Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520-8103
The Problem: An Organelle whose Assembly Site Is
Distant from the Site of Protein Synthesis
Unlike most organelles, which are surrounded by cytoplasm, the flagellum protrudes from the cell surface extending tens or even hundreds of microns into the external
medium. This elongated organelle must import all the
macromolecules required for its assembly, maintenance,
and function including >200 polypeptides that make up
the microtubular axoneme (Dutcher, 1995 A dramatic example of the delivery of molecules into
the flagellum is seen during flagellar regeneration in the
biflagellate alga Chlamydomonas: flagella 10 µm long are
assembled in ~1 h. As the organelle elongates, flagellar
precursors must reach the site of assembly at the distal tip
(Rosenbaum and Child, 1967 Extensive transport also occurs into nonmotile sensory
cilia of certain neuronal cells. The outer segments of retinal rods (ROS),1 for example, are modified cilia replete
with the membrane-associated photoreceptor machinery
for receiving and transducing light signals. These stacks of
membranes are constantly being replenished: in mouse,
the membranes of the ROS are completely replaced every 2 wk. The only connection between the ROS and the rod
inner segment (RIS), in which synthesis of all the components occurs, is the connecting cilium composed of a membrane bound 9+0 axoneme. All the materials required for
continual turnover of the ROS must pass through the connecting cilium.
The following briefly reviews recent literature that addresses how the cell rapidly mobilizes over 200 polypeptides required for flagellar assembly, and how it transports
these polypeptides to the flagellar tip assembly site.
The Solution: Intraflagellar Transport of Preassembled
Flagellar Complexes
Many flagellar proteins exist as complexes of multiple
polypeptides, e.g., the radial spokes contain 17 polypeptides and the outer dynein arms contain >15 polypeptides
(Dutcher, 1995 Intraflagellar transport (IFT) appears to be the mechanism that moves flagellar precursors to the flagellar tip.
IFT, visualized with high-resolution video-enhanced differential interference-contrast (DIC) microscopy, is a motility located between the flagellar membrane and axoneme (Kozminski et al., 1993 Thin sectioning and electron microscopy indicate that
the material moving beneath the flagellar membrane by
IFT consists of "lollipop-shaped" particles occurring in
groups of varying particle numbers, called "rafts" (Fig. 1;
Kozminski et al., 1993
ARTICLE
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Article
References
), all the constituents of the flagellar membrane, as well as a prodigious amount of ATP to supply the thousands of dynein motors
that drive flagellar motility.
; Johnson and Rosenbaum,
1992
), which grows farther and farther away from the site
of protein synthesis. The site of tubulin addition during
flagellar assembly was identified by fusing cells with half-length flagella to cells containing epitope-tagged tubulin: all the tagged tubulin incorporated into the growing flagella at their distal tips. When cells with full-length flagella
lacking radial spokes were fused to wild-type cells, radial
spokes from the wild-type cytoplasm entered the spokeless flagella, assembled at the distal tips of the flagella, and
gradually continued assembly toward the base (Johnson
and Rosenbaum, 1992
). Similar results were obtained with
inner dynein arms (Piperno et al., 1996
). Thus, there appears to be a mechanism for transporting axonemal precursors to the distal tip of the flagellum, whether or not it
is elongating.
); it would be advantageous for the cell to
preassemble these axonemal structures in the cytoplasm
rather than to send individual polypeptides into the flagella for assembly. In keeping with this idea, assembled radial spoke complexes have been found in the cytoplasm
of Chlamydomonas (Diener, D.R., D.G. Cole, and J.L.
Rosenbaum. 1996. ASCB Meeting, San Francisco. Abstract 273). Similarly, flagellar outer dynein arms from
Chlamydomonas and Paramecium also preassemble in the cytoplasm (Fok et al., 1994
; Fowkes and Mitchell, 1998
).
Because during flagellar assembly these flagellar precursor complexes assemble at the flagellar tip, a mechanism
to transport large protein complexes through the flagellum
was hypothesized.
, 1995
, 1998
). Particles of
variable size travel to the flagellar tip (anterograde transport) at 2.0 µm/s and smaller particles return from the tip
to the base (retrograde transport) at 3.5 µm/s (Kozminski
et al., 1993
). The movement is continuous and linear along
the entire flagella. IFT is neither affected by mutations
that cause immotile flagella, e.g., mutations affecting dynein arms, radial spokes, or central pair microtubules, nor
does it appear to be related to other motilities associated
with the flagellar membrane, e.g., gliding of whole cells on
surfaces by means of their outstretched flagella or the movement of polystyrene beads on the flagellar surface
(Bloodgood, 1992
; Kozminski et al., 1993
). IFT continues
unabated in cells in which gliding or bead movement has
been blocked chemically (Kozminski et al., 1993
) or genetically (Kozminski, 1995
). All three motitities, however,
are reversibly inhibited by increasing the osmolarity with
NaCl (100 mM) or sucrose (6%) (Kozminski et al., 1993
).
