The physiology of cilia and mucociliary interactions.

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Quick links to online content Annu. Rev. Physiol. 1990. 52:137-55 Copyright © 1990 by Annual Reviews Inc. All rights reserved

Annu. Rev. Physiol. 1990.52:137-155. Downloaded from www.annualreviews.org Access provided by University of California - Davis on 02/11/15. For personal use only.

THE PHYSIOLOGY OF CILIA AND MUCOCILIARY INTERACTIONS Peter Satir Department of Anatomy and Structural Biology, Albert Einstein College

of Medicine

,

Bronx, New York

Michael A. Sleigh Department of Biology, University of Southhampton, United Kingdom KEY WORDS:

respiratory epithelium, mucus, ciliary motility, signal transduction, dynein

INTRODUCTION Mucociliary transport in the respiratory tract is necessary for health and normal function of the tissue, particularly in resistance to respiratory infec tion. Transport depends upon the characteristics of the cilia and the thin layers of fluid and mucus that form the interface between the respiratory epithelium and air. Understanding of the basic processes involved in mucociliary clear ance requires a clear picture of how cilia generate motion, of how the respiratory epithelium controls and coordinates this motion, and of how the cilia interact with periciliary fluid and with mucus. Recent progress has been made because the cell biology and biophysics of ciliary motility studied with a variety of classic nonmammalian systems has proven applicable to mamma lian ciliated epithelial function. Genetic approaches to ciliary function have also been uncovered both in human 'immotile cilia syndrome' (1, 75, 45) and in other systems. Because of the complexity of ciliary structure and composi tion, mutations in many different pathways can produce immotility or abnor mal beat. Although such approaches support and augment the functional conclusions discussed here (cf 64), detailed discussion is beyond the scope of this article. This article will review features of the physiology of cilia and

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ciliated tissues derived from basic studies and the application of such approaches to mucociliary transport in the respiratory tract. Pertinent earlier reviews include

(59, 64, 87, 70).

STRUCTURE AND FUNCTION OF CILIA Structure and Biochemistry of the Axoneme Annu. Rev. Physiol. 1990.52:137-155. Downloaded from www.annualreviews.org Access provided by University of California - Davis on 02/11/15. For personal use only.

Respiratory cilia are virtually identical in structure and closely related in biochemistry to motile cilia of nonmammalian cells. The cilium is an exten sion of the free cell surface, whose motor is a tubulin-based axoneme. The axoneme is surrounded by a specialized extension of the cell membrane. Axonemal composition and response are studied directly by removing the membrane via detergent treatment, after severing the axoneme from the cell.

(24) including mammals (16, 44, 26) are available, many of the details reported below relate to

Although preparations of tracheal cilia of vertebrates nonvertebrate axonemes. AXONEMAL MICROTUBULES

The nine outer doublet microtubules and two

single central pair microtubules of the fromheterodimers of

a

9+2 axoneme are constructed mainly

and �-tubulin, arranged in protofilaments. The doub

let microtubules consist of subfiber A comprised of

13 protofilaments upon

which subfiber B (l0-11 protofilaments) assembles. The a-tubulin is acety lated as is true for many long-lived stable microtubules. Some portion of the doublet, probably the midwall common to both subfibers, is composed of tektin, an intermediate-filament-like protein that resists detergent extraction (cf

89).

In mammalian cells, assembly of the axoneme occurs in a fixed pattern at the cell surface above basal bodies

(15). The basal body or centriole is also an

organizational site for cytoplasmic microtubules. Although the same pool of tubulin may be used, axonemal assembly is fundamentally different from

(a) assembly (b) complex doublets--rather

assembly of cytoplasmic microtubules in two related respects: occurs directly on the basal body microtubules,

than single tubes-are eventually polymerized. Like other microtubules, axonemal doublets are polarized such that the fast polymerizing

(+) end is

most distal to the basal body, which corresponds to the tip of the axoneme. Doublet stability probably relies on multiple interactions with other axonemal proteins, of which there are several hundred in two-dimensional gels

(34);

doublet length is probably controlled or at least influenced by capping struc tures

(13) and by specific membrane interactions. Most axonemes of mamma

lian respiratory cells grow to about 5-10 /Lm in length; human respiratory cilia are about

6 /Lm. Length is under genetic control. Indeed, if too long, the (2) (see below). At the tip,

axoneme is less effective in mucociliary transport

CILIA AND MUCOCILIARY TRANSPORT

139


the doublets simplify and only subfiber A is seen. In mammalian cilia, all nine subfiber As insert into a disc that usually forms the cytoplasmic surface of a transmembrane complex-the ciliary crown. Near its base, the axoneme is also connected to a special transmembrane complex, the ciliary necklace. Just above the necklace region, there is a zone where Ca2+ shock produces severing of the axoneme, possibly by activating a calcium contractile protein localized to this region. DYNEIN STRUCTURE AND MECHANOCHEMISTRY

