Department of Botany, University of Cologne, Gyrhofstr. 15, D-50931 Cologne, Germany
* Author for correspondence (e-mail: karl.lechtreck{at}uni-koeln.de)
Accepted 19 March 2003
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Summary |
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Key words: Centrosome, Centriole, Flagella, RNAi, Spindle pole
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Introduction |
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Several mechanisms can explain aberrant bb/centriole numbers. Centriole
duplication usually occurs once per cell cycle at the transition from G1- to
S-phase (Pennisi, 1999) and
failed duplication or overproduction would lead to deviant numbers. Too many
centrioles can also be the result of failed cell division
(Meraldi et al., 2002
).
Furthermore, abnormal numbers of bbs can be the result of errors during
mitotic segregation. Several studies suggest that centrin is involved in
bb/centriole assembly and centrosome duplication. This calcium-binding protein
is present in centrosomal structures over a broad range of species
(Salisbury, 1995
). Centrin is
located in the lumen of mammalian centrioles, in the half-bridge of the yeast
spindle pole body (spb), and in various contractile fibers associated with the
bbs of protists. Knockout of the gene encoding Cdc31p, a centrin homologue in
Saccharomyces cerevisiae, is lethal and cells fail to duplicate the
spb (Huang et al., 1988
;
Spang et al., 1995
). Silencing
of the centrin gene in the water fern Marsilea effectively inhibits
the formation of motile cells (Klink and
Wolniak, 2001
). Moreover, silencing of human centrin 2 impairs
centriole duplication in HeLa cells, emphasizing that centrin is required for
the replication of bbs and centrioles
(Salisbury et al., 2002
). A
point mutation in the single-copy centrin gene of the biflagellate green alga
Chlamydomonas causes the vfl2 phenotype (for `variable
flagellar number'), suggesting that centrin is also involved in the
segregation of bbs (Wright et al.,
1989
). In Chlamydomonas, centrin is located in the
stellate structure of the flagellar transition region, which represents the
border between the bb and the axoneme, in the distal connecting fiber (dCF)
between bbs, and in the two nucleus-bb connectors (NBBCs), which link the bbs
to the nucleus (Salisbury,
1995
). These structures are defective in vfl2 cells and
it has been assumed that the missing link between bbs and the nucleus causes
mistakes in bb segregation during cell division
(Wright et al., 1989
). Thus,
centrin seems to be involved in both the production of bbs and their
distribution to daughter cells, raising the question of how centrin performs
these multiple tasks.
Recently, a method to repress gene expression based on RNAi has been
established in Chlamydomonas
(Fuhrmann et al., 2001;
Lechtreck et al., 2002
).
Constructs consisting of a partial genomic DNA with introns linked to the
corresponding intron-free DNA in antisense orientation are thought to cause
the formation of hairpin RNA molecules that effectively repress homologous
genes (Smith et al., 2000
).
Here, we used this method to suppress centrin and obtained strains containing
only about 5% of the amount of centrin present in wild-type cells. Centrin
deficiency resulted in a high number of cells without bbs or flagella, as well
as additional defects such as elevated numbers of binucleated cells, mistakes
in bbs segregation during mitosis, and removal of bbs from the spindle poles.
Over-expression of centrin-GFP in wild-type cells disrupted the NBBCs and
removed bbs from the spindle poles but did not cause a bbs segregation defect.
We assume that centrin deficiency interfered with bb maturation as indicated
both by the frequent absence of flagella and the defects in the flagellar root
system that, in consequence, cause segregation errors.
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Materials and Methods |
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Plasmid DNA and transformation protocol
Centrin-GFP: The coding region of centrin including 40 bp of the
5'-UTR was amplified using genomic DNA of C. reinhardtii and
Cenf1 (5'-GCGTCTAGAGCTAGCATCATTTATCAAAACCGTTCTAGC-3') as a forward
primer containing a XbaI and a NheI restriction site, and
CenR1w (5'-GCGTCTAGAGCGGCAGCCGCGGCGAAGAGCGA-GGTCTTCTTCATG-3') as a
reverse primer including a XbaI site and introducing five alanines
between centrin and GFP. The amplified product of 1840 kb was digested
with XbaI and cloned into the XbaI site of pCRGFP
(Fuhrmann et al., 1999
). The
resulting plasmid was cut with NheI and EcoRI to isolate a
fragment consisting of the centrin PCR product upstream of the GFP gene and
the 3'UTR of pCrGFP. This fragment was ligated downstream of the
HSP70A/rbcS2 promotor into pCB740 digested with NheI and
EcoRI (Schroda et al.,
2000
).
