University of Texas Southwestern Medical School, 5323 Harry Hines Blvd, Dallas, TX 75235-9039, USA
* Author for correspondence (e-mail: william.snell{at}utsouthwestern.edu)
Accepted 21 February 2003
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Summary |
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Key words: Chlamydomonas, Kinesin II, Regulated translocation, Aurora protein kinase, Flagella
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Introduction |
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In addition to their role in cell motility, the flagella of
Chlamydomonas are also central signaling organelles during
fertilization. When vegetatively growing mt+ and
mt cells are transferred into nitrogen-free medium
they undergo gametogenesis to become mt+ and
mt gametes. The gametes are in a fusion-incompetent
(unactivated) state until they are mixed with gametes of the opposite mating
type. Unactivated gametes are surrounded by a cell wall, their mating
structures (cell fusion organelles) are not activated, and they have not yet
undergone other cellular changes that render the cells capable of cell fusion
(Pan and Snell, 2000b). When
unactivated mt+ and mt gametes
are mixed together, interactions between their mt+ and
mt flagellar adhesion molecules, called
agglutinins, cause the cells to adhere to each other via their flagella. In
addition to bringing the cells into close contact, interactions between the
flagellar agglutinins also initiate a signal transduction pathway leading to
activation of a gamete-specific flagellar adenylyl cyclase. The resulting
increases in cAMP bring about multiple changes in the cells as part of a
process called gamete activation, which prepares the gametes for cell fusion.
Incubation of gametes of either mating type alone with the cAMP analog
dibutyryl cAMP (db-cAMP) also induces gamete activation
(Pan and Snell, 2000b
;
Pasquale and Goodenough, 1987
;
Pijst et al., 1984
).
Recently, we made the surprising discovery that a cell body cytoplasmic
protein, the Chlamydomonas aurora kinase CALK, is rapidly
translocated into the flagella during gamete activation
(Pan and Snell, 2000a;
Pan and Snell, 2000b
). In
vegetative cells, Chlamydomonas CALK, which is expressed as 78 kDa
and 80 kDa isoforms, is present exclusively in the cell bodies. Even after
gametogenesis, during which time mt+ and
mt vegetative cells differentiate into
mt+ and mt gametes, neither the
78 kDa nor the 80 kDa form of CALK were present in the flagella of the
(unactivated) gametes. Only after gamete activation, induced either by mixing
mt+ and mt gametes together or
by adding dibutyryl cAMP to gametes of a single mating type, did we find that
the 78 kDa isoform translocated into flagella. The 78 kDa isoform of CALK was
detectable in flagella within 5 minutes; after 30 minutes of gamete
activation, substantial amounts were present. The 80 kDa form continued to be
detectable only in the cell body. To our knowledge, this is one of the first
examples of a cytoplasmic protein whose translocation into a cilium or
flagellum has been shown to be regulated.
Because kinesin II is known to be essential for constitutive transport of flagellar components required for flagellar structure and motility, we used fla10 mutant cells, which have a lesion in the motor domain of kinesin II, to test the idea that this plus-end-directed microtubule motor protein plays a role in the regulated movement of proteins during signal transduction. By use of cell fractionation and immunoblotting, we show that, like wild-type vegetative cells, fla10 vegetative cells contain CALK only in their cell bodies. On the other hand, and unlike wild-type cells, the 80 kDa isoform of CALK is present in the flagella of unactivated gametes in an unregulated fashion and is associated with the axonemal fraction of the organelles. Moreover, cAMP-induced transport of the 78 kDa form of CALK into the flagella is disrupted in fla10 gametes, indicating that kinesin II is essential for its regulated transport.
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Materials and Methods |
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Isolation of flagella
Flagella were isolated essentially as described in Zhang et al.
(Zhang et al., 1991). Briefly,
3-4 l of cell culture was concentrated to 30 ml by centrifugation at 3500
g for 5 minutes at 4°C, ice-cold 25% sucrose in 10 mM
Tris, pH 7.2, was added to yield a final concentration of 7% sucrose, the
cells were subjected to pH shock and the flagella samples were purified by
underlaying/centrifugation twice with 25% sucrose in 10 mM Tris, pH 7.2.
Flagella were harvested by centrifugation at 9000 g for 8
minutes. The sedimented flagella were resuspended in HMDEK buffer (20 mM
HEPES, pH 7.2, 5 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA, 25 mM
KCl) containing a 1/100 dilution of the Sigma protease inhibitor cocktail for
plant cells (Sigma catalogue number P9599) and flash frozen in liquid
nitrogen. To obtain cell bodies, the cell bodies that were in the lower phase
after the first underlaying/centrifugation were harvested by centrifugation at
3500 g for 5 minutes. The cell body pellet was resuspended in
HMDEK buffer containing protease inhibitors and flash frozen in liquid
nitrogen.
