- Email: [email protected]
Cilia, flagella and the basal apparatus
Cilia, flagella and the basal apparatus U.W. Goodenough Department of Biology, Washington University, St Louis, Missouri, USA Current Opinion in Cell Biology 1989, 1:58-62
Introduction
Ciliary dynein
Cilia and flagella (the term 'cilia' is used generically here) are complex ( > 200 polypeptide species) and highly conserved organelles of eukaryotic motility (Gibbons, J Cell Biol 1978, 91:107--124; Huang, Int Rev Cytol 1986, 99:181-215). They are synthesized under the aegis of basal bodies, organelles identical to, and often interconvertible with, the centrioles of the centrosome; the basal bodies and their associated fibrillar structures (the 'basal apparatus') also contain numerous unique polypeptides. It is generally agreed that ciliary microtubules slide past one another during bend formation, a motion mediated by the adenosine triphosphate (ATP)ase activity of the outer and inner dynein arms, but little is understood about the mechanics of this motion or about the role played by such accessory structures as the central pair, radial spokes and nexin links. Much current research on these organelles is focused on the regulation of motility by cyclic nucleotides and calcium, and on control of their biosynthesis. A two-volume book has just gone to press which contains numerous up-to-date articles and reviews on these subjects (Warner et al., eds, Cell Movement. New York. Alan Liss, 1988).
Certainly the most important recent methodological ad vance in the study of ciliary dynein has been the development of in vitro assays for its force-transducing activity, assays that parallel those for cytoplasmic motors such as kinesin (reviewed by Gelfand in this issue, pp 63~56). Paschal et al. [1] and Vale and Toyoshima [2] have shown that sea urchin and Tetrahymena outerarm dyneins, adsorbed to glass coverslips, will translocate bovine brain microtubules in the presence of MgATP. These findings prove that the doublet structure of ciliary microtubules is not necessary for effective ciliary dynein/microtubule interaction [3], that outer arms can function independently of inner arms and that outer arms move towards the minus ends of the microtubules ('retrograde motility'), as they do in vivo [4] (Fig. 1).
(+)
(+)
Vale and Toyoshima [2] also report an intriguing property of the 14S dynein fraction from Tetrahymena: not only does it induce smooth microtubule gliding; it also uniquely causes the microtubules to rotate clockwise. The 14S fraction contains two dynein-like molecules [5], one of which is morphologically similar to an inner-
(+)
N+ (a)
(-)
(b)
(-)
N
(c) (--)
Fig. 1. Direction of active microtubule sliding generated by the dynein arms. The distal end of the microtubules is designated with a plus ( + ) sign and the proximal end with a minus ( - ) sign. The doublets are viewed from the inside of the axoneme toward the outside. Doublet N is the doublet with the active arms and doublet N + I is the adjacent doublet. As illustrated in (a) and (b), doublet N always slides proximally. More generally (c), dyneinmediated motility is toward the minus ( - ) end of the cytoplasmic microtubule along which dynein, and associated structures such as a membrane vesicle, move.
Abbreviations AMP--adenosinemonophosphate;ATP--adenosinetriphosphate;GMP--guanosinemonophosphate 58
(~ Current Science Ltd ISSN 0955-0674
Cilia, flagella and apparatus Goodenough 59 arm dynein from Chlamydomonas (Goodenough et al., JMolBio11987, 194:481--494); rotation may therefore be induced by one or both species or, conceivably, by a non-dynein protein present in the preparation. The phenomenon is of particular interest in that the central-pair microtubules of the axoneme rotate clockwise during the ciliary beat (Fig. 2) [6] (Omot 9 and Rung, J Cell Biol 1980, 87:33-46), apparently because of an interaction between the radial spokes and the central pair [6], and the spoke-central pair system appears to dictate the shape of the ciliary wave form [6] (Brokaw et al., J Cell Biol 1982, 92:722-732; Ishijima et al., Cell Motil Cytoske11988, 9:264-267).
Rotation
194:481-494). Since the inner arms move towards the minus ends of microtubules and will support ciliary beating at half the rate of intact cilia [4], their heterogeneity is not obviously related to force generation and may serve more complex regulatory functions. The close association of inner arms with the radial spoke system (Goodenough and Heuser, J Cell Biol 1985, 199:2008-2018) is therefore particularly interesting. The mode of force transduction generated by dynein arms continues to elude researchers. Intuition dictates that the arms should make contact with the B microtubules during force generation and detach themselves during inactivity, and Spungin et al. [10] have obtained intriguing images of subsets of outer arms attached to B microtubules in the presence of the non-hydrolysable analogue AMP-PCP. These subsets alternate with groups of unattached arms, consonant with the notion of a complex regulation of arm activity.
Regulation of motility Swimming direction
Fig. 2. Schematic representation of the swimming direction E~> of an axoneme from which a central pair has been extruded, and the clockwise rotating direction of a central pair (~). Tip of axoneme is to the left.