Centrin sense-antisense hybrid construct: The sense arm was amplified using a forward primer Cenfor (5'-GCGTCTAGAATGTA-CAAGGCAAAGACC-3') containing a XbaI restriction site and a reverse primer CensenseR1 (5'-GCGCAAGCTTCTGCATGTATAG-AAGTCAGG-3') containing a HindIII restriction site. For the amplification of the antisense arm, CenantiF1 (5'-GCGGGATCCA-TGAGCTACAAGGCAAAGACC-3') containing a BamHI site and CenantiR1 (5'-CGCCAAGCTTAGGTCGAATGCCTCGCGGATC-3') containing a HindIII restriction site were used. After digestion, both products were cloned into the pCrGFP digested with XbaI and BamHI, which removed the GFP gene. The resulting plasmid was cut with XbaI and EcoR1 to obtain a fragment consisting of the sense-antisense hybrid DNA and the 3'UTR of pCRGFP, which was ligated into pCB740 as described above. The resulting plasmids contained the arg7 gene, a selectable marker for arginine-requiring strains. Alternatively, the HSP70A/rbcS2 promoter was cloned into pCRGFP in front of the centrin sense-antisense construct resulting in the plasmid pCR-CenAS.
Nuclear transformation was performed as described earlier
(Kindle, 1990). In brief,
5x107 cells were agitated in the presence of 1-2 µg
plasmid DNA linearized with EcoRI, 5% polyethylen glycol 8000, and
0.3 g of 0.5 mm glass beads. Transformants were selected on solid medium
without arginine. Individual colonies, visible after 7-10 days, were
transferred to liquid media for further analysis. Strains were maintained in
liquid medium (50 ml volume), but slowly growing stocks were also prepared in
solid TAP medium. As a control, we used a strain transformed with an empty
pCB740 (digested with NheI and EcoRI). Transformation of
strains expressing GFP-SFA with pCR-CenAS was performed by co-transformation
using the ble gene (Stevens et
al., 1996
) and selection on plates containing zeocin. A
description of the N-terminally GFP-tagged SFA will be reported elsewhere
(J.S. and K.-F.L., unpublished)
Western blotting analysis
Cells from 10 ml culture were pelleted at 500 g for 2
minutes, resuspended in 500 µl MT buffer (30 mM HEPES, 15 mM KCl, 5 mM
MgSO4, 5 mM EGTA, 100 µM DTT, pH 7), and lysed by the addition
of 500 µl MT buffer containing 3% Triton X-100. After 15 minutes at room
temperature, cytoskeletons were pelleted at 18,320 g for 15
minutes at 4°C. The pellets were dissolved in 4x sample buffer,
denaturated at 95°C for 10 minutes, subjected to SDS-PAGE, and transferred
onto PVDF membrane. Western blotting was carried out as described previously
(Lechtreck and Geimer, 2000).
After transfer, the membrane was fixed by a glutaraldehyde incubation step
(Karey and Sirbasku, 1998
).
Blots were documented using a digital camera and processed using Adobe
Photoshop and Illustrater (Adobe Systems, San Jose, CA). The intensity of
immunoreactive bands was determined using Metamorph.
Indirect immunofluorescence
Cells were pelleted at 500 g for 2 minutes, resuspended in
MT buffer, and lysed by an additional volume of MT buffer containing 3% Triton
X-100 or 0.5% Nonidet P-40, respectively. After 45 seconds, the cytoskeletons
were fixed with 3% paraformaldehyde (final concentration in MT) and were
allowed to settle onto poly-L-lysine-treated multiwell slides for 10-15
minutes. Alternatively, cells were concentrated in MT buffer, transferred onto
the poly-L-lysin-treated multiwell slide and, after 5-10 minutes, were
permeabilized by rinsing in -20°C methanol for 7 minutes (20 seconds for
centrin-GFP strains). Immunolabeling was carried out as described
(Lechtreck and Geimer, 2000).