For cell fractionation, whole cells or cell bodies in HMDEK buffer were sonicated on ice (three times for 10 seconds each separated by 1 minute rests, output control 90%, Vibra Cell Sonicator; Sonics & Material, Danbury, CT, USA). The disrupted cell sample was centrifuged at 600 g for 3 minutes to remove unbroken cells, and the supernatant was then centrifuged at 170,000 g for 10 minutes at 4°C (TLA 100.3 rotor; TLX Ultracentrifuge, Beckman) to obtain the freely soluble cell fraction. The sedimented material was resuspended in HMDEK buffer plus 1% NP-40, kept on ice for 15 minutes and centrifuged as above to yield the NP-40-soluble and NP-40-insoluble cell fractions. Freely soluble and NP-40-soluble and -insoluble fractions were obtained from flagella by use of the same procedures.
Sucrose gradient analysis
Samples were loaded onto 2 ml 5-20% linear sucrose gradients prepared
according to the method described in Beckman products application bulletin
(DS-640A) for the tabletop ultracentrifuge (TL-100, Beckman). Samples (0.1 ml)
were loaded on top of the gradient followed by centrifugation in rotor TLS-55
at 200,000 g for 4 hours at 4°C. After centrifugation,
fractions were collected from above and analysed as described in the Results.
Bovine serum albumin (BSA) and aldolase run in parallel tubes were used as
standards. Protein concentration was determined by use of a Bio-Rad protein
assay kit with BSA (Albumin Standard from Pierce) as a standard.
SDS-PAGE and immunoblotting
SDS-PAGE on 9% polyacrylamide gels and immunoblotting were carried out as
previously described (Pan and Snell,
2000a). Typically 15-30 µg of protein was loaded in each lane.
The rabbit polyclonal anti-Fla10N antiserum
(Cole, 1999
) was provided by D.
Diener and J. Rosenbaum (Yale University, New Haven, CT), and the anti-LC8
antibody (Pazour et al., 1998
)
was provided by G. Pazour and G. Witman (University of Massachusetts Medical
School, Worcester, MA). NIH Image (v. 1.62) was used to estimate the amount of
78 kDa CALK on scans of immunoblots of flagella isolated from activated
wild-type and fla10 gametes. The results for flagellar 78 kDa CALK in
each sample were normalized to the amounts of 78 kDa cell body CALK.
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Results |
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|
Fig. 2A shows that, during
gamete activation, the 78 kDa isoform of CALK was translocated into the
flagella (Pan and Snell,
2000a). In this experiment, unactivated and activated gametes were
deflagellated, and cell bodies and flagella were analysed by immunoblotting.
Fractionation of activated gamete cell bodies indicated that gamete activation
did not alter the properties of cell body CALK. Both isoforms of CALK in cell
bodies were distributed between the freely soluble fraction and the
detergent-insoluble fraction, and neither isoform was present in the
detergent-soluble fraction (Fig.
2B).
Quite different results were obtained, however, when we examined the properties of the translocated 78 kDa flagellar CALK. As shown in Fig. 2B, only a small portion of the 78 kDa CALK that was translocated into the flagella during gamete activation was freely soluble. In addition, little was associated with the NP-40-insoluble, or axonemal, fraction of flagella. Instead, most of this form of CALK required detergent to become soluble. These properties were consistent with the idea that the translocated 78 kDa flagellar CALK became associated with the flagellar membrane.
We also studied the properties of 78 kDa flagellar CALK by use of velocity
sedimentation to determine whether the CALK that translocated into the
flagella had formed new associations with cellular components. Detergent
extracts of whole flagella from activated gametes were subjected to
fractionation on sucrose gradients and CALK location in the gradient was
determined by immunoblotting as above (Fig.
2C). Whereas cell body CALK sedimented near the top of the
gradient in the form of a single peak, 78 kDa flagellar CALK sedimented as two
broad peaks with significant overlap. One component peaked in fraction 3 and
the other in fraction 6 (Fig.
2C). Based on the sedimentation of protein standards on identical
gradients, the larger CALK complex sedimented at 6-7 S.