Biochemical analysis of dynein proteins was given an important boost several years ago by the discovery that; when the molecules are irradiated at 365 nm in the presence of vanadate, their component 'heavy' polypeptide chains are cleaved at two sites which apparently occur on separate loops of the ATP-binding domain (Lee-Eiford et al., J Biol Chem 1986, 261:2337-2342; Tang and Gibbons, J Biol Chem 1987, 262:17728--17734). King and Witman [7] and Mocz et al. [8] have combined photolysis with proteolysis to produce extensive maps of the Chlam3* domonas 0t and ~ and the sea-urchin ]Bheavy chains, respectively, and several of the peptides have been further identified by monoclonal antibody probes. It is interesting that for the sea urchin, none of the monoclonal antibodies binds to the central 100 000 Mr region of the chain where ATP binding and hydrolysis appear to occur (i.e. the domain flanked by the two photolytic sites), and only one binding site has been identified in this region for Cblamydomonag suggesting that this is the more conserved and hence less antigenic region of the protein. Each outer arm is a complex of two or three dynein subunits, each with a unique heavy chain (Toyoshima, J Cell Bio11987, 105:897-901), but there is no evidence for any heterogeneity from one outer arm to the next. By contrast, the inner arms display at least two discrete morphologies (Goodenough and Heuser, J Cell Biol 1985, 199:2008-2018), and biochemical and structural analysis of inner-arm or putative inner-arm proteins also reveals heterogeneity [5,9] (Goodenough et al., J Mol Bio11987 ,
Dynein arms function in a constant ionic environment, in contrast to actin-myosin systems where contraction is stimulated by Ca 2 + and relaxation by Ca2 + withdrawal. However, certain cilia fail to beat at all unless effectors are provided, and the pattem of the beat is clearly subject to exogenous regulation; moreover, sustained beating is apparently dependent on the phosphorylation state of key proteins. The two agents most often implicated in these regulatory events are Ca 2+ and cyclic AMP (cAMP), but as stressed in a review by Brokaw [11], the observations are complicated by potential interactions between the two. Moreover, different ciliary systems give seemingly contradictory results: cAMP stimulates sperm motility in some systems [12] but not others [13] and actually inhibits motility in Chlamydomonas [14]; Ca 2+ stimulates an adenylate cyclase in Paramecium [15] and a protein phosphatase in sperm [16]. It seems probable that despite the apparent conservation of axonemal hardware, many patterns of ciliary regulation have evolved. This is, of course, true for muscle contraction as well, where neural and hormonal factors have come to govern tissue-specific patterns of activity. By a conserved regulatory activity, Ca2+ is to reverse the beat direction of cilia, a response first observed in Paramecium (Naitoh and Kaneko, Science 1972, 176:523-524) and occurring as well in complex ciliated epithelia [17]. Ca2+ influx occurs via voltage-sensitive Ca2+ channels activated by membrane depolarization; membrane depolarization, in tum, is induced by neural stimulation (epithelia) or environmental stimuli (protozoa). Moss and Tamm present electrophysiological evidence [17] that in the ciliary comb plate of ctenophores, the Ca2 + channels are distributed over most of the length of the cilia. Since calmodulin, the putative Ca 2+ sensor in this system, has been shown to be tightly bound to the axoneme along its entire length (Maihle et al., J Cell Bio11981, 89:695-699), Ca 2+ influx and the subsequent
60 Cytoplasmcell motility modulation of beat direction appear to occur along the entire cilium and not, for example, via channels at the ciliary base. The effectors responsive to Ca-calmodulin have not yet been identified, but given the apparent role of the central pair/radial spoke system in governing patterns of ciliary beating (see above), this system is a likely target. A more unusual sample of regulation is documented by Bonini and Nelson [18], who show that cyclic nucleotides can antagonize the Ca2 + stimulation of ciliary reversal, and that the helical path traced by swimming Paramecium is modulated in one direction by cAMP and in another direction by cyclic guanosine monophospahte (cGMP; Fig. 3). Since numerous enzymes involved in cyclic nucleotide generation and utilization have been identified in Paramecium cilia [15], this system appears to be particularly promising for identifying the regulatory circuits involved.
Biosynthesis of cilia The numerous genes that specify ciliary components are apparently under coordinate regulation. The pioneering studies of this regulation used Chlamydomonas (Lefebvre and Rosenbaum, A n n u Rev Cell Biol 1986, 2:517-546), but studies using other organisms are now demonstrating a similar coordination (Eldon et al., Genes Devel 1987, 1:1280-1292; Harlow and Nemer, Genes Devel 1987, 1:1293-1304). An important complementary system is provided by Naegleria, an amoeba that differentiates into a flagellate within 1 h of nutrient starvation. In contrast to Chlamydomonas, where amputation of existing flagella stimulates flagellar gene transcription and as(a)
(d)
Control (RHH)
(b)
cAMP (RHH)
(c)
Ca
(e)
Ca + cAMP (RHH)
(f)
(RHH, bkwd)
sembly of new flagella, the Naegleria amoeba has no flagella, and gene induction results in the de novo synthesis of basal bodies and rootlets as well as flagella. Shea and Walsh [19] and Lai et al. [20] have begun exploiting this system at a molecular level, identifying complementary DNA for 0~ and [3-tubulin and for a flagellar calmodulin, all of which are coordinately expressed. Here, as in the Chlamydomonas system, the challenge is now to identify upstream genomic sequences shared by these genes, the factors that presumably initiate coordinate transcription by binding to these sequences, and the stimuli provided by starvation (Naegleria) or deflagellation (,Chlamydomonas) that activate these factors.
