, with the free medium to prevent contraction of the NBBCs (Wright stellate fibers appearing to hold the cylinder in place in the et al. 1985). A wild-type example is shown in Fig. 4. The center of the transition region. Perhaps the stellate fibers observation of NBBCs in wild-type cell ghosts was serve as rigid struts that keep the transition cylinder in common, with one or two connectors apparent in about position. Alternatively, they could serve as tensile el- 40 7o of all sections that contained the nucleus and both ements, all pulling on the transition cylinder to keep it basal bodies. In identically prepared ufl-2 cells, however, centered. Given what is known about the physical NBBCs were never observed, despite examination of many properties of centrin and centrin-containing fibers (Salis- dozens of appropriate sections. bury et al. 1984), the latter possibility seems the more likely. Indeed, if the stellate fibers are continuously in vfl-2 lacks distal striated fi.bers tension, then the observation of transverse contraction of The ufl-2 basal body apparatus was completely devoid of the axoneme in association with flagellar autotomy makes the prominent centrin-containing distal striated fibers immediate sense, since, once the microtubules have been

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liolr',,1' llu,gt,l ltt t' I rrr ttsi I iott t-t,,g'iott -.)-t,)l severed, they ought to bend at their ends in response to generally present in some form or another. Even a known transverse force. counter-example can be viewed as an 'exception that There exists a reasonable, and rather obvious, reason for proves the rule': in certain euglenoids the transition keeping the central pair microtubules out of the basal region is free of internal structures, but here the axoneme body. As the flagellum beats, any central pair micro- also lacks central pair microtubules in its cell-proximal tubules that pass into the basal body would move about portion (Dynesius and Walne ,1975), ma}

738 J. W. Jaruik and J. P. Sultan then what is? One intriguing possibility is that there Keurya, R. eNo WrrvraN, G. B. (1984). Subbicromolar levels of calcium exists a microtubule-severing activity in the transition control the balance between the beating of the two flagella in demembranated models of Chlamydomonas reinhardtii. J. Cell BioI. region that is activated by the deflagellation signal (Lewin 98, 97 -\07 . and Lee, 1985). The substrate for such an activity - the KeNnsnrno, E. (1989). Lipids of ciliary and flagellar membranes. In outer doublet microtubules at the abscission point - might Ciliary and Flagellar Membranes (ed. R. Bloodgood), pp. 24L-263. contain a distinct form of tubulin (Oakley et al. 1990) or Plenum Publishing, New York. (Linck KpNNnoy, J. R. nNo BnrmrNcHAM, E. (1968). Fine structure changes tektin and Langevin, L982), or they could be during chloral hydrate deciliation of Paramecium caudatum. 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740 J. W. Jaruik and J. P. Suhan