Botanisches Institut, Universität zu Köln, Germany
* Author for correspondence (e-mail: karl.lechtreck{at}uni-koeln.de )
Accepted 6 January 2002
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Summary |
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Key words: Coiled-coil, Flagella, RNAi, Striated roots, Microtubule
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Introduction |
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Striated fiber assemblins from flagellate green algae are about 20%
identical to ß-giardin, one of the proteins in the microribbons
(Weber et al., 1993). Both
proteins share a similar domain structure with a short, nonhelical,
proline-rich head domain and a coiled-coil domain of approximately 250
residues. Striated fiber assemblin is phosphorylated in vivo and the head
domain of green algal SFAs contains several potential phosphorylation sites
for the p34cdc2 kinase. It is not known whether
phosphorylation at these sites is responsible for the disassembly of the SMAFs
observed during mitosis (Lechtreck and
Silflow, 1997
). The rod domain of ß-giardin/SFA is
characterized by a distinctive series of heptads arranged into blocks of four,
followed by a skip residue interrupting the heptad pattern
(Holberton et al., 1988
;
Weber et al., 1993
). A study
of the in vitro assembly properties of Chlamydomonas-SFA showed that
headless molecules were assembly incompetent, whereas large deletions and
insertions in the rod domain were tolerated and resulted in a shift in the
axial repeat of the striated fibers
(Lechtreck, 1998
). SFA
polymers are polar and consist of 2 nm protofilaments that are thought to be
composed of parallel dimers overlapping with the N- and C-terminal parts of
their rod domains (Patel et al.,
1992
; Lechtreck,
1998
).
Because SMAFs and similar fibers are rigid, they are thought to function as stabilizing elements in the basal apparatus. Noncontractile striated fibers, often with differing periodicities to the SMAFs, have been described in a broad range of organisms, including mammals, suggesting that these fibers have an important function in the basal apparatus. Detailed studies on in vivo function of SMAFs and similar roots are lacking, mainly because of the absence of mutants with defects in these fibers. Here, we present in vivo data on the expression of green fluorescent protein (GFP)-tagged SFA molecules and the repression of SFA expression by RNA interference. Our results indicate that the amount of SFA in Chlamydomonas is controlled by a complex mechanism balancing synthesis, degradation and polymerization of the protein, and that SMAFs are needed for correct flagellar assembly.
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Materials and Methods |
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Plasmids and transformation
A genomic library of Chlamydomonas
(Schnell and Lefebvre, 1993)
was screened using a cDNA-clone coding for SFA of C. reinhardtii (or
CRA8) (see Lechtreck and Silflow,
1997
). The screen resulted in three clones (BA1, BA5 and BA10);
BA1 was used as a template for subsequent PCR reactions. The coding region of
SFA including 88 bp of the 5'-UTR was amplified using Taq polymerase
(GeneCraft, 488163 Münster, Germany), a forward primer UTRf4
(5'-GCGTCTAGAGCTAGTTCTCATACATATTACTC-3'), which contained an
overhanging XbaI site followed by a NheI site, and the
backward primer GFPr1 (5'-GCTCTAGAGCTGCGCTGACCAGCTTGAGG-3'), which
contained a XbaI site, omitted the stop codon of SFA and adjusted the
reading frame to that of GFP. The 2.2 kb product was digested with
XbaI and ligated into pCr-GFP (BioCAT, Heidelberg, Germany)
(Fuhrmann et al., 1999
), which
contained a synthetic gfp-gene codon-adjusted for
Chlamydomonas and the 3' UTR of the rbcS2 gene as a
terminator sequence. A clone (pCr-SFA-GFP2) containing the sfa gene
in the correct orientation was partially sequenced, digested with
NheI and EcoRI, and the fragment containing the
sfa::gfp::rbcS2 gene was ligated into pCB740
(Schroda et al., 2000
)
digested with NheI and EcoRI to remove the hsp70A
gene. Thus, the GFP-tagged sfa gene was downstream of the strong
HSP70B/rbcS-fusion promoter in a vector containing also the arg7
gene, a selectable marker for transformation into arginine-requiring strains.