The basal apparatus Since studies of basal bodies and centrioles appear only infrequently, two 1987 studies are worth examination. One [21] describes a high-yield purification of centrosomes and the other [22] the inhibitory effects of benzodiazepines on the migration of basal bodies to the cell surface during ciliogenesis. A further paper of general importance to the field is the characterization of the socalled 'uni' linkage group in Chlamydomonas (Ramanis and Luck, Proc Natl Acad Sci USA 1986, 83:423-426), a group of genes that show an unusual pattern of inheritance and specify a variety of traits associated with basalbody/flagellar function. Of particular interest are several papers describing a novel calcium-binding protein associated with basal bodies and centrioles, called centrin by Salisbury et al. [23] and caltractin by I-Iuang et al. [24,25]. The protein poly-
cGMP (LHH)
Ca + cGMP (LHH)
Fig. 3. Effects of cyclic nucleotides and Ca 2+ on ciliary beat direction of permeabilized cells. Arrows represent the direction of the power stroke of the cilia that cover the cell body. Cells swim forward except in condition d, with Ca 2+ and no added cyclic nucleotide. In vivo Paramecium swims forward with the beat direction at 5 o'clock. (a) Control (Mg-ATP alone). Beat direction is 7 o'clock; (b) cyclic AMP (cAMP), beat direction is 7 o'clock; (c) cGMP, beat direction shifts toward 5 o'clock; (d) Ca 2+, beat direction is shifted toward 1 o'clock and the cell swims backward; (e) Ca 2+ + cAMP, beat direction shifts from 1 o'clock to 7 o'clock; (f) Ca 2+ + cGMP, beat direction shifts from 1 o'clock to 4 or 5 o'clock. RHH, righthanded helix; LHH, left-handed helix; bkwd, backward swimming of the cell.
Cilia, flagella and apparatus Goodenough 61
Chlamydomonas 20 kD CaBP NH2 [~?]
COOH
Rat calmodulin NH2
Fig. 4. The predicted secondary structure of the Chlamydomonas20kD calcium-binding protein (CaBP) conforms to the helix-loop-helix arrangement for the potential calcium-binding domains. ([]), a-helical segments; (17), p-sheet; (11), [8-turn or random coil conformations. Potential calcium-binding loops are indicated (--).
COOH
Rat calmodulin (crystal structure NH2
COOH 10 Residues
merizes to form fibrous basal body-associated structures that have variously been called 'rootlets', 'rhizoplasts' and 'striated fibers' [23,24]. It has also been localized to mammalian centrosomes (Salisbury et al., Cell Motil Cytoskel 1986, 6:193-197), in particular to pericentriolar satellites and basal feet (Baron and Salisbury, J Cell Biol 1987, 105:205a) and to the mitotic spindle [23,24]. Huang and colleagues have cloned the gene f6r this protein and have shown that it is related to calmodulin (Fig. 4). They also report a 50% sequence identity with the CDC31 gene product of S. cerevisiae which is required for spindle pole body duplication. Salisbury and colleagues have pursued several functional studies, showing that the rhizoplast (basal body-nucleus connector) of Chlamydomonas contracts in response to deflagellation, pulling the nucleus up to the cell anterior [26], and that the striated fiber interconnecting the basal booties of Spermatozopsis similis contracts in response to light, generating flagellar reversal (the 'photophobic' response) [27[. Both contractions require Ca2+, and the rhizoplast can subsequently be relaxed by ATP. Collectively, these papers introduce a new eukaryotic illament system. While the system is often associated with basal bodies and centrioles, this association may not be obligatory; for example, centrin is apparently homologous with the 'spasmin' filament system of vorticellid ciliates (Amos, J Cell Sci 1975, 19:203-213; Salisbury et al., J Cell Bio11984, 99:962-970). There will be keen interest in leaming how the protein undergoes Ca-induced contractions and in its additional roles in eukaryotic cell biology.
arm dynein translocates brain microtubules in vitro. Nature 1987, 330:672-674. Sea urchin outer-arm dynein can now be assayed for functional activity in vitro. VALE RD, TOYOSHIMAYY: Rotation and translocation of microtubules in vitro induced by dyneins from T e t r a h y m e n a cilia. Cell 1988, 52:459-469. A similar in vitro assay to [1] indicates that the 14S dynein of Tetrahy mena rotates microtubules as they translocate. 2. ••
3, HAIMOLT, FENTON RD: Interaction of C h l a m y d o m o n a s • dynein with tubulin. Cell Motil Cytoskel 1988, 9:129-139. Dyneins are shown to be capable of binding to tubulin subunits as well as potya~aerized microtubules.
FOX LA, SALEWS: Direction of force generated by the inner r o w of dynein arms on flagellar microtubules. J Cell Biol 1987, 105:1781-1788. The inner arms induce microtubule sliding in the same direction (tipward) as do axonemes having both inner and outer arms. 4. •
5. •
MARCHESE RAGONA SP, WALLJS, JOHNSON KA: Structure and mass analysis of 14S dynein obtained from T e t r a h y m e n a cilia. J Cell Biol 1988, 106:127-132. The 14S dynein fraction contains two distinctive dynein proteins. HOSOKAWAY, MIKI-NOUMURAT: Bending motion of Chlamyd o m o n a s axonemes after extrusion of central-pair microtubules. J Cell Biol 1987, 105:1297-1302. The central pair system appears necessary to generate asymmetric bends. 6.
•
KING SM, WITMANGB: Structure of the ct and ~ heavy chains of the outer arm dynein from C h l a m y d o m o n a s flagella. Location of epitopes and protease-sensitive sites. J Biol Chem 1988, 263:9244-9255. Monoclonal antibody epitopes and protease sensitive sites are identified with respect to ultraviolet-induced vanadate cleavage sites to produce detailed peptide maps. 7. ••
8. ••
Annotated references and recommended reading
MOCZ G, TANG W-J Y, GIBBONS IR: A map of photolytic and tryptic cleavage sites on the [3 heavy chain of dynein ATPase from sea urchin sperm flagella. J Cell Biol 1988, 106:1607-1614.
see [7]. 9. •
• ••
Of interest Of outstanding interest
Pwee,NO G: Isolation of a sixth dynein subunit adenosine triphosphatase of C h l a m y d o m o n a s axonemes. J Cell Biol 1988, 106:133-140. An additional inner arm component is identified and characterized.