Images were acquired with a CCD-camera (RT Monochrome spot 2.1.1., Diagnostics
Instruments, Sterling Heights, MI) on a Nikon-Eclipse fluorescence microscope
and processed using Adobe Photoshop and Illustrator. For measurements of the
average fluorescence intensity of the bbs, we used the region statistics tool
of Metamorph with the constant region depicted in
Fig. 8C.
|
Antibodies
The following antibodies were used for indirect immunofluorescence:
monoclonal anti-centrin BAS6.8 (1:20), polyclonal
anti-Spermatozopsis-centrin (1:200, pCen2), polyclonal
anti--tubulin (1:800), monoclonal anti-acetylated tubulin (clone
6-11B-1, 1:600; Sigma), monoclonal anti-
-tubulin (clone DM 1A; Sigma)
and monoclonal GT335 (1:1200) (Wolff et
al., 1992
) [for sources of these and secondary antibodies, see
Grunow and Lechtreck (Grunow and
Lechtreck, 2001
), and references therein]. For western blot
analyses, we used a polyclonal anti-Centrin (pCen1)
(Salisbury et al., 1984
)
diluted 1:3000 in blocking buffer, polyclonal anti-GFP 290 (1:3000; Abcam,
Cambridge, UK) and secondary antibodies directed against rabbit-IgG conjugated
with alkaline phosphatase (Sigma).
Standard EM
Whole cells were resuspended in 0.5 ml TAP and fixed by addition of 0.7 ml
TAP containing 5% glutaraldehyde and 1% OsO4 on ice for 30 minutes.
To prepare cytoskeletons, cells were harvested by centrifugation and washed
once in MT buffer. Cell lysis was carried out by adding MT buffer containing
3% Triton (1:1) and cytoskeletons were immediately prefixed with 2.5%
glutaraldehyde (final concentration in MT buffer) and pelleted (5 minutes).
After fixation in 0.5% OsO4 and 2% glutaraldehyde on ice for 30
minutes, the cytoskeletons were washed twice, dehydrated and embedded as
previously described (Grunow and
Lechtreck, 2001).
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Results |
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Prolonged observation showed that, in the centrin-RNAi strains, the
repression of the centrin gene was not stable
(Fig. 2). The loss of the
centrin-RNAi effect was accompanied by increasing amounts of centrin on
western blots (Fig. 2B),
reappearance of the centrin-structures
(Fig. 2C), and increasing
numbers of biflagellate cells (Fig.
2A). The intensity of centrin-RNAi decreased more rapidly in
cultures of N41 in liquid medium, whereas slowly growing N41 stocks maintained
in solid media preserved the centrin-deficient phenotype for several months
(Fig. 2A). Finally, all
isolates of a given strain lost the centrin-RNAi effect, even those maintained
in solid medium or raised from isolated nonflagellate single cells. We assume
that the transgene itself was silenced, as it has been reported earlier for
other transgenes (Wu-Scharf et al.,
2000). All experiments depicted in this study were performed while
the strains showed a relatively stable RNAi effect, as indicated for N41 in
Fig. 2A.
|
Centrin deficiency interferes with flagellar assembly on bbs
Counts of centrin dots and flagella indicated a discrepancy between the
number of centrin dots, presumably representing bbs, and that of flagella in
the centrin-RNAi strains (Fig.
1D, Table 1).
Double labeling using anti-centrin and anti--tubulin showed centrin
dots without attached flagella (Fig.
3Aa-c). To test whether these centrin signals represent genuine
bbs, we performed immunofluorescence using antibodies to polyglutamylated or
acetylated tubulin, which are enriched in bbs
(LeDizet and Piperno, 1986
;
Lechtreck and Geimer, 2000
).
Both antibodies identified nonflagellate bbs in the centrin-RNAi strains
(Fig. 3Ae,f), and also
confirmed the absence of bbs from many cells
(Fig. 6Ad, not shown).
Furthermore, EM analysis of N41 cells showed numerous bbs without or with
extremely short flagella revealing that centrin knockdown interfered with
flagellar assembly, which has not been reported from vfl2 cells
(Fig. 3Bb-e). Some bbs were
improperly docked to the plasma membrane or located within the cytoplasm
(Fig. 3Bc-e). Similar to
earlier observations in the vfl2, a strain with a point mutation in
the centrin gene (Jarvik and Suhan,
1991
), the transition region of flagellate and nonflagellate bbs
of N41 was defective (Fig. 3B;
a wild-type flagellum is shown in a) and central pair microtubules were
penetrating the bb (Fig.