Dynein structure and mechanochemistry are reviewed by Warner et al (90). Ciliary dynein is a two or three-headed bouquet-like molecule of molecular weight 1-2 million dal tons (31). Each head contains a heavy chain ATPase of 4-500 kd. The molecule also contains polypeptides of lower molecular weight (intermediate and light chains). Dynein isolated from tracheal cilia is two-headed (27) and resembles sea urchin flagellar dyncin. Dyncin extracted from bovine or porcine tracheal cilia sediments at 18-19S and at 12S (26, 44), and appears comparable in polypeptide composition to nonmammalian dynein. As assembled on the axonemal microtubules, the dynein molecule is compacted into an arm. Although there is controversy regarding the exact in situ appearance of the arms, a model consistent with most of the structural evidence has been constructed (5, 63). Each arm projects across the in terdoublet gap and is attached, more or less permanently after assembly, to subfiber A of one microtubule (conventionally, N) with at least one head projecting toward and capable of attaching to subfiber B of the next doublet (N+ 1). Dynein is a (-) end microtubule motor; that is, it moves the structure to which it is attached in an ATP-insensitive manner (e.g. subfiber A of doublet N) toward the base of the axoneme. By Newton's third law, the microtubule along which the dynein walks by its ATP-sensitive heads (e.g. subfiber B of doublet N + 1) moves in a (+) direction toward the tip of the axoneme. Microtubule movement generated by dynein has been studied in sliding axonemes (77, 53, 78, 18) or completely in vitro by using isolated dynein attached to glass and taxol-stabilized microtubules (81, 46). A single-head fragment of dynein is sufficient to give motility (51, 82). Although the polarity of force generation by mammalian ciliary dynein is still un determined, cytoplasmic dynein from mammalian epithelial or nerve cells (47, 89) translocates vesicles and microtubules with the usual polarity. PERIODIC LINKS Two types of linkages integrate the individual microtu buIes into a functioning axoneme: radial and circumferential. The radial spokes connect the doublet microtubules to the central pair complex. They are a self-assembling, multipolypeptide structure (34) consisting of a cylindrical


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stalk and an expanded spoke head. They usually occur in groups of three (37) (S1-S3) along subfiber A with a 96 nm period and spacings of S 1-S2 of 32 nm; S2-S3, 24 nm; S3-S1, 40 nm. Their function is incompletely un derstood, but they probably act to limit microtubule sliding of active doublets by converting such sliding into bending; they may help to maintain proper spacing of the doublets. They may also be involved in the activation or inhibition of dynein arm cycling on a specific doublet. Mutants of motile cilia whose spoke assembly is defective are paralyzed, and their axonema1 arrays may collapse (74), but restoration of motility does not require restoration of spoke activity (30). The circumferential linkages connect adjacent doublets. One type of in terdoublet link persists after near total extraction of dynein arms by high salt (88, 2 1). These links are paired, one pair per spoke group. They lie near the base of the inner arm and are relatively inextensible, probably breaking and reforming as the doublets move past one another. In the absence of dynein, they are sufficient to hold the axoneme together. There may be more than one type of circumferential linle THREE-DIMENSIONAL RECONSTRUCTION OF THE AXONEME

A major activity of the past decade has been reconstruction of the axoneme in three dimensions at resolution better than 10 nm. Data for such reconstruction have been derived from negative stain electron micrographs (3, 6, 5), thin section images (63), freeze-etch replicas (21), and provisionally from frozen, hy drated specimens (40). Reconstructions differ somewhat depending on the technique used to generate the data. Computer modeling provides for con sistency of structure from different perspectives and rapid comparison of models. Tomographic reconstruction is also useful (37). For most of its length, axonemal structure is repetitive; only the tip and base vary significant ly. The basic repeat of 96 nm contains four outer arms per doublet, four (63), or possibly three (2], 52), inner arms per doublet, one spoke group, six central sheath projections, and one pair of nonelastic interdoublet links per doublet. This can be considered the unit of motile activity of the axoneme.

The Axonemal Basis of Motility CURRENT STATUS OF THE SLIDING MICROTUBULE MODEL

It is firmly established that axonemal motility is based on microtubule sliding as a consequence of the mechanochemistry of the dynein arms (65, 3 1, 90, 10). In vitro sliding of microtubules in isolated, partially digested axonemes (77) has been demonstrated directly for vertebrate sperm (93) and for mammalian tracheal and oviduct cilia ( 16). Dentler & LeCluyse ( 14) have reinvestigated the geometrical consequences of sliding for mammalian tracheal cilia, whose tip structure differs from that of invertebrate cilia. Axonemes are reactivated


141

with ATP and bends vs tip configuration studied, using the equation of Satir

(60). The results indicate that the relationship of sliding to bending may be more complex for tracheal cilia, where the subfiber As of all nine doublets end in a cap, than for invertebrate cilia. Bend formation may require the generation of compensating bends and axonemal twisting as a consequence of sliding.