1. • e
PASCHALBM, KING SM, MOSS AG, COLLINS CA~ VALISE RB, WITMANGB: (Letter to the Editor) Isolated flagellar outer
10. •
SPUNGINB, AVOLIO J, ARDEN S, SATIR P: Dynein arm attachment probed with a non-hydrolyzable ATP analog - - struc-
62
Cytoplasm cell motility tural evidence for patterns of activity. J Mol Biol 1987, 197:671~678. AMP-PCP induces a subset of outer arms to remain attached to the B microtubule after sliding disintegration. 11. •
BROKAWCJ: Regulation of s p e r m flagellar motility by calcium and cAMP-dependent phosphorylation. J Cell Biochem 1987, 35:175. A review of sperm motility regulation written by a leader in the field. 12. •
BROKAWCJ: A lithium-sensitive regulator of s p e r m flagellar oscillation is activated by cAMP-dependent phosphorylation. J Cell Biol 1987, 105:1789-1798. The primary effect of phosphorylation is to activate a regulatory mechanism that governs flagellar oscillation. 13. e
ISHIJIMAS, WITMANGB: Flagellar m o v e m e n t of intact and de-
membranated, reactivated ram spermatozoa. Cell Motil C3, toskel 1987, 8:375. High-speed high-resolution video microscopy is able to capture details of flagellar beating. 14. •
HASEGAWAE, HAyASH1 I-l, ASAKURA S, KAMIYAR: Stimulation of in vitro motility of Chlamydomonas a x o n e m e s by inhibition of cAMP-dependent phosphorylation. Cell Motil CO* toskel 1987, 8:302-311. Cyclic AMP inhibits motility in this organism, an effect correlated with the phosphorylation of 2 polypeptides.
SHEADK, WALSHCJ: IlIRNAs for ¢{-and ~-tubulin and flagellar calmodulin are a m o n g t h o s e coordinately regulated w h e n Naegleria gruberi amebae differentiate into flagellates. J Cell Biol 1987, 105:1303-1310. Complementary DNA to several flagellar proteins are used to probe de nov• gene transcription. 19. •
20. •
[.M EY, REMILLARDSP, FULTON C: T h e ~-tubulin gene family expressed during cell differentiation in Naegteria gruberi. J Cell Biol 1988, 106:2035-2046. Several ~ tubulin complementary DNAs are analysed to define the gene family and its transcriptional regulatjon. 21. •
BORNENSM, PAINTRANDM, BERGES J, MARTYM-C, KARSENTIE: Structural and chemical characterisation of isolated centro, somes. Cell Motil Cytoskel 1987, 8:238-249. A procedure for high-yield preparations of active lymphoid centrosomes. 22. •
BOISVIEUX-ULRICHE, LAINE MC, SANIX)Z D: In vitro effects of benzodiazepines on aliogenesis in t h e quail. Cell Motil Cytoskel 1987, 8:333-344. Cytoskeletal drugs inhibit basal body migration as well as ciliary motility. SALISBURYJL, BARON A, SANDERS MA: The centrin-based cytoskeleton of Chlamydomonas reinhardti: distribution in interphase and mitotic cells. J Cell Biol 1988, 107:635~41. The Ca-binding protein centrin is immunolocalized to various filamentous systems and to the mitotic apparatus. 23. • e
15. •
GUSTIN MC, NELSON DL: Regulation of ciliary adenylate cyclase by Ca 2+ in Paramecium. Biochem J 1987, 246:337-345. The cyclase is shown to be sensitive to Ca 2 + and inhibited by calmod ulin antagonists.
HUANGB, WATI'ERSONDM, LEE VD, SCHIBLERmJ: Purificatiorl and characterization of a basal body-associated Ca 2 + -binding protein. J Cell Biol 1988, 107:121-131. A 20 kD Ca2 + -binding protein is immunolocalized to various filamentous systems and to the mitotic apparatus of Chlamydomonas.
16. •
TASHJS, KRINKS M, PATELJ, MEANS RL, KLEE CB, MEANS AR: Identification, characterization, and functional correlation of calmodulin-dependent protein phosphatase in sperm. J Cell Biol 1988, 106:1625 1634. A calmodulin dependent protein phosphatase is implicated in the Ca2+ -dependent regulation of motility.
HUANGB, MENGERSENA, LEE VD: Molecular cloning of cDNA for caltractin, a basal body-associated Ca 2 + -binding protein: homology in its protein s e q u e n c e with calmodulin and the yeast CDC31 gene product. J Cell Biol 1988, 107:133 140: The Ca2+-binding protein caltractin is shown to be related to a yeast protein that also localizes to the spindle poles.
17. e
Moss AG, TAMM SL: A calcium regenerative potential controlling ciliary reversal is propagated along the length of c t e n o p h o r e c o m b plates. Proc Natl Acad Sci USA 1987, 84:64764480. Voltage-dependent channels that mediate increases in ciliary Ca 2 + are distributed over most of the ciliary length in this organism.
SALISBURYJL, SANDERS MA~ HARPST L: Flagellar root contraction and nuclear m o v e m e n t during llagellar regeneration in Chlamydomonas reinhardtii. J Cell Biol 1987, 105:1799-1806. Deflagellation induces a Ca-dependent contraction of the centrin fibers connecting the nucleus to the basal bodies.
18. •
27. •
BONININM; NELSON DL: Differential regulation of Paramecium ciliary motility by cAMP and cGMP. J Cell Biol 1988, 106:1615-1624. cAMP and cGMP stimulate forward motility but have distinct synergistic effects on Ca 2 + -regulated motility.
24. ••
25. ••
26. e
MCFADDENGI, SCHULZE D, SUREK B, SALISBURYJL, MELKONIAN M: Basal body reorientation mediated by a Ca 2+- modulated contractile protein. J Cell Biol 1987, 105:903412. Ca2+ influx induces a change in the angle between basal bodies which is dependent on a centrin fiber.