). The rafts, initially observed by
Ringo (1967)
during ultrastructural analysis of the Chlamydomonas flagellum, are attached by a thin connection
to the B-subfibers of the outer-doublet microtubules and
to the overlying flagellar membrane (Kozminski et al.,
1993
). Electron microscopy of IFT particles observed by
DIC in the flagellum of a single embedded cell confirmed
that the particles observed by DIC are, indeed, the rafts
observed by electron microscopy (Kozminski et al., 1995
).
View larger version (163K):
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Fig. 1.
Transmission electron micrographs of a Chlamydomonas flagellum (micrograph courtesy of Karl A. Johnson) and a
vertebrate rod connecting cilium (reprinted with permission from
Sandborn, 1970 ). Note the presence of a raft (arrows) in both organelles.
Chlamydomonas flagella contain several kinesins (Bernstein et al., 1994; Fox et al., 1994
; Johnson et al., 1994
;
Kozminski et al., 1995
; Walther et al., 1994
). One of these,
known as FLA10 or khp1 (Walther et al., 1994
), is located
between the flagellar membrane and axoneme where IFT
occurs (Kozminski, 1995
). A temperature-sensitive mutant
defective in this flagellar kinesin, fla10, cannot assemble flagella at the restrictive temperature; in fact, existing flagella shorten and disappear at the restrictive temperature
(Huang et al., 1977
; Adams et al., 1982
; Lux and Dutcher,
1991
). Just before flagellar shortening, IFT decreases
and disappears in these cells; simultaneously, ~70% of
the rafts observed by electron microscopy also disappear
(Kozminski et al., 1995
; Vashishtha et al., 1996
). Together,
these results indicate that (a) the IFT particles observed by DIC microscopy are the rafts observed in thin sections
by electron microscopy; (b) the movement of the rafts is
dependent on FLA10; and (c) the movement of the rafts,
IFT, is essential for assembly and maintenance of the flagella.
Sequence analysis reveals that FLA10 is most closely related to the kinesin-II subfamily of kinesins; some of these
kinesins have been purified as heterotrimeric complexes
consisting of two distinct, though related, motor subunits
and one nonmotor subunit (Scholey, 1996). FLA10 is part
of a similar heterotrimeric complex, FLA10 kinesin-II
(Cole et al., 1998
). As is typical for kinesins and their cargoes, FLA10 kinesin-II does not copurify with IFT particles; however, a small amount of FLA10 coprecipitates
with immunoprecipitates of IFT particle polypeptides,
suggesting a weak interaction between FLA10 kinesin-II
and IFT particles (Cole et al., 1998
).
The IFT particles were purified from flagellar extracts
taking advantage of the fact that they are greatly decreased in flagella of fla10 cells maintained at the restrictive temperature. 15 polypeptides sedimenting at ~16 S
are reduced in flagella of fla10 cells incubated at 32°C
(Piperno and Mead, 1997; Cole et al., 1998
). These 15 polypeptides form two complexes: complex A, composed
of 4 polypeptides; and complex B, composed of 11 polypeptides. Analysis of new mutants with defects in IFT
have identified a fifth polypeptide in complex A (Piperno
et al., 1998
). Considering the size of the IFT particles seen
in the electron microscope, there are probably multiple
copies of the 16 S complexes in each single IFT particle,
several of which compose the rafts.
Perhaps the strongest evidence that the 16 S particles
are, indeed, the IFT particles observed in DIC, came from
the work of Pazour et al. (1998). They isolated a Chlamydomonas mutant, fla14, that lacks LC8, a component of
both flagellar (Piperno and Luck, 1979
) and cytoplasmic
dyneins (King et al., 1996
). In fla14, kinesin-powered anterograde IFT is normal, but retrograde particle movement is missing (Pazour et al., 1998
). The flagella of fla14
are immotile, approximately half length, and deficient in
dynein arms and radial spokes. Most importantly, these
flagella contain massive accumulations of the rafts, and,
biochemically, contain 10-20-fold the amount of IFT particle polypeptides and FLA10 found in wild-type flagella
(Pazour et al., 1998
). Apparently, the rafts, composed of
the 16 S particles, are brought into the flagella by the
FLA10 kinesin-II, and accumulate because they cannot be
moved out in the absence of retrograde IFT.
Although analysis of fla14 suggested that cytoplasmic
dynein was the retrograde IFT motor, this interpretation
was clouded by the fact that LC8 is also a component of
myosin-V (Espindola, F.S., R.E. Cheney, S.M. King, D.M.