3Cb).
|
|
Centrin tethers bbs to the spindle poles
Whereas the average number of bbs decreased with the amount of centrin, we
observed a considerable number of cells that contained more than two centrin
dots/bbs (Table 1). In A10, for
example, 12.7% of cells contained three or more centrin dots, indicating a bbs
segregation defect (Table 1,
Fig. 4A). In wild-type cells,
centrin and bbs are located at the poles of the spindle which, in
Chlamydomonas, is intranuclear with fenestrae at the poles (e.g.
Johnson and Porter, 1968;
Coss, 1974
;
Wright et al., 1989
)
(Fig. 4Ba-c). It was assumed
that, in Chlamydomonas vfl2, proper segregation of bbs fails due to
the absence of intact NBBCs (Wright et
al., 1989
). Thus, we determined the position of bbs with respect
to the mitotic spindle in centrin-depleted cells. Indirect immunofluorescence
revealed that centrin dots/bbs were not located at the spindle poles in
mitotic cells of A10 (7 of 7 cells) and, more surprisingly, of F11 (18 of 19
cells). The latter strain was similar to A7 with respect to average flagellar
and bb number per cell (Table
1), but displayed a somewhat stronger bb segregation defect (19.3%
of cells with aberrant numbers of centrin dots compared with 5.5% in A7, which
was no longer available for this analysis). Bbs mostly resumed a lateral
position with a distance of up to 3 µm to the poles
(Fig. 4Bd-j). The data suggest
that centrin fibers are needed to tether bbs to the spindle poles. Mitotic A10
often had aberrant bb numbers and spindles without bbs, whereas F11 cells
mostly contained two bb pairs that were interconnected by the microtubules of
the so-called metaphase band (Johnson and
Porter, 1968
; Doonan and Grief,
1987
; Gaffal and el-Gammal,
1990
). This extranuclear system of microtubules present in mitotic
cells of Chlamydomonas develops from two of the four microtubular
roots surrounding the bbs in interphase
(Fig. 6Ac). It originates near
the bb pairs and consists of two bands, each of four microtubules. The two
bands run towards each other, parallel to the spindle and overlap above the
nucleus in an antiparallel fashion (arrow in
Fig. 4Bd). Then, both bands
make a turn and continue down into the cell on the sides of the nucleus
perpendicular to the spindle microtubules (arrow in
Fig. 4Ba). In A10, the
metaphase band seemed unordered or absent
(Fig. 4Bh-j).
Fig. 4Bk-m show examples of
dividing A10 and N41 cells in which bbs were not correctly segregated. Thus,
segregation errors contributed to aberrant bb numbers in
Chlamydomonas centrin-RNAi strains.
|
Over-expression of centrin-GFP disrupted the NBBCs
Despite the absence of NBBCs, A7 or F11 displayed only a weak vfl
phenotype (Table 1) and we
wondered whether the displacement of bbs from the mitotic poles is sufficient
to explain the observed mistakes in segregation. To address this question, we
took advantage of an earlier observation
(Ruiz-Binder et al., 2002),
which showed that expression of GFP-tagged centrin interfered with a system of
delicate centrin fibers in the stellate structure of the flagellar transition
region. We used the strong HSP70A/rbcS2 fusion promoter to transform control
cells with a centrin-GFP construct (Fig.
5). Moderate expression of centrin-GFP (e.g. strain Cen-GFP3)
allowed incorporation of the tagged protein in the dCF and the distal parts of
the NBBCs (Fig. 5Ab), whereas
higher expression levels (e.g. strain Cen-GFP8) disrupted the centrin system
of the NBBCs and centrin-GFP accumulated in clumps on the nuclear surface and
at the bbs (Fig. 5Ac). Staining
of Cen-GFP8 with anti-centrin confirmed the absence of centrin-based NBBCs
(Fig. 5Ac'). Furthermore,
cytoskeletons isolated from Cen-GFP8 or the centrin-RNAi strain A7 exhibited a
reduced stability: about 30% of the nuclei were detached from the basal
apparatus, which was not observed with cytoskeletons isolated from Cen-GFP3 or
wild-type cells (Fig. 5C,D).
Interestingly, bbs were not located at the poles in most mitotic Cen-GFP8
cells (23 of 25) as determined by indirect immunofluorescence using
anti-centrin or GT335 (Fig.
5B). In contrast to centrin knockdown cells, some residual centrin
was present at the acentric spindle poles
(Fig. 5Ba-c). Despite the
abnormal mitotic position of bbs, Cen-GFP8 cells were more than 99%
biflagellate and contained two bbs. Thus, disruption of the NBBCs via
over-expression of centrin-GFP detached the bbs from the spindle poles but did
not cause a conspicuous phenotype, indicating that NBBCs are not required to
maintain a constant bb number.
Effect of aberrant bb numbers on the microtubular cytoskeleton
Centrin deficiency caused cells with too many bbs or without bbs, allowing
us to study the effect of aberrant bb numbers on the microtubular system. In
Chlamydomonas, the flagellar basal apparatus with its two
flagellar-bearing bbs functions as an MTOC, giving rise to four acetylated
microtubular bundles, two consisting of four and two consisting of two
microtubules. On these, numerous non-acetylated microtubules originate
(LeDizet and Piperno, 1986)
(Fig. 6Aa-c). Additional bbs of
the centrin-RNAi cells were surrounded by microtubules and were often
associated with acetylated microtubular bands
(Fig. 6Ae,g,
Fig. 3Af). In A10 and N41, we
observed numerous cells (21% or 67%, respectively) without bbs. In such cells,
we mostly observed an unordered microtubular system
(Fig. 6Af) and acetylated
microtubules were either present or absent
(Fig. 6Ad, cells 3, 4). Also,
we observed cells with an incomplete set of microtubular roots
(Fig. 6Ad, cell 2,
Fig. 3Af). The four
microtubular flagellar roots are associated with striated fibers composed
predominately of striated fiber assemblin (SFA)
(Lechtreck et al., 2002
). We
transformed cells expressing GFP-SFA with a plasmid inducing centrin-RNAi to
study the effect of centrin deficiency on this flagellar root system. In a
wild-type background, GFP-SFA uniformly (98.8% of 252 cells) assembled into
cross-like structures with prominent fibers attached to the four-stranded
microtubular roots and smaller ones associated with the two-stranded roots
(Fig. 6Ba). After induction of
centrin deficiency, the GFP-SFA system was altered: additional bbs caused
aberrant GFP-SFA fiber formation and, in cells without bbs, GFP-SFA fibers
were either absent or one or two giant GFP-SFA fibers were formed
(Fig. 6Bc-e, not shown).
Interestingly, cells with a low amount of centrin but two bbs mostly displayed
an altered GFP-SFA system with additional fibers and/or fibers of unusual size
(Fig. 6Bb). The data show that
bbs are critical to establish the focused microtubular system and that centrin
deficiency interferes with the ability of bbs to organize the usually
stereotyped flagellar root system of Chlamydomonas.
In N41, and to a lesser degree in A10, we observed cells with additional non-wild-type characters that, we assume, were caused by the defects of the microtubular system described above. These include multiple eyespots and pryrenoids (Fig. 7a) indicative for several plastids or incomplete plastid division, a higher variation in cell size and shape, and the occurrence of incompletely divided cells (not shown). Nuclei and pyrenoids were often positioned incorrectly with respect to each other and the cell surface (not shown). Furthermore, we observed that A10 and N41 contained elevated numbers of multinucleated cells (7% and 17%, respectively). Cells with multiple nuclei did not contain elevated numbers of bbs (not shown) and we assume that too many as well as no bbs increased the rate of errors during cell division of Chlamydomonas (Fig. 7b-d).
|
Bbs of the centrin-RNAi strains varied in the amount of associated
centrin
In Chlamydomonas, bbs duplicate in late mitosis
(Gaffal and el-Gammal, 1990).
We attempted to analyze when centrin is recruited to the bbs by using
antibodies to acetylated tubulin (6-11B-1), a posttranslational modification
acquired early during bb development, and to polyglutamylated tubulin (GT335)
which is characteristic for mature, usually flagellate bbs
(Lechtreck and Geimer, 2000
).
Probasal bodies were observed in positions lateral of mature bbs between the
four microtubular roots and frequently cells contained aberrant numbers of
probasal bodies. These were not stained by anti-centrin
(Fig. 8Aa,b), indicating that
bbs acquire centrin during their subsequent development. In double-staining
experiments with GT335, all centrin dots analyzed were polyglutamylated, but
about 5% of the GT335 signals of N41 contained no or only very small amounts
of centrin (Fig. 8B).
Interestingly, some of these centrin-free bbs had assembled a flagellum
(8Bb,c). The data indicate that, under conditions of strong centrin knockdown,
some bbs mature, as judged by the presence of polyglutamylated tubulin and
axonemes, without acquiring considerable amounts of centrin. In green algae,
centrin is not a component of the bbs itself but is restricted to the stellate
structure in the flagellar transition region
(Schulze et al., 1987
). The
centrin dots observed at the base of flagella in vfl2 or the
centrin-RNAi cells represent remnants of the dCF and NBBCs on the surface of
bbs (Taillon et al.,
1992
).
We measured the intensity of signals obtained by indirect immunofluorescence with monoclonal anti-centrin (Fig. 8C) to analyze whether the amount of centrin at the bbs varied within a strain or between strains. A7 cells showed only small differences (7%) in the staining intensities between the two flagellate bbs of a cell, which presumably reflect developmental differences. Centrin dots in A10 showed about 30-50% of the fluorescence intensity of those in A7. In N41, some dots were of very low intensity (see above) but most bbs showed 10-45% of the intensity in comparison with A7 bbs (not shown). In uniflagellate cells of A10 with two bbs, the signal observed at the base of the flagellum was on average 36% stronger than that at the nonflagellate bb. We assume that a lower centrin content and the absence of a flagellum are indicative for a delay in bb development under conditions of centrin deficiency.
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Discussion |
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Centrin and bb segregation
In wild-type cells of Chlamydomonas, centrin and bbs are located
at the spindle poles. Since NBBCs are defective in the Chlamydomonas
mutant vfl2, it has been assumed that these linkers are needed for
proper segregation of bbs during cell division
(Wright et al., 1989;
Marshall et al., 2001
). Here,
we show that centrin deficiency detaches bbs from the spindle poles, but
nevertheless provide evidence against a crucial role of NBBCs in the equal
distribution of bbs. Indirect immunofluorescence and EM failed to detect NBBCs
in the centrin-deficient strains and we observed a decreased stability of
isolated nucleo-flagellar-apparatuses similar to earlier observations on
vfl2 cells. Despite the apparent absence of NBBCs in all analyzed
strains, these differed considerably in their ability to maintain a constant
bb number (e.g. 94.5% of A7 cells contained bb pairs). It seemed possible that
cells suffering from mild centrin knockdown were able to assemble NBBC-like
fibers during mitosis that link the bbs to the spindle poles and thereby
ensure proper segregation. The analysis of mitotic cells demonstrated that bbs
are not at the poles in most cells regardless of whether the strains showed a
mild or a strong vfl phenotype. We conclude that centrin and NBBCs
are needed to tether bbs to the spindle poles. However, the data also
suggested that correct distribution of bbs in Chlamydomonas neither
requires NBBCs nor an association of bbs with the spindle poles. To test this
assumption further, NBBCs were disrupted by over-expresssing centrin-GFP,
which resulted in a decreased stability of the nucleo-flagellar apparatus
complexes. Indeed, bbs were detached from the poles in most cells expressing
centrin-GFP, emphasizing the role of the NBBCs in tethering bbs to the spindle
poles. Interestingly, these strains were perfectly able to segregate their
bbs. We conclude that NBBCs or an association of bbs with the spindle poles
are not crucial for segregation of bbs in Chlamydomonas. Observations
from other green flagellates related to Chlamydomonas support this
conclusion. In Polytomella agilis, NBBCs were not observed
(Schulze et al., 1987
) and bbs
are not at the poles, but nevertheless centriole/bb numbers are maintained
constant in these cells. Bbs are not at the poles of the mitotic spindle in
Dunaliella bioculata, which possesses NBBCs in interphase
(Grunow and Lechtreck, 2001
).
In metazoan cells, the poles of acentric spindles often appeared less focused
than those with centrioles, suggesting a role of these microtubular cylinders
in spindle assembly or maintenance
(Compton, 2000
). By contrast,
spindles appeared mostly normal in centrin-deficient cells of
Chlamydomonas despite the absence of bbs at the poles, suggesting a
fundamental difference between metazoan centrioles and green algal bbs, the
latter apparently not essential for mitotic spindle assembly.