The plasmid pCB740-SFA-GFP was linearized with EcoRI and transformed
into Chlamydomonas CC3395 using the glass bead method
(Kindle, 1990
). A gene coding
for SFA with a C-terminal truncation of 18 residues was constructed similarly
using the reverse primer GFPr2
(5'-GCTCTAGACCTGCACGATCTCGTCGTCCTC-3'). Constructs with
single-residue exchanges or a deletion of six residues were performed
accordingly. As a control we used a cell line transformed with pCB740 digested
with NheI and EcoRI.
To obtain the antisense gene AS1, parts of the genomic clone BA1
and the cDNA clone CRA8 were amplified using the primers f1UTR
(5'-CGCGCATGCTAGCTTCTCATACATATTACTC-3') and r1AS
(5'-GCGTCTAGACGTTCGGTGACATGCTCAAGC-3') containing a SphI
and a XbaI site, and primers f1AScDNA
(5'-GCGGATCCTTCTCATACATATTACTC-3') and r1AS containing a
BamHI and XbaI, respectively. After digestion with the
enzymes indicated, the two fragments of 350 and 200 bp were ligated into
pCr-GFP digested with SphI and BamHI, which removed the
GFP gene. The resulting plasmid was digested with SphI and
EcoRI and the fragment containing the AS1 construct was ligated into
pCB740 downstream of the HSP70B/rbcS2 promoter. Double transformants were
obtained by cotransformation of AS5 with pCB740-SFA-GFP and the pJN4 plasmid
(Nelson et al., 1994
).
Western blotting analysis
Chlamydomonas cells, 10 ml, were pelleted at 500 g
for 3 minutes, resuspended in 500 µl MT buffer, and lysed by the addition
of 500 µl MT/3% Triton X-100. Cytoskeletons were immediately pelleted at
15.300 g and 200 µl of the supernatant were precipitated
with 1.4 ml of methanol at -20°C for several hours. Whole cells were
pelleted for 2-4 minutes at full speed in a table-top centrifuge. All pellets
were boiled in 2x sample buffer, subjected to SDS-PAGE and transferred
onto PVDF membrane. Western blotting was carried out as described previously
(Lechtreck and Geimer, 2000)
and blots were documented using a digital camera. Cycloheximide (Sigma) was
used at 2 µg/ml and removed by repeated washes (3x) in culture medium
(500 g, 5 minutes).
Nothern blotting
Total RNA was isolated from frozen cells (-80°C) using a guanidine
thiocyanate-phenol solution and precipitation with LiCl
(Ausubel et al., 1995),
separated by denaturating agarose electrophorese, and transferred to Biodyne B
membrane (PALL, Dreieich, Germany). Probes (HindIII fragment of
pQE-SFA or SphI/BamHI fragment of pCR-AS1) were labeled with
32P-ATP by random oligonucleotide-primed synthesis using the Klenow
fragment of DNA polymerase I. For northern hybridization, we followed the
protocol of Church and Gilbert (Church and
Gilbert, 1984
) and membranes were scanned using a PhosphorImager
(Storm).
Antibodies and microscopy
The following antibodies were used for western blotting (WB), indirect
immunofluorescence (IF) and immunogold electron microscopy (EM): polyclonal
anti-SFA (WB 1:2000-5000, IF 1:100-200, EM 1:200)
(Lechtreck, 1998), monoclonal
anti-centrin BAS6.8 (WB 1:50), polyclonal anti-GFP 290 (WB 1:3000, EM 1:100,
Abcam, Cambridge, UK), monoclonal anti-acetylated tubulin 6-11B-1 (IF 1:400,
Sigma) and monoclonal anti-
-tubulin (IF 1:400, clone DM 1A, Sigma).
Secondary antibodies were obtained from Dianova (anti-rabbit-IgG-Cy3,
anti-mouse-IgG-Cy3; Hamburg, Germany), Sigma (anti-rabbit-IgG-FITC,
anti-rabbit or anti-mouse-IgG conjugated to alkaline phosphatase) or British
BioCell (anti-rb-IgG 10-nm gold; Cardiff, UK) and used as recommended by the
manufacturer.
For fluorescence microscopy and indirect immunofluorescence,
Chlamydomonas cells were immobilized on poly-L-lysine-treated slides
and submerged in methanol at -20°C for 10-25 seconds. Cytoskeletons were
isolated by detergent treatment (1.5% Triton X-100) in MT buffer (30 mM Hepes,
15 mM KCl, 5 mM, MgSO4, 5 mM EGTA, pH 7) as described previously
(Lechtreck and Geimer, 2000).
