Control of Cilia in the Branchial Basket of Ciona intestinalis (Ascidacea)

9 Pages·1992·4 MB·English

by  D Bergles

Reference: Biol Bull 182: 382-390. (June. 1992)
Control of Cilia in the Branchial Basket
of Ciona intestinalis (Ascidacea)
DWIGHT BERGLES* AND SIDNEY TAMM
Boston UniversityMarine Program, Marine Biological Laboratory,
Woods Hole. Massachusetts 02543
Abstract. We investigated arrest and inactivation re- portion and vigor ofreactivated beating. Addition of 100
sponses of stigmatal cilia in the branchial basket ofthe M^/Catothe RS + cAMP + IBMX solution caused reac-
ascidian, Ciona intestinalis. Using an improved prepa- tivated cilia to vibrate or twitch in an upright position,
ration of living tissue for microscopic imaging ofciliary suggesting that Ca and cAMP have antagonistic effects
responses, we found that Ca-ionophore A23187 in sea- on stigmatal cilia.
mM
water+ 50 Cacausedactivelybeatingciliatoassume
the upright inactive posture, while A23187 in seawater Introduction
+ 100 mA/Ca caused transient (5-10 s) stigma-wide ar-
rests in which the cilia lie flat against the stigmatal walls. The activity ofcilia and flagella is regulated in many
Both responses are therefore Ca dependent, but the in- organisms, reflecting the important roleoftheseorganelles
active state has a lowerthreshold forCa than does arrest. in locomotion, suspension feeding, gas exchange, mucous
Membrane permeant cAMPanalogues induced >40% of andgametetransport,andsperm chemotaxis. Well-known
the quiescent cilia within a stigma to begin beating. examples ofciliary and flagellar responses to stimuli in-
Saponin-extracted models of stigmatal cilia were de- clude reorientation of beat direction in ciliate protozoa
veloped to study the ionic and molecularcontrol ofciliary (Eckert ct a/.. 1976; Machemer. 1986), arrest of lateral
activity in Ciona. Extracted cilia were stimulated to beat cilia of lamellibranch gills (Murakami and Takahashi,
vigorously for >45 min by ATP-containing reactivation 1975:Tsuchiya, 1977; Walterand Satir, 1978), inhibition
M
solution (RS). Addition of 10~5 to 10"' Ca to reacti- ofvelarcilia ofmolluscan larvae (Carter, 1926), activation
vation solution caused the cilia to stand upright (inacti- ofAfytilitsabfrontal cilia (Stommel, 1984), Beroemacro-
vate), but not to arrest. The calmodulin antagonists tri- cilia (Tamm. 1988). and sperm flagella (Brokaw. 1987),
fluoperazine and calmidazolium (100 nM) restored active changes in waveform ofsea urchin sperm flagella (Brokaw
beating when included in RS + 50-100 /iMCa, thereby el u/-. 1974; Brokaw, 1979) and Chlamydonionas flagella
reversing Ca-dependent inactivation. Addition ofbovine (Hyams and Borisy, 1978; Bessen ct a/.. 1980), and re-
obrfairneaccatilvmaotdeudlicinlitao. RRSS++101000nMftCMacdAidMnPot+cau1semaMrres3t- vfleargsealllao(fHdoilrweicltlioanndofMcwGarveegoprro,pa1g9a76t)i.onMainnytroyfptahneosseoamxe-
isobutyl-1-methyl-xanthine or the catalytic subunit ofc- onemal responses are known to be triggered by depolar-
AMP-dependent protein kinase increased both the pro- ization-induced changes in intracellular Ca oralterations
in cyclic nucleotide levels (Machemer, 1986; Brokaw,
*RePcreeisveendt a1d3drDeescs:emDbeeprar1t9m9e1:ntacocfepMtoeldec2u7laMrar&chCe1l9l9u2l.ar Physiology. a1n98d7;SaOitmtie,r,1919908;9;BSotneipnhienctsaain.d1S9t91o)m.mel, 1989; Preston
Beckman Center. Stanford University School of Medicine. Stanford, Tunicates show periodic interruptions in the beating of
California94305-5426. cilia that line openings (stigmata) of the branchial basket
Abhrcvmnony B-cAMP. N6-benzoyl-cAMP; M-cAMP. N6-mono- andgeneratethe feedingcurrent(Fedele, 1923; MacGinitie,
butryl-cAMP; ES, extraction solution; WS. wash solution; RS. reacti-
vation solution; TFP, tnfluoperazine: IBMX, 3-isobutyl-l-methyl-xan- 1939). These temporary ciliary arrests occur sponta-
thine;TAME,Na-p-tosyl-L-argininemethylester,PICA,c-AMP-depen- neously, in response to general disturbances, or when
dent protein kinase. undesirable material enters the branchial siphon (Bone
382
CONTROL OF ClONA CILIA 383
and Mackie, 1982), andare usuallyaccompanied byquick further subdivided into small rectangular pieces, about 1
contractions of siphons and mantle ("squirts") (Mac- X 5 mm, by cutting between the longitudinal bars with
Ginitie, 1939). Previousstudies on the ascidian branchial fine iridectomy scissors. Tissue was used within 2-4 h
basket showed that ciliary arrests are: ( 1 ) induced by de- after its removal from the animal.
