The control of ciliary beat frequency

To avoid being eaten, eukaryotic microorganisms sometimes need to move much more quickly than usual. This is nicely seen when Paramecium escapes from its predator Dileptus in a burst of rapid swimming several times its normal speed, powered by its thousands of cilia. Cellular control of ciliary beat frequency is a universal phenomenon that is found in metazoan ciliated epithelia as well as in protozoa. A classical metazoan example is the isolated gill epithelium of mussels, where addition of serotonin (S-HT) to the medium increases beat frequency of the lateral cilia; the ciUa are quiescent at 10-9 M 5-HT, but beat frequency rises by 4 Hz per tenfold increase in 5-HT concentration up to 10-5 ML Similarly, tracheal respiratory cilia of mammals increase their beat frequency when stimulated either via the nervous system or mechanically by mucus2,3. Ciliary motility depends on the '9 + 2' axoneme, the specialized microtubule cytoskeleton of the organelle, which moves by meam of the mictotubule motor axonemal dynein~. How can an external stimulus change the activity of axonemal dynein? The signal transduction pathway leading to the regulation of microtubule motor activity is initiated at the cell membrane of the cilLiatedcell; it involves transmembrane receptors and a number of mechanosensitive ion channels either in the membrane surrounding the cell body or in the ciliary membrane that surrounds the axonemes. The second messenger molecules implicated in ciliary control - Ca 2÷ and cyclic AMP (cAMP) - af[ect different cilia in various ways4. In Paramecium and in mussel gill lateral cilia, beat frequency increases are accompanied by an increase in cAMP in the cytoplasm. However, an increase in Ca2÷ first slows ciliary beat and then, at higher concentrations, a~rests the gill lateral cilia, but causes reversal of the beat direction in Paramecium6. Mammalian cilia respond to increases in both Ca2÷ and cAMP by increasing their beat frequency2''~. In Panune¢ium, a K~ channel seems to be directly coupled to adenylate cyclase; opening of this channel during hyperpolarization leads to increased intracellular cAMP and to fast forward swimming of the cell7. The addition of monobutyryl cAMP or drugs that elevate intracellular cAMP increase the Paramecium swimming rate two- to threefold8.

The control of ciliary beat

frequency Ciliary movement is powered by axonemal dynein. This article considers how a signal transduction cascade initiated at the cell membrane may activate outer dynein arms to change the velocity of microtubule sliding and the swimming speed of ciliated cells. For Paramecium, a critical event in the cascade is the cAMPdependent phosphorylation of a 29 kDa polypeptide that is associated with the outer dynein arm.

have important implications for the mechanism by which cAMP increases beat frequency: for example, aside from small molecules and proper mechanical anchorage to the remaining cytoskeleton, only axonemal components are required for normal motility and the behavioural response. If a cAMPdependent protein kinase (CAPK) is involved in the transduction mechanism, the kinase must be firmly attached to the axoneme, since soluble proteins are washed away during the various solution changes that accompany permeabilization and reactivation. CAPKtype I and type 1Iare both found in Paramecium cilia and co-sediment with isolated axonemes lz.

Sliding and bending in the ciliary axoneme

Most experimental protocols for dissecting the signal transduction pathway controlling ciliary beat frequency involve permeabilization of the cell and cilium by detergents9. Destruction of the membrane in this way causes the loss of the signal-generating system, and of the small molecules that are part of the cytosol and the matrix around the axoneme. The cilium naturally stops moving, but normal movement can be restored by replacing the cytosol around the axoneme with simple buffers containing Mg2÷-ATP. If the appropriate second messenger is added, ciliary behaviou~ returns as well ~°,l~ (Fig. 1). For Paramecimn, when care is taken to prevent deciliation, the demembranated cells swim when reactivated and swimming speed increases as a function of cAMP concentration from 0-30 IJM. These findings