Introduction
Ciliary dynein
Cilia and flagella (the term 'cilia' is used generically here) are complex ( > 200 polypeptide species) and highly conserved organelles of eukaryotic motility (Gibbons, J Cell Biol 1978, 91:107--124; Huang, Int Rev Cytol 1986, 99:181-215). They are synthesized under the aegis of basal bodies, organelles identical to, and often interconvertible with, the centrioles of the centrosome; the basal bodies and their associated fibrillar structures (the 'basal apparatus') also contain numerous unique polypeptides. It is generally agreed that ciliary microtubules slide past one another during bend formation, a motion mediated by the adenosine triphosphate (ATP)ase activity of the outer and inner dynein arms, but little is understood about the mechanics of this motion or about the role played by such accessory structures as the central pair, radial spokes and nexin links. Much current research on these organelles is focused on the regulation of motility by cyclic nucleotides and calcium, and on control of their biosynthesis. A two-volume book has just gone to press which contains numerous up-to-date articles and reviews on these subjects (Warner et al., eds, Cell Movement. New York. Alan Liss, 1988).
Certainly the most important recent methodological ad vance in the study of ciliary dynein has been the development of in vitro assays for its force-transducing activity, assays that parallel those for cytoplasmic motors such as kinesin (reviewed by Gelfand in this issue, pp 63~56). Paschal et al. [1] and Vale and Toyoshima [2] have shown that sea urchin and Tetrahymena outerarm dyneins, adsorbed to glass coverslips, will translocate bovine brain microtubules in the presence of MgATP. These findings prove that the doublet structure of ciliary microtubules is not necessary for effective ciliary dynein/microtubule interaction [3], that outer arms can function independently of inner arms and that outer arms move towards the minus ends of the microtubules ('retrograde motility'), as they do in vivo [4] (Fig. 1).
(+)
(+)
Vale and Toyoshima [2] also report an intriguing property of the 14S dynein fraction from Tetrahymena: not only does it induce smooth microtubule gliding; it also uniquely causes the microtubules to rotate clockwise. The 14S fraction contains two dynein-like molecules [5], one of which is morphologically similar to an inner-
(+)
N+ (a)
(-)
(b)
(-)
N
(c) (--)
Fig. 1. Direction of active microtubule sliding generated by the dynein arms. The distal end of the microtubules is designated with a plus ( + ) sign and the proximal end with a minus ( - ) sign. The doublets are viewed from the inside of the axoneme toward the outside. Doublet N is the doublet with the active arms and doublet N + I is the adjacent doublet. As illustrated in (a) and (b), doublet N always slides proximally. More generally (c), dyneinmediated motility is toward the minus ( - ) end of the cytoplasmic microtubule along which dynein, and associated structures such as a membrane vesicle, move.
Abbreviations AMP--adenosinemonophosphate;ATP--adenosinetriphosphate;GMP--guanosinemonophosphate 58
(~ Current Science Ltd ISSN 0955-0674
Cilia, flagella and apparatus Goodenough 59 arm dynein from Chlamydomonas (Goodenough et al., JMolBio11987, 194:481--494); rotation may therefore be induced by one or both species or, conceivably, by a non-dynein protein present in the preparation. The phenomenon is of particular interest in that the central-pair microtubules of the axoneme rotate clockwise during the ciliary beat (Fig. 2) [6] (Omot 9 and Rung, J Cell Biol 1980, 87:33-46), apparently because of an interaction between the radial spokes and the central pair [6], and the spoke-central pair system appears to dictate the shape of the ciliary wave form [6] (Brokaw et al., J Cell Biol 1982, 92:722-732; Ishijima et al., Cell Motil Cytoske11988, 9:264-267).
Rotation
194:481-494). Since the inner arms move towards the minus ends of microtubules and will support ciliary beating at half the rate of intact cilia [4], their heterogeneity is not obviously related to force generation and may serve more complex regulatory functions. The close association of inner arms with the radial spoke system (Goodenough and Heuser, J Cell Biol 1985, 199:2008-2018) is therefore particularly interesting. The mode of force transduction generated by dynein arms continues to elude researchers. Intuition dictates that the arms should make contact with the B microtubules during force generation and detach themselves during inactivity, and Spungin et al. [10] have obtained intriguing images of subsets of outer arms attached to B microtubules in the presence of the non-hydrolysable analogue AMP-PCP. These subsets alternate with groups of unattached arms, consonant with the notion of a complex regulation of arm activity.
Regulation of motility Swimming direction
Fig. 2. Schematic representation of the swimming direction E~> of an axoneme from which a central pair has been extruded, and the clockwise rotating direction of a central pair (~). Tip of axoneme is to the left.