Cells were observed and documented using a Nikon Eclipse E800 equipped with a
RT monochrome Spot camera (Diagnostics Instruments). Images were captured
using Metamorph, converted to 8-bit, exported to Photoshop (Adobe) and mounted
with Adobe Illustrator. For in vivo observations, Chlamydomonas cells
were immobilized in TAP/0.75-1.5% low melting agarose, adopted from a
previously described method (Reize and
Melkonian, 1989
). All methods for standard or postembeddding
immunogold electron microscopy have been previously described
(Lechtreck and Geimer,
2000
).
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Results |
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SFA-GFP assembled into striated fibers
Fluorescence microscopy revealed that SFA-GFP assembled into cross-like
structures centered at the basal bodies
(Fig. 1C). In some strains
(e.g. GFP3) the size of the cross was similar to that visible in control cells
after staining with anti-SFA (1 µm length) but, in GFP8, one or two
branches of the cross were much longer and up to 4 µm in length
(Fig. 1C,E). Often, GFP8 cells
developed one especially long fiber. These were often irregular in thickness
and even had discontinuities in them breaks that were not bridged by
wild-type SFA (Fig. 1Ea-c). In
western blots using anti-SFA, the GFP-tagged protein was visible in isolated
cytoskeletons as one or several bands at approximately 60 kDa, in addition to
the 34 kDa band representing SFA (Fig.
1D).
Chlamydomonas SMAFs run along the four bundles of microtubular
flagellar roots, which, in contrast to the other cytoskeletal microtubules,
contain acetylated tubulin (Fig.
2, top) (LeDizet and Piperno,
1986). The SMAFs along the two-stranded bundles sit end-to-end and
were originally described as one continuous fiber, whereas those along the
four-stranded roots sit laterally on the axis forming fibers
(Fig. 5a,b)
(Goodenough and Weiss, 1978
;
Weiss, 1984
). The length of
the proximal acetylated region of the four microtubular roots differed and the
prominent fluorescent fibers in GFP8 cells were attached to the roots with
short acetylated regions (Fig.
2), with the longest branch mostly (>90%, n=30)
associated with the root having the shortest acetylated region. Standard
electron microscopy showed that GFP8 contained striated fibers, especially
along the two-stranded microtubular roots, which exceeded control SMAFs by
several fold in length, thickness and width
(Fig. 3d-f,h). The
cross-striation periodicity of the SFA-GFP fibers was determined with 27.5 nm
(n=5) similar to that of wild-type SMAFs (27.8 nm in whole mount
microscopy) (Lechtreck, 1998
).
We were able to decorate the fibers with anti-SFA and anti-GFP in immunogold
electron microscopy (Fig.
3a-c), confirming that SFA-GFP assembled into
microtubule-associated striated fibers. Thin sections of GFP8 frequently
showed microtubules radiating from the oversized SMAFs
(Fig. 3d,e,h) and emphasized
the close association of SMAFs and probasal bodies
(Fig. 3f,h).
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Heat shock induced disassembly of SFA-GFP fibers
Expression of SFA-GFP was controlled by the strong, constitutive
HSP70B/rbcS2 fusion promoter, the activity of which can be modulated by heat
shock or culture conditions (Schroda et
al., 2000). In fact, the size of the fluorescent fibers was
reduced when GFP8 cells were cultivated in the dark for 48 hours
(Fig. 1C). Also, signal
strength decreased in cultures of high cell density (not shown) and therefore
all experiments were carried out with cultures
106 cells/ml.
Heat shock (60 minutes at 40°C) caused the disassembly of the SFA-GFP
fibers, with the exception of a dot-like core region near the basal bodies
(Fig. 4A, T60). A similar
SFA-GFP core structure was also observed in dividing cells when SFA fibers
were largely disassembled (see below). Anti-SFA staining of cells after heat
shock revealed fibers exceeding the corresponding GFP signals in size,
indicating that remnant wild-type SFA fibers were present after heat shock
(Fig. 4C). Reassembly of the
SFA-GFP fibers was evident 1 hour after the heat shock, when we observed up to
four thread-like extensions of the central bright dot
(Fig. 4A, T120). These thin
fibers became longer and thicker and after 5-6 hours reached the size of those
in untreated controls. Heat shock also induced the formation of globular
SFA-GFP aggregates in Chlamydomonas. These were transient in nature,
visible in phase contrast microscopy (Fig.