Suter, and M.S. Mooseker. 1996. ASCB Meeting, San
Francisco. Abstract 2160) and flagellar inner and outer
arm dyneins (Piperno and Luck, 1979; Harrison et al., 1998
). The importance of cytoplasmic dynein in IFT has
now been more convincingly shown by mutations in
DHC1b, a cytoplasmic dynein heavy chain. The effect of
these mutations is similar to, though even more severe than,
fla14: the flagella of these mutants, dhc1b, stfl-1, and stfl-2
are only 1-2 µm long and the space between the axoneme
and the flagellar membrane is filled with rafts (Pazour et
al., 1999
; Porter et al., 1999
). Therefore, IFT represents one of the only microtubule-based motility systems in
which there are mutants in the motors responsible for both
the anterograde and retrograde transport of defined, isolable particles.
The Cellular Localization of IFT Particles and Motors
Because FLA10 kinesin-II and IFT polypeptide antigens
were obtained from isolated Chlamydomonas flagella, it
was a surprise to find that their principal location in wild-type cells was not in the flagella but, rather, in the region
of the flagellar basal bodies. In wild-type cells, immunofluorescent localization of IFT polypeptides and FLA10 appears to be in a tripartite or ring-like structure around the
basal bodies (Vashishtha et al., 1996; Cole et al., 1998
). In
dhc1b the IFT particle proteins are redistributed from the
peri-basal body region to the flagella (Pazour et al., 1999
).
Apparently, the IFT particles can enter the flagella in these retrograde transport mutants but cannot exit, leading to a buildup in the flagella and a decrease around the
basal bodies. In a Chlamydomonas mutant, bld2, lacking
basal bodies (Goodenough and St. Clair, 1975
), the IFT
particle polypeptides continue to localize around the position where the basal bodies would have been, but FLA10
does not (Cole et al., 1998
). Furthermore, in fla10 cells
maintained at the restrictive temperature, the IFT particle
polypeptides still accumulate around the basal bodies. Therefore, the basal body region appears to be a holding
area for IFT particles and the motors that move them;
however, delivery and accumulation of IFT particles to
this region are not dependent on FLA10 kinesin-II.
IFT in Other Motile Cilia
Several lines of evidence suggest kinesin-II is involved in
ciliogenesis in a wide variety of organisms. Injection of an
antibody against a motor subunit of kinesin-II into sea urchin embryos disrupts formation of cilia that normally appear in the blastula stage (Morris and Scholey, 1997). In
Tetrahymena, simultaneous genetic knockout of two kinesin-II motor subunits completely blocked formation of
cilia (Brown, J., C. Marsala, R. Kosoy, and J. Gaertig. 1998. ASCB Meeting, San Francisco. Abstract 173). Disruption of a kinesin-II motor subunit, KIF3B, in mice resulted in the lack of cilia in the nodal cells of 7.5-d postcoitum embryos, leading to the randomization of left-right
asymmetry of the embryo (Nonaka et al., 1998
), a characteristic associated with immotile cilia syndrome in humans
(Afzelius, 1976
). Expression of DHC1b increases during
ciliogenesis in sea urchin embryos (Gibbons et al., 1994
) and rat tracheal epithelial cells (Criswell et al., 1996
), suggesting that this cytoplasmic dynein heavy chain plays a
role in assembly of cilia in these organisms.
IFT in Nonmotile Sensory Cilia
Homologues of several Chlamydomonas IFT particle proteins have been identified in C. elegans through peptide
microsequencing (Cole et al., 1998). The C. elegans mutants osm1 and osm6 have mutations in the genes encoding homologues of IFT polypeptides p172 and p52 (Cole
et al., 1998
), respectively. In these C. elegans mutants, the
ability to sense osmotic gradients and chemoattractants is
lacking. The basis for the mutant phenotype at the cellular level is that the sensory cilia found at dendritic ends of
sensory neurons do not assemble properly (Perkins et al.,
1986
).
Interestingly, similar phenotypes are found in two additional C. elegans mutants, osm3 and che3, which have defects in a subunit of heterotrimeric kinesin-II (Shakir et al.,
1993; Tabish et al., 1995
) and in the cytoplasmic dynein
heavy chain DHC1b (Grant, W., personal communication), respectively. Thus, IFT particle proteins and the motors that move them, kinesin-II and cytoplasmic dynein
DHC1b, are essential for the assembly of neuronal nonmotile sensory cilia of C. elegans.