Biochemical analysis of dynein proteins was given an important boost several years ago by the discovery that; when the molecules are irradiated at 365 nm in the presence of vanadate, their component 'heavy' polypeptide chains are cleaved at two sites which apparently occur on separate loops of the ATP-binding domain (Lee-Eiford et al., J Biol Chem 1986, 261:2337-2342; Tang and Gibbons, J Biol Chem 1987, 262:17728--17734). King and Witman [7] and Mocz et al. [8] have combined photolysis with proteolysis to produce extensive maps of the Chlam3* domonas 0t and ~ and the sea-urchin ]Bheavy chains, respectively, and several of the peptides have been further identified by monoclonal antibody probes. It is interesting that for the sea urchin, none of the monoclonal antibodies binds to the central 100 000 Mr region of the chain where ATP binding and hydrolysis appear to occur (i.e. the domain flanked by the two photolytic sites), and only one binding site has been identified in this region for Cblamydomonag suggesting that this is the more conserved and hence less antigenic region of the protein. Each outer arm is a complex of two or three dynein subunits, each with a unique heavy chain (Toyoshima, J Cell Bio11987, 105:897-901), but there is no evidence for any heterogeneity from one outer arm to the next. By contrast, the inner arms display at least two discrete morphologies (Goodenough and Heuser, J Cell Biol 1985, 199:2008-2018), and biochemical and structural analysis of inner-arm or putative inner-arm proteins also reveals heterogeneity [5,9] (Goodenough et al., J Mol Bio11987 ,
Dynein arms function in a constant ionic environment, in contrast to actin-myosin systems where contraction is stimulated by Ca 2 + and relaxation by Ca2 + withdrawal. However, certain cilia fail to beat at all unless effectors are provided, and the pattem of the beat is clearly subject to exogenous regulation; moreover, sustained beating is apparently dependent on the phosphorylation state of key proteins. The two agents most often implicated in these regulatory events are Ca 2+ and cyclic AMP (cAMP), but as stressed in a review by Brokaw [11], the observations are complicated by potential interactions between the two. Moreover, different ciliary systems give seemingly contradictory results: cAMP stimulates sperm motility in some systems [12] but not others [13] and actually inhibits motility in Chlamydomonas [14]; Ca 2+ stimulates an adenylate cyclase in Paramecium [15] and a protein phosphatase in sperm [16]. It seems probable that despite the apparent conservation of axonemal hardware, many patterns of ciliary regulation have evolved. This is, of course, true for muscle contraction as well, where neural and hormonal factors have come to govern tissue-specific patterns of activity. By a conserved regulatory activity, Ca2+ is to reverse the beat direction of cilia, a response first observed in Paramecium (Naitoh and Kaneko, Science 1972, 176:523-524) and occurring as well in complex ciliated epithelia [17]. Ca2+ influx occurs via voltage-sensitive Ca2+ channels activated by membrane depolarization; membrane depolarization, in tum, is induced by neural stimulation (epithelia) or environmental stimuli (protozoa). Moss and Tamm present electrophysiological evidence [17] that in the ciliary comb plate of ctenophores, the Ca2 + channels are distributed over most of the length of the cilia. Since calmodulin, the putative Ca 2+ sensor in this system, has been shown to be tightly bound to the axoneme along its entire length (Maihle et al., J Cell Bio11981, 89:695-699), Ca 2+ influx and the subsequent
60 Cytoplasmcell motility modulation of beat direction appear to occur along the entire cilium and not, for example, via channels at the ciliary base. The effectors responsive to Ca-calmodulin have not yet been identified, but given the apparent role of the central pair/radial spoke system in governing patterns of ciliary beating (see above), this system is a likely target. A more unusual sample of regulation is documented by Bonini and Nelson [18], who show that cyclic nucleotides can antagonize the Ca2 + stimulation of ciliary reversal, and that the helical path traced by swimming Paramecium is modulated in one direction by cAMP and in another direction by cyclic guanosine monophospahte (cGMP; Fig. 3). Since numerous enzymes involved in cyclic nucleotide generation and utilization have been identified in Paramecium cilia [15], this system appears to be particularly promising for identifying the regulatory circuits involved.
Biosynthesis of cilia The numerous genes that specify ciliary components are apparently under coordinate regulation. The pioneering studies of this regulation used Chlamydomonas (Lefebvre and Rosenbaum, A n n u Rev Cell Biol 1986, 2:517-546), but studies using other organisms are now demonstrating a similar coordination (Eldon et al., Genes Devel 1987, 1:1280-1292; Harlow and Nemer, Genes Devel 1987, 1:1293-1304). An important complementary system is provided by Naegleria, an amoeba that differentiates into a flagellate within 1 h of nutrient starvation. In contrast to Chlamydomonas, where amputation of existing flagella stimulates flagellar gene transcription and as(a)
(d)
Control (RHH)
(b)
cAMP (RHH)
(c)
Ca
(e)
Ca + cAMP (RHH)
(f)
(RHH, bkwd)
sembly of new flagella, the Naegleria amoeba has no flagella, and gene induction results in the de novo synthesis of basal bodies and rootlets as well as flagella. Shea and Walsh [19] and Lai et al. [20] have begun exploiting this system at a molecular level, identifying complementary DNA for 0~ and [3-tubulin and for a flagellar calmodulin, all of which are coordinately expressed. Here, as in the Chlamydomonas system, the challenge is now to identify upstream genomic sequences shared by these genes, the factors that presumably initiate coordinate transcription by binding to these sequences, and the stimuli provided by starvation (Naegleria) or deflagellation (,Chlamydomonas) that activate these factors.
The basal apparatus Since studies of basal bodies and centrioles appear only infrequently, two 1987 studies are worth examination. One [21] describes a high-yield purification of centrosomes and the other [22] the inhibitory effects of benzodiazepines on the migration of basal bodies to the cell surface during ciliogenesis. A further paper of general importance to the field is the characterization of the socalled 'uni' linkage group in Chlamydomonas (Ramanis and Luck, Proc Natl Acad Sci USA 1986, 83:423-426), a group of genes that show an unusual pattern of inheritance and specify a variety of traits associated with basalbody/flagellar function. Of particular interest are several papers describing a novel calcium-binding protein associated with basal bodies and centrioles, called centrin by Salisbury et al. [23] and caltractin by I-Iuang et al. [24,25]. The protein poly-
cGMP (LHH)
Ca + cGMP (LHH)
Fig. 3. Effects of cyclic nucleotides and Ca 2+ on ciliary beat direction of permeabilized cells. Arrows represent the direction of the power stroke of the cilia that cover the cell body. Cells swim forward except in condition d, with Ca 2+ and no added cyclic nucleotide. In vivo Paramecium swims forward with the beat direction at 5 o'clock. (a) Control (Mg-ATP alone). Beat direction is 7 o'clock; (b) cyclic AMP (cAMP), beat direction is 7 o'clock; (c) cGMP, beat direction shifts toward 5 o'clock; (d) Ca 2+, beat direction is shifted toward 1 o'clock and the cell swims backward; (e) Ca 2+ + cAMP, beat direction shifts from 1 o'clock to 7 o'clock; (f) Ca 2+ + cGMP, beat direction shifts from 1 o'clock to 4 or 5 o'clock. RHH, righthanded helix; LHH, left-handed helix; bkwd, backward swimming of the cell.