Kinesin II is essential for proper localization of 80 kDa CALK in
unactivated gametes
Having established the localization and properties of CALK in wild-type
cells, we next studied the protein in fla10 mutant cells, which
contain the fla10-1 allele of kinesin II. The mutation in the
fla10-1 allele produces a kinesin II with a lysine instead of an
asparagine at residue 329, which is in the motor domain of the protein
(Walther et al., 1994).
Although the cellular effects of the mutation are especially noticeable when
fla10 cells are incubated at elevated temperature, being associated
with a complete cessation of IFT and resorption of flagella, the mutation has
significant effects on cells cultured under standard culture conditions. At
room temperature, vegetative fla10 cells produce substantially less
Fla10 protein than the wild type (Cole et
al., 1998
; Kozminski et al.,
1995
; Walther et al.,
1994
), contain fewer IFT particles in their flagella and exhibit
reduced IFT (Cole et al., 1998
;
Iomini et al., 2001
;
Kozminski et al., 1995
).
Because the above studies were carried out on vegetative cells, we
determined whether gametes of fla10 cells also showed reduced amounts
of Fla10 protein compared with wild-type cells, and we also tested for
differences between wild-type and fla10 gametes in their
fertilization phenotype. As shown in Fig.
3A, and consistent with previous studies on vegetative
fla10 cells, the flagella from fla10 gametes cultured at
23°C contained substantially less Fla10 protein than wild-type gametes.
The lower panel of Fig. 3A is a
loading control showing that the samples contained equivalent amounts of LC8,
a dynein light chain. We evaluated the fertilization phenotype of
fla10 gametes by mixing them with mt+ gametes and
assessing flagellar adhesion and zygote formation. Examination of the mixed
samples by phase-contrast microscopy indicated that the extent of flagellar
adhesion in the fla10 samples was similar to that of the wild-type
samples, consistent with previous results
(Pan and Snell, 2002).
However, the quality of the adhesive interactions was different. The
fla10 gametes formed looser, more tenuous associations with each
other than wild-type gametes (not shown). Analysis of the rate of zygote
formation of fla10 gametes at 23°C
(Fig. 3B) indicated that the
mutant cells exhibited a lower rate of gamete fusion than their wild-type
counterparts. These results were consistent with the idea that fla10
gametes exhibit alterations in flagellar signal transduction at 23°C.
|
Examination of total cellular CALK in fla10 cells showed that the total amounts of protein and the relative amounts of the 78 kDa and 80 kDa isoforms were similar to the wild type in fla10 vegetative cells and fla10 gametes (Fig. 4A). Likewise, just as both isoforms were absent from the flagella of wild-type vegetative cells and gametes, both were also missing from the flagella of vegetative fla10 cells (Fig. 4B). However, the flagella of unactivated fla10 gametes contained substantial amounts of CALK, but only of the 80 kDa isoform; as with wild-type gametes, the 78 kDa isoform was not present in the flagella of unactivated fla10 gametes. These results indicated that wild-type levels of kinesin II are essential for excluding the 80 kDa form of CALK from the flagella of unactivated gametes. Moreover, the results showed that gametes contain mechanisms for interactions between CALK and kinesin II that are not present vegetative cells.
|
We also examined the properties of the 80 kDa CALK that appeared in the flagella of fla10 gametes. To our surprise, this inappropriately localized 80 kDa CALK in unactivated gametes exhibited properties quite distinct from the 78 kDa CALK that translocated into the flagella of activated wild-type gametes. Whereas most of the translocated 78 kDa CALK in activated wild-type gametes was detergent soluble (Fig. 2B), none of the 80 kDa flagellar CALK in the unactivated fla10 gametes was soluble in the absence or presence of detergent, but was associated with the axonemal (detergent-insoluble) fraction (Fig. 4C).
Kinesin II and regulated translocation of 78 kDa CALK into
flagella
Having established the dependence on kinesin II for exclusion of the 80 kDa
CALK from gamete flagella, we next investigated the role of kinesin II in
translocation of the 78 kDa CALK during gamete activation. fla10
gametes were activated by incubation in db-cAMP, the cells were deflagellated
and the cell body and flagellar fractions were analysed by SDS-PAGE and
immunoblotting to assess CALK distribution. As expected, in control wild-type
gametes, the 78 kDa CALK translocated into the flagella upon activation
(Fig. 5). However, in the
activated fla10 gametes, translocation of the 78 kDa CALK was
dramatically reduced (Fig. 5).
Densitometric analysis (see Materials and Methods) indicated that the amount
of 78 kDa CALK translocated into flagella during activation of fla10
gametes was 10% that of wild-type gametes. Interestingly, the levels of
kinesin II present in the flagella of fla10 cells was
10% of
that found in the flagella of wild-type cells
(Fig. 3A).
