Jpn. J. Protozool. Vol. 36, No. 2. (2003)

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Review
Control Molecules in Protozoan Ciliary MotilityPeter Satir
Dept. of Anatomy & Structural Biology, Albert Einstein College of Medicine, Bronx,
NY 10461 USA
Keywords: Paramecium, Tetrahymena, cAMP, Ca2+, dynein, ciliary reversal

Ciliated protozoa such as Tetrahymena or
Paramecium have long been important organisms for the study of ciliary motility. The coordinated activity of many hundreds of cilia is
responsible for the movement of these ciliates
through water. Furthermore, changes in that
activity are responsible for the behavior of thecells, in particular chemotactic and mechanotactic responses. Almost a century ago,
Jennings (1906) described the avoiding reactionof Paramecium, whereby the cell backs awayfrom an obstacle by changing the direction ofciliary beat, a so-called ciliary reversal. It wasthen shown that this response was produced
when a voltage sensitive Ca2+ channel of theciliary membrane opened, permitting Ca2+ toenter the cilium (Eckert, 1972; Eckert et al.
1976).
Ultrastructural studies beginning with
Fawcett and Porter (1954) showed that virtually
all motile cilia are comprised of a microtubulebased 9+2 axoneme surrounded by the ciliary

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membrane, an extension of the cell membrane.
The ciliary membrane contains the receptors
and channels for the transduction of environmental events into ciliary, and consequently,
cell behavior. The signaling molecules, second
messengers such as Ca2+ or cyclic AMP(cAMP), act on axonemal proteins to change
ciliary beat characteristics. This can be demonstrated experimentally by removing the ciliary
membrane with a detergent such as Triton X100; the result is a naked axoneme directly exposed to the surrounding solution. In Paramecium, the cortical epiplasmic network maintains
cell shape and ciliary position after detergent
treatment (Lieberman et al 1988). When ATP is
added, the cilia beat and the membranelessmodel swims just as the living cell (Fig. 1).
When the appropriate second messenger, forexample Ca2+, is added the cilia change theirbeat characteristics, again mimicking the response of the living cell, with one exception. In
the living cell, the cell adapts spontaneously.
For example, when Ca2+ pumps in the ciliary
membrane remove Ca2+ from around theaxoneme, the cell resumes forward swimming.
In the membraneless models, ciliary reversal
continues until the ATP is depleted or the model
falls apart. Naitoh and Kaneko (1972, 1973)
and later Lieberman et al. (1988) carefully

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Control Molecules in Protozoan Ciliary Motility

1aFig. 1. Motility and control molecules are built into the axoneme. A: Permeabilization dissolves the ciliary and cell
membranes of Paramecium. Ciliary membrane at arrowhead in (a), missing in (b). Arrows show width of cortical ridge;
cell membrane present in (a), absent in (b). Bar 0.25 µm (from Lieberman et al., 1988 with permission). B: Fixed metachronal wave. (Above) Intact Paramecium swimming forward just before fixation. (Below) Membraneless Paramecium
reactivated by ATP, swimming after permeabilization . Bar 10 µm (Satir et al., 1993 with permission).

Jpn. J. Protozool. Vol. 36, No. 2. (2003)

1b

89

Paramecium tetraurelia
intact cell

extracted modelFig. 1b. See p. 88 for explanation.

measured the Ca2+ responses of detergent
treated Paramecium. When Ca2+ was 10-7M(pCa7), the models swam forward; forward
swimming speed decreased as Ca2+ rose toward
10-6M (pCa6), until the model virtually stopped,
and the individual ciliary axonemes beat in an
uncoordinated helical fashion. Further increases in Ca2+ reactivated the swimming, but
now the models swam backward with increasing
speed as Ca2+ increased toward 10-5M (pCa5).
The responding axoneme functions as acellular nanomachine comprised of approximately 250 different protein components arranged into structural complexes including theaxonemal microtubules, inner and outer dyneinarms (IDAs and ODAs), spokes etc. Axoneme
structure can be shown accurately in threedimensional computer reconstruction (Taylor etal. 1999) (Fig. 2).
The structure and mechanism of motion of
the axoneme is evolutionarily conserved fromprotozoa to man. All proteins necessary for90

Control
Control Molecules
Molecules inin Protozoan
Protozoan Ciliary
Ciliary Motility
Motility

Subfiber A
Subfiber B

A

Outer dynein arm (ODA)

Radial Spoke
Spoke HeadCentral Pair

Inner dynein arm (IDA)

Interdoublet link

AKAP97

B
Spoke Head
ODA

IDA

Fig. 2. A: Computer reconstruction of Tetrahymena axoneme with major structures labeled (modified
from Taylor et al., 1999). B: Enface view of Tetrahymena doublet subfiber A with attachments showing
arrangement of ODA and IDA subunits, radial spokes and AKAP97 (modified after Taylor et al., 1999)

motility and control of motility are built into theaxoneme at specific locations. The mechanismof ciliary motion depends on a series of molecular motors built into the axoneme. These
are the IDAs and ODAs, which act to produce a
vectorial force that causes the doublet microtubules to slide relative to one another; thespoke and links transduce the force into a defined bend. The central pair divides theaxoneme into opposite halves. The arms on
doublets on one side of the axoneme are predominantly active during the effective stroke ofthe cilium; the arms on the other side are mainly
active during the recovery stroke, and activity

Jpn. J. Protozool. Vol. 36, No. 2. (2003)

cAMP

A

cAMP –PK (associated with axoneme)
pp29

p29

22A
Dynein (outer(outerarm)arm)
22S Dynein

22S Dynein *Faster sliding
decrease in time for aconstant amount ofsliding per ciliary stroke
Increased beat frequency
Faster swimming

B

Ca2+

IDA3 Centrin

IDA3 Centrin-Ca2+
Faster sliding
Increase in amount ofsliding for a constant time
taken for ciliary stroke
Increased bend amplitude
Change of Beat Form
Change of Swimming direction
Fig. 3. Signal transduction cascades in the axoneme.

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Motility
Fig. 4. Localization ofcentrinin
Tetrahymena
axonemes. A: In permeabilized cells, centrin localizes tobasal bodies; not evident alongaxoneme. Bar 10 µm B:
Isolation of axonemes reveals
centrin. Co-localization with
α-tubulin along axonemal
length shown by displacementof images. Addition of competing peptide (TcN peptide)abolishes centrin but not atubulin localization. Bar 5 µm.
C: Immunogold labeling ofaxoneme using centrin primary
antibody. Bar 0.1 µm (From
Guerra et al. 2003 with permission).

A

TcN Centrin - red, α-tubulin - green

B

C(+) TcN peptideJpn. J. Protozool. Vol. 36, No. 2. (2003)

Fig. 5. Ca2+ binding to centrin speeds up IDA activity. Measurements of IDA translocation velocity (v0) in
the absence (control) and presence of Ca2+ (10 mM CaCl2),
the Ca2+ mimicking peptide CALP1 and/or an antibody to
Tetrahymena centrin (from Guerra et al., 2003 with permission).

switches back and forth. The current state ofunderstanding of this switch point mechanism issummarized in Satir (1997). Studies withChlamydomonas swimming mutants show thatODA mutants have approximately normal beatform, while beat frequency falls to between 1/3
to 1/2 normal (Brokaw and Kamiya, 1987); IDA
mutants, on the other hand, have almost normalbeat frequency, but altered beat form. This implies that IDAs primarily control beat form,
while the ODAs primarily control beat frequency. The end point of a response that alters
cell swimming behavior by changing ciliary
beat characteristics is a change in the sliding
velocity of the axonemal microtubules causedby changes in dynein arm activity. In the absence of a change in beat form, beat frequency is
directly proportional to ODA produced microtubule sliding velocity; likewise, in the absenceof a change in beat frequency, beat form, as
measured by bend amplitude in the effective andrecovery stokes, is directly proportional to IDA
produced microtubule sliding velocity.

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cAMP is a signal transduction moleculethat increases ciliary beat frequency. In Paramecium, cAMP is generated via a hyperpolarizing membrane K+ channel (Schultz et al.,1992). When a cell is stimulated appropriately,cAMP rises and beat frequency increases. In
permeabilized cells, cAMP applied directly tothe axoneme speeds up swimming by 60%. In
the absence of cell shape or viscosity changes,
swimming speed increases correlate with beatfrequency increases. An ODA light chain, p29,
is phosphorylated in the presence of cAMP
(Hamasaki, et al., 1988). High Ca2+ abolishes
the swimming speed increase by inhibiting p29
phosphorylation. 22S outer arm dynein can beextracted from cAMP or cAMP+Ca2+ treatedaxonemes and used for in vitro motility assays
to measure microtubule sliding rate. The cAMP
treated dynein with phosphorylated p29 produces a 40% increase in microtubule sliding rate(comparable to the increase in swimming speed)
while cAMP+Ca2+ treated dynein without phosphorylated p29 shows no increase in microtubule sliding rate compared to controls. p29
can be extracted and readded to the 22S dynein(Barkalow et al., 1994; Hamasaki et al., 1995).
When phosphorylated p29 is readded, microtubule sliding rate is increased in the same way
as before, while addition of p29 which has notbeen phosphorylated produces no increase. It
can concluded that cAMP works in a signaltransduction cascade that produces phosphorylation of p29 of the 22S ODAs, which in turncauses faster microtubule sliding, increased beatfrequency and faster swimming (Fig. 3A). An
ortholog of p29, p34, present in Tetrahymena
thermophila, is also a phosphorylatable ODA
light chain that becomes phosphorylated in thepresence of cAMP and causes an increase in
microtubule sliding velocity (Christensen et al.,2001). A similar ortholog is present in mammalian cilia.

94

ControlP. tetraurelia
MoleculesAutogamy-related
in Protozoan Ciliary
Genes
MotilitycAMP functions as a co-factor that activates a protein kinase (PKA) that is incorporated
into the structure of the axoneme. The regulatory subunits of the protein kinases of Paramecium have been studied extensively by Nelson's
group (Bonini et al., 1991). In the axoneme, theregulatory subunits of PKAs are docked by Akinase anchoring proteins (AKAPs). In Chlamydomonas, AKAP97 is a radial spoke component (rsp3) located near the base of the spoke(Fig. 2B), while AKAP240 is a component of
the central pair (Reviewed in Porter and Sale,
2000). Protein phosphatases can also be built
into the axoneme, but the phosphatase that removes phosphate from phosphorylated p29 isextracted by detergent treatment, so that permeabilized Paramecium cannot adapt after cAMP
treatment.
Although p29 is not the exclusive substrate
for the axonemal PKAs and phosphatases, it is
the substrate most closely linked to beat frequency and swimming speed changes wherebeat form is largely unchanged. Ca2+ affects
ODA function in some measure by interaction
with the PKA-phosphatase system that controls
p29 phosphorylation. Between pCa7 and pCa6
in Paramecium, Ca2+ primarily affects beat frequency. However, between pCa6 and pCa5,
Ca2+ causes changes in beat form and in microtubule activation patterns, resulting in ciliary
reversal, independently of phosphorylation
changes.
Ca2+ normally acts via Ca2+ binding proteins that activate specific enzymes. One classof such binding proteins are the 'EF hand' proteins, such as calmodulin. EF hand proteins
could be part of the axonemal response mechanism responsible for changes in beat form inciliate swimming. The obvious candidate enzyme for activation by a Ca2+ binding EF handprotein is inner arm dynein, since regulation of

IDA sliding rate would affect bend amplitude,
as discussed previously. Accordingly, we haverecently examined the EF hand proteins of Tetrahymena with this possibility in mind (Guerra
et al., 2003). There are four closely related EF
hand proteins in Tetrahymena: TCBP23 and
TCBP25 are found in the cell cortex; centrin andcalmodulin are found in cilia.
Tetrahymena centrin is particularly interesting. LeDizet and Piperno (1995) found that
centrin was a component of the Chlamydomonas inner dynein arm I3. Although centrin
could be shown to be present in Tetrahymena
and Paramecium basal bodies (Fig. 4) and elsewhere in cortical fibers (in Paramecium as acontractile cortical network), the protein was
thought not to be present in the ciliary axonemesof these ciliates. Guerra et al. (2003) havecloned and sequenced Tetrahymena centrin andprepared an antibody to the unique N-terminal
region of molecule. The antibody does not recognize calmodulin. Although centrin cannot bedemonstrated by immunofluorescent or immunogold antibodies techniques in cilia of fixed
cells (Fig. 4A), in isolated axonemes it is clearly
localized all along the axoneme; immunofluorescent localization along the axoneme is abolished by preteatment with the N-terminal peptide to which the Tetrahymena centrin antibody
has been made (Fig. 4B,C). Immunoblots
against isolated dynein fractions show that centrin is localized to 14S IDA fractions, whilecalmodulin is not localized to dynein.
In in vitro motility assays using 14S IDA
fractions, Ca2+ produces a highly significant
increase in microtubule sliding velocity, measured as V0 (Fig. 5) (Guerra et al., 2003). A
similar increase is seen when CALP1, a synthetic peptide that mimics Ca2+ binding to EF
hand proteins, is used in place of Ca2+. The

Jpn. J. Protozool. Vol. 36, No. 2. (2003)

increase is abolished by first treating the IDA
fraction with the antibody to the N-terminal
region of Tetrahymena centrin. These results
indicate that centrin containing IDAs responddirectly to the binding of Ca2+ by changing thevelocity of microtubule sliding in the axoneme,such that the amount of sliding during a constanttime of the ciliary stroke is increased (Fig. 3B).
To provide ciliary reversal, Ca2+ must also work
asymmetrically on the phases of the stoke;speculatively, this might be accomplished by
Ca2+ binding to calmodulin associated with thecentral pair of microtubules, which could affectthe switching mechanism.
We have come to a partial understanding of
the signal transduction cascades governed bycAMP and Ca2+ that produce ciliary behavior,defining principal control molecules involved inregulating the dynein arms in each case: p29 or
its ortholog, a regulatory light chain of the outerarms, whose phosphorylation state regulates
ODA activity, and centrin, the Ca2+ binding EF
hand protein of a group of inner arms, whereCa2+ binding directly regulates IDA activity,
much as Ca2+ binding myosin light chains regulate myosin activity. IDA and ODA activity is,of course, regulated in a coordinated way thatnecessitates feedback between the two systems;
how such co-ordination is achieved is unknown.
Nevertheless, this approach has begun to indicate how specific molecules interact with theaxonemal mechanism of motion to generate
behavior and response in ciliated microorganisms.
Acknowledgement
Work reported in this manuscript was donein collaboration with many helpful students andcolleagues. I especially thank T. Hamasaki, S.
T. Christensen, M.E.J. Holwill, and C. Guerra,

95

who also provided assistance in preparing themanuscript for publication. Some current support was provided by NIDDK (DK41918 andDK41296) and NCI (CA09475).

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