519Commentary

IntroductionFlagella (and cilia) are organelles of eukaryotic cells that producemotility by repetitive episodes of bending. Flagella and cilia arefunctional in diverse cell types: the beating of cilia in the bronchiof the lungs keeps airways clear of mucus and debris; theflagellum of a sperm cell propels the cell to the egg and is anessential step in the life cycle of humans and most complexorganisms; and cilia and flagella are used for feeding andreproduction in animals and plants as diverse as clams and algae.The cycle of bending and bend propagation in a cilium or flagellumis called the flagellar beat, and is the subject of this Commentary.

Cilia and flagella are two forms of the same cell organelle.Historically, they were called flagella when present singly or in pairs,and called cilia when many were present. They both originatethrough the assembly of proteins onto a centriole-like basal bodyand they have the same internal arrangement of microtubules, motorproteins and accessory structures.

We already know a great deal about the inner workings of aflagellum. The internal cytoskeletal arrangement of a flagellum iscomposed of nine doublet microtubules in a ring surrounding a pairof single microtubules [the central pair (CP)]; these structurescollectively compose the axoneme. This structural arrangement isillustrated in Fig. 1. Each of the outer doublets is linked to itsneighbors by strands of protein called the nexin links. Each doubletalso has a series of projections, called the radial spokes, that seemto act as spacers to position the doublets in a circle around the centralpair of microtubules. The CP itself is enclosed in a sheath of proteinsthat form a series of projections that are well positioned to interactwith each of the spoke heads. The spokes in turn are anchored ontoeach outer doublet near a complex of proteins called the dyneinregulatory complex (DRC), which in turn is in close contact withthe inner row of dynein arms. Both the spokes and the DRC are

known to contain calcium-binding proteins (centrin in the DRC andcalmodulin in the spokes) and all cilia and flagella respond to freecalcium by altering the beating pattern.

In 1965, Gibbons and Rowe identified dynein, the major proteinof the arm-like projections found on each of the nine doublettubules of the axoneme, as the provider of molecular motive forcefor the flagellar beat (Gibbons and Rowe, 1965). The base of theflagellum, which usually terminates in a centriole-like basal bodyor in the connecting piece in mammalian sperm, anchors thedoublets. The nexin links are elastic and resist the free sliding ofthe microtubules with respect to each other. Summers and Gibbonsshowed that sea urchin flagella broken off at their base andsubjected to brief tryptic digestion to cut the nexin links wouldslide apart in the presence of Mg-ATP (Summers and Gibbons,1971). Therefore, the basic movement-inducing interaction in theflagellar structure is the sliding of the microtubule doublets alongeach other’s length as a result of the action of dynein in the presenceof Mg-ATP. This sliding is converted to bending through therestraining influence of the basal anchor and the nexin links, as wasfirst demonstrated by the modeling work of Brokaw (Brokaw, 1971;Brokaw, 1972a; Brokaw, 1972b).

Sale and Satir showed that the direction of sliding is uniformaround the axoneme, with the dyneins of each doublettranslocating that doublet base-ward by acting on the neighboringdoublet (Sale and Satir, 1977). Therefore, the dyneins on one sideof the axoneme tend to bend the flagellum in one direction of thebeat cycle, and the dyneins on the opposite side contribute tobending in the opposite direction. In most flagella and cilia, thedoublets numbered 5 and 6 in the standard numbering conventionare permanently linked to one another and cannot slide relativeto each other (Afzelius, 1959), and the CP is positionedperpendicularly to the major plane of the beat (Gibbons, 1961;

Flagellar and ciliary beating: the proven andthe possibleCharles B. Lindemann* and Kathleen A. LesichDepartment of Biological Sciences, Oakland University, Rochester, MI 48309, USA*Author for correspondence (lindeman@oakland.edu)

Journal of Cell Science 123, 519-528© 2010. Published by The Company of Biologists Ltddoi:10.1242/jcs.051326

SummaryThe working mechanism of the eukaryotic flagellar axoneme remains one of nature’s most enduring puzzles. The basic mechanicaloperation of the axoneme is now a story that is fairly complete; however, the mechanism for coordinating the action of the dyneinmotor proteins to produce beating is still controversial. Although a full grasp of the dynein switching mechanism remains elusive,recent experimental reports provide new insights that might finally disclose the secrets of the beating mechanism: the special role ofthe inner dynein arms, especially dynein I1 and the dynein regulatory complex, the importance of the dynein microtubule-bindingaffinity at the stalk, and the role of bending in the selection of the active dynein group have all been implicated by major new evidence.This Commentary considers this new evidence in the context of various hypotheses of how axonemal dynein coordination might work.

This article is part of a Minifocus on cilia and flagella. For further reading, please see related articles: ‘The primary cilium at a glance’ by PeterSatir et al. (J. Cell Sci. 123, 499-503), ‘Sensory reception is an attribute of both primary cilia and motile cilia’ by Robert A. Bloodgood (J. Cell Sci.123, 505-509), ‘The perennial organelle: assembly and disassembly of the primary cilium’ by E. Scott Seeley and Maxence V. Nachury (J. Cell Sci. 123,511-518) and ‘Molecular mechanisms of protein and lipid targeting to ciliary membranes’ by Brian T. Emmer et al. (J. Cell Sci. 123, 529-536).

Key words: Dynein, axoneme, Geometric clutch, Radial spokes, Central pair, Hydin, Model

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Afzelius, 1961). These structures provide landmarks that definethe beat plane, particularly in metazoan flagella. Doublet 1, whichis located approximately 90 degrees to the plane of the CP on oneside of the axoneme, is the first of the group of doublets 1-4 whosedyneins bend the axoneme in one direction of the beat cycle. Theopposing group are the dyneins on doublets 6-9, which bendthe flagellum in the opposite direction. Satir (Satir, 1985; Satir,1989; Satir and Matsuoka, 1989) proposed that the beat consistsof alternate episodes of activation of the two opposing dynein-bridge sets, which is regulated by some means of switching oneset ‘on’ and the other set ‘off’ at appropriate mechanical set points.This was postulated as the ‘switch-point’ hypothesis and isgenerally believed to be a valid summary of the known data intoan orderly picture of the events in the beat.