THE SWITCH POINT HYPOTHESIS

Since the axoneme is a cylinder, and all

dynein arms produce sliding with a single polarity, in order to produce motion, some asynchrony of arm activity must be present. A simple hypoth esis-the switch point hypothesis-assumes that half of the doublets of the axoneme have active arms when the axoneme is moving in its effective stroke and that the other half has active arms during the return stroke

(62). Activity

then switches from one set of arms to another during a ciliary beat and back again at the beginning of the next beat. With slight modifications, this model can be used to correlate specific doublet activity with ciliary beat form. The model has some important features.

Translocation rates imply a non-cycling state of the dynein arm

Transloca

tion rates of microtubules on dynein in vitro or in sliding assays after protease digestion in the axoneme are comparable to sliding rates for microtubules in the intact axoneme i. e.

=10 JLm/sec (78, 46). However, maximum displace =0.1-0.4 /.Lm per

ment of a dOUblet by sliding in the intact axoneme is only half beat

(61). According to the switch point hypothesis, in an axoneme 50 Hz, arms on doublet N in the active half of the axoneme would switch off after 10 msec and would become non-cycling and refractory for = 10 msec before resuming activity. During this refractory beating at a frequency of

period, the doublet would move passively in the opposite direction. This implies that the dynein arm exists in two states: cycling and non-cycling. Spungin et al

(71) have produced negative stain images with two differently

appearing arm distributions, one which may correspond to cycling arms and one to the non-cycling state. In preliminary reports,

R. D. Vale & colleagues

(personal communication) note that in vitro translocation by axonemal dy neins is discontinuous with periods of apparent inactivity and consequently, reversed movement of the microtubules being propelled.

Two switches

There are at least two different axonemal switches. One of

these turns the arms of one set of doublets on and off, the other turns the complementary set on and off.. When one switch is blocked, the cilia will come to rest in one specific position, no matter where in the beat cycle the block is applied (49). Blocking the second switch will lead to ciliary arrest in \

a second position. In mussel gill cilia

(86) one arrest position is near the

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beginning of the effective stroke and the second is near the beginning of the recovery stroke, and the two positions have been labeled 'hands up' and 'hands down' respectively. If the blocking agent is changed, cilia can be moved from one arrest position to another without restarting beat

Radial spoke function

The switch point hypothesis provides a rationale for

the importance of the radial spokes and central complex of


(49).

9 + 2 axonemes.

The opposite acting halves of the axoneme are determined by those doublets

(em) and its projections 1-4 that interact with cm 3 to move the axoneme to the hands up position (62) and doublets 6-9 that interact with em 8 to move the axoneme to the hands down position. Presumably, whose spokes interact with one central microtubule and not the other. These correspond to doublets

coordinated spoke-central sheath attachment in the active half of the axoneme is used to regulate sliding and convert sliding into bending. In axonemes where mutations in spoke or central sheath proteins produce immotility, the

sliding system is intact and operational. Dynein arm activity along a doublet is regulated in a redundant manner, so

that the spoke cen tral sheath signal can -

be bypassed by appropriate modifications in other controls. In metazoan cilia, the position of the central pair seems fixed (80,

18) but in protistan axonemes,

the central pair may rotate (42) either as a causal factor in switching of arm activity in the axoneme, or as a consequence of such switching. Timing and comparative physiology

Although ciliary structure, biochemis

try, and mechanism of motility have been conserved during animal evolution, cilia from different organisms or cilia and sperm tails from the same organism have quite disparate beat phenotypes. Moreover, ciliary beat is under cellular controL For example, respiratory ciliary beat frequency can be slowed down

(79) or speeded up (57). Other cilia-especially of swimming cells-can drastically change their beat form so that the cell swims backwards (cf 33) or turns toward a chemotatic stimulus (9). These changes are readily explained by the switch point hypothesis if the timing of the switches controls beat form of the axoneme. Where dynein arms are actively sliding for equal times in the two half-axonemes (while the opposite half is inactive), the bends generated would be symmetrical; where timing was unequal, the principal bend would correspond to the longer on time, the reverse bend to the shorter, since: and where

1.

2.