polarizing stimuli, (2)controlled by identified neurons in In the final preparation, apieceofbranchial basketwas
thecentral ganglion, (3) mediated bycholinergic synapses, pipetted onto a microscope slide that had been ringed by
(4) correlated with action potentials recorded from the a 15 X 50 mm rectangular ridge ofpetroleumjelly (Vase-
stigmatal ciliated cells, and (5) dependent on external Ca line). The tissue was stretched out near the center ofthis
(Takahashit'M/., 1973; Mackie etal.. 1974;Arkett, 1987; rectangular well with the pharyngeal side facing upward.
Arkett ct ai, 1989). Ascidian stigmatal cilia also undergo Theendsofthe piece were then pressed down against the
an "inactive" state in which they stand in an upright po- slide by fine stainless steel pinsanchored in dabs ofVase-
sition before resuming normal beating (Takahashi et al., line that had previously been placed on the dry slide. A
1973; Mackie etal.. 1974; Arkett, 1987). squarecoverslipwasplacedoverthetissue, mounted near
Despite the evidence suggesting that ascidian cilia are the center ofthe rectangular well, leaving room on either
controlled by mechanisms similar to those operating in side ofthe coverslip to add and withdraw solution from
other systems, neither Ca dependence ofarrest or inac- the well during perfusion.
tivation responses, nor the possible role ofcyclic nucleo-
tides in ciliary activity has been investigated directly in Cell models
any tunicate.
We have addressed these questions in the ascidian Small rectangularpiecesofbranchial basketwereplaced
dona intestinalis by devising an improved method for in 0.1% saponin, 1% DMSO, 20 mAI EGTA, 150 m.\/
microscopic imaging of motile responses of living cilia, KC1, 10 mAI MgCl:, 30 mAI PIPES, pH 7 (extraction
and by developing the first detergent-extracted, ATP- solution, ES) in a glass well for 4-9 min at room tem-
reactivated cell models ofstigmatal cilia. These new ad- perature. Tissue wasmtMransferred toma sMecondwell contaminM-
vances enable direct tests of the ionic and biochemical ing 2 mAI ATP, 1 DmTMT, 10 EGTA, 150
basis ofcontrol ofciliary motility in Ciona and are valu- KC1, 10 mAI MgCl:. 30 PIPES, pH 7 (reactivation
ablecomplementstootherstudieson regulation ofsperm solution, RS). ortothe same solution without ATP(wash
flagellarmotility in Ciona(Brokaw, 1987). A preliminary solution. WS). The tissue pieces were then mounted on
report of this work has appeared (Bergles and Tamm, perfusion slides for observations. Extraction and reacti-
1989). vation of living pieces mounted on perfusion slides was
not feasible due to distortion of the tissue by muscular
Materials and Methods contractions induced by ES.
The effects of ions or reagents on ciliary reactivation
Organism were estimated as follows (Table I). Twenty-five to 100
Specimens of Ciona intestinalis (5-10 cm long) were stigmata with clearly observable cilia were observed for
obtained from Marine Resources at the Marine Biological each tissue piece. A rating of++++ indicates that >95%
Laboratory and kept in baskets immersed in runningsea- ofthecilia in each stigmawerebeatingvigorously. Ratings
water,orweresimplyremoved from thesidesoflaboratory of+++, ++. and + indicate that 75-95%, 50-75%., and
ssepae-ctiambleenssiwnerwehiucshedthweiythhiandaswetetelkedaaftnedr rgreomwonv.alCfiroonma t1i5ve-l5y,0%tyopfictahlelycilaitadienceraeacshinsgtigfmreaquweenrceiebse.atAingr,atriensgpeco-f
their substrate, because the branchial basket often dete- indicates that 5-15% of the cilia were active, and a
riorated after the animals were detached. negative ratingindicatesthat <5%werebeating. Alltreat-
mentswere repeatedonat least fivedifferentpreparations
ofbranchial basket pieces.