4A, T240) and often located at the ends of the two long SFA-GFP
fibers (Fig. 4A, T240). The
proportion of cells with aggregates post heat treatment varied between
experiments (not shown). Using cells immobilized in low-melting agarose, it
was possible to observe fiber disassembly and reassembly in living cells
(Fig. 4B). After heat shock,
Schroda et al. (Schroda et al.,
2000
) observed a strong increase in the amount of mRNA and protein
from genes expressed under the control of the HSP70A/rbcS2 fusion promoter. On
northern blots probed with a partial cDNA of SFA, a transcript of
approximately 3.2 kb and several smaller transcripts were abundant in a GFP8
sample taken 45 minutes after heat shock, whereas these transcripts were not
detected in GFP8 cells before or 3 hours after heat shock or in the control
strain (Fig. 4D). Despite this
obvious increase in the amount of mRNA coding for SFA-GFP, the amount of
protein detected on western blots of whole cells showed only minor variations
during heat shock (Fig. 4E,
top). Fractionation of cells after heat shock confirmed the heat instability
of SFA-GFP fibers: SFA-GFP increased in the soluble fraction but was largely
removed from the cytoskeletal fraction
(Fig. 4E, middle). During
recovery, the amount of SFA-GFP in the cytoskeletons increased again. In
heat-shocked control cells the amount of SFA showed only minor variations
(Fig. 4E, top and bottom).
Often, SDS-PAGE revealed a shift in the apparent molecular weight between the
soluble and insoluble forms of SFA-GFP and SFA
(Fig. 4E), suggesting that a
modification like phosphorylation could be responsible for dissolving SFA
fibers. Further, anti-SFA detected additional bands near 60 kDa in some
westerns (Fig. 4E;
Fig. 8A,B), which were also
present when protease inhibitors had been added during cell lysis and could be
caused by phosphorylation.
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Analysis of SFA-GFP in dividing cells
The distribution of SFA during the cell cycle of C. reinhardtii
has been studied previously by indirect immunofluorescence. Briefly, two of
the four SMAFs disassemble prior to mitosis. The remaining fibers fall apart
and are then reduced to dot-like structures. One or two dots positive with
anti-SFA were present at each spindle pole and in telophase one prominent
fiber was formed before the small cross-like structure typical for cells in
interphase was re-established (Lechtreck
and Silflow, 1997). During mitosis SFA-GFP fibers were
disassembled (Fig. 5b, living
cells), but we noted that SFA-GFP was removed more rapidly from the fibers
along the two-stranded roots than SFA. Furthermore, the remaining dot-like
signals contained only small amounts of SFA-GFP, whereas brighter signals were
observed after antibody staining (Fig.
5, methanol-fixed cells). To analyze living cells embedded in
agarose, we mostly used short exposure times (<1 second), 4x to
16x excitation filters to prevent photodamage, and controlled so that
the contractile vacuoles were pumping rapidly indicative for cell viability
(Fig. 5e, top). Before mitosis
we observed changes in the angle between the SFA-GFP fibers
(Fig. 5a, top). Then, the
fibers separated and disassembled (Fig.
5b, top). Towards the end of the first division the distance
between the dot-like structures present at the apex of both cell halves
decreased and often, additional dots appeared
(Fig. 5c,d, top). These were
either positioned near the cell apex and thus might be located in the
duplicated basal apparatus, or deeper in the cell body. Some of these dot-like
structures moved with a velocity of up to 0.4 µm/minute. Similar dots were
observed previously in control cells
(Lechtreck and Silflow,
1997
).