As discussed in the article by Asai and Brokaw in this issue, axonemal dynein is packaged in rows of outer arms and inner arms of several types, which are attached to one side, the A subfibre, of each of the nine doublet microtubules. It has been known for some time that during ciliary motility the doublet microtubules slide with respect to one another 4. Sliding has recently been confirmed by direct visualization on moving reactivated axonemes of sea urchin sperm 13. Sliding is produced by a mechanochemical cycle of the arm and can be reproduced in vitro using purified dynein and microtubules~4. Both inner and outer arms function as minus-enddirected microtubule motors. In the axoneme this . . . . . . . means that the doublet to which the arms are per- Theauthorsare at manently attached moves baseward, towards the cell the Departmentof body, relative to its neighbour on which the arms Anatomyand transiently 'walk'Is (Fig. 2). It is not exactly clear how StructuralBiology, sliding is converted into bending, but signals gener- AlbertEinstein ated by various constraining linkages within the CoUegeof axoneme, including the radial spokes 16, are probably Medicine,Bronx, important in this conversion. In any event, the NY10461,USA.

TRENDS IN CELL BIOLOGY VOL. 3 NOVEMBER 1993

© 1993 ElsevierSciencePublishersLtd (UK)0962-8924/93/$06.00

A permeabllized cillated cell system

409

(a)

2 mM ATP + 1001ULMcAMP

2 mM ATP

' ~". '., ', I,~,'~',

The velocity of microtubule sliding (VE) can be calculated for the same cilium beating at two different frequencies (F1 and Fz), assuming that there is no change in beat form. The formation of a bend (ccE)from a straight position dudng the effective stroke takes about a quarter of the stroke time. The amount of sliding (ALE)is calculEted from the equation AIE = dnT.¢¢E= VEt Ewhere d n is a constant related to the distance between adjacent doublets N and N+I and t E is tile duration of bend formation.

~"

.x

X

eo

c 0 .(,.,

(;.-,r~,)',,.

if) 0

=--,~=

0

BOX 1 - SLIDING VELOCITY AT DIFFERENTBEAT FREQUENCIES

._~

0

_=nr~ "J;~!l= d¢ d~

/"1

240 0

256 Horizontal position (pixels)

240

,6_r~0

256 Horizontal position (pixels)

(b) Paramecium tetraure/ia Intact ceil

Triton-X-100-permeabilizod cell: reac'dvated at pCa 7

i

FIGURE 1

Quantitative considerations At

Fj = 16.7 Hz

Fz = 25 Hz

Beat duraUon

60 ms

40 ms

o~

100o

100 o

tE

-15 ms

-10 ms

AIE

0.1 p.m

VE

6.7 p-m s-1

0.1 ~m 10 pms -I

ues irreversibly to completion, producing a displacement of 10 I~m or more. The doublets end up overlapping each other only slightly. The velocity of sliding of a given doublet, maximally about 20 ilms-1, is constant as the axoneme elongates to this point~L Over a wide range, sliding velocity is independent of the number of dynein arms present or of the load. At any instant, one dynein arm is probably sufficient to push the entire microtubule. However, dynein remains attached to the microtubule for only a small fraction of each arm cycle ~8, perhaps 1-2 ms in a cycle of perhaps 33 ms. To produce continuous sliding at maximum velocity, enough dynein arms need to be working so that one dynein is actively pushing the microtubule at all times. Where this is not so, sliding velocity will fall proportionately. The velocity of sliding in bending, intact axonemes at normal beat frequency (a velocity of 10 pms -~) is similar to rates of unconstrained sliding in unlinked axonemes, since both the displacement and duration of sliding during any given phase of the beat cycle are proportionately much smaller in the intact axoneme. This suggests that the production of sliding in viva during repetitive beating of the axoneme is not intrinsically different to that observed after proteolysis or in in vitro experiments.

(a) Swimming tracks of reactivated permeabilized Paramecium. The paths represent distance travelled by the cells in 1 s; only the 1st, 4th, 7th and 10th frames are displayed. Arrows indicate the direction of the path. In 2 rnM Mg2+-ATP, pCa>7, without cAMP, most cells swim slowly forward (average speed: 115 p.ms-1). With 100 pM cAMP, cells on average swim faster (average speed: 205 I~ms"l) and some cells (*) swim several times faster than any control cell. (b) Scanning electron micrographs of quick-fixed intact (top) and Triton-permeabilized reactivated (bottom) cells. Both cells are oriented with their anterior end to the left. The cilia beat out of phase with metachronal waves, with an effective stroke towards the posterior of the cell. The wave pattem, and therefore the form of the beat, of the permeabilized cells is similar to that of the living, forward-swimming cells. Bar, 10 p.m. See Ref. 9 for further details.

The locus o f beat frequency control Ciliary beat can be changed by second messengers

amount of sliding and the amount of bend in the beating axoneme are related by simple geometry (Box 1), and a normal bend of 100 ° requires a sliding displacement of about 0.1 pm between adjacent daub. lets. When the constraining linkages are broken by protease digestion, in the presence of MgZ+-ATP, the axoneme elongates as the doublets rapidly move apart. In contrast to the situation in the beating cilium, here sliding between adjacent doublets contin-

in three distinct ways: the beat can be abruptly arrested, the beat form can change, or the beat frequency can change. Changes in beat form and bequency are independently controlled. Certain mutants of Chlamydomonas missing inner dynein arms beat at wild-type frequencies but with highly modified beat patterns, while other mutants, missing outer dynein arms, can beat with normal bend patterns, but at frequencies that are greatly reduced ~9. This suggests that, as a first approximation, the inner d ~ e i n arms primarily influence beat form, while the outer dynein arms regulate beat frequency.

410

TRENDS IN CELL BIOLOGY VOL. 3 NOVEMBER 1993

Let us now consider what actually happens within the cilium of a cell when, without significant change in beat form, beat frequency increases from 16.7 Hz to 25 Hz, the sort of increase that is seen when I mM 5-HT is added to perfused mussel gill epithelium (see Box 1). Because beat form is constant, the magnitude of the bend formed during the effective stroke (c~) remains constant, and therefore the amount of sliding (A/E) associated with the bend remains constant. The time (rE) for this bend to form, estimated to be about a quarter of the total beat cycle time, obviously decreases. This means that the velocity of sliding (V0 increases in proportion to the increase in beat frequency, the percent increase being identical for the two parameters. The implications of these conclusions are that doublet sliding velocity is set primarily by the mechanochemical properties of outer-arm dynein, and that this in turn determines beat frequency. The transduction question then becomes: how does an increase in a second messenger such as cAMP lead to increased microtubule sliding velocity by outer-arm dynein?

i

cAMP-dependent phosphorylMion of axonemal proteins A number of studies2°-24have used the Paramecium model to explore this question. In Paramecium, the outer dynein arm is a three-headed molecule that sediments at 22S upon sucrose density gradient centrifugation2L The heads represent parts of the dynein heavy chains with ATPase activity; each head is probably a slightly different isoform22,23. In some cases, Paramecium axonemes have been isolated and incubated with radiolabeUed ATP in the presence of various concentrations of cAMP to determine which axonemal proteins become phosphorylated in a cAMP-dependent manner. A 29 kDa polypeptide (p29) was among those prominently labelled in the presence of mlcromolar concentrations of cAMP. The cAMP-dependent phosphorylation or thiophosphowlafion of this protein, but not other proteins, was inhibited in the presence of high Ca 2÷ (Ref. 20; Fig. 3). p29 copunfies with the intact three-headed outer arm z~,24. Preliminary evidence suggests that association is specific to one of the thTee heads (K. Barkalow et al., unpublished).