Perfusion slides oflivingstigmata! cilia
Ciona were pinned laterally against a Sylgard-coated Reagents andsolutions
dish, and the tunic was sliced open and pulled back. The
branchial cavity was opened by cutting longitudinally Calmidazolium and norepinephrine were obtained
alongthe length oftheendostyle, then acrossbetween the from Calbiochem-BehringCorp. (San Diego, California):
twosiphons. The branchial basketwasremovedbycutting sodium metavanadate was obtained from Mallinckrodt
the many trabeculae connectingthe baskettothemantle. Inc. (St. Louis, Missouri). Calmodulin (bovine brain),
Theexcisedbranchial basketwaspinned flatin aSylgard- cAMP (bovine brain), protein kinase catalytic subunit
lined petri dish of normal seawater and kept on ice at (bovineheart), and all otherchemicalswereobtained from
0C. Prior to experimentation, the branchial basket was Sigma Chemical Co. (St. Louis, Missouri). Ca-EGTA
384 D. BERGLES AND S. TAMM
B
BEATING ARREST INACTIVE
QUIESCENT
Figure 1A-C. Diagram ofthreedifferentstatesofCiomistigmatal cilia, as viewed in crosssection ofa
stigmatoshowciliaryprofiles. Forclarity,onlyoneoftheciliaarisingfromthesevenciliatedcellsisshown.
Thebranchialcavity(P)istotheleft,theatrial(cloacal)cavity(A) istotheright inall figures. Basedonour
observationsandTakahashi elal. (1973), Mackieelal. (1974),andArkett(1987). (A)Activeciliabeatwith
theeffective strokedirected towards theatrial chamber(arrows), propellingwaterout ofthe pharynx. The
recovery strokeoccurs in three dimensions, out ofthe plane ofthe powerstroke. Metachronal waves (not
shown) travel at right angles to the effective stroke. (B) Arrested cilia lie flat against the stigmatal walls,
inclinedinaposturebeyond theendoftherecoverystroke(stigmataopen).(C)Followingarrest,ciliastand
upright in an inactiveposture(stigmaclosed)beforeactive beatingresumes. Quiescentciliaalsoremain in
thisposition.
buffers were prepared according to Salmon and Segall previously (MacGinitie. 1939; Takahashi et al.. 1973;
(1980). Mackieetal.. 1974;Arkett. 1987),andarereviewedbriefly
here.
Light microscopy
Perfusion slides were viewed with Zeiss brightfield or The branchial slits or stigmata are lined with seven
jpehcatsieve-sc)o,ntarnadstiompatgiecss(w1e6rXe/0r.e4c0orNdeAdowrit4h0Xa/0D.A75GENA67obM- rroowwsofofcillaitae(rFailgl.y 1f)l.atTtheenedcilciealltse,d ecealclhs abreearsitnagckaedsisnigdle-e
videocamera(Dage-MTI, Michigan City, Indiana46360) by-side in clusters that abut end-to-end, formingacon-
on a VHS videocasette recorder allowing still-field play- tinuous ciliated band around the inside of a stigma.
back (GYYR model 2051, Anaheim, California 92802). Neurons run within the blood sinus and make synaptic
Beat frequency was determined by repetitive counting of contacts onto the bases ofthe ciliated cells, which are
the number ofvideo fields (1/60 s) per beat cycle. Pho- coupled by gap junctions (Mackie el al., 1974; Arkett
tographs ofstill-fields from a video monitor were taken et ul.. 1989).
with an Olympus OM-2N camera on Kodak Tech Pan The stigmatal cilia beat outward from the branchial
(2415)35 mm film. cavity towards the atrial cavity, generatinga watercurrent
that enters the branchial (incurrent) siphon, passes
Results
through the branchial basket, and flows out the atrial si-
Stigmatal (///(,'] system phon (Fig. 1A). Ciliary beating is coordinated into dex-
The anatomy and motility ofthe stigmatal ciliary sys- ioplectic metachronal waves that travel unidirectionally
tem of dona and other ascidians have been described around the stigmatal openings (Fig. 2A).
CONTROL OF CIOKA CILIA 385
In responseto mechanical, electrical, orchemical stim-
ulation, all the cilia lininga stigma perform a single rapid
reverse stroke and lie flat against the stigmatal walls for
1-2 s in an arrest position inclined beyond the end ofthe
normal recover, stroke (Fig. IB). Ciliary arrest haltswater
flow into the pharynx and leavesthe stigmata completely
open, allowing muscular contractions to "squirt" water
out ofthe branchial siphon.
Followingan arrest, theciliagradually risetoan upright
position,closingthestigmatal opening(Fig. 1C). Thecilia
remain in this straight "inactive" state for a few seconds
before beating resumes and normal metachrony is re-es-
tablished.
In both excised piecesofbranchial basket and exposed
intact baskets, some stigmata are always observed with
cilia that stand upright in an "inactive" posture for long
periods. Itisnot knownwhetherthislong-lastinginactive
state was, in any case, preceded by an arrest; but a direct
transition from beatingto the inactive position has never
been reported (Takahashieffl/., 1973; Mackie et a!.. 1974:
Arkett, 1987). Upon stimulation, inactive cilia as well as
beating cilia perform an arrest response together (Taka-
hashi et ui. 1973: Mackie et a/.. 1974).