If not the NBBCs, what else might ensure the distribution of bbs to the
daughter cells? A study in 1968 (Johnson
and Porter, 1968) first reported two bands, each of four
microtubules, that persist throughout mitosis and arc over the mitotic
nucleus. These `metaphase band' microtubules are identical with the two
four-stranded microtubular flagellar roots associated with the bbs during
interphase. During bb segregation, each pair of bbs keeps one root and the two
roots overlap in an antiparallel fashion. A central overlapping region of the
metaphase band is maintained while the roots elongate and the bbs migrate
towards the nuclear poles (Gaffal and
el-Gammal, 1990
; Ehler et al.,
1995
). This sequence of events has lead to speculations that the
metaphase band moves the bbs into both cell halves in a process analogous to
that of spindle elongation with a motor situated in the zone where the
microtubules overlap (Sluiman and
Blommers, 1990
). In this study, we established experimental
conditions in which bbs are no longer attached to the spindle poles via
centrin fibers. Cells were still able to segregate bbs properly and we
postulate that the extranuclear spindle-like microtubule system of the
metaphase band provides the force and structure for this process. Accurate
centriole/bb segregation is fundamental and therefore might be ensured by more
than one mechanism, which would explain the centrin-dependent association of
bbs with the spindle poles in Chlamydomonas and other systems. In
Chlamydomonas, the two mitotic assemblies of microtubules (spindle
and metaphase band) can be distinguished due to the persisting nuclear
envelope, but one might speculate that a subpopulation of microtubules in the
spindle of metazoan cells serves to separate the pairs of centrioles more than
in chromosome movements or spindle elongation.
Centrin and bb duplication
A strong reduction in the amount of centrin as in N41 caused a decrease in
the average number of bbs. It has been shown that the repression of centrin
expression in Marsilea inhibits the differentiation of motile cells
(Klink and Wolniak, 2001) and,
more recently, that siRNAs to human centrin2 inhibit centriole replication in
HeLa cells (Salisbury et al.,
2002
). Thus, bb assembly in Chlamydomonas and many other
systems seems to depend on centrin. What could be the role of centrin during
bb/centriole replication? One possibility is that centrin is an essential
component of bbs/centrioles. Indeed, centrin is located in the distal lumen of
centrioles e.g. in metazoan cells. In green algal bbs, however, centrin was
observed only in the stellate structure between the bb and the axoneme but is
otherwise restricted to bb-associated structures. Furthermore, we report here
that the amount of centrin attached to bbs can be very low under conditions of
centrin deficiency, indicating that centrin is not required to maintain bbs.
In various systems, centrin has been observed in structures on which bbs
subsequently assemble. These include the blepharoblast of Marsilea or
`fibrous granules' during the differentiation of ciliated epithelial cells
(Laoukili et al., 2000
).
Similarly, new bbs develop on the end of centrin fibers in the green
flagellate Spermatozopsis similis
(Lechtreck and Bornens, 2001
).
Centrin structures precede centriole assembly in metazoan cells
(Middendorp et al., 1997
) and
the de novo assembly of bbs in the amoeboflagellate Naegleria
(Levy et al., 1998
). Finally,
it is noteworthy that Cdc31p is not a component of the spb itself but of the
half-bridge, a structure on which material for the new spindle pole is
deposited (Spang et al., 1995
;
Adams and Kilmartin, 1999
).
Centrin-containing structures might provide assembly sites for bbs and other
centrosomal organelles over a broad range of species
(Adams and Kilmartin, 2000
).
Centrin knockdown seriously reduced the amount of centrin and we assume that
many bbs failed to recruit enough centrin to ensure the formation of probasal
bodies within one cell cycle. Subsequent cell divisions will then dilute the
bbs, resulting in cells without them.
Centrin and bb maturation
We observed bbs without attached flagella in centrin-deficient cells.
Nonflagellate bbs can be caused by defects in flagellar assembly (e.g. the
bald1 mutation) (Brazelton et al.,
2001) or by defects in the bbs that hinder them to template an
axoneme (e.g. bald2 or uni3)
(Dutcher and Trabuco, 1998
).
However, in the centrin-RNAi strains, ultrastructural defects in bb triplets,
typical for bald2 or uni3, were not observed. All strains
assembled at least a few motile, full-length flagella qualified by the
observation that N41 (and more rarely A10) often had shorter than wild-type
flagella, resulting in a reduced average length (for N41: 9.4
µm±2.85 µm, n=67; control: 12.1 µm±0.98 µm,
n=44). It is unlikely that flagellar assembly is blocked by defects
of the transition region because these also occurred in flagellate bbs of
vfl2 and centrin-RNAi cells
(Taillon et al., 1992
).