The C-terminal domain of SFA is essential for fiber formation
On the basis of the observation that heat shock largely dissolved SFA-GFP
fibers, we assumed that the GFP-tag deformed the C-terminus of the protein and
thereby destabilized the striated fibers. Attempts to extract SFA-GFP in vitro
from isolated cytoskeletons by heat treatment were not successful, indicating
that fiber disassembly depended on cellular activities. To examine further the
role of the C-terminus in fiber stability, we constructed a GFP-tagged SFA
with a C-terminal deletion of 18 residues (SFAC18-GFP)
(Fig. 6A). In overexpressing
strains, for example,
C18-11 cells, weak green fluorescence was only
detected in a dot-like region near the basal bodies
(Fig. 6B). Wild-type SFA was
largely insoluble, whereas full length SFA-GFP was present in both the
cytoskeletons and the supernatant (Fig.
6C). Densitometric analysis of western blots stained with anti-SFA
indicated that about 60% of the total SFA-GFP was insoluble in cells
cultivated at 25°C. By contrast, western blot analysis of
C18-11
after cell fractionation showed that SFA
C-GFP was mostly soluble
(Fig. 6C). The results
suggested that the C-terminal domain of SFA is necessary for fiber formation.
To further determine the region necessary for fiber formation, we expressed a
construct coding for a SFA-GFP with a C-terminal deletion of six residues,
which showed similar properties to those of
C18 (not shown). Next, we
exchanged the penultimate serine with either alanine (SFA-GFP-SA) or glutamic
acid (SFA-GFP-SE). The latter mimics phosphoserine and resulted in a mostly
soluble protein, as documented by western blotting
(Fig. 6C, lower panel), and
fluorescence microscopy, which showed a dot or small cross near the flagellar
base (not shown). By contrast, SFA-GFP-SA was hardly detected in the
supernatant obtained from overexpressing strains (e.g. SA11). Fluorescence
microscopy of SFA-GFP-SA strains showed large cross-like structures, often
with additional branches and additional fibers that were not connected to the
basal apparatus or associated with microtubules
(Fig. 6D, not shown).
Interestingly, the serine-to-alanine mutation conferred heat stability (1
hour, 40°C) to the fibers (Fig.
6D).
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SFA-GFP interfered with the expression of SFA
We noted that cytoskeletons isolated from GFP8 cells contained less SFA in
comparison to control cells (Fig.
6C). A similar reduction of untagged SFA was noticed in cells
expressing the assembly incompetent SFAC18-GFP, suggesting that soluble
SFA-GFP might repress the expression of SFA. To analyze further the assumed
interference between the expression of SFA-GFP and SFA, we took advantage of
the observation that SFA-GFP was mostly soluble at higher temperatures: only
short fibers were observed by fluorescence microscopy at 32°C, whereas
large signals were observed at 25°C and especially at 15°C when
fluorescence was often prominent, even along the proximal parts of the
four-stranded microtubular roots (Fig.
7A). On western blots of whole cells cultivated at 15, 25 or
32°C, decreasing amounts of SFA-GFP were detected
(Fig. 7B). This decrease was
caused by a loss of polymeric SFA-GFP, whereas almost constant amounts of
SFA-GFP and only traces of SFA were detected in the soluble fractions
(Fig. 7C). The results suggest
that soluble SFA and SFA-GFP are maintained at constant low levels by
degradation, although other explanations are possible. Interestingly, the
amount of SFA increased with rising temperatures
(Fig. 7B,C), which could
represent a mechanism by which cells try to balance the temperature-induced
loss of polymeric SFA-GFP. Thus, striated fibers were mostly composed of
SFA-GFP at 15 or 25°C but contained more SFA than SFA-GFP at 32°C.
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Because cells assemble fibers with different ratios of SFA to SFA-GFP depending on the environmental temperature, temperature shifts should induce synthesis of SFA and degradation of SFA-GFP. Indeed, increased levels of SFA and decreased levels of SFA-GFP could be observed within 6 hours after increasing the temperature from 15 to 32°C (Fig. 8A, lanes 1,5). To test whether SFA was synthesized de novo, we added 2 µM cycloheximide to cells before shifting them to 32°C. In comparison to controls, the cytoskeletons of cells treated with cyclohexmide for 6 hours contained less SFA but more SFA-GFP, which was observed either in cells heat-shocked before the temperature shift to dissolve the fibers (Fig. 8A, lanes 3,4) or in cells only shifted from 15 to 32°C (Fig. 8B, lanes 2,3). We presume that cycloheximde does not only block SFA synthesis but also interferes with the degradation of soluble SFA-GFP. Assuming further a balance between soluble and insoluble SFA-GFP, a block in the degradation of soluble SFA-GFP should force some protein to be re-incorporated into striated fibers in the presence of cycloheximide at 32°C. Accordingly, fluorescent fibers in cells treated with cycloheximide after the temperature shift were larger than in controls without cycloheximide (Fig. 8C, part 1,3). Interestingly, 2 hours after cycloheximide was removed from temperature-shifted cultures, elevated amounts of SFA and SFA-GFP were detected on western blots, and large fibers were observed by fluorescence microscopy (Fig. 8B, lane 4, C4). In cells that were permanently cultivated at 15°C neither cycloheximide treatment nor its removal caused significant changes in the ratio or amount of SFA and SFA-GFP (not shown).