Effects ofcalcium ionophore Figure2. Caicnophore-inducedarrestofstigmatalcilia.(A)Insea-
Perfusion of pieces of branchial basket with 100 nM wwaaltler(,amrertowahcehardo)n.al(Bw)avPeersfousficoinliaorfyAac2t3i1vi8t7yairneseevaiwdaetnetro+nt1h0e0stmi.gmUatCaal
A23187 in normal seawater for 15-30 min had no no- causesciliary arrest(arrowhead). Video prints; scalebar, 20Mm.
ticeable effect on stigmatal ciliaryactivity. Addition of50
m\lCaCl toboth thebath and the ionophore suspension
:
caused most of the cilia to assume an upright inactive cAMP) or N6-monobutyryl-cAMP (M-cAMP) in normal
position (stigmata closed) within 5 s after perfusion of seawater caused many ofthe quiescent cilia to beat with
ionophore. Cilia remained in this posture for as long as normal metachronal coordination within 1-3 min. In
observed (up to 5 min). The addition of 100 m.U CaCl: most cases, more than 40% ofthe cilia became active. A
to the bath and ionophore suspension resulted in stigma- greater number ofquiescent cilia became active in stig-
wide ciliary arrests throughout the field ofview within 5 mata where some ofthe cilia were already beating prior
sleasfsttehrapner5f-u1sios,naoffteAr2w3h1i8ch7t(hFeigc.il2i)a.mCoilvieadrytoartrheestisnalcatsitevde atoctipveartfiuosnioonfcwiiltiahwBa-scoAbMsePrvoerdMaf-tcerApMePr.fusNioonsoifgntihfeicsaonlt-
position and remained upright for 15-30 s before resum- vent carrier (0.1% ethanol) in seawater without cAMP
ing beating. When larger pieces ofbranchial basket were
analogues.
used, arrests were accompanied by vigorous muscular
contractions.
Perfusion of normal seawater containing 50 mAI or Cell models
100 m.U Ca without A23187 but with solvent (0.1% Extraction and reactivation. Treatment of pieces of
ethanol/DMSO) did not elicit arrests or inactivations. branchial basket in ES for4-9 min stopped moststigmatal
cAMP ciliain amoreorless upright position. Theciliaprojected
Membrane-permeant analogues astufts from clusters ofswollen stigmatal cells, and there
To investigate the possible role of cAMP-regulated weregapsbetween theciliary tuftsofadjacent cellgroups.
WS
processes (i.e.. activation ofPKA) in stigmatal ciliary re- Transferoftissue to did not activatebeating; thecilia
sponses, we applied membrane-permeant cAMP ana- remained in arelatively upright posture(Fig. 3A). Shorter
logues to branchial basket pieces on perfusion slides. extraction times (2-3 min) resulted in very slow ciliary
We directed our attention to stigmata where most of beating (<2 Hz) in WS; the cilia usually stopped in the
the cilia were standing upright in a long-lasting inactive inactive position within 5-10 min. Thin-section electron
state. Perfusion of 1-10 mM N6-benzoyl-cAMP (B- microscopy oftissue extracted for 7 min showed partial
386 D BERGLES AND S. TAMM
or complete removal ofciliary membranes from most of reactivation wasconsistentlybetterinsolutionscontaining
the stigmatal cilia, while non-extracted tissue prepared by KC1 rather than K acetate (Table I).
the same procedure had intact ciliary membranes (not Calinociiilin. Addition of calmodulin antagonists, 100
shown). \iM trifluoperazine (TFP) or 100 nAl calmidazolium to
Transfer ofextracted branchial basket tissue to RS re- RS + 50-100fiAlCarestoredreactivatedbeating,thereby
sulted in vigorousbeatingof50-70% ofthestigmatal cilia. reversing Ca-dependent inactivation (Table I). TFP typ-
Ciliary reactivation typically lasted more than 45 min. ically gave more consistent results than did calmidazo-
Beatingsometimesdislodged ordisplaced theciliated cells lium. Chlorpromazine (100 ^M) did not significantly re-
from the fragile stigmatal wall, causing them to "swim" verse the inactivation ofcilia in RS + Ca. These results
through the solution. Normal metachronal waves were indicate that calmodulin mediates Ca-dependent inacti-
not present. Separated tufts of cilia often beat indepen- vation ofstigmatal cilia.
dently, and displayed unicellular metachrony as reported Addition of65 /ug/ml bovine brain calmodulin to RS
for reactivated lateral cilia on separated cell groups of + 100 n.M Ca did not elicit an arrest of cilia in tissue
Modiohts demissus gills (Child and Tamm. 1963). initially bathed in WS or RS (Table I).
Reactivated beatingwasalso observed by perfusing RS
through a slide ofextracted branchial basket in WS (Fig. cAMP and PK.4
f3iBr)st.wRiSthpaerrfeusstiroinctecdaursaendgethoef cmioltiaiotno,btehaetnhreaspitiadnltylyanadt RS + 100 nM cAMP and 1 m.U IBMX. a cyclic nu-
fully within 10-20 s. Reactivated cilia often reached a cleotide phosphodiesterase inhibitor substantially in-
s1t4eaHdzy)-.stLaotnegfrsterqeutecnhceysosfimciilliaarstoomethtaitmeofslbievaitngnecaelrllsy(s1y0n-- cvraetaisonedcboomtphartehde tporotpiosrsuteioinncaunbdatveidgoirn oRfScaillioanrey (reTaacbtlie-
chrIoaniohuisdlaylctoinflioirhimticono.mRmSon+w2a0veffirMonvtasna(dFaigt.e.3Ba).potent rI)e.acTthiuvasticonA.MIPn RaSndcoCnataeixneirntgo1p0p0osninMgceAffMecPts+on10c0ilinarMy
inhibitor ofdynein ATPase (Gibbons el a/., 1978), did Ca, cilia vibrated or twitched rapidly in a rigid inactive
not reactivate ciliary beating. Norepinephrine (5 mA/) in position. Addition of28 Mg/ml catalytic subunit ofPK.A
RS + vanadate restored reactivated beating, reversing to RS likewise improved theextent ofciliary reactivation
vanadate inhibition ofmotility (Table I). to more than 95% in most cases (Table I).
Ca sensitivity. ES-treated pieces of branchial basket
showed normal reactivatioMn ofciliary beatingwhen placed Discussion
in wells of 10 7 to 10 b frMee Ca (Ca-EGTA buffer). Calcium andarrest
However, RS + 10~5 to 10 3 Ca caused the majority
ofcilia to assume an upright inactive posture, but never The role of Ca in triggering a variety of ciliary and
an arrest position (Table I). A gradual decrease in Ca sen- flagellar motor responses iswell documented (Eckert and
sitivity forelicitinginactivation wasobserved with longer Murakami, 1972; Naitoh and Kaneko, 1972; Tsuchiya,
times in ES; extraction times of more than 9 min often 1977; Hyams and Borisy. 1978; Walter and Satir. 1978;
yielded cilia that did not inactivate in response to Ca. Gibbons and Gibbons, 1980; Brokaw and Nagayama,
To check whether a transient Ca-induced arrest re- 1985;NakamuraandTamm, 1985; Satir, 1985;Stommel
sponse might have been missed in depression wells, RS and Stephens. 1985: Machemer, 1986: Brokaw. 1987,
+ 10 4to 10 3 A/Cawas perfused into a slide containing 1991; Tamm. 1988; Otter, 1989).
a piece ofextracted branchial basket in WS. No momen- Although arrest ofstigmatal cilia in ascidians has long
taryarrest responsewasobserved beforetheciliaassumed been suspected to be Ca-dependent (Takahashi el a/..
a rigidly straight inactive position. 1973; Mackie el ai, 1974). direct evidence for this has
Various approaches were tried to elicit arrests in RS been lacking. Our finding that Ca ionophore in the pres-
+ 10 5 to 10 3 A/Ca. Forexample, a cocktail ofprotease ence of 100 m.U Ca elicits arrest ofdona stigmatal cilia
inhibitors ( 1 mg/ml trypsin inhibitor; 0.5 mg/ml leupep- strongly argues for the Ca-dependency ofthis response.
tin: I mA/TAME; 0.2 mg/ml PMSF. 1 mg/ml BSA)were However, these experiments were performed on piecesof
included in both ESand RSto prevent possible proteolysis branchial basket tissue, and stigmatal cilia have been
of putative Ca sensors or proteins required to mediate shown to be under neuronal control (Mackie et a/.. 1974;
arrest. Differentdetergents(Brij-58, Brij-35, Triton-X 100) Arkett, 1987). Therefore, our results could also be ex-
were tried in place ofsaponin to preclude the extraction plained byionophore-mediated influx ofCaat presynaptic
ofCa-binding proteins(i.e.. calmodulin). In anotherseries sites mediating nervous control ofciliary arrest, without
ofexperiments, Kacetatewassubstituted for KC1 in both requiring Ca influx into the ciliated cells themselves.
ES and RS. None ot thMese modifications resulted in ciliary To directly test whether ciliary motility in ascidians is
arrests in RS + 10 J Ca. We did note, however, that regulated by Ca, we prepared the first ATP-reactivated
CONTROL OF CIONA CILIA 387
IB.
Figure 3. Stigmatal cell models. Cilia lining a stigma are shown on the left and diagrammed on the
right.(A)A singlestigmainWS. Tuftsofimmotilecilia(black)stand upright, projectingfromtherefractile
wall ofthestigma intotheStigmatal space. In thediagram, the wall (edge)ofthestigma is indicated bythe
irregular horizontal lines. (B) The same stigma after perfusion ofRS. The cilia beat vigorously and, to a
largeextent,synchronously,givingrisetocommonwavefronts(arrowheads). Asaresult,thestigmaismore
open than in A. TheStigmatalwalldoesnotchange(compareoutlinesofstigma indiagrams). Scalebar. 1(1
fim.A. B
modelsofStigmatal ciliated cells. Wewere unabletoelicit However, our attempts to restore presumed Ca sensi-
ciliary arrest in oursaponin-permeabilized modelsat any tivity ofciliary arrest in models by trying different deter-
Caconcentration used(10 5 to 10 \UCain RS). Instead, gents, extraction times, protease inhibitors, oraddition of
the axonemes stopped in an upright inactive position exogenous bovine brain calmodulin, were uniformly un-
without passing through an arrest. successful. Nevertheless, the variable sensitivity ofthe in-
We were concerned that our permeabilization proce- activation response of models to Ca, particularly after
dure, or possibly subsequent proteolysis, might have re- longerextraction times(more than 9 min in ES), suggests
moved ordestroyed critical control factors orCa-binding that the absence ofCa-sensitive arrest in our models may
proteins (i.e.. calmodulin) necessary for demonstrating be due to loss or modification ofan as yet unidentified
Ca-sensitivity ofarrest in cell models. For example, ex- factor.
traction ofcalmodulin from sea urchin sperm and protist
cilia leads to modification or loss ofCa control ofaxo- Calcium and inaclivalion
nemal motor responses (Brokaw and Nagayama, 1985; The upright inactive postureofStigmatal cilia isclearly
Izumi and Miki-Noumura. 1985; Izumi and Nakaoka, Ca-dependent: reactivated cilia are inactivated by 10~5 to
M
1987). Extraction and incubation procedures may also 10 3 Ca, and cilia on living tissue are inactivated by
modify the calmodulin-binding affinity of the axoneme Ca ionophore in thepresenceofalowerCaconcentration
(Brokaw. 1991). In addition, some detergents commonly (50 mA/)than that leadingtoarrest ( 100 mM. seeabove).
used to make cell models (i.e., Triton-X 100) are potent These findings suggest that inactivation has a lower
inhibitorsofboth calmodulin and calmodulin-dependent threshold to intracellular Ca than does arrest.
cyclic nucleotide phosphodiesterase (Sharma and Wang, Inactive cilia ofcell models in RS -f- Ca were induced
1981). to beat by the addition ofa calmodulin antagonist, either
388 D. BERGLES AND S. TAMM
Table I state, or frequency minimum, also intervenes between
/:"//('i7v ni nirn>n\ compounds un rcactivatum ofciliarymotility normal beating and the onset ofstimulus-induced ciliary
in<c reversal. In Paramecium, atransient inactivation response
therefore precedes and follows the Ca-dependent ciliary
Ciliary reversal response, suggesting that inactivation may be
Solution activity caused by aCaconcentration slightlyelevated above nor-
WS mal restinglevel(Machemer, 1986). Thetransient inactive
RS(KCI)* ++(+) state following the arrest of dona cilia may also reflect
RS(K. Acetate) ++ an intermediate level of internal Ca concentration. The
RRSS ++ 2200nfi.M\lVVaannaaddaattee + 5 m.\INorepinephnne +++ epaulette cilia ofechinoplutei larvae sometimes undergo
RS + 10//;UCa:+ + asimilaruprightinactivestateafterCa-dependent reversed
RS + 50 beating (Mogami ct a/.. 1991).
RS+ Finally, a recent study of Ca-induced asymmetry of
RS + lOOfiA/Ca2* + Pis ATP-reactivated flagellar bending waves of sea urchin
RRSS ++ 110000fniMMCCaa2:++ ++ 110000Mp.MUTCrailfmliudoapezroalziiunme +++++ sperm indicatestheexistenceoftwoseparateCa responses,
RS + 100nMCa2+ + 100rflChlorpromazine mediated by high-affinity and lower-affinity Ca sensors
RS + 100n.MCa:+ + Calmodulin (65 Mg/ml) (Brokaw. 1991).
RS + 100/u.UcAMP + mMIBMX ++++
RS + 10011MCa:+ + 1010pMcAMP - vibrating cAMPandquiescence
RS + PKAcs(28 ++++ Cyclic nucleotides (cAMP, cGMP) also play a role in
* Standard RS used below. regulating ciliary and flagellar motility: in particular.
Plusandminusratingsindicaterelativedegreesofciliaryactivity(see cAMP is typically involved in initiating and maintaining
Materialsand Methods); minus indicates that nonbeatingcilia were in ciliary and flagellar beating (Opresko and Brokaw, 1983;
an upright inactiveposition. Stommel and Stephens. 1985; Takahashi ct ul.. 1985:
Abbreviations: WS, wash solution; RS, reactivationsolution; IBMX, Murofushi ct ul.. 1986; Brokaw, 1987; Murakami,
isobutylmethyKanthine;Pis.proteaseinhibitors;PKAcs.catalyticsubunit
ofprotein kinase. 1987a,b: Stephens and Stommel. 1989; Tash. 1989; Bo-
nini ci til.. 1991).
Neuronalactivation ofquiescent lateral cilia on Mytilus
TFPorcalmidazolium. Thissuggests that Ca-induced in- gill is due to 5 HT-triggered augmentation of cellular
activation of dona stigmatal cilia is mediated by cal- cAMPlevels, leadingtocAMP-dependent protein kinase-
modulin. Other Ca-dcpcndcnt ciliary motor responses, mediated phosphorylation of axonemal dynein light
such asarrest ofmussel gill lateral cilia (Reed el a/.. 1982; chains (Stephens and Prior. 1990). Quiescence oflateral
Stommel, 1984), activation ofMytilux gill abfrontal cilia cilia is thus believed to reflect lowered cAMP concentra-
(Stommel, 1984), and reorientation ofciliary beat direc- tion and resultant dephosphorylation ofdynein polypep-
tion in some cell models of Pcirdiuec/uni (Otter ct til.. tides. Phosphorylation ofaxonemal dynein polypeptides
1984; Izumi and Nakaoka, 1987) and Tetrahymcna has also been reported in other systems (Hamasaki and
(Izumi and Miki-Noumura, 1985), are also partially or Satir, 1989; ChilcoteandJohnson. 1990; Deyand Brokaw
completely inhibited by anti-calmodulin drugs. 1991; Stephens and Prior, 1991 ).
Ca may exert itseffectson ciliary motility byactivating We found that membrane-permeant cAMP analogs
calmodulin-dependent protein kinase (C kinase) orphos- stimulate the beating of Cioiui stigmatal cilia held in a
phatase (calcineurin), thus changingthe phosphorylation long-lasting inactive state (termed quiescence). Moreover,
levels ofaxonemal regulatory proteins (Nakaoka and Ooi, reactivated beatingofstigmatal ciliary models is improved
1985; Tash, 1989; Hamasaki ct til.. 1989; Bonini ct til.. by cAMPand IBMX, ortheaddition ofthecatalytic sub-
1991). Because the catalytic subunit of PICA did not in- unit ofprotein kinase to RS. Reactivation ofMytilus lat-
activate reactivated dona stigmatal cilia, but rather en- eral ciliary models is also improved by the presence of
hanced motility, the mechanism by which Ca-calmodulin thecatalytic subunit. which can overrideCaarrest (Stom-
is presumed to inactivate stigmatal cilia may involve a mel, 1984; Stommel and Stephens, 1985; Stephens and
dephosphorylation reaction. Stommel, 1989). These findings suggest that the quies-
The transient inactive state exhibited by dona cilia cence (long-lasting inactivation) ofdona cilia is physio-
after every arrest resembles the transient inactivation of logically similar to the quiescence ofMyti/nslateral cilia;
Paramecium cilia that occursafterdepolarization-induced i.e.. that increased cAMP levels may also be responsible
Ca-dependent reversal ofbeat direction, before the beat for maintaining the activity of dona stigmatal cilia via
cycle is renormalized (Machemer, 1986). Voltage-clamp cAMP-dependent phosphorylation of regulatory axone-
experiments with Puninwciwn showed that an inactive mal polypeptides.
CONTROL OF CIONA CILIA 389
Although their underlying biochemical mechanisms andcyclicnucleotides. Pp.227-272inAdvancesinSecondMessenger
seem tobesimilar,theposturesofquiescent Myliluslateral aisnodn.Pehdoss.pRhaovpeminePirnesRse,sNeaerwch,Yovrokl.23. P.GreengardandG. A. Rob-
cilia and quiescent Ciona sigmatal cilia arequite different. Brokaw,C.J. 1979. Calcium-inducedasymmetricalbeatingofTriton-
Lateralciliarestattheendoftherecoverystroke, whereas demembranated seaurchinsperm flagella.J CellBiol. 82:401-41 1.
stigmatal ciliastand upright, midwaybetween theeffective Brokaw,C.J. 1987. Regulationofspermflagellarmotilitybycalcium
and recovery strokes. and cAMP-dependent phosphorylation. / Cell Biochem. 35: 175-
Ciona cilia thus remain upright during both the tran- 184.