Naturally, the next question that needs to be resolved is: whatcontrols the switching of activation of the two dynein-bridge sets?We know that the control must reside within the axoneme. Brokawdemonstrated that a flagellum removed from an intact cell wascapable of autonomous functioning (Brokaw, 1961). Later, we(Lindemann and Rikmenspoel, 1972a; Lindemann andRikmenspoel, 1972b) showed that isolated pieces of flagellum retainthe capacity for bend propagation and sustained rhythmic beatingif supplied with Mg-ATP and ADP. A fascinating observation ofour early studies was that cut pieces of flagella lost coordinationand stopped beating, but could be restarted by bending the flagellumwith a microprobe (Lindemann and Rikmenspoel, 1972a). Thisobservation seems to be a universal characteristic of the axoneme(Okuno and Hiramoto, 1976; Omoto et al., 1996; Hayashibe et al.,1997).

In this Commentary, we examine some of the ideas that havebeen advanced to explain how the flagellar axoneme works as anorganelle of motility. Rather than providing a general review of thesubject, we will focus on recent experimental findings that lendnew insight into the nature of the beating mechanism.

Competing views on how switchingis controlledCurvature controlThe first mechanistic proposal for the control of switching was the‘curvature control’ hypothesis of Brokaw (Brokaw, 1971; Brokaw,1972a; Brokaw, 1972b). Curvature is a mechanical parameter of theaxoneme. The curvature control hypothesis maintains that, whenthe flagellum bends to a sufficient curvature, it triggers theinactivation of one set of dyneins and the activation of the oppositeset. To make such a scheme work, a time delay is required betweenreaching the triggering curvature and switching the two sets ofmotors. Flagellar bending waves appear to maintain a nearlyconstant curvature, especially in very long flagella (>200 mm) suchas those of quail sperm (Woolley, 2007). However, Brokaw surmisedthat ‘simple curvature-controlled models are incompletely specified’to be able to account for many flagellar behaviors (Brokaw, 1985).

Cibert suggested a geometric mechanism that might serve as aregulator in a curvature control scheme. When a bend forms ona flagellum, the periodicity (spacing) of the motor proteins relativeto the periodicity of the binding sites on the adjacent doublets mustbe altered by curvature (Cibert, 2008). This periodicity relationshipmight serve as a cue for initiation and termination of dynein action.This is an interesting proposal, although no plausible mechanismhas yet been elaborated to link the periodicity relationship to thecontrol of dynein function. The geometric-clutch hypothesis, whichcan be thought of as a type of curvature control, is discussed indetail in the following section.

Geometric clutchThe ‘geometric clutch’ hypothesis (Lindemann, 1994a; Lindemann,1994b) is based on the mechanics of the axonemal scaffold. Theidea resulted from our studies of the behavior of wooden modelsof the axoneme (for details, see Lindemann, 2004). The hypothesisis predicated on the following idea: in an intact axoneme, motorproteins are positioned just far enough from their binding siteson the adjacent doublet that, when the flagellum is straight and atrest, the dynein heads have a very small, but finite, probability offorming a bridge to the next doublet. The probability of forming abridge increases as the interdoublet distance decreases. Thus, a bendin the flagellum stretches the nexin links that hold the nine outerdoublets in a ring and results in the development of a force transverseto the bend (the t-force) that squeezes certain doublets towards eachother. Moving the doublets together increases the likelihood that adynein bridge will be formed. This idea is illustrated in Fig. 2.

When dynein bridges attach in the presence of Mg-ATP, theytranslocate upon the adjacent doublet and apply a longitudinal forceto both of the doublets involved. The longitudinal force exertedacross the diameter of the axoneme provides the torque that isrequired to actively bend the flagellum, and generates a t-force thatprises the doublets apart. When this t-force component is sufficientlylarge, the dynein motors no longer function in a processive mannerbecause they are pulled away from their binding sites on the adjacentdoublet. This is the switching mechanism that, according to thegeometric-clutch hypothesis, terminates dynein action (Fig. 2).

The main component of the transverse force acting on theaxoneme is defined by multiplying the longitudinal force onthe active doublets by the local curvature (Lindemann, 1994a). Thus,in the geometric-clutch mechanism, curvature is a primary controlparameter in switching, and one can consider the geometric-clutchmechanism as a mechanistically more specified type of curvaturecontrol. This mechanism has the advantage over other

Journal of Cell Science 123 (4)

Outer doublets

Basal body

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Central pairprojection

Outer-doubletA subtubule 5–6 bridge

Radialspoke

Beatplane

DRC

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Outerdynein arm

Flagellaraxoneme

Fig. 1. Schematic diagram of the flagellar axoneme in cross-section.Structures that are discussed in this Commentary are highlighted. The detailsof the CP complex are based on the work of Mitchell (Mitchell, 2003a).Reprinted from Lindemann (Lindemann, 2007) with permission. DRC, dyneinregulatory complex.

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curvature control schemes in that switching can occur over a rangeof curvatures, depending on the amount of total tension on thedoublets. This allows considerable variation in the appearance ofthe beat, as is found in nature.

The switching mechanism of the geometric clutch utilizes thedynamic balance between t-force and dynein binding affinity.Consequently, such factors as the adhesiveness of the dynein stalksto their binding sites on the adjacent doublet will alter the switch-point (the point at which dynein bridges are inactivated). Also,because the switching mechanism depends on the total stress actingacross the axonemal scaffold, imposed external forces that stressthe axoneme will also alter the switch-point. Cibert and Heck havesuggested that, when the doublets are bent, they also experience atorsional force because they are not radially symmetric (Cibert andHeck, 2004). They suggested that the torsional force might play arole in breaking the dynein attachments and in terminating episodesof activity in a geometric-clutch-type stress-based regulatoryscheme. Stress-based models have the advantage that theyautomatically provide an explanation for the intrinsic mechanicalsensitivity of flagella and cilia.