IFT particles and their motors also appear to be present
in vertebrate cilia-containing sensory neurons. Using antibodies to KIF3A, kinesin-II has been localized by fluorescence and immunogold electron microscopies in the connecting cilium between the RIS and ROS of fish retinal
rod cells (Beech et al., 1996). Rafts similar to those observed in flagella of Chlamydomonas can also be seen in
the connecting cilia in the vertebrate eye (Fig. 1; Sandborn, 1970
). Mouse and human expressed sequence tags
with homologies to IFT particle polypeptides have also
been identified (Cole, D., unpublished observations), so
IFT may play a similar role in mammals. Recently, targeted knockout of a kinesin-II (KIF3A) in the mouse retina resulted in degeneration of ROS, starting at the proximal portion where new material is added (Marszalek, J.R.,
X. Liu, E. Roberts, D. Chui, J. Marth, D.S. Williams, and L.S.B. Goldstein. 1998. ASCB Meeting, San Francisco.
Abstract 756). Thus, kinesin-powered IFT is probably
present in the connecting cilium between the RIS and the
ROS of vertebrates and ROS assembly and maintenance
is apparently dependent on IFT. The implications of this
for studies of retinal pathologies leading to blindness are clear.
Hypothesis: The Role of IFT in the Assembly and Maintenance of Flagella
One possible role of IFT in flagellar assembly is to transport flagellar precursors to the flagellar tip where assembly occurs. Flagellar proteins appear to be synthesized on
mRNAs localized close to the basal bodies (Han, 1997; W. Marshall, unpublished results), and proteins that form various flagellar substructures, such as the dynein arms and
radial spokes, preassemble in the cytoplasm (Fok et al.,
1994; Fowkes and Mitchell, 1998
; D.R. Diener, D.G. Cole,
and J.L. Rosenbaum. 1996. ASCB Meeting, San Francisco. Abstract 273). The molecular motors, FLA10 kinesin-II
and cytoplasmic dynein DHC1b, responsible for transporting these precursors into and out of the flagella and the
IFT particle polypeptides are concentrated around the
basal bodies (Vashishtha et al., 1996
; Cole et al., 1998
; Pazour et al., 1999
) and may become associated with each
other as well as with the flagellar precursors in this region.
The associations between the IFT particles and the precursors are likely to be weak to facilitate release of the precursors in the flagellar compartment. The IFT particles
and flagellar precursors are moved into the flagella by the
anterograde motor FLA10 kinesin-II and are recycled to
the cell body by cytoplasmic dynein DHC1b. How the
switch is made at the flagellar tip from the anterograde to
the retrograde motor remains a provocative question.
Although the above discussion has emphasized the importance of IFT in transporting flagellar axonemal precursors into the flagella, it is important to note that (a) the
IFT rafts are clearly associated with the flagellar membrane as well as the B tubule of the outer doublets; (b) axonemal components, e.g., radial spokes, dynein arms, and
central pair microtubules, are absent from immotile sensory cilia of C. elegans and vertebrate photoreceptors; and (c) the first phenotype that appears (in ~30 min) in fla10
cells at the restrictive temperature is a membrane defect:
the cells lose the ability to mate (Piperno et al., 1996) by
use of their flagella. It is known that the flagellar mating
molecules must be moved onto the flagellar surface and
activated before mating (Hunnicutt et al., 1990
). Therefore, IFT is almost certainly involved in flagellar membrane maintenance and function.
IFT is required for the assembly and maintenance of
Chlamydomonas flagella and probably functions in a similar manner in the formation of the ciliated sensory neurons in C. elegans and other higher organisms including
vertebrates (Kozminski et al., 1995; Cole et al., 1998
; J.R.
Marszalek, X. Liu, E. Roberts, D. Chui, J. Marth, D.S.
Williams, and L.S.B. Goldstein. 1998. ASCB Meeting, San
Francisco. Abstract 756). Exactly how IFT functions in
the assembly and maintenance of motile cilia and nonmotile sensory cilia and what roles the individual IFT particle
polypeptides play in this process are not yet known.
![]() |
Footnotes |
---|
Address correspondence to Joel L. Rosenbaum, Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520-8103. Tel.: (203) 432-3472. Fax: (203) 432-5059. E-mail: joel.rosenbaum{at}yale.edu
Received for publication 9 December 1998 and in revised form 21 January 1999.
Douglas G. Cole's current address is Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Moscow, ID 83844-3052.
The authors would like to thank R. Bloodgood, J. Gaertig, W. Grant, L. Goldstein, K. Kozminski, G. Pazour, M. Porter, J. Scholey, and G. Witman for sharing unpublished data and/or for comments on the manuscript. Thanks also to K. Johnson for providing the micrograph of Chlamydomonas shown in Fig. 1.
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Abbreviations used in this paper |
---|
DIC, differential interference-contrast; IFT, intraflagellar transport; RIS, rod inner segment; ROS, rod outer segment.
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