� lp.r is the amount of sliding of a given doublet during the principal or reverse bend, respectively, which is proportional to tpm the on time of dynein



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arm activity of that doublet, and to uP'" the amount of bend generated (60, 61). Evidence for asynchronous doublet activity has been summarized by Satir (62). One important finding is that reactivation of hamster sperm flagella by local application of ATP generates a predictable pattern of bending, depend ing on initial pos. ition (95). Sliding restricted to specific subsets of doublets has been seen directly in ctenophore macrocilia (80). In some cases doublets 9, 1, 2 were consistently extruded after reactivation of these axonemes; in others 5-7 were extruded. Identical patterns have recently been shown in mussel gill axonemes (36); the former pattern occurs when ATP reactivates tethered protease-treated hands down cilia, while the latter occurs in hands up cilia. Sale (50) has also shown that in sea urchin axonemes arrested in a specific position, ATP addition permits one subset of doublets, probably 5-7, to slide away from the remain der of the axoneme. These consistent observations in diverse ciliated cells support the universality of the switch point mechanism.

Qualifications and remaining problems Several modifications of the switch point hypothesis may be necessary to fit actual beat form. In particular, (a) all arms along a particular doublet may not activate synchronously, but rather in progression with bend propagation, and (b) the arms on all doublets within a half axoneme may not activate simultaneously, but rather with a defined phase relationship. In this way, one could envision nine separate activation events rather than two. This might be particularly useful in explaining helical beat in certain sperm tails. Some experimental evidence suggesting that these modifications may be necessary has been provided by Sugino & Machemer (76). Another qualification is that axonemal bending may sometimes arise by mechanisms different from axonemal sliding or at least the sliding that is necessary for cyclic bending (17). The switch point hypothesis does not specify the manner of bend propaga tion during beat, yet major studies indicate that there are extensive feedback systems in the axoneme relating bend generation and bend propagation (19, 11, cf also 66), and propagation must be understood as part of the description of the axonemal mechanism. Furthermore, the nature of the intrinsic oscillator of arm activity that underlies the switching is unspecified so that it is unclear whether the intrinsic switching of this oscillator is mechanically as well as biochemically controlled. Curvature control models, where physical forces regulate arm activity, are successful to a point, particularly in explaining wave propagation along sperm tails (11). There are very limited possibilities of biochemical controls of arm activity because of time constraints, and also because the controls must be fully operative in the presence of only ATP and simple ionic buffers without other non-axonemal constitutents. One possibil ity is that the non-cycling state of the arm is intrinsically part of each

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SATIR & SLEIGH

mechanochemical event. The length of this non-cycling state might be in fluenced by ATP concentration alone or by phosphorylation of some specific dynein constituents, much as the 'latch state' of smooth muscle is influenced by phosphorylation of myosin. Changes in the phosphorylation state could easily be supposed to influence the intrinsic arm cycle in various ways

(62).


MECHANISMS OF PROPULSION BY CILIA Water Transport and Hydrodynamic Considerations The propulsion of water by cilia is a low Reynolds number phenomenon, where viscous forces are more important than inertial forces. Reynolds num ber (Re) for a cilium can be defined by:

Re =

where w

fluid density fluid viSCOSity :::::

X wLr

angular frequency,

L

3.

=

ciliary length, and r

=

ciliary radius. Re

for a cilium is low because the linear dimensions of cilia are so small. A capture zone of water is dragged along around the cilium as it moves, and in the absence of any appreciable inertial effects, the motion of the water stops as soon as the cilium stops moving. This is evolutionarily significant, since it permits rapid behavioral responses of ciliated cells by switching off ciliary motility. The propulsive effect of the cilium on the water is about twice as high when the motion of the cilium is perpendicular to its long axis as when its motion is parallel to the long axis (cf

29) and, the faster the cilium moves, the

faster the water in the capture zone moves. The water tends to adhere to the cell membrane, at the scale of size of the cilium, opposing the tendency of the moving cilium to propel the fluid; the extent of the fluid zone carried around the cilium is limited near the base and increases towards the ciliary tip; the radius of the capture zone at any point along the cilium is approximately half the height of that point above the cell surface (7). During a typical beat cycle, the cilium moves through a large angle in an effective stroke, moving fairly quickly and perpendicular to its long axis, but it moves more slowly along its axis in an unrolling motion close to the cell surface in the recovery stroke. A net movement of water occurs because a larger volume of water is moved to one side in the effective stroke and a smaller volume is carried back in the recovery stroke; indeed, the volume of water carried in the recovery stroke is reduced further because the cilium tends to bend low to one side or the other, close to the cell surface, in this stroke. In the absence of inertial forces, it is easier to think of water being scooped across the cell surface by a ciliary power stroke than being swept along, as if by an oar. The rate of propulsion of water by a cilium, therefore, depends on ciliary


145

length and beat frequency. The force generated by a cilium is related to the number of active dynein arms and to ciliary length, but stiffness depends upon passive mechanical properties such as the Young modulus of the axoneme, as well as on active dynein arm attachment

(29). Efficiency in transmitting force

to the surrounding water during the effective stroke will be lost if the cilium is not stiff enough to remain reasonably straight. Such loss can be reduced by cooperation between adjacent cilia, which either stand so close together that


they form compound structures or beat in close coordination in metachronal waves where each cilium provides some mutual assistance to the motion of neighboring cilia. The formation of metachronal waves is usually important in water propul sion. Adjacent cilia experience forces of viscous interaction if their zones of captured water overlap one another as they move. The strength of this viscous-mechanical interaction between cilia depends on their positions rela tive to the beat direction and their separation relative to their length

(69).

Interactions between adjacent cilia in the plane of the effective stroke tend to result in synchrony of beating, whereas interactions perpendicular to this plane produce metachrony, with metachronal waves moving in the direction towards which the cilium swings sideways in the recovery stroke. It must be emphasized that metachronism is a property of hydrodynamic coupling be tween closely packed, relatively synchronously beating axonemes, and it can be reconstituted in single cells

(33) or on respiratory epithelial cells (91)

after detergent treatment when membraneless axonemes are reactivated by Mg2+-ATP. The importance of metachronism to water propulsion is that at any instant there are adjacent cilia involved in different stages of their effective stroke; each cilium does not accelerate water from rest during its effective stroke, but adds impetus to water already being moved by adjacent cilia. A continuous flow can therefore be maintained at a level (near

1 mm S-I), which

approaches the ciliary tip speed, and the lack of inertial momentum is overcome by use of continuously overlapping viscous paddles. In most ciliary systems specialized for water propulsion, the cilia form narrow bands per pendicular to the water flow. Because the propulsion of water by a cilium is only a local viscous phenomenon, only a shallow zone of water some two cilium lengths deep is transported across the ciliated surface

(7); the total

volume transported is therefore small, unless the surface is extensive, with many ciliary tracts in parallel. The overall propulsive effect depends upon the arrangement of the cilia, their metachronal relationships and pattern of beat ing, as well as on ciliary length and beat frequency.

Mucus Transport Mucus is a non-Newtonian, viscoelastic fluid. It is secreted in concentrated form and rapidly hydrates to a remarkable extent

(83), and then only very

SATIR & SLEIGH

146

slowly disperses in water

(38). When mucus is secreted onto a ciliated

epithelium and hydrates, it spreads as droplets or strings that may coalesce into larger rafts or sheets that are carried along by the cilia at the level of the ciliary tips above a layer of periciliary fluid (cf

39, 58). The ciliary tips

penetrate the mucus during their effective stroke, but moye beneath it in their recovery stroke. The cilia experience a strong resistance ib movement at their


extreme tips when they penetrate a mucous layer, and because of their limited stiffness they can only provide effective propulsion of the mucus if they are very short (usually

5-7 /-tm).· The length of mucus-propelling cilia is a

compromise between the need to shorten the cilium to minimize backward bending when the cilium meets mucus at its tip and the need to lengthen the cilium both to maximize tip speed at a reasonable beat frequency and to maximize the difference in height between power and recovery strokes

(68).

Because the cilia are short, the tip speed achievable at the common beat frequencies of

12-20 Hz is modest, at around 600--1000 /-tm S-1 in the

absence of mucus. While the effective stroke propels the overlying mucus forward, the underlying periciliary fluid merely oscillates to and fro during

the beat cycle. Metachronal coordination of the cilia maintains a continuous

forward thrust on the mucus, and the presence of several or many metachronal waves under a raft of mucus spreads the propUlsive effect so that the whole raft moves as a unit.

Movement and Coordination of Respiratory Tract Cilia Between beat cycles, respiratory tract cilia normally rest in the hands down position

(58, 35). The beat cycle therefore begins with a recovery stroke.

Bending begins at the cilium base and propagates up the shaft. At the same time the cilium is drawn backwards and sideways in a clockwise sweep (as seen from above), moving through 1800 until it is inclined in the opposite direction from its starting position. From here it performs an effective stroke, in a plane perpendicular to the cell surface that brings it back to the rest position. The cilia are packed closely together on mucus-propelling epithelia, and in spite of their short length, the cilia interact with their neighbors as they beat. This interaction results in metachronal coordination, as in water propelling cilia but, because the beat cycle has a rest phase and commences with a recovery stroke, the form of metachronism is a little different. As a cilium commences its recovery stroke and moves backwards and sideways, it presses against other cilia at that side and excites them to commence their recovery stroke

(58); these then excite others, and so on. This recruitment of cilia into a

coordinated wave proceeds, and the wave moves across a few ciliated cells before it dies away

(58, 35), presumably because of a break in the ciliated

surface (at a cell boundary, perhaps) sufficient to disrupt transmission of

147


excitation to commence a recovery stroke. If cilia are beating at low frequen cies the waves tend to cover a small area and propagate across only

2 or 3

cells; but cilia that beat more vigorously form longer metachronal waves that propagate for longer distances. Because the ciliary beat is not as regularly rhythmic, and the cilia do not have such tightly coupled coordination, the metachronal waves of mucus-propelling cilia are less conspicuous than those of water propelling cilia. The metachronal waves on respiratory epithelia do


not sweep across the surface in long lines, but appear as numerous transient islands in a sea of quiescent resting cilia

(58).

Mucociliary Transport Mucus propulsion rates seldom exceed

200 /Lm s -1, but this speed is relative

ly independent of the load, apparently because of the recruitment of additional cilia into the propulsive parts of metachronal waves when necessary. This can happen automatically because when cilia in their stroke encounter increased resistance from the mucus and slow down, the cilia following behind will catch them up and add their propulsive force to the effort exerted by the wave until the mucus is pressed forward. In fact, once the mucus is moving, it will be kept in motion by the simultaneous action of numerous cilia, with rather larger numbers of cilia active at any instant if the load is higher. Any cilium whose power stroke is prolonged by reduced speed of swing may show a shortened rest phase in compensation. Mucus is an incipient gel composed principally of a macromolecular meshwork of glycoprotein molecules in a watery fluid (38, 39,

12). This

composition permits it to show slow distortion by viscous flow over a time scale of the order of many seconds, but to act as a relatively solid elastic structure at the size and time scales of the propulsive swing of a cilium

(20, 67). Thus, while a cilium penetrates and pushes forward a section of a mucus

sheet, energy is stored elastically in the mucus, and the mucus will recoil slowly, if allowed to, unless other cilia propel it further forward; normally the latter occurs and sustained forces are transmitted laterally to regions of the mucus sheet that are not directly propelled. During propulsion, with mucus moving at about

200 p,m S-I, a cilium

commencing its effective stroke will swing upwards with a tip speed faster than the mucus flow, the tip will penetrate the mucus, engage in the elastic gel and be slowed down somewhat so it transmits force to the mucus. As the stroke continues, the ciliary tip moves downwards once more towards the cell surface and its speed falls below that of the mucus, so that the mucus is pulled away from the ciliary tip

(70). During the ensuing rest phase the cilia are bent

low, with tips pointing in the direction of mucus flow; they do not interfere with forward flow of the mucus, but could provide roughness restricting reverse flow. The cilia complete their recovery stroke beneath the mucus. Not

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SATIR & SLEIGH

only is the effective stroke well matched to mucus propulsion, but other features of the beat cycle are also well adapted for this function. When mucus is present, it can only be transported if the depth of the periciliary layer is within certain limits. If the periciliary layer is too deep, the cilia will not penetrate the mucus and will be ineffective in mucus propulsion; normally, however, the cilia will propel away surplus periciliary fluid beneath the mucus and bring mucus within the reach of cilia to recommence propul


sion. If the periciliary layer becomes too shallow, the cilia will be prevented from completing their beat and mucus propUlsion will stop; in this case it is assumed that additional fluid will be released from the epithelium as part of the regulation of fluid flux achieved by chloride secretion and sodium absorp tion

(41, 92). In the absence of mucus the depth of the layer of periciliary

fluid is thought to be maintained by capillary action between the shafts of the close set cilia

(39).

Cilia on the frog palate cease activity if unstimulated, and mucus secretion stops, but if a particle is dropped onto the ciliated surface, it becomes surrounded by mucus, the cilia are stimulated to beat in its vicinity,

and the

patch of mucus gets carried away along a strip of epithelium that is stimulated by its presence (72). Stimulation with a wire probe also induces ciliary beating. In mammals, some cilia on unstimulated epithelia appear to continue to beat, albeit slowly, even in the absence of mucus, but can be stimulated to beat more rapidly by mechanical stimulation

(55). Because of the recruitment

mechanism of the respiratory tract cilia (see above), local mechanical stimula tion of a few cilia, physiologically related to the presence of mucus, will initiate coordinated mucociliary transport. In addition to a local activation of beating in response to mechanical stimulation, a more general activation of the cilia may be promoted by nervous or hormonal mechanisms.

CILIATED EPITHELIAL CELLS AND THE MECHANISM OF CILIARY RESPONSE Ciliated Cell Organization in Relation to Ciliary Function In discussing ciliary activity of the respiratory tract, it is instructive to consider the organization of the apical cell surface of a ciliated epithelial cell in detail. The organization is conservative, possibly because it is useful in influencing cytoskeletal orientation throughout the cytoplasm for vesicular trafficking (cf

89). In the primitive condition, a single motile cilium is

surrounded by a ring of microvilli, but epithelial cells, where transport efficiency of water or mucus seems important, are often multiciliated. Multi ciliarity requires an unusual form of organellogenesis

(15).

The pattern of organization of the apical surface has been carefully studied in an invertebrate epithelium

(48), and a similar organization is found in


tracheal epithelium

149

(25, 4, 22). On a single, fully differentiated, 5-10 /-tm 200 cilia are arranged in a hexagonally-packed semi

long cell, up to about

crystalline array, where each cilium is surrounding by six shorter (ca 1-3 /-tm long) microvilli. The microvilli form at the vertices of a grid of microtubules and actin-based microfilaments that form two trabeculae that underlie the apical cell surface and are integrated into an actin-containing contractile belt, the belt desmosome, at the lateral cell surface. Fodrin has been identified in


the web of filaments between basal bodies

(32); intermediate filaments are

also components of this area. Each basal body is also underpinned by striated rootlets. All these interconnections lead to a mechanically integrated cell cortex where contractile and elastic elements act to resist distortions at the basal end of the beating cilia

(28). The epithelial cells are in mechanical

communication with other cell types and with one another at their actin containing belt and in ionic contact via gap junctions

(49, 54). In cell cultures

of respiratory epithelium, both ciliated and nonciliated cells are electrically coupled, so that changes in ion concentrations or small messenger molecules in one cell spread through the epithelium for short distances

(54).

Mechanism of the Mechanosensitive Response The ciliated cells of the respiratory tract epithelium are mechanosensitive. When the cell surfaces or cilia of cultured ciliated epithelial cells from rabbit trachea are stimulated with a small glass microneedle, beat frequency in creases by

20% or more in a transitory manner (55). The ciliary responses are

lost if extracellular Ca2+ is removed and restored when Ca2+ is replaced.

A23187 is added (57). After ionophore stimulation, mechanical

Further, beat frequency is increased if the Ca2+ -ionophore under similar circumstances

stimulation results in little or no additional increase in beat frequency. Thus ionophore stimulation and mechanical stimulation are thought to work by an identical mechanism, namely an increase in cytoplasmic Ca2+ concentration, caused by Ca2+ entering the cell from the exterior. Mechanical stimulation, which physiologically is initiated by the presence of mucus (72), probably permits extracellular Ca2+ to enter via cell membrane channels. The Ca2+ channel blocker verapamil, albeit at high concentration, added in the presence of external Ca2+ inhibits the response to microneedle stimulation. An increase in Ca2+ concentration in the cytoplasm can be visualized if the epithelium has previously been loaded with dyes, such as fura-2 (56). The spread of increase in Ca2+ from the point of stimulation to adjacent cells can also be directly visualized. In ciliated unicellular organisms and invertebrates, mechanosensitive cell membrane channels induce depolarization to which voltage-gated Ca2+ chan nels in the ciliary membrane itself respond, by opening to permit Ca2+ entry around the axoneme. The time course of this response is rapid compared to

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that of the response of respiratory cell cilia (55), and it may be that voltage gated ciliary Ca2+ channels are absent or reduced in number in the respiratory

tract ciliary membranes. Then, mechanical stimulation might let Ca2+ only into the cell body proper, so that the increase in concentration around the axonemes would be slow. Ca2+ apparently acts directly on the axoneme, as


can be demonstrated with permeabilized cells of respiratory tract epithelia

(85) as well as with similar preparations of invertebrate or protistan cilia (33). The mechanism of Ca2+ interaction within th e axoneme is probably medi ated by calmodulin (CaM) that acts as an axonemal Ca2+ sensor. Some CaM is firmly bound to the axoneme, and the addition of CaM antagonists reverses Ca2+ responses in permeabilized cells (62, 43, 85). In relation to the switch point hypothesis discussed above, formation of Ca2+ -CaM complexes could activate appropriate kinases or phosphatases that migh t affect the timing of arm activity.

Mechanisms of Hormonally Based Responses Ciliary beat frequency is increased in human and other species upon applica tion of some neurotransmitters and certain adrenergic or cholinergic drugs (cf 57,

94). In particular, J3-adrenergic compounds, such as isoproterenol, in

crease beat frequency of mammalian respiratory cilia in vivo and in vitro. The

ef fe ct probably occurs through J3-adrenergic receptors, since propranolol, a J3-antagonist, blocks the response (84). Although J3-adrenergic drugs will stimulate an increase in beat frequency when applied to either the ciliated

surface or the basal (serosal) surface of the tissue, stimulation is primarily at the latter (94). Cholinergi c drugs, however, may stimulate the ciliated surface slightly more effectively. J3-adrenergic receptors act via a signal transduction pathway involving G proteins that activate adenylate cyclase to raise in tracellular cAMP. Presumably, serotonin increases the beat frequency of invertebrate cilia by comparable mechanisms (73). cAMP like Ca2+ probably acts directly on the axonemes (62, 73). For example, cAMP applied directly to ATP-reactivated permeabilized paramecia causes the cells to swim faster (8). The effect is most likely achieved through phosphorylation of specific axonemal polypeptides, mediated by cAMP dependent kinases built into the axonemal structure. Hamasaki et al (23) have recently identified a 29-kd polypeptide in paramecium axonemes that re sponds appropriately to increases in cAMP around the reactivated per meabilized cells. This polypeptide is extracted by procedures that extract dynein arms and might be a regulatory dynein light chain. This result has not yet been demonstrated in o th er species . Tamaoki et al (79) have shown that in cultured rabbit tracheal epithelium, ciliary beat frequency is suppressed by adenosine and related substances. A high affinity

receptor for adenosine, the AI receptor, inhibits adenylate


151

cyclase actIvIty in tissues. In the presence of adenosine, the intracellular cAMP of the respiratory epithelium is decreased; the decreases in beat frequency and cAMP are reversed by 8-phenyltheophylline, an adenosine receptor antagonist. Adenosine-modulated ciliary inhibition may be regulated by uptake or catabolism of adenosine.

Integration of Stimuli Annu. Rev. Physiol. 1990.52:137-155. Downloaded from www.annualreviews.org Access provided by University of California - Davis on 02/11/15. For personal use only.

The normal quiescence of most respiratory cilia at the end of the effective stroke may be due to the fall of intracellular cAMP below a critical level, which temporarily reduces phosphorylation of a critical axonemal polypeptide such that one of the axonemal switches (discussed above) is temporarily inhibited. Mechanical stimulation at the apical surface of the cell by mucus impinging on the cilia would elevate cytoplasmic and axonemal Ca2+ and overcome the block, thus leading to beat in quiescent cilia and increasing beat frequency in beating cilia. This local response would have a limited spread from cell to cell and would be dependent on mucus load

(57). Alternatively,

hormonal or neurotransmitter-based stimulation, mainly at the basal side of the epithelium, would result in an increase in intracellular cAMP. This would lead to a more global response, a generally increased number of beating cilia throughout the epithelium, independent of mucus load and local factors. Sanderson

& Dirksen (57) have demonstrated that the beat frequency response

of tracheal cilia to isoproterenol and mechanical stimulation are additive. This suggests that there is dual control

(57) where Ca2+ and cAMP influence the

axoneme via independent pathways-that is, Ca2+-CaM does not primarily activate an adenylate cyclase, and cAMP does not work, for example, by releasing intracellular stores of Ca2+. It seems likely that, as in other organ isms, both agents will work directly on axonemal polypeptides, probably via changes in their phosphorylation patterns. In a beating cilium, the switch point hypothesis suggests that during each beat every

arm

is temporarily converted to a non-cycling state and that this

occurs asynchronously in opposite half axonemes. A simple, although not unique, explanation of dual control consistent with the switch point hypoth esis may be that cAMP-dependent changes in phosphorylation of axonemal components or of dynein regulatory light chains control the rate of progres sion from non-cycling to cycling state of the dynein

arm,

while Ca2+ -CaM

dependent changes in phosphorylation control the reverse step, namely the conversion of cycling to non-cycling state. In respiratory cilia, increases in either messenger would work to increase the appropriate rate constant and to increase beat frequency; in other cilia, this may not necessarily be the case, even though the axonemal locus of action might be identical. If changes in phosphorylation of axonemal components are a key to our deeper understand ing of the control of ciliary movement and to further exploration of this or

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similar hypotheses, we need to know more specifically which polypeptides change phosphorylation levels under conditions where ciliary behavior is clearly understood in respiratory cilia and in a variety of model systems. ACKNOWLEDGMENT A portion of this work was supported by a grant from the United States Public

Health Service (HL22560). We thank M. Ann Holland for help with the


manuscript.

Literature Cited 1. Afzelius, B. A. 1979. The immotile cilia syndrome and other cil iary dis eases. Int. Rev. Exp. Parhol. 19:1-43 2. Afzelius. B. A. , Gargani, G., Romano. C. 1985. Abnormal leng th of cilia as a pos sibl e cause of defective mucociliary clearance. Eur. J. Respir. Dis. 66:17380 3. Amos, L. A. , Linck, R. W., Klug, A. 1976. Molecular structure of fla gell ar microtubules. In Cell Motility. ed. R. Goldman, T. Pollard, J. L. Rosenbaum, pp. 847-67. New York: Cold Spring Harbor Lab. 4. Arima, T., Sh iba ta , Y., Yamamoto, T. 1985. Three dimensional visualization of basal body st ructur e and s om e cyto skeletal components in the apical zone of tracheal ciliated cells. J. Ultrastruct. Res. 93:61-70 5. Avolio, J., Glazzard, A. N., Hol will, M. E. J., Satir, P. 1986. Structures attacbed to doublet microtubules of cilia: computer modeling of thin section and negat ive stained stereo im ages. Proc. Natl. Acad. Sci. USA 83:4804-8 6. Avolio, J., Le bduska , S. , Satir, P . 1984. Dynein a rm su bstruc ture and the orientation of arm-microtubule attach ments. J. Mol. Bioi. 173:389--401 7. Blake, J. R., Sleigh, M. A. 1974. Me

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