CP-spoke axisThe most widely held conception of axonemal coordination is thatit is controlled through the CP-spoke apparatus. In the earliestversion of this hypothesis (Omoto and Kung, 1980), the CP acts asa rotor that, during its rotation, is the trigger for switching selectedsets of outer doublet pairs into action (as illustrated in Fig. 3A).There is evidence that, in some cilia and flagella, the CP does rotateduring the beat cycle (Omoto and Kung, 1979; Omoto and Kung,1980; Omoto and Witman, 1981; Kamiya et al., 1982). The mostdirect demonstration of the rotation of the CP was presented by

Mitchell (Mitchell, 2003b). In this report, the author looked at theorientation of the CP in Chlamydomonas flagella that had beenrapidly fixed to preserve the waveform of the beat. However,Mitchell and Nakatsugawa analyzed the mechanism of CP rotationand concluded that the CP is a helically curved structure inChlamydomonas, and that its orientation follows the beat, ratherthan being the driver for the beat (Mitchell and Nakatsugawa, 2004).

It is well established that the CP does not rotate in metazoans(Gibbons, 1961; Tamm and Horridge, 1970; Tamm and Tamm,1981; Sale, 1986), which have functional flagella and cilia. Thismakes the postulate that CP rotation is the main element of beatcoordination untenable as a universal mechanism. In mammaliansperm, the CP is rather solidly anchored in a partition that traversesthe axoneme from doublets 3 to 8. This partition is so sturdy thatit is often the last structure that remains intact when the axonemedisintegrates by partial digestion and ATP-induced sliding (Olsonand Linck, 1977; Lindemann et al., 1992; Kanous et al., 1993). Thisis most probably the case in other metazoan flagella as well, asshown in elastase digestion studies in sea urchin sperm (Shingyojiand Takahashi, 1995). In bull sperm, when flagellar motion of theproximal part of the flagellum is stalled by an obstacle, theunobstructed distal part of the flagellum continues to beat (Holcomb-Wygle et al., 1999; Schmitz et al., 2000). This is an impossibleresult in a CP-regulated beat cycle because the CP is continuousalong the length of the flagellum and cannot be both stopped androtating.

The major contributors to the rotating CP hypothesis alsoconcluded that there are many instances in which coordinatedmovement is produced without the CP. In a review in 1999, theystate: “Thus we propose that the nine outer doublets exhibit a defaultmovement in the absence of the central pair-radial spoke complex”(Omoto et al., 1999). Nonetheless, the idea that the spokes are theregulators of the beat cycle presently holds favor with manyresearchers in the field. This is because of the accumulated evidencefor spoke-mediated regulation of microtubule sliding (Wargo andSmith, 2003; Smith and Yang, 2004; Yang et al., 2006; Dymekand Smith, 2007).

A

B

Compression

Longitudinalforce

t-force actsas ‘off’ switch

t-force

Force transfer

Distension

Inactivedynein bridges

Activedynein bridges

Fig. 2. Geometric-clutch hypothesis. (A)The dynein ‘off’ switchingmechanism of the geometric-clutch hypothesis. Accumulated tension from theactive dyneins bends the flagellum and produces a transverse tension in thebent region. When the t-force felt by individual dyneins becomes large enoughto overcome the adhesive capacity of the dynein bridges, the doublets separateand dynein action is terminated. (B)Active bridges on one side of the axonemeexert a negative bias on the formation of active bridges on the opposite side ofthe axoneme. This effect is also enhanced by the spokes, which act as spacerelements.

A B

Nexin

DyneinSliding

Hydin

Radial spoke

Microtubules

Dynein

CaM on spoke

Sliding

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CProtation

Switch-point

Fig. 3. CP-spoke control hypotheses. (A)Control of dynein switching by arotating CP. Some elements of the CP complex, such as hydin, interact with aspecific spoke head to either activate or deactivate the dyneins on theassociated outer doublet, leading to creation of an active zone of doublets (inthis example, between doublets 7, 8 and 9) that bend the flagellum as the CProtates. (B)Alternatively, in flagella in which the CP does not rotate, shearbetween the CP and the outer doublet might act as a trigger to tilt the spokes ofthe doublet and initiate an episode of dynein engagement. Location of hydin,calmodulin (CaM) and the dynein regulatory complex (DRC) are indicated.

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Smith and Sale produced convincing evidence that the spokeapparatus can modulate doublet sliding and therefore might mediatecoordinated sliding, possibly through interaction with the CP(Smith and Sale, 1992). The action of the spokes in wild-typeChlamydomonas appears to be an inhibitory action that the paralyzedflagella (pf) suppressor mutations can override (Porter et al., 1992;Piperno et al., 1994). Nakano and colleagues provide similarevidence for an inhibitory action of the CP complex when associatedwith the spokes in sea-urchin sperm flagella, so the effect might beuniversal (Nakano et al., 2003). If so, the CP-spoke axis would keepthe doublet it controls inactive until some de-inhibiting mechanicalor chemical signal is produced, either through CP rotation or arelated mechanism.

The most recent candidate for a mechanical trigger is thephenomenon of spoke tilting. Spoke tilting is induced by interactionof the spoke heads with the CP, and occurs when shear is presentas a result of flagellar bending. It was proposed that this might bethe trigger mechanism that initiates a signalling pathway actingvia the kinase assemblage that resides on the spokes (Porter andSale, 2000; Smith and Yang, 2004; Yang et al., 2006). In theoverview of this control, interaction of the spoke heads with theCP projections [including, most importantly, hydin on C2 (Lechtreckand Witman, 2007)] leads to the activation of elements of this kinaseassembly that control the activation state of the dyneins on theassociated doublet. Consequently, this mechano-chemical view,which is illustrated in Fig. 3B, is now a contending mechanism forbeat control. In contrast to direct control through CP rotation, spoketilting incorporates a bending-dependent step in the control sequencethat is more compatible with the mechanical feedback that all ciliaand flagella are known to exhibit. It is an attractive and plausiblehypothesis in that it is consistent with the many studies (Pipernoet al., 1994; Piperno, 1995; Porter and Sale, 2004; Smith and Yang,2004; Yang et al., 2006; Yang et al., 2008; Lechtreck and Witman,2007) that implicate individual protein constituents on the CP,spokes and DRC as being essential to flagellar beating. In this view,beating depends on an enzymatic regulatory cascade that beginswith the spoke-CP interaction and sends a signal through the spoke-DRC linkage to the inner-arm dynein.