Reduction of SFA expression by RNA interference
We constructed a gene consisting of the 5' region of the sfa
gene, including parts of the 5' UTR and the first two introns, to which
the corresponding region of the cDNA was fused in the antisense orientation
(Fig. 9A). This construct is
predicted to form dsRNA with a hairpin that has been shown to effectively
induce post-transcriptional gene silencing in plants
(Smith et al., 2000) and
Chlamydomonas (Fuhrmann et al.,
2001
). Indeed, western blots with anti-SFA of cells transformed
with pCB-AS1 identified strains (five out of 20 tested) with significantly
reduced amounts of SFA. In two of these cell lines, AS5 and AS8, the
expression of the antisense construct was verified by northern blotting, which
showed a transcript of
900 bp in the total RNA isolated from cells 45
minutes after heat shock (Fig.
9B). Furthermore, Southern blotting showed that AS8 contained a
single copy of the transformed plasmid (not shown).
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The amount of residual SFA varied between cell lines, and for further
analyses we chose strains (AS3, AS5, AS6 and AS8) in which SFA was barely
detectable on western blots of isolated cytoskeletons
(Fig. 9C) and was not detected
on westerns of whole cells (not shown). In these cell lines, indirect
immunofluorescence with anti-SFA still showed a dotted signal near the basal
bodies and, in a few cells, cross-like structures were observed
(Fig. 9D). We assume that these
signals were caused by residual SFA in the cytoskeletons. Most antisense
strains showed elevated numbers of uniflagellate or flagella-less cells, and
AS3 had no flagella (Table 1).
The latter could be the result of an independent mutantion, for example,
caused by insertion of the transformed DNA into a gene necessary for flagellar
assembly. In control or GFP8 cells approximately 98% of cells were
biflagellate, whereas in the antisense strains only 51- 86% showed wild-type
flagellar numbers, which included up to 10% of cells with one fully grown
flagellum and one shorter or stumpy flagellum
(Fig. 9D). In AS8 cells,
flagellar regeneration after mechanical deflagellation was delayed and slow
compared with control or GFP8 cells (not shown). Several counts of flagellar
numbers in AS5 and AS8 over several months showed a variation in the
distribution of cells with zero, one or two flagella
(Table 1). On western blots,
variable but always significantly reduced amounts of SFA were detected (not
shown). We also generated double transformants by introducing pCB740-SFA-GFP
into AS5 by contransformation with pJN4, containing the cry1 gene,
which confers emetine resistance (Nelson
et al., 1994). The phenotype of AS5-SFA-GFP transformants was
similar to that of AS5 and strains overexpressing SFA-GFP were not detected
(Table 1). Fluorescence
microscopy revealed weak globular signals scattered in the cytoplasm, in
addition to a weak dot at the apical cell pole
(Fig. 10A). Western blotting
with anti-SFA and anti-GFP revealed that SFA-GFP mostly remained in the 15,300
g supernatant after cell lysis, when expressed in the AS5
background (Fig. 10B). Presumably, the crucial concentration for polymerization of SFA-GFP, which
could be much higher than that for SFA, was not reached when expressed in the
antisense background.
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Discussion |
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The C-terminal GFP-tag of SFA conferred temperature sensitivity to the
chimeric protein. We assume that GFP, which is about 2.4x4.2 nm in
dimension, deformed the C-terminal domain and thereby destabilized SFA-GFP
fibers. Constructs with C-terminal deletion of six or 18 residues were mostly
soluble, emphasizