Cilia, Flagella, and Microtubules

LEAH T. HAIMO and JOEL L. ROSENBAUM

In 1676 Leeuwenhoek sent to the Royal Society of London a flagella (10) and resulted in the recognition of the "9 + 2
letter describing his discovery of protozoa and their cilia and axoneme" as the common structural organization of cilia and
flagella . He wrote, "I also discovered a second sort of animal- flagella . Later studies demonstrated, however, that there were
cules, whose figure was an oval, . . . provided with diverse notable departures from this format, particularly among the

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incredibly thin little feet, or little legs [cilia], which were moved spermatazoa of insects (13, 14) .
very nimbly . . ., and wherewith they brought off incredibly With the development of improved fixation and staining
quick motions" (cf. translation of letter 18 [1]) . techniques, accessory structures within the axoneme could be
Cilia and flagella were observed on a variety of cells during visualized . By use of a high percent osmium solution to fix sea
the next two hundred years, and by the end of the 19th century urchin sperm flagella, Afzelius (15) was first to describe the
several theories had been proposed to explain flagellar beating . arms, later termed dynein (16), present in two rows along the
For example, it was postulated that flagella were lifeless and larger fiber of each outer doublet . Noting their orientation
were moved by elements within the cell body or were hollow toward the smaller fiber of the adjacent outer doublet, Afzelius
structures into which fluid was injected and withdrawn . Alter- (15) postulated that the dynein arms might be active in a sliding
natively, it was suggested that flagella contained a central filament model of flagellar beating. The next 15 years were to
contracting fiber or possessed a fibrillar substructure . This last prove this hypothesis correct . Afzelius also observed that the
theory had been formulated in 1868 by Engelmann, who dynein arms imparted an asymmetry to the axoneme . Subse-
proposed that the flagellum contained aligned fibrillar elements quently, Gibbons and Grimstone (17) demonstrated in several
which shortened into a globular form during beating . Although species that the arms always pointed clockwise around the
Jensen in 1887 and Ballowitz in 1888 observed numerous fibrils axoneme when viewed from base to tip. They introduced the
in the fraying tips of sperm tails, the prevailing belief at the nomenclature terming the fiber bearing the dynein arms the
turn of the century was that the flagellum contained a solid A-subfiber, the other the B-subfiber .
contracting core (see reference 2) . Studies of axonemes in longitudinal sections demonstrated
In later studies, the fibrillar substructure of cilia and flagella that the dynein arms, as well as the other major accessory
was described in a variety of cells (2, 3), and these observations structures, were regularly spaced along the length of the axo-
were confirmed with the development of the electron micro- neme . The dynein arms in both the inner and outer rows had
scope (4-7) . The number of fibrils was variously reported as a center-to-center spacing of approximately 24 nm along the
between nine and twelve, and the diversity of ciliary and length of the A-subfiber (18-22) . In addition, the inner and
flagellar wave forms suggested no reason that the number of outer rows of arms were axially staggered with respect to each
fibrils would be constant among species . other (23, 24), and superimposition of the two rows of arms
may have accounted for earlier reports of an arm spacing of
Axonemes : the 9 + 2 Pattern 12-16 nm (17, 25) .
Radial spokes were visualized in thin sections as projections
Based on electron-microscope studies of plant sperm in a from each A-subfiber to the central sheath (15) . These spokes
number of species (8-11), a diagrammatic reconstruction of the terminated in an enlarged head that was incorrectly identified
flagellum was proposed (10) . Considering the resolution of the as a fiber running the length of the axoneme (17) . More recent
shadow-cast, whole-mount preparations used in these studies, studies verified the "spokelike" ultrastructure and revealed that
the model was remarkable in its accuracy . During this same the radial spokes occurred in pairs in Chlamydomonas (19, 22,
period, techniques were developed for embedding and section- 25, 26) or triplets in Elliptio gill and Tetrahymena cilia (18, 27),
ing biological material for electron microscopy, and in 1954 which were grouped at intervals of about 96 nm along the
Fawcett and Porter published the first ultrastructural study of lengths of the A-subfibbes.
cilia (12) . Regardless of origin, all cilia were observed to possess The central sheath observed in whole-mount preparations
the same configuration, nine hollow, doublet fibrils equidistant by Manton and Clarke (10) and described in cross sections of
from and radially surrounding a central pair of single fibrils. cilia by Gibbons and Grimstone (17) consisted of two rows of
This structure was identical with that proposed for plant sperm projections spaced at 16-nm intervals along each of the two
central fibrils (22, 25, 27, 28) . The final axonemal structure to
LEAH T . HAIMOand JOEL L . ROSENBAUM Department of Biology, Yale be described were the nexin links (29, 30), which connected the
University, New Haven, Connecticut A-subfiber of one outer doublet to the adjacent outer doublet

THE JOURNAL OF CELL BIOLOGY " VOLUME 91 NO . 3 PT . 2 DECEMBER 1981 125s-130s

©The Rockefeller University Press - 0021-9525/81/12/125x/06 $1 .00 125s
and were axially spaced at approximately 96-nm intervals (21, The Sliding Mechanism
22,31).
Theoretical analysis of the mechanism generating the motive
Optical diffraction patterns of negatively stained axonemal
force within the flagellum indicated that shearing between
fibers, microtubules, revealed the existence of a strong 4-nm as
adjacent outer doublets that resulted in microtubule sliding
well as an 8-nm axial periodicity (23, 32-34) corresponding to
could account for the uniform propagation of waves along the
the monomer and heterodimer subunit composition of the axoneme (63, 64) . Sliding between outer doublets was demon-
microtubules (33, 34) . The relative intensity of the 8-nm repeat strated first in sectioned axonemes (65, 66) and more directly
compared with the 4-nm repeat varied considerably with dif- by dark-field visualization of trypsin-digested axonemes sup-
ferent types of microtubules, and suggested that accessory plied with ATP (67). These and later studies indicated that the
structures on microtubules might contribute to an amplification
sliding motions were mediated by dynein arms cyclically at-
of the 8-nm diffraction pattern (23). The observations that the
taching to and causing a shearing force between adjacent outer
dynein arms, central pair projections, radial spokes, and nexin
doublet microtubules (68, 69) exerted toward the tip of the
links, all bound to the flagellar microtubules at approximate axoneme (70).
multiples of 8 nm, supported this hypothesis, and it has been Dynein arms present on intact axonemes or reattached to
proposed that the axial spacing between adjacent binding sites
dynein-extracted axonemes projected from the wall of the
on microtubules is 8 nm (20).
microtubules at an angle of approximately 55° . In Chlamydo-
monas, the arms tilted toward the tip of the flagellum (19),
Dynein whereas in mollusc gill and Tetrahymena the dynein arms tilted
toward the base of the cilium (57, 70-72) . To exert a directional