Dynein cross-bridge cycleOf the other mechanistic proposals to explain the beat cycle, themost noteworthy are those that can generally be grouped as beingdependent on the dynein cross-bridge cycle (that is, the cycle ofdynein attachment to microtubules, power stroke and detachment).This control scheme, which has been proposed in somewhatdifferent forms by Sugino and Naitoh (Sugino and Naitoh, 1982)and by Murase and Shimizu (Murase and Shimizu, 1986), holdsthat the beat cycle of the cilium or flagellum is fundamentally adirect outcome of the dynein cross-bridge cycle. In other words,each dynein undergoes one cycle of attachment, power stroke anddetachment as the cilium undergoes one effective and recoverystroke (illustrated in its simplest form in Fig. 4).

The proposal of Sugino and Naito suggested a metachronal(sequential) scheme of dynein activation, in which every dynein isactivated in succession to produce the beat (Sugino and Naitoh,1982). Consequently, the beat reflects a collective dynein cross-bridge cycle, but dyneins activate in sequence rather thansimultaneously. A recent synthesis developed by Seetharam andSatir provides support for this model (Seetharam and Satir, 2008).In such a metachronal scheme, the number of dyneins thatsimultaneously contribute force is small; therefore, the dyneins must

be very powerful. However, unless such a metachronal scheme isused, the amplitude of the beat is limited by the step-length of thedynein cross-bridge cycle, which is not more than 16 nm, and yieldsa beat that is too shallow to correspond to those that are found innature. Modifications of the idea have been proposed to overcomethis limitation. In one modified form of the hypothesis, the ‘onebridge cycle, one beat’ idea is replaced with multiple cycles thatterminate when shear velocity falls to zero against a bendingresistance (Murase, 1991).

A similar idea has been carried further by Camelet and colleagues,who treated whole groups of dyneins as tuned oscillators in whicha harmonic-like resonance of the group results when the action ofthe group is resisted by elastic stress (Camelet et al., 1999). Thebeat of the whole flagellum results as an emergent behavior of anexcitable ensemble of interacting dyneins (Kruse and Julicher,2005). Energy from the power stroke of many dyneins is transferredinto storage in an elastic resistance, which, when it accumulatessufficiently, acts to terminate the action of the group (reviewed byRiedel-Kruse et al., 2007). Consequently, this view incorporateselements of both an excitable-dynein cross-bridge cycle and stress-mediated inactivation. The inactivation by the accumulated stressrenders the switching shear dependent, in a way that is somewhat

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Stalk

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Stem

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456C

A Beat cycle

S

B Dynein cross-bridge cycle

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56 C

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AAAdomainhead

Fig. 4. Hypotheses based on the dynein cross-bridge cycle. (A)The mostbasic dynein-bridge-cycle-based beat with attached dyneins executing a cross-bridge cycle that results in a cilium-like beat. In this simple version, onecross-bridge cycle of dynein is converted to one episode of ciliary bending.In the original version of the excitable-dynein hypothesis, dynein does notnecessarily detach from the adjacent microtubule. (B)The current conceptionof the dynein power stroke and recovery (Burgess et al., 2003, Roberts et al.,2009). The various models of flagellar beating that are based on the kinetics ofthe dynein cross-bridge cycle incorporate considerable variation in the detailsof coupling between the bridge cycle and the beat, including metachronal(sequential) activation, multiple cycle activation and cooperative groupactivation with stress-based termination. These are discussed in the text. TheAAA domain heads 1-6 and C are labeled.

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similar to the inactivation by zero shear velocity in the modifiedexcitable-dynein proposal of Murase (Murase, 1991). The idea ofinactivation of dynein by a threshold of accumulated stress also hassomething in common with the geometric-clutch hypothesis,although the vector direction of the stress is not transverse butlongitudinal with respect to the direction of dynein translocation.

In summary, there are currently three quite different views ofhow the axoneme might work as an organ of motility. The firstview is that the structure of the axoneme is itself a mechanical devicethat regulates the operation of the dynein motor proteins bychanging the spacing of the doublets. The second view is that themotors are enzymatically regulated through a cascade of molecularand mechanical events that originate at the CP and are transmittedthrough the spokes and DRC to the inner-arm dyneins. The thirdview is that the beating is the result of an intrinsic oscillatoryproperty of the dynein motors, which is expressed as the flagellarbeat when the motors act in cooperative groups.

Recent findingsDynein I1 and the role of the CP-spoke axisThe inner row of dynein arms (see Fig. 1) consists of seven differentdynein heavy chains arranged in a linear pattern that repeats alongeach outer doublet every 96 nm. One of the inner-arm dyneins, calledI1 or dynein f, has two motor domains and therefore is referred toas double headed. The inner-arm dyneins, especially I1, seemto occupy a special place in the regulation of the flagellar beat.Inner-arm-dynein mutations of Chlamydomonas result in eithercomplete or partial loss of coordinated beating. The 138 kDintermediate chain (IC138) is a protein that is associated with theinner-arm dyneins (Habermacher and Sale, 1997) and is activelyphosphorylated by casein kinase 1 (Yang and Sale, 2000). Wirschelland colleagues state: ‘The current model for regulation of I1-dyneinis that chemical and/or mechanical signals from the central pair aretransmitted through the radial spokes to affect IC138phosphorylation on specific outer doublets (e.g. outer doublets N,N+1, N–1). Thus, phosphorylation of IC138 in an asymmetricalmanner is predicted to result in local inhibition of sliding’ (Wirschellet al., 2007). Their view of the importance of dynein I1 is basedon reports that indicate that dynein activity is modulated by thepresence or absence of spokes (Smith and Sale, 1992), thatphosphorylation of the IC138 component of I1 can inhibit doubletsliding (Smith, 2007), and that the phosphorylation is controlled bythe action of the CP-associated protein hydin (see Fig. 3) that iscrucial for motility (Lechtreck and Witman, 2007; Smith, 2007).

To make the IC138 phosphorylation site doublet specific, so thatit can produce alternation of dynein action in the beat, requires someform of control. In species such as Chlamydomonas, in which theCP is known to rotate, this might be accomplished through the CP-spoke interaction (see above). However, to propose this mechanismas the universal coordination mechanism that regulates beating, evenin organisms in which the CP apparently does not rotate, wouldrequire the operation of another selection criterion, such as themechanical tilting of the spokes during interdoublet sliding (seebelow). Such a view has been proposed, and assigns to the spokesthe role of stress transducers (Smith and Yang, 2004).

Several important observations run counter to the hypothesis thatthe CP-spoke axis controls the activation and deactivation of thedyneins to produce the beat cycle. In nature, there are functionallymotile flagella that have no CP or spoke apparatus (Woolley, 1998;Mencarelli et al., 2001). As discussed above, Chlamydomonas pfmutants, which lack the spokes and/or CP, are motile if they also

carry a suppressor mutation (Brokaw et al., 1982; Huang et al., 1982;Porter et al., 1992). Some of the suppressor mutations lead to defectsin the regulatory chains of both inner and outer dyneins (Porteret al., 1992; Porter et al., 1994; Rupp et al., 1996). Other mutationscause the absence of regulatory proteins and/or dynein subsets(Piperno et al., 1994; Piperno, 1995; Rupp et al., 1996). Therefore,we must conclude that the absence of regulatory components of thehypothetical beating mechanism actually restores a beat.Furthermore, IC138 is hyperphosphorylated in the CP-spoke-defective pf mutants (Hendrickson et al., 2004). As phosphorylationof IC138 blocks sliding, the suppressor mutations must overcomethis inhibition of sliding.

The suppressor mutation data pose a serious problem to thecontention that the CP-spoke axis is the fundamental mechanismresponsible for beating. They indicate that, as long as the dyneinsare not actively inhibited, another mechanism can take over in theabsence of the CP-spoke control and institute a complete beat cycle.The question then becomes: is this other mechanism a default back-up mechanism, or is it the normal mechanism for coordinatingbeating, which is fine-tuned by CP-spoke-dependent regulation?

It is possible that control by the CP-spoke axis is part of an on-off switch that has a global effect on all axonemal dyneins. Thiswould allow a complex organism such as Chlamydomonas to turnits flagella on and off, which it is known to do. Yang and colleagueshave presented evidence to show that control by radial spokes playsa role in pausing flagellar beating to maintain coordination betweenthe two flagella of Chlamydomonas (Yang et al., 2008). In thisway, control by the CP-spoke axis might act to limit or terminatethe action of selected dyneins, shaping the beat and altering thewaveform for the different modes of swimming that are observedin Chlamydomonas. This might explain the role of Ca2+-bindingelements on the spoke shafts (Yang et al., 2001) in controlling dyneinactivity (Dymek and Smith, 2007), as Ca2+ can alter the waveformof the Chlamydomonas beat and change the swimming pattern. Theresults obtained by Nakano and colleagues using sea urchin spermshow that Ca2+ can alter which specific dyneins are inhibited byassociation with the CP-spoke system (Nakano et al., 2003), andfit nicely with the interpretation that the CP-linked regulation ofsliding is an element of the Ca2+ response pathway.

Spoke tilting and spoke-head–CP interactions are topics thatdeserve serious investigation because so little is known aboutthe functions of the spokes and CP. The dyneins that do most of thework of the beat cycle are those attached to doublets 2, 3 and 4 onone side of the axoneme and doublets 7, 8 and 9 on the opposingside. Most of the shear developed in the beat (~80%) results fromactive sliding between these doublets. The kind of spoke tilting thatis the result of inter-doublet sliding, as observed by Warner andSatir (Warner and Satir, 1974), has a minimal influence on the spokesof these doublets because their spokes project nearly perpendicularlyto the beat plane. Instead, spoke tilting is greatest on doublets 1, 5and 6, in which the spokes project from their doublets in a directionthat is nearly parallel to the beat plane. It is these doublets thatwould reach a crucial threshold of spoke-tilt first, and wouldtherefore be the probable site of regulation.

To invoke spoke tilting as the principal coordinating mechanismof the beat requires a better understanding of how such an activationscheme could control the dyneins on doublets 2, 3, 4, 7, 8 and 9,which contribute most of the motive force of the beat. Perhaps asystem for signal transfer through the CP proteins is possible. Inany case, better information is needed on the functional interactionsof the spokes and CP.

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Dynein I1 and models of beat controlA special role of the inner arms and dynein I1 is compatible withthe geometric-clutch model and possibly with dynein-cycle-basedmodels as well. Experiments in a number of systems have shownthat extraction of the outer dynein arms reduces the beatingfrequency but leaves the waveform of the beat relatively unaltered(Brokaw and Kamiya, 1987). The latest version of the geometric-clutch model (Lindemann, 2002) can duplicate the results of theouter-arm extraction experiment, but only if inner arms contributealmost all of the interdoublet adhesion at low sliding velocity. Thisis consistent with the idea put forward by Brokaw that the innerarms contribute most of the force at low sliding velocity (Brokaw,1999). There is good experimental support for the idea that the innerarms are more processive and can hang on more securely to anadjacent doublet (Shingyoji et al., 1998; Sakakibara et al., 1999).In the case of dynein I1, this has been specifically investigated byOiwa’s laboratory, who have shown that dynein I1 might bespecially designed to hold on rather than to translocate (Kotani et al.,2007). This is exactly the property that would give it a special rolein the beat cycle, as viewed from the perspective of the geometric-clutch model. The inner arms would provide the interdoubletadhesion to keep an active group of dyneins attached and resistantto the t-force until a sufficient bend develops.

The experiments of Oiwa and co-workers showed that isolateddynein I1 also provided a resistance that slowed down the rate ofmicrotubule translocation by other dyneins (Kotani et al., 2007).This might provide the linear resistive element that the model(dyneins as tuned oscillators; see above) proposed by Camelet andcolleagues (Camelet et al., 1999) requires. Consequently, the specialrole of dynein I1 is not very hypothesis selective; all of themechanistic hypotheses for control of the beat cycle seem to requirethat a dynein-I1-like function exists.

Nucleotide regulation of dyneinThere are numerous molecular studies that dissect the functions ofthe AAA (ATPases associated with diverse cellular activities)domain of the dynein motor (Silanovich et al., 2003; Kon et al.,2004; Reck-Peterson and Vale, 2004; Sakato and King, 2004; Choet al., 2008; Numata et al., 2008). These experimental studieshave revealed the functional complexity of the AAA ring and haveprovided evidence that the multiple ATP-binding sites play a rolein nucleotide regulation of dynein, as proposed by Kinoshita andcolleagues (Kinoshita et al., 1995). They also support the idea thatdynein-ADP is the force-generating intermediate in the powerstroke, as proposed by Tani and Kamimura (Tani and Kamimura,1999).

We have demonstrated that reactivated, de-membranated bullsperm show a significant increase in the t-force at the switch-pointof the beat in the presence of ADP (Lesich et al., 2008). This is inagreement with experimental studies on the nucleotide regulationof dynein, which assert that the binding affinity of dynein for tubulinis directly regulated through a long-residence ADP-binding site onthe dynein head (Inoue and Shingyoji, 2007; Cho et al., 2008). Ourresult is consistent with the geometric-clutch hypothesis, in that theswitch-point of the beat should depend on the balance betweendynein-microtubule binding affinity and t-force. Our study withreactivated bull sperm shows that an independently identifieddiscrete factor (ADP) alters the dynein-binding affinity and changesthe dynamics of switching. The change in switching curvature iseasily observable (Fig. 5) and is predicted by the geometric-clutchmodel. The result can also be seen as compatible with the

dynein-cycle group of hypotheses because a distinct change inthe dynein cross-bridge cycle results in a distinct change in the beatcycle. It is difficult to reconcile the data with beat-coordinationschemes that rely on CP rotation or the degree of spoke tiltingbecause these parameters should not be directly influenced by achange in dynein-binding affinity (if the switching event is regulatedby a threshold of spoke tilt, then the switching event should takeplace at a conserved interdoublet shear).

The increase in t-force in the presence of ADP shows that eachepisode of dynein engagement is terminated at a higher t-forcethreshold when the dynein-binding affinity is increased by ADP.This directly supports the concept that dynein-tubulin affinity mustbe overcome by t-force at the switch-point of the beat (as illustratedin Fig. 5). At the same time, the interdoublet shear (which shoulddetermine spoke tilting) is not conserved but increases substantiallyat the switch-point. These results provide an explanation forobservations that ADP (in the presence of low ATP concentrations)can reinstitute a beat in some pf Chlamydomonas mutants (Freyet al., 1997). It is not the basic coordination mechanism that isdefective in those pf mutants, but the binding properties of theremaining active dynein. Increasing the processive behavior ofthe functional dyneins that remain restores the capacity forcoordinated beating.

In conclusion, it appears that the nucleotide regulation ofthe dynein-tubulin binding affinity is directly connected to themechanism of switching in the beat cycle. As we gather a morecomplete understanding of the parameters that govern the tubulin-binding affinity of the dynein molecule, it will be possible to see

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B

0.1 mM ATP only 0.1 mM ATP + 4 mM ADP

t-force t-force

Microtubules

Low binding affinity High binding affinity

AAA bindingdomains

Stalk

Stem

Fig. 5. Nucleotide regulation of dynein affinity for microtubules. (A)Wehave recently shown that the curvature of the principal bend in bull spermincreases dramatically in the presence of 1-4 mM ADP (Lesich et al., 2008).Tracings of Triton-extracted, reactivated bull sperm are shown with theprincipal bends identified by arrows. (B)A large body of evidence nowsupports the view that ADP has a direct effect on the binding affinity andprocessivity of dynein (as reviewed in the text). In the geometric-clutchframework, an increase in the binding affinity of the dynein to the B-subtubuleof the outer doublet should increase the t-force threshold for the ‘off’switching event (indicated by a thicker arrow). This is consistent with ourobservation. The dynein illustration is based on the proposal put forth by Inoueand Shingyoji (Inoue and Shingyoji, 2007). Figure adapted from Lesich et al.(Lesich et al., 2008).

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how changes in the binding affinity produce predictable changesin the observable characteristics of the flagellar beat cycle. In thisway, the nucleotide regulation of dynein might provide a touchstonefor linking theory and experimental results.

Bending-induced slidingThe observation that bending isolated pieces of bull sperm flagellacan re-institute a beat cycle (Lindemann and Rikmenspoel, 1972a)supports the idea that a passive (externally imposed) bend can assistin the engagement of the dynein motors. To invoke a mechanicalmechanism such as the geometric clutch to explain the completebeat cycle there must also be a mechanism to disengage the actionof the dyneins. Kamiya and Okagaki provided a proof of principlefor the assertion that the dyneins can act to terminate their ownaction (Kamiya and Okagaki, 1986). By looking at individualdoublet pairs sticking out from the frayed end of a Chlamydomonasflagellum, they showed that the pairs of doublets set up a limitedbeat of their own by sliding and bending to a critical bend angleand then separating near the base where the t-force is greatest.

Recent important studies have demonstrated that imposedbending can initiate and reverse episodes of sliding in sea urchinsperm flagella when the doublets have been freed to slide byenzymatically cutting the nexin links with elastase (Morita andShingyoji, 2004; Hayashi and Shingyoji, 2008); moreover, imposedbending can initiate beating at very low ATP concentrations(Ishikawa and Shingyoji, 2007). These results demonstrated thatthe curvature imposed on the partially digested structure controls thedirection of dynein-driven sliding as the axoneme disintegrates. Thisis a direct confirmation of the hypothesis that initial curvature playsa role in governing the pattern of dynein engagement.