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Discussion |
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After gamete activation, only a small proportion of the 78 kDa CALK that had translocated into flagella was associated with the freely soluble fraction or the detergent-insoluble (axonemal) fraction of flagella. Most of the translocated 78 kDa flagellar CALK was associated with the detergent-soluble membrane fraction of these organelles. Furthermore, sucrose gradient analysis showed that, in contrast to the soluble cell body CALK, a sizeable proportion of the 78 kDa flagellar CALK was part of a higher molecular weight complex (Fig. 2) that was not observed in cell body samples.
These results suggested that translocated 78 kDa flagellar CALK formed new associations during the process of moving into the flagella. The observation that 78 kDa CALK required detergent to become soluble was consistent with the idea that it was associated with the flagellar membrane. The fact that it was part of a more rapidly sedimenting entity on sucrose gradients suggested that it had formed a complex with a flagellar membrane protein or that it had undergone a conformational change. It will be interesting to determine whether formation of this altered form of CALK plays a role in the presence of 78 kDa CALK in the flagella.
Kinesin II is required for exclusion of the 80 kDa form of CALK from
the flagella of gametes
Our studies to test for a relationship between Chlamydomonas CALK
and kinesin II indicated that kinesin II influences the cellular location of
CALK. Unlike in wild-type cells, the 80 kDa isoform was present in the
flagella of unactivated and activated fla10 gametes. Moreover, the
mislocalization did not occur in vegetative fla10 cells, and the 80
kDa flagellar CALK of fla10 gametes was associated with the
detergent-insoluble, axonemal fraction. Although these results do not easily
lend themselves to a simple explanation, they document a genetic interaction
between the two proteins and a role for kinesin II in the localization of CALK
in gametes. The gamete specificity of the altered localization also suggests
that gametes contain CALK-interacting molecules not found in vegetative cells,
which would allow gametes to exploit CALK for as-yet-unidentified functions.
One possible explanation, suggested above for 78 kDa CALK, is that (even
though the steady-state flagellar levels are very low) the 80 kDa CALK
normally cycles through the flagella of gametes and that kinesin II is
essential for the exit of 80 kDa CALK from the flagella.
Regulated translocation of 78 kDa CALK into flagella during gamete
activation requires kinesin II
Our studies to test the idea that kinesin II is the motor for regulated
translocation of the 78 kDa isoform of CALK showed that
gamete-activation-induced movement of 78 kDa CALK into flagella was
significantly disrupted in fla10 cells
(Fig. 5). Compared with
wild-type cells, movement of IFT particles is substantially reduced in
fla10 cells maintained under standard culture conditions (room
temperature) and movement ceases at elevated temperatures. Cells might have a
requirement for increased kinesin II at elevated temperature or the mutation
in fla10 causes kinesin II to be somewhat unstable at room
temperature and even more unstable at elevated temperature
(Cole et al., 1998;
Kozminski et al., 1995
). It
was the subsequent disassembly of the flagella in fla10 cells
transferred to elevated temperature that originally led to the idea that
kinesin II is the motor for anterograde IFT
(Kozminski et al., 1995
;
Lux and Dutcher, 1991
).
Apparently, even though kinesin II is considerably diminished in
fla10 cells at room temperature compared with wild-type cells
(Cole et al., 1998
;
Kozminski et al., 1995
;
Walther et al., 1994
), the
reduced amount of kinesin II activity is still above a threshold needed to
maintain flagella of full length. We also showed recently that activation of
the flagellar adenylyl cyclase during flagellar adhesion between
mt+ and mt gametes occurs in
fla10 gametes at room temperature but is disrupted at elevated
temperatures (Pan and Snell,
2002
). Different circumstances hold with regulated translocation
of 78 kDa CALK. Our results show that the low amount of kinesin II in
fla10 cells at room temperature is associated with a disruption of
induced movement of 78 kDa CALK into the flagella. Thus, the thresholds for
constitutive movement of IFT particles and regulated translocation of 78 kDa
CALK are different.
Our experiments do not address the question of the role of IFT particles in
CALK translocation. It is possible, for example, that 78 kDa CALK does not
interact with IFT particles but binds directly to kinesin II. Alternatively,
78 kDa CALK could move into flagella as cargo on IFT particles, and the
reduced levels of IFT particles present in fla10 gametes
(Cole et al., 1998;
Iomini et al., 2001
;
Kozminski et al., 1995
) might
be insufficient to move the CALK.
We should note that the requirement shown here for kinesin II in regulated
translocation of 78 kDa CALK contrasts with recent studies on agglutinin, a
flagellar adhesion molecule that also undergoes cAMP-induced movement from the
cell body to the flagella during gamete activation. We and others have shown
that substantial amounts of the agglutinins, which are responsible for
flagellar adhesion between mt+ and
mt gametes, are stored in an inactive form at the
cell body. During gamete activation, the pre-existing, inactive agglutinins
are recruited from the cell body to the flagella, where they become active
(Goodenough, 1989;
Hunnicutt et al., 1990
;
Saito et al., 1985
). We found
that, independent of temperature, gamete-activation-induced recruitment of
flagellar agglutinins was intact in fla10 gametes
(Pan and Snell, 2002
).
It will be interesting to determine whether the ability of fla10
gametes to recruit active agglutinins, but not 78 kDa CALK, reflects a
difference in the amount of kinesin II required for these two activities or a
difference in the motor molecules used for movement. Studies from Scholey's
group have shown that, in addition to the heterotrimeric CeKinesin II,
Caenorhabditis elegans cilia contain a dimeric kinesin, CeOsm-3,
which is essential for assembly of the distal portions of the cilia in C.
elegans (Perkins et al.,
1986; Signor et al.,
1999b
). Perhaps Chlamydomonas uses Fla10 kinesin II for
flagellar assembly and maintenance, initiation of signal transduction, and
CALK translocation in gametes, and uses an as-yet-unidentified
Chlamydomonas CeOsm-3-related protein for agglutinin recruitment.
CALK, kinesins and signal transduction
To our knowledge, the work reported here is the first evidence for a role
of kinesin II in protein translocation induced during sensory transduction
(Solter and Gibor, 1977) in
cilia or flagella. Chemosensation in C. elegans has been shown to
require a functional IFT system (Cole et
al., 1998
; Haycraft et al.,
2001
; Qin et al.,
2001
; Signor et al.,
1999a
; Signor et al.,
1999b
), but the requirement is indirect: the C. elegans
IFT mutants fail to undergo sensory transduction because of their inability to
assemble structurally intact cilia, not because of the failure of their
assembled cilia to function (Collet et al.,
1998
; Haycraft et al.,
2001
; Perkins et al.,
1986
). Some of the chemosensory IFT mutants in C. elegans
do assemble cilia of sorts, but the cilia are misshapen and their centers are
filled with amorphous material (Perkins et
al., 1986
; Qin et al.,
2001
).
Although gamete-activation-induced CALK translocation is the first example
of kinesin-II-dependent regulated movement of proteins into cilia or flagella,
this motor protein has been shown to be involved in regulated movement of
membrane vesicles on cytoplasmic microtubules. In Xenopus
melanophores, pigment granule dispersion is driven by kinesin II and
cytoplasmic dynein is implicated in granule aggregation
(Nilsson and Wallin, 1997;
Tuma et al., 1998
). Recent
work has shown that regulation is at the level of interaction of kinesin II
with microtubules. The motor protein is constitutively present on the granules
but can bind to microtubules only upon a change in its phosphorylation state
(Reese and Haimo, 2000
). If
Chlamydomonas used a similar mechanism for regulating CALK movement,
and kinesin II and CALK were pre-associated in the cell body, it is likely
that cell body CALK would have behaved as a much larger entity in the sucrose
gradients. It will be interesting to learn more about the
gamete-activation-induced events that mediate its kinesin-II-dependent
movement.
The observations reported here on the role of kinesin II in regulation of
CALK localization during gamete activation are part of an emerging concept
that microtubule motors are important players in signal transduction
(Gundersen and Cook, 1999;
Verhey and Rapoport, 2001
).
Recently, we showed that kinesin II is essential for activation of signal
transduction during fertilization in Chlamydomonas
(Pan and Snell, 2000b
). In
other systems, kinesin and kinesin-related proteins have been shown to
interact directly or indirectly with signal-transducing proteins, including
protein kinases. In some cases, the interaction influences the activity of the
motor protein and, in others, the motor protein is crucial for the delivery of
signaling molecules to their sites of action
(Bowman et al., 2000
;
Day et al., 2000
;
Donelan et al., 2002
;
Morfini et al., 2002
;
Nagata et al., 1998
;
Setou et al., 2002
;
Verhey et al., 2001
). The
relationships documented here between kinesin II and CALK represent a useful
system to learn more about the mechanisms that regulate the spatial and
temporal localization of proteins in cilia and flagella during signal
transduction.
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Acknowledgments |
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