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Concurrent with a description of axonemal fine structure has
force resulting in sliding (70), it has been postulated that the
been an elucidation of the molecular basis for ciliary and
orientation of the dynein arms changes during the cross-bridge
flagellar motility . Spermatozoa were shown to contain both
cycle (69), and a recent study supports this suggestion (73).
measurable amounts of adenosine triphosphate (ATP) and
Such a change in the orientation might account for the above
ATPase activity (35), which later studies demonstrated was differences in arm-tilt direction .
concentrated in the flagellum (36-38) . Using techniques devel-
Transient ATP-dependent bridging between adjacent outer
oped to study muscle contraction (39), Hoffman-Berling (40)
doublets was predicted from the orientation of the dynein arms
discovered that addition of ATP to glycerinated sperm resulted and by their role in sliding, but these bridges were preserved
in the reactivation of beating . The wave form resembled that
and, therefore, visualized only when axonemes were fixed in
of live sperm and indicated that the energy for motility was
rigor (74). The existence of a rigor state had been suggested by
supplied by ATP hydrolysis. Reactivation specifically required
observations that bull sperm flagella became plasticized after
ATP and Mg" and was inhibited by EDTA (38, 41, 42) .
ATP addition (75). Subsequent studies demonstrated that re-
After the development of methods to isolate cilia in large
moval of ATP from reactivating axonemes caused them to
quantities (43), Gibbons (29, 44) demonstrated by selective
enter rigor, characterized by a wave form frozen at the time of
solubilization of Tetrahymena axonemes that the ATPase activ-
ATP depletion and maintained by dynein arm cross-bridges
ity was localized in the two rows of arms on the A-subfiber .
between adjacent outer doublets (74, 76) . Release of the B-
The enzyme was named "dynein," force protein, for its postu-
subfiber of the adjacent outer doublet by dynein as manifested
lated role in the mechanochemical transduction of energy
by relaxation of the rigor wave required ATP binding, whereas
required for motility (16). Dynein specifically hydrolyzed ATP
subsequent reactivation required ATP hydrolysis (69, 76, 77) .
in preference to other nucleotides, required Mg" or Ca" for
In other studies, however, dynein cross-bridges between adja-
its activity, and was inhibited by EDTA, characteristics that
cent outer doublet microtubules could be produced by fixing
correlated closely with those necessary for axonemal reactiva- axonemes in appropriate buffers containing divalent cations
tion (45, 46). Although the inner and outer rows of dynein
(78). Nevertheless, addition of ATP to these cation-induced
arms appeared to be functionally equivalent (47), they were rigor axonemes resulted in relaxation of dynein cross-bridges
morphologically distinct (48), had different solubilities (49), between particular outer doublets (79). Although these obser-
and were composed of different polypeptides (26, 50) . The vations conflict with studies demonstrating that reactivating
existence of more than one axonemal dynein has been dem- axonemes entered rigor when either the Mgt+ (80) or ATP (76)
onstrated in a number of studies (29, 51-55). concentration of the reactivation medium was rapidly lowered,
A specific association between the dynein arms and the A- they indicate that the dynein arms do, in fact, cross-bridge the
subfiber was indicated by the observations that the solubilized
adjacent outer doublet microtubule, and that the cross-bridges
arms rebound to their original sites on the A-subfiber (29, 56).
are ATP dependent.
Recently, it was demonstrated that solubilized dynein arms
also rebound to the B-subfiber, presumably to those sites with
Microtubules
which the arms would normally interact during beating (57).
The role of dynein arms in motility, implicated both by their Based on the observations of Van Beneden that protoplasmic
ATPase activity and orientation toward the adjacent outer fibrillae existed within the spindle and of Ballowitz that sperm
doublet microtubule, was confirmed in studies that demon- tails contained minute fibrils, Wilson (81) postulated that the
strated that the beat frequency of reactivation was directly substance and outgrowth of the flagellar fibrils were compa-
proportional to the number of dynein arms present within the rable with those of the fibrils within the spindle . Moreover, he
axoneme (47, 56, 58) . Furthermore, antibody prepared against suggested that the contractile behavior of the spindle fibers
dynein inhibited both its ATPase activity and axonemal reac- noted by Boveri might also apply to the flagellum . It was not
tivation (59-61) . The existence of paralyzed flagellar mutants until the 1960s, however, that the relationship between cyto-
lacking dynein arms in both Chlamydomonas (26) and humans plasmic and flagellar microtubules was to be established, and
(62) also suggested a motile role for the dynein arms . it has not yet been determined if motility associated with
12 6s THE JOURNAL OF CELL BIOLOGY " VOLUME 91, 1981
cytoplasmic microtubules is elicited by a mechanism similar to inent class of polypeptides having a high molecular weight (ca .
that of the flagellum . 300,000 daltons) copurified stoichiometrically with tubulin
Although early researchers using the light microscope had through several cycles of assembly (110, 111). Separation of the
observed fibers within the spindle, the periphery of red blood 6S tubulin from these proteins inhibited its ability to polym-
cells, and in neurites, the existence of these structures had been erize into microtubules except at high protein concentration
a matter of controversy that was finally resolved with the (112) or unphysiological solvent conditions (113-115) . Read-
advent of electron microscopy. By use of osmium as a fixative, dition of the high molecular-weight MAPs (microtubule-asso-
fibrous structures were observed in the mitotic apparatus (82, ciated proteins) to 6S tubulin stimulated both the rate and
83) and in nerve axons (84), but their widespread distribution extent of polymerization by lowering the critical concentration
was only fully appreciated after the development of glutaral- of tubulin necessary for assembly (110, 116) . MAPs stabilized
dehyde fixation (85) . In one of the earliest uses of this tech- the microtubules (117) by lowering the reverse rate constant
nique, the fibers were described as microtubules whose simi- for assembly (118) . Another class of proteins that copurified
larity in morphology to the fibrils within the axoneme was with tubulin, termed tau, was also shown to stimulate micro-
apparent (86, 87) . tubule assembly (109, 119) .
Ultrastructural analysis revealed the presence of 13 longitu- By examining the incorporation of radioactive precursors
dinal protofilaments comprising the walls of both cytoplasmic into regenerating flagella, it was determined that flagella assem-
and axonemal central pair and A-subfiber microtubules (88- bled principally at their distal tips (120, 121) . Tubulin obtained
91) . Moreover, optical diffraction studies of cytoplasmic micro- from cytoplasmic microtubules also exhibited directional as-
tubules demonstrated that they have both a 4- and an 8-nm sembly . For example, addition of brain tubulin onto isolated
axial periodicity (92), as had been observed previously for basal bodies (122), centrioles (123-125), or axonemes (19, 126)