These observations are generally consistent with the idea thatcurvature control or some modified form of curvature control, suchas the geometric-clutch mechanism, is responsible for activating aselect group of dyneins. It can also be viewed as consistent witha centrally mediated mechanism, spoke tilt or spoke-CP interaction(as proposed by the authors). Presently, there is no easy discriminatorto decide between these possibilities because the elastase-digestedsystem is of necessity mechanically compromised in order to makeit possible to view the sliding event. It might represent normalcontrol in the intact flagellum, but it might also be an artifact of apartially incapacitated regulation system.

What is the flagellar mechanism for dynein engagement in theseexperiments? It might be as simple as activation of the first pair ofdoublets that is forced together by the stress imposed on theaxoneme. Although the axonemes in these studies are structurallycompromised and probably lack most of the interdoublet elasticlinkages, there is still no way to bend the nine-fold ring withoutimposing a distortion that will push certain doublets together. Thedoublet microtubules are known to be fairly resistant to stretch orcompression (Brokaw, 1991). Conservation of length, combinedwith the fact that enough interdoublet attachments must remain tokeep the axoneme intact, requires that the axoneme will compressin the bending plane (Fig. 6A,B). If all the doublets that are initiallyforced together are activated, there is an immediate leveragedisadvantage to the dyneins on one side, as compared with thoseon the other. The action of one group will create a transverse stressthat pushes those doublets tighter together and ensures continuationof the activity, whereas the action of those on the opposite side willcreate a transverse stress that immediately moves the doublets apartfrom each other and inhibits further attachment (Fig. 6C).Consequently, the selection of the dyneins on one side by the

bending event could be viewed as a direct outcome of the sign ofthe t-force that is generated by the action of the dyneins on the twosides of the axoneme. This is not only consistent with the geometric-clutch mechanism but can be viewed as a demonstration of thet-force switching principle, which maintains that dyneins willbecome activated when the doublets are pushed together and bedeactivated when the doublets are prised apart.

It is equally possible that the action of the bending is mediatedthrough a mechanism that senses the direction and degree of spoketilting and relays an activating signal to the dyneins on one side,

A B

C Doublet pairs viewed from opposite sides of a bent axoneme

Compressiont-force Tension

t-force

Imposition of force

Dynein Microtubule

Positive t-forcefacilitates

dynein attachment

Negative t-forceimpedesdynein attachment

Sliding

Fig. 6. Hypothetical mechanism for doublet pair selection by bending.Recent reports (Morita and Shingyoji, 2004; Hayashi and Shingyoji, 2008)have shown that bending can initiate microtubule sliding and can determinewhich doublets are activated. The illustration shows a mechanism that mightbe responsible for the selection, but does not invoke CP- or spoke-dependentregulation. (A,B) When an axoneme is bent, the internal elements are pushedtogether in the plane of bending. The image in B was modified in PhotoshopCS4 to simulate the collapse of the axoneme. Doublets 3 and 8 and the CP arecolored red. The remaining doublets (blue) were moved closer to that group.(C)In the geometric-clutch formulation, bringing doublets closer togetherengages the dyneins by allowing them to attach to the adjacent doublet.Initially, this would happen equally on both sides of the compressed axoneme;however, the dynamics of stress from dynein activation are different on thetwo sides of the axoneme. On one side, the activated doublets will be pushedcloser together by the resulting t-force, which acts basally to the bent area.On the other side of the axoneme, the action of the dyneins will tend toseparate the activated doublet pair and put them at a mechanical disadvantage.The side with the mechanical advantage will engage more and more dyneinsuntil disintegration of the structure allows sliding to occur. The opposite side,which is at a mechanical disadvantage, does not experience a cascade ofdynein engagement and does not slide.

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and an inhibitory signal to those on the other side, through thespoke-DRC enzymatic linkage. This is an attractive hypothesis,given all of the indisputable discoveries of the control-element-likecomponent proteins associated with the spokes and DRC. It mightbe that an inhibition of certain doublets is mediated when the spokesand CP are pressed together by bending stress. The same conceptualissue as addressed earlier remains applicable here: there still mustbe a coordinating mechanism that can explain how beating ispossible in the absence of the spokes and CP. Even the more limitedinterpretation that the spokes and CP, when present, select and inhibitcertain dyneins requires a much better understanding of the CP-spoke interaction. At this point, it is not possible to say with absolutecertainty that the spoke heads actually have a physical bindinginteraction at the CP apparatus.

Structural considerationsThe structural arrangement of the microtubules and proteins thatmake up the axoneme is a marvel of natural engineering. To convertthe action of thousands of molecular motors into useful work, theouter doublets must be strong, yet flexible, and they must beanchored solidly at the base of the flagellum or cilium in order togenerate the torque to bend it. In this section we examine somerevealing work that has aided understanding of the role of the basalanchor in the beat, the forces that the axoneme must carry, and theingenious tektin fiber system that reinforces the load-carryingstructures.

In the geometric-clutch hypothesis, the basal body of theflagellum has a dual role. In addition to providing an anchor forthe development of torque, it also allows the accumulation of tensionon the doublets at the basal end of the flagellum, which is crucialfor generating a large t-force for switching the dyneins (Lindemannand Kanous, 1995; Lindemann and Kanous, 1997). Fujimura andOkuno recently showed, by photochemical cross-linking, thatforming an artificial anchor at either end of a cut sea urchin spermflagellum restores spontaneous beating to fragments of flagella(Fujimura and Okuno, 2006), consistent with the proposed role ofthe basal anchor in the coordination of the beat.

The magnitude of the t-force at the switch-point of the beat insea urchin and bull sperm flagella is about 0.5-1.0 nN/micron(Lindemann, 2003). This magnitude of force is consistent with theidea that switching might occur when the magnitude of the t-forceis approximately equal to the aggregate tension that the dyneinscan carry (Lindemann, 2003).

Such a large t-force is also likely to produce substantial distortionof the axoneme during the beat. Distortion of the axonemal ringduring the beat is a necessary tenet of the geometric-clutchhypothesis and has been elusive to demonstrate. Sakakibara andcolleagues provided direct measurement of diameter oscillations insea urchin flagella that correlated with dynein activity (Sakakibaraet al., 2004). Other evidence that the diameter of the flagellumdistorts during the beat comes from David Mitchell’s transmissionelectron microscopy (TEM) images of Chlamydomonas flagella thatwere rapidly fixed while beating (Mitchell, 2003b). In acollaborative study using Mitchell’s TEM plates, we quantifiedthe degree of distortion of axoneme diameter in the fixedChlamydomonas beat cycle and compared it to predictions of thegeometric-clutch model (Lindemann and Mitchell, 2007). The dataallowed us to derive a preliminary estimate of 0.02 MPa for thetrans-axonemal Young’s modulus (the ratio of stress to strain thatdefines how a structure will distort under tension). If this estimatecan be confirmed by direct measurement on intact axonemes, it will

mean that the axoneme must always experience considerabledistortion during the beat cycle.

Before concluding, we note with special interest that a moredetailed picture of the mechanical construction of the axoneme isemerging. It is known that axonemes are reinforced by a networkof tektin protofilaments that, together with the nexin links, maintainthe ninefold integrity of the axoneme, even when tubulin issolubilised (Stephens et al., 1989). A recent synthesis of the dataon the tektin network provides a convincing case that the nexinlinks, spokes and inner-arm dyneins are all located so as to bestrategically supported by the tektin network (Setter et al., 2006).Thus, these proteins all appear to be anchored to an embedded cablesystem that is strategically arranged to redistribute the tension thatresults from the action of the dyneins. This makes the tektin skeletonof the axoneme the major system for carrying and redistributingthe forces generated in the beat.

The nexin links are ideally positioned to sense and react tointerdoublet shear. If interdoublet shear and reactive elasticresistance are the key elements in dynein switching, as in the modelproposed by Camelet and colleagues (Camelet et al., 1999), thenit is imperative to learn more about the regulatory components ofthe nexin link complex. The recent structural work on the axonemeusing cryoelectron tomography does show a large aggregate ofprotein with the correct periodicity and placement for the nexinlinks (Nicastro et al., 2006). The identity of the proteins in thiscomplex and their mechanical properties need to be examined.

The spokes are optimally positioned to carry a large componentof the transverse stress across the center of the axoneme. This tensionwould be redistributed differently according to whether the spokeheads bind to, or are free of, the elements of the CP apparatus.Consequently, this is an important point for investigation. The detailsof the mechanical interaction of the spoke heads at the CP mightsignificantly impact the feasibility of both the geometric-clutchmechanism and the CP-spoke control of dynein. Therefore, this issueis pivotal in discriminating between possibilities. It might well bethat the t-force determines the interaction of the spokes with theCP apparatus, and that only when the axoneme is ‘pinched’ byt-force acting across the axoneme diameter do the spokes interactstrongly enough with the CP to engage the CP-spoke axis. Afterall, nature is not constrained to pick from only one of our hypotheses.

Conclusions and perspectivesAt present, there are three contending views of how the axonememight generate the flagellar beat cycle: the mechanical view, whichis elaborated in the geometric-clutch hypothesis; the enzymatic view,which relies on the action of components of the CP-spoke axis; andthe view that it is the behavior of the dynein motors that contributesspontaneous oscillatory behavior. Each view has considerableconceptual merit and all have at least some observational support.

The control of dynein function and microtubule sliding by meansof axoneme components located on the CP, spokes and DRC hasthe largest base of experimental support. We think it is certain thatthis regulatory pathway is a key mediator of the calcium responseof cilia and flagella. It is also likely that it can act to turn the motilityoff by rendering a subset of the inner-arm dyneins inactive,particularly I1. From the available experimental evidence it isconsiderably less certain that the CP-spoke axis is fundamental tothe mechanism that generates repetitive beating.

There is strong evidence for the position that the primarymechanism that underlies the beat cycle is bending activated andmechanical, and is intrinsic to the axoneme structure and to the

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dyneins themselves. The exact mechanism that imposes this controlis still inconclusive, but many of the recent experimentalobservations are consistent with the geometric-clutch hypothesis.Yet, there are several specific points that still need to be resolved.First, to what extent does the axoneme distort during the beat cycle,and does the observed distortion support the geometric-clutchinterpretation, which maintains that interdoublet spacing is theprincipal modulator of dynein activity? Second, what is the natureof the spoke-CP interaction? (Do the spoke heads actually bind tothe CP projections? Does spoke-CP binding directly alter theactivation state of the associated dyneins, and does this process havea means of reciprocation between the two dynein sets that bend theflagellum in opposite directions?) These are the key issues ifthe CP-spoke axis hypothesis is to be considered part of the primarymechanism that generates the beat cycle.

Finally, the dynamics of the dynein motor itself need to be betterunderstood. The latest experimental evidence from our laboratory(Lesich et al., 2008) suggests that the microtubule-binding affinityof the dynein motors is crucial to the switch-point of the beat cycle.This supports the idea that the load-bearing capacity of the dynein-tubulin bridges is essential to the mechanism of beat-cyclegeneration. However, it does not discriminate between a beat-cycle mechanism based on a t-force load limit (i.e. the geometricclutch) and models based on the dynein cross-bridge cycle,especially if loading is invoked as the mechanism that turns thedyneins ‘off’ [as proposed by Camelet et al. (Camelet et al., 1999)].Ultimately, experimental investigation of the response of the dyneinmotor to external loading might be able to settle this issue.

Our thanks to David Mitchell for the excellent micrograph used inFig. 6 and to Mary Porter for helpful consultation on pf mutants. Thiswork was supported by grant MCB-0516181 from the National ScienceFoundation.

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