In vitro motility assays The development of in vitro microtubule-transiocation assays~4has made it possible to investigate the role of p29. In such assays, without ATP, some microtubules attach to Paramecium 22S dynein coating a glass surface. When MgZ÷-ATP is added, the dynein causes the mic~otubules to slide ('glide') along the substratum. For axonemal dyneins, the rates of gliding, while reproducible for any given dynein, are perhaps only 20-50% of those measured in the living cilium or in the telescoping axoneme. This is possibly because orientation and alignment of the operative dyneins on the glass is not optimal, or because handling the dynein during extraction and washing perturbs it. However, in the presence of the thiophosphorylated p29 a 40°,6 increase in velocity from 1.6 ~ms-~ was observed (Ref. 21; Fig. 3). This sort of TRENDS IN CELL BIOLOGY VOL. 3 NOVEMBER 1993

•

FIGUR$2 Sliding ,~f axonemal microtubules visualized by negative..stain electron microscopy; active arms on s~Jbfibre A of doublet N push neighbou~ing doublet N+I in a tipward direction, while doublet N moves baseward. Bar, 0.1 ~m. See Ref. 26 for further details. Reproduced,. ~vith permission, from Ref. 26.

change corresponds to a physiologicall~y relevant 40% increase in beat frequency from S to 7 Hz or from 16 to 22 Hz.

Role of p29 It seems likely that phosphorylation of p29 is the critical event necessary for the increase in gliding velocity in response to increased cAMP in Paramecium, since a cAMP.dependent increase in microtubule translocation was not detected using 22S dynein preparations treated with cAMP and high Ca 2., conditions that uniquely inhibit cAMP-dependent phosphowlation of p29 (Fig. 3). Furthermore, in the presence of p29 thiophosphorylated in response to cAMP, but after effective removal of cAMP, swimming speeds of permeabilized cells increased 40-135% 21. The action of p29 on 22S dynein and microtubule gliding may be considered analogous to the role of myosin light chain in the activation of smooth muscle myosin. The proposed cascade from cAMP signal to ciliary response is shown in Fig. 4. Although the basic dynein-based sliding mechanism and structure of the axoneme have been conserved during evolution, it is not yet clear whether the mechanism of beat frequency regulation is also conserved. A similar cascade almost certainly operates for some other ciliates, such as Tetrahymena, and probably also operates for mussel gill lateral cilia where similar cAMP-dependent phosphorylation of dynein-associated polypeptides has been demon-

Acknowledgements This work was .~upported in part IW a grant from the American Heart Association. T. h. is a fellow of the I~lewYork Affiliate of the AHA; I~, B. isa predoct~ral fellow supported by USPHSgrant CA09457$ We thank N. Isaacfor photographi'. assistance. 411

1.6

,.: 1.4 II o

I

E

,5.~ 1'2

--~1.0 or.

6.8. - -

control

cAMP

o A M P + C a 2+

FIGURE3 Relativevelocity (+ SEM) of in vitro microtubule translocation in studies using 1 mM ATP and Paramecium 22S dynein pretreated with cAMP or cAMP and Caz+. Autoradiogram (insert) shows p29 region of three 22S dynein fractions used for these experiments, p29 is thiophospho~ylated (arrowheads) after cAMP pretreatment (lane 2), but not in control (no cAMP) or cAMP plus Caz+ pretreatments. Each bar averagesover 200 individual measurements. Average control velocity is 1.6 Hms-1. See Ref. 21 for further details.

strated2s. For other systems, such as Chlamydomonas, sperm and mammalian epithelial cilia, the regulatory mechanism described here may be modified to a greater or lesser extent, just as the behavioural response to second messengers ,varies. The physiological situation of beat frequency control is, of course, complicated by factors such as changes in beat form, interactions of outer and inner arms, and crossta.lk between Ca 2. and cAMP. Even for the relatively straightforward cascade illustrated

cAMP

cAMP.PK (associated with axoneme) p29

pp29

22S Oynein (outer arm)

22S Dynein+'

L~

Fastersliding a) cycle time decrease

b) step size increase Increased beat frequency Faster s w i m m i n g

FIGURE4 The proposed transduction cascade by which cAMP activates 22S outer-arm dynein to increase microtubule sliding velocity, beat frequency and speed of ciliate swimming. 412

in Fig. 4, the resultant changes in 22S dynein mechanochemistry that result in faster sliding are not well understood. In an in vitro translocation assay, not every 22S dynein molecule will have a phosphorylated p29 associated with it, so that a small number of activated dyneins would have to increase gliding velocity of the microtubules. To do this, the activated dyneins would have to change their mechanochemical properties; an individual molecule might cycle faster or produce a bigger translocation step, so that the few activated dyneins pushing the microtubule at any short time would act as pacemakers to determine the overall gliding velocity. It would not be overly surprising if some controls of activity similar to those discussed for Paramecium 22S dynein applied to signal transduction nechanisms affecting cytoplasmic dynein, and regulated microtubulebased motility in mitosis and membrane trafficking.

References 1 SANDERSON,M. J., DIRKSEN,E. R. and SATIR,P. (1985) Cell Motil. 5, 293-309 2 SANDERSON,M. J. and DIRKSEN,E. R. (1989) Am. Rev. Respir. Dis. 139, 432-440 3 SATIR,P.and SLEIGH,M. A. (1990)Aflnu. Rev.Physiol.52,131-155 4 WARNER,F. D., SATIR,P. and GIBBONS,I. R., eds (1989) Cell Movement Vol. 1: The Dynein ATPoses,Alan R. Liss 5 BLOODGOOD,R. A., ed. (1990) Ciliary and Flagellor Membranes, PlenumPress 6 SATIR,P. (1985) in Modem Cell Biology (Satir, B. H., ed.) 4, pp. 1-46, Alan R. Liss 7 SCHULTZ,J. E., KLUMPP,S., BENZ,R., SCHURHOFF-GEOTERS, W. H. and SCHMID,A. (1992) Science255, 600-603 8 BONINI,N. M., GUSTIN,M. C. and NELSON,D. L (1986) Cell Motil. Cytoskel. 6, 256-272 9 LIEBERMAN,S. J., HAMASAKI,T. and SATIR,P. (1988) CellMotiL Cytoskel. 9, 73-84 10 BONINI,N. M. and NELSON,D. (1988)1.CdlBiol. 106,1615-1623 11 STOMMEL,E. W. and STEPHENS,R. E. (1985) 1. Camp. Phl~iOL A 157, 451-459 12 BONINI,N. M., EVAN,T. C., MIGLIE'B'A,L. A. P. and NELSON, D. L. (1991)Adv. 2nd Mess. Phosphopmt. Res.23, 227-272 13 BROKAW,C. I. (1991)1. CellBiol. 114, 1201-1215 14 VALE,R. D. and TOYOSHIMA,Y. Y. (1988) Cell 52, 459-469 15 SALE,W. S. and SATIR,P. (1977) Proc. NatlAcad. SoL USA 74, 2045-2049 16 SMITH,E. F. and SALE,W. S. (1992)I. Cell Biol. 117, 573-581 17 TAKAHASHI,K., SHINGYOJl,C. and KAMIMURA,S. (1982) Syrup. Sac. Exp. Biol. 35, 159-177 18 VALE,R. D., MAUK, F. and BROWN,D. (1992) 1. CellBiol. 119, 1589-1596 19 BROKAW,C. J. and KAMIYA,R, (1987) CellMotil. Cytoskel. 8, 68-75 20 HAMASAKI,T., MURTAUGH,T., SATIR,B. H. and SATIR,P. (1989) Cell Motil. Cytoskel. 12, 1-11 21 HAMASAKI,T., BARKALOW,K., RICHMOND,J. and SATIR,P. (1991) Proc.Natl Acad. Sci. USA 88, 7918-7922 22 WALCZAK,C. E., MARCHESE-RAGONA,S. P. and NELSON, D. L. (1993) Cell Motil. Cytosket. 24, 17-28 23 BECKWlTH,S. M. and ASAI,D. J. (1993) Cell MotiL Cytoskel. 24, 29-38 24 BONINI,N. M. and NELSON,D. L. (1990)J. Cell. Sci. 95, 219-230 2.5 STEPHENS,R. E.and PRIOR,G. (1992)J. CegSd. 103, 999-1012 26 SATIR,P. (1989) Camp. Riochem. Physiol. 94A, 351-357 TRENDS IN CELL BIOLOGYVOL 3 NOVEMBER 1993