Centrin has been identified in the flagella of Chlamydomonas
(LeDizet and Piperno, 1995
)
and Tetrahymena (Guerra et al.,
2003
). Thus, flagellar assembly could be perturbed because centrin
is an essential component of the flagellum. Alternatively, many bbs in the
centrin-RNAi strains could be in an immature condition not allowing flagellar
assembly. Similar to the formation of a primary cilium on the mother centriole
in metazoan cells, bbs of Chlamydomonas need to have a certain age
before flagellar assembly occurs: between the formation of new bbs during the
end of mitosis (Gaffal and el-Gammal,
1990
) and the assembly of axonemes after the next mitosis, bbs
rest in an immature nonflagellate state. Bb maturation involves an increase in
size, docking to the plasma membrane, development of a flagellar root system,
assembly of the transition region and flagella, accumulation of centrin, and
biochemical modifications of bb tubulin. Indeed, several observations suggest
improper bb maturation in the centrin-RNAi strains: bbs had a reduced centrin
content, some failed to dock to the plasma membrane, and the flagellar root
system was often defective (e.g. missing roots and an altered distribution of
GFP-SFA, a marker for microtubular roots). We assume that centrin deficiency
delays bb development and/or disturbs the pace of steps occurring during bb
maturation. It is important to note that improper bb maturation could be
related to segregation defects: the metaphase band develops from the
microtubular roots and it is reasonable to assume that defects in the
flagellar root system will affect the metaphase band and thereby might cause
mistakes in bb segregation. However, other scenarios are possible: root
microtubules are nucleated not directly on the bbs but at the dCF, which
thereby provides a link between bbs and the microtubular cytoskeleton
potentially related to segregation. Furthermore, proper segregation of bbs
might require a physical link between the new and the old bb of each pair in
the form of a centrin-based new dCF ensuring that all bbs, including new ones
without roots, are attached to the metaphase band. The metaphase band has also
been implicated in the determination of the cleavage plane and the assembly of
the cytokinetic apparatus of Chlamydomonas
(Johnson and Porter, 1968
;
Ehler et al., 1995
). Therefore,
defects in the flagellar root system could be responsible for the formation of
multinucleated cells. Moreover, probasal bodies of green algae develop in
association with the flagellar roots [Lechtreck et al.
(Lechtreck et al., 1997
); and
references therein] and therefore defects in the roots could even interfere
with bb replication.
Differential sensitivity of centrin structures and functions
The amount of residual centrin varied considerably between transformants,
allowing us to analyze the differential sensitivity of centrin-based processes
and centrin-based structures. Residual centrin was concentrated near the bbs,
which apparently have the highest affinity for centrin. In A7 and other
strains, we observed a dotted staining on the cell nucleus, whereas linkers to
the bbs were absent, indicating that the fimbriae are less sensitive to
decreasing amounts of centrin than the proximal parts of the NBBCs. The
strains analyzed in detail are representative of different levels of impaired
centrin function: A7 displayed a mild defect, F11 an intermediate segregation
defect, whereas bb replication seemed unaffected as indicated by an average
number of bbs per cell of near 2. In A10, the number of cells with aberrant bb
numbers was increased and the average number of bbs was slightly decreased,
suggesting beginning impairment of bb duplication in addition to segregation
defects. In strains like N41, bb duplication was strongly disturbed with in
average of only 0.53 bbs per cell; segregation defects and bbs without
flagella were frequent. Our analysis indicated further that about 40% of the
wild-type amount of centrin is sufficient to restore
(Fig. 2) or maintain
(Fig. 1D) a mostly biflagellate
phenotype and that flagellar assembly onto bbs is more sensitive to centrin
deficiency than bb assembly. Thus, in a condition of centrin deficiency,
flagella number cannot be used to determine bb number. In a broad view, the
various tasks of centrin in Chlamydomonas were affected by decreasing
amounts of this protein in the following order: segregation, ability to
assemble flagella, replication of bbs. This differential sensitivity seemed
unrelated to the presence or absence of certain centrin-based cytoskeletal
elements: NBBCs and the dCF were defective even in strains displaying only a
mild phenotype. Thus, centrin functions such as bb segregation appear to be
independent of the known centrin-based structures like the NBBCs, a view
supported by the analysis of strains having normal levels of wild-type centrin
but over-expressing centrin-GFP, which disrupted the NBBCs without causing
segregation defects. However, the increasing impairment of centrin functions
correlated with the amount of residual centrin on bbs, which was high in A7,
lower in A10, and frequently strongly reduced in N41. We suggest that centrin
is essential for bb maturation and, when disturbed, causes defects in
duplication and segregation of bbs.
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Acknowledgments |
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References |
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