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flagellar microtubules. In addition, the subunit dimers were resulted in microtubule polymerization predominantly onto
axially staggered in adjacent protofilaments in both the A- the distal ends of these organelles . Similarly, addition of brain
subfiber and cytoplasmic microtubules, indicating that they tubulin onto microtubule pieces resulted in preferential assem-
were structurally similar. On the other hand, in the B-subfiber bly onto one end of these pieces (108, 127) . Recent studies of
the dimers were lined up in adjacent protofilaments, thereby microtubule assembly in vitro have indicated that polymeri-
indicating a distinct surface lattice (23) . zation occurred at one end of the microtubule and depolym-
The use of the drug colchicine has been basic to the under- erization occurred at the opposite end (128) . These studies
standing of the chemical composition of microtubules . Noting suggest that the two ends of the microtubule have different
that colchicine caused a reversible loss in birefringence of critical concentrations for assembly, and at polymerization
spindle fibers, Inoue (93) postulated that the drug bound to the equilibrium the rate of tubulin addition onto one end of the
subunit of these fibers . Later, Taylor (94) demonstrated that microtubule would equal the rate off the other end . Other
colchicine was reversibly bound by a substance within the cell, experimentation, however, has demonstrated that microtubules
and in subsequent studies a colchicine-binding protein was assembled from kinetochores or centrosomes polymerized and
isolated and shown to be the subunit of both cytoplasmic (95, depolymerized at the same end (129) .
96) and flagellar (97, 98) microtubules . Characterization of the The polarity of microtubules, as manifested in their direc-
purified colchicine-binding protein from brain tissue revealed tional polymerization, may permit them to function in direc-
it to bind 2 mol of guanosine triphosphate (GTP) per mol of tional intracellular movements . Of these movements, those
protein and to have a sedimentation coefficient of 6S and a exhibited during mitosis have generated the most interest and
native molecular weight of about 110,000 daltons (99), prop- have been the subject of several different models (130-135) .
erties identical with those of the colchicine-binding protein in Both chromosomes (123, 136, 137) and centrosomes (124, 125,
the mitotic apparatus and axoneme . The protein was given the 136) served as nucleation sites for microtubule assembly in
name "tubulin" (100) . Electrophoretic analysis revealed tubu- vitro. Studies of the direction of this assembly have indicated
lin to be composed of two closely migrating 55,000-dalton that both kinetechore (129, 138) and centriolar (129, 139)
polypeptides present in equal amounts (101) . Recent experi- microtubules added tubulin subunits at the microtubule end
mentation has shown that 6S tubulin is a heterodimer com- distal to the organizing center . These observations suggest that
posed of these two components (102). Although similar in each half-spindle of the mitotic apparatus is composed of
subunit composition and structure, flagellar microtubules dif- microtubules present in an antiparallel array .'
fered from cytoplasmic microtubules in their ability to form While axonemes could be reactivated to beat in vitro and,
doublets and in their relative stability ; the biochemical basis accordingly, their movements analyzed biochemically, no
for these differences has not been determined . equivalent assay has been developed to study the movements
associated with cytoplasmic microtubules, although work on
Assembly In Vitro : the Role of Accessory the reactivation of mitotic movements is progressing (140) .
Proteins and Microtubule Polarity Nevertheless, recent studies have provided some insight into
the mechanism by which motility is elicited within the cyto-
With the discovery of conditions that permitted the in vitro plasm .
assembly of tubulin into microtubules (103), it was possible to
study their biochemistry . Microtubule assembly occurred at MAPs, Arms, and Movement
37°C from homogenates of brain in buffers containing Mg",
GTP, and a calcium chelator . Tubulin has subsequently been The high molecular-weight MAPs, which copurified with
assembled from a number of other sources, including flagellar brain tubulin, had an electrophoretic mobility similar to that
outer doublet microtubules (104-106) . of flagellar dynein, leading to speculation that they might be
Electrophoretic analysis of the protein composition of brain
microtubules assembled in vitro revealed the presence of sev- ' Direct visualization of microtubule polarity has recently revealed the
eral protein species in addition to tubulin (107-109) . A prom- half-spindle to be composed of parallel microtubules (139a, 1396) .

HAIMO AND ROSENBAUM Cilia, Flagella, and Microtubules 12 7s

 functionally . This
containing the .
brain review
flagellar the
of . beginning .
low several
assembled the .
ATPases . attempts
have tubules .
cytoplasm development
with . procedure
that and
microtubules of .
with microtubules
(147) . other
MAPs of
112) flagella .
microtubules . development
observed . of
arms microtubules
has

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tibody
ically has
the . moving .
however,
function .
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130s THE JOURNAL OF CELL BIOLOGY " VOLUME 91, 1981