sient inactive state and the quiescent or long-lasting in- Brokaw,C.J. 1991. Calciumsensorsinseaurchinsperm flagella. Cell
Moid. Cywskei 18: 123-130.
active state. However, quiescence differs from inactiva- Brokaw,C.J.,R.Josslin,and L.Bobrow. 1974. Calciumionregulation
tion. not only by its longer duration, but also by the ap- offlagellar beat symmetry in reactivated sea urchin spermatozoa.
parent absence ofa preceding arrest. Biochem. Biopln-s. Re*. Commim. 58: 795-800.
cAMP and Ca act antagonistically on Mytilus lateral Brokaw, C. J., and S. M. Nagayama. 1985. Modulation ofthe asym-
cilia, aswellasonseveralotherciliaryand flagellarsystems metryofsea urchin sperm flagellarby calmodulin. / CellBiol 111(1:
(elseael.B.r1o9k9a1w),. M1o9r8e7:ovSetre.phceAnsMaPndorSctAoMmPm-edle.p1e9n8d9e;nBtonpirnoi- Cartn1eur8,d7i5Gb-.r1a8Sn8.c3h.19v2e6li.ger.O/n Etxhpe.nBeirovlo.u4s:c1o-n2t6r.ol ofthe velar cilia ofthe
tein kinasecan overridetheCaeffectand activatebeating Chilcote, T. J., and K. A. Johnson. 1990. Phosphorylation of Tetra-
of Ca-arrested cilia (Murakami and Takahashi, 1975: hymena 22 Sdynem. ./ Biol. Chem 265: 17257-17266.
Murakami. 1983: Stommel and Stephens, 1985). Our Child,F.M.,andS.I,.Tamm. 1963. Metachronalciliarycoordination
finding, that adding Ca and cAMP to RS causes rapid in ATP-reactivated models ofModiolus gills. Biol Bull 25: 373-
374.
ivniabcrtaitvieonpoosrititowni,tcihnidnigcatoefssatisgimmaitlaalracniltiaagoinnitshmebsettrwaiegehnt Dey,phCo.spS.h,oarynldaCt.ioJn.oBfrdoyknaewi.n1p9o9l1y.peptAicdteisvaatnidonefoffecCtisoonfaastpyerorsminmeotkiilniatsy:e
cAMP-mediated activation of beating and Ca-induced inhibitor./ CellSci. 100: 815-824.
inactivation (rather than arrest) ofcilia. Eckert, R., and A. Murakami. 1972. Calcium dependence ofciliary
In conclusion, we have developed an in vitro prepara- activity in the oviduct ofthe salamanderNectiirus. J. Physiol. 226:
tion of Ciona branchial basket cilia which allows inves- 699-711.
tigationforthefirsttimeoftheionicand molecularcontrol Eckeerlte,ctrRi.c,aVn.dNmaoittoohr,faunndctHio.nsMaocfhPeamreamre.ci1i9i7m6..SyCmapl.ciSuocm.iEnxpth.eBbiiolo.-
ofciliary motility in tunicates. Further studies using im- 30: 233-255.
proved models should provide more detailed and quan- Fedele,M. 1923. Leattivitadinamicheedirapportinervosi nellavita
titative information for comparison to other systems. dei Dolioli. Puhl. Sla:. Zoo/. Napoli4: 129-239.
Gibbons, I. R., M. P. Cosson, J. A. Evans, B. H. Gibbons. B. Houck.
Acknowledgments K. H. Martinson. \V. S. Sale, and \\.-J. V. Tang. 1978. Potent
inhibitionofdyneinadenosinetnphosphataseandofthemotilityot
We thank Dr. Ray Stephens, MBL, for helpful discus- ciliaandsperm flagellaby vanadate. Proc. Nail. Acad. Sci. USA 75:
sions and advice, and Signhild Tamm for electron mi- 2220-2224.
croscopy. Dorothy Hahn patientlyandskillfully processed Gibbons,B.H.,and I.R.Gibbons. 1980. Calcium-inducedquiescence
these words. This research fulfilled the requirements for in reactivated sea urchin sperm. J CellBiol 84: 13-27.
IUnnidveepreGsniMdteynMtaWrionrekPorfogDriasmti,ncatnidonwabsy sDuBppoartttehdebByosNtIoHn HampaChesolaslkMpioh,toirTly..,laTCt.yitJoo.nsMkoeuflr.Ptaa1ru2ag:mhe1,c-1iB1.i.iHm.aSxatoinr,emaensdPa.ndSatpierr.me1a9b8i9l.izeIdncveiltlrs.o
grant 27903 to SLT. Hamasaki, T., and P. Satir. 1989. Parameciiim dynein contains a
phosphorylatable29kDalightchain.CellMotil. Cytoskel. 11: I89A.
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