Cilia, flagella and apparatus Goodenough 61
Chlamydomonas 20 kD CaBP NH2 [~?]
COOH
Rat calmodulin NH2
Fig. 4. The predicted secondary structure of the Chlamydomonas20kD calcium-binding protein (CaBP) conforms to the helix-loop-helix arrangement for the potential calcium-binding domains. ([]), a-helical segments; (17), p-sheet; (11), [8-turn or random coil conformations. Potential calcium-binding loops are indicated (--).
COOH
Rat calmodulin (crystal structure NH2
COOH 10 Residues
merizes to form fibrous basal body-associated structures that have variously been called 'rootlets', 'rhizoplasts' and 'striated fibers' [23,24]. It has also been localized to mammalian centrosomes (Salisbury et al., Cell Motil Cytoskel 1986, 6:193-197), in particular to pericentriolar satellites and basal feet (Baron and Salisbury, J Cell Biol 1987, 105:205a) and to the mitotic spindle [23,24]. Huang and colleagues have cloned the gene f6r this protein and have shown that it is related to calmodulin (Fig. 4). They also report a 50% sequence identity with the CDC31 gene product of S. cerevisiae which is required for spindle pole body duplication. Salisbury and colleagues have pursued several functional studies, showing that the rhizoplast (basal body-nucleus connector) of Chlamydomonas contracts in response to deflagellation, pulling the nucleus up to the cell anterior [26], and that the striated fiber interconnecting the basal booties of Spermatozopsis similis contracts in response to light, generating flagellar reversal (the 'photophobic' response) [27[. Both contractions require Ca2+, and the rhizoplast can subsequently be relaxed by ATP. Collectively, these papers introduce a new eukaryotic illament system. While the system is often associated with basal bodies and centrioles, this association may not be obligatory; for example, centrin is apparently homologous with the 'spasmin' filament system of vorticellid ciliates (Amos, J Cell Sci 1975, 19:203-213; Salisbury et al., J Cell Bio11984, 99:962-970). There will be keen interest in leaming how the protein undergoes Ca-induced contractions and in its additional roles in eukaryotic cell biology.
arm dynein translocates brain microtubules in vitro. Nature 1987, 330:672-674. Sea urchin outer-arm dynein can now be assayed for functional activity in vitro. VALE RD, TOYOSHIMAYY: Rotation and translocation of microtubules in vitro induced by dyneins from T e t r a h y m e n a cilia. Cell 1988, 52:459-469. A similar in vitro assay to [1] indicates that the 14S dynein of Tetrahy mena rotates microtubules as they translocate. 2. ••
3, HAIMOLT, FENTON RD: Interaction of C h l a m y d o m o n a s • dynein with tubulin. Cell Motil Cytoskel 1988, 9:129-139. Dyneins are shown to be capable of binding to tubulin subunits as well as potya~aerized microtubules.
FOX LA, SALEWS: Direction of force generated by the inner r o w of dynein arms on flagellar microtubules. J Cell Biol 1987, 105:1781-1788. The inner arms induce microtubule sliding in the same direction (tipward) as do axonemes having both inner and outer arms. 4. •
5. •
MARCHESE RAGONA SP, WALLJS, JOHNSON KA: Structure and mass analysis of 14S dynein obtained from T e t r a h y m e n a cilia. J Cell Biol 1988, 106:127-132. The 14S dynein fraction contains two distinctive dynein proteins. HOSOKAWAY, MIKI-NOUMURAT: Bending motion of Chlamyd o m o n a s axonemes after extrusion of central-pair microtubules. J Cell Biol 1987, 105:1297-1302. The central pair system appears necessary to generate asymmetric bends. 6.
•
KING SM, WITMANGB: Structure of the ct and ~ heavy chains of the outer arm dynein from C h l a m y d o m o n a s flagella. Location of epitopes and protease-sensitive sites. J Biol Chem 1988, 263:9244-9255. Monoclonal antibody epitopes and protease sensitive sites are identified with respect to ultraviolet-induced vanadate cleavage sites to produce detailed peptide maps. 7. ••
8. ••
Annotated references and recommended reading
MOCZ G, TANG W-J Y, GIBBONS IR: A map of photolytic and tryptic cleavage sites on the [3 heavy chain of dynein ATPase from sea urchin sperm flagella. J Cell Biol 1988, 106:1607-1614.
see [7]. 9. •
• ••
Of interest Of outstanding interest
Pwee,NO G: Isolation of a sixth dynein subunit adenosine triphosphatase of C h l a m y d o m o n a s axonemes. J Cell Biol 1988, 106:133-140. An additional inner arm component is identified and characterized.
1. • e
PASCHALBM, KING SM, MOSS AG, COLLINS CA~ VALISE RB, WITMANGB: (Letter to the Editor) Isolated flagellar outer
10. •
SPUNGINB, AVOLIO J, ARDEN S, SATIR P: Dynein arm attachment probed with a non-hydrolyzable ATP analog - - struc-
62
Cytoplasm cell motility tural evidence for patterns of activity. J Mol Biol 1987, 197:671~678. AMP-PCP induces a subset of outer arms to remain attached to the B microtubule after sliding disintegration. 11. •
BROKAWCJ: Regulation of s p e r m flagellar motility by calcium and cAMP-dependent phosphorylation. J Cell Biochem 1987, 35:175. A review of sperm motility regulation written by a leader in the field. 12. •
BROKAWCJ: A lithium-sensitive regulator of s p e r m flagellar oscillation is activated by cAMP-dependent phosphorylation. J Cell Biol 1987, 105:1789-1798. The primary effect of phosphorylation is to activate a regulatory mechanism that governs flagellar oscillation. 13. e
ISHIJIMAS, WITMANGB: Flagellar m o v e m e n t of intact and de-
membranated, reactivated ram spermatozoa. Cell Motil C3, toskel 1987, 8:375. High-speed high-resolution video microscopy is able to capture details of flagellar beating. 14. •
HASEGAWAE, HAyASH1 I-l, ASAKURA S, KAMIYAR: Stimulation of in vitro motility of Chlamydomonas a x o n e m e s by inhibition of cAMP-dependent phosphorylation. Cell Motil CO* toskel 1987, 8:302-311. Cyclic AMP inhibits motility in this organism, an effect correlated with the phosphorylation of 2 polypeptides.
SHEADK, WALSHCJ: IlIRNAs for ¢{-and ~-tubulin and flagellar calmodulin are a m o n g t h o s e coordinately regulated w h e n Naegleria gruberi amebae differentiate into flagellates. J Cell Biol 1987, 105:1303-1310. Complementary DNA to several flagellar proteins are used to probe de nov• gene transcription. 19. •
20. •
[.M EY, REMILLARDSP, FULTON C: T h e ~-tubulin gene family expressed during cell differentiation in Naegteria gruberi. J Cell Biol 1988, 106:2035-2046. Several ~ tubulin complementary DNAs are analysed to define the gene family and its transcriptional regulatjon. 21. •
BORNENSM, PAINTRANDM, BERGES J, MARTYM-C, KARSENTIE: Structural and chemical characterisation of isolated centro, somes. Cell Motil Cytoskel 1987, 8:238-249. A procedure for high-yield preparations of active lymphoid centrosomes. 22. •
BOISVIEUX-ULRICHE, LAINE MC, SANIX)Z D: In vitro effects of benzodiazepines on aliogenesis in t h e quail. Cell Motil Cytoskel 1987, 8:333-344. Cytoskeletal drugs inhibit basal body migration as well as ciliary motility. SALISBURYJL, BARON A, SANDERS MA: The centrin-based cytoskeleton of Chlamydomonas reinhardti: distribution in interphase and mitotic cells. J Cell Biol 1988, 107:635~41. The Ca-binding protein centrin is immunolocalized to various filamentous systems and to the mitotic apparatus. 23. • e
15. •
GUSTIN MC, NELSON DL: Regulation of ciliary adenylate cyclase by Ca 2+ in Paramecium. Biochem J 1987, 246:337-345. The cyclase is shown to be sensitive to Ca 2 + and inhibited by calmod ulin antagonists.
HUANGB, WATI'ERSONDM, LEE VD, SCHIBLERmJ: Purificatiorl and characterization of a basal body-associated Ca 2 + -binding protein. J Cell Biol 1988, 107:121-131. A 20 kD Ca2 + -binding protein is immunolocalized to various filamentous systems and to the mitotic apparatus of Chlamydomonas.
16. •
TASHJS, KRINKS M, PATELJ, MEANS RL, KLEE CB, MEANS AR: Identification, characterization, and functional correlation of calmodulin-dependent protein phosphatase in sperm. J Cell Biol 1988, 106:1625 1634. A calmodulin dependent protein phosphatase is implicated in the Ca2+ -dependent regulation of motility.
HUANGB, MENGERSENA, LEE VD: Molecular cloning of cDNA for caltractin, a basal body-associated Ca 2 + -binding protein: homology in its protein s e q u e n c e with calmodulin and the yeast CDC31 gene product. J Cell Biol 1988, 107:133 140: The Ca2+-binding protein caltractin is shown to be related to a yeast protein that also localizes to the spindle poles.
17. e
Moss AG, TAMM SL: A calcium regenerative potential controlling ciliary reversal is propagated along the length of c t e n o p h o r e c o m b plates. Proc Natl Acad Sci USA 1987, 84:64764480. Voltage-dependent channels that mediate increases in ciliary Ca 2 + are distributed over most of the ciliary length in this organism.
SALISBURYJL, SANDERS MA~ HARPST L: Flagellar root contraction and nuclear m o v e m e n t during llagellar regeneration in Chlamydomonas reinhardtii. J Cell Biol 1987, 105:1799-1806. Deflagellation induces a Ca-dependent contraction of the centrin fibers connecting the nucleus to the basal bodies.
18. •
27. •
BONININM; NELSON DL: Differential regulation of Paramecium ciliary motility by cAMP and cGMP. J Cell Biol 1988, 106:1615-1624. cAMP and cGMP stimulate forward motility but have distinct synergistic effects on Ca 2 + -regulated motility.
24. ••
25. ••
26. e
MCFADDENGI, SCHULZE D, SUREK B, SALISBURYJL, MELKONIAN M: Basal body reorientation mediated by a Ca 2+- modulated contractile protein. J Cell Biol 1987, 105:903412. Ca2+ influx induces a change in the angle between basal bodies which is dependent on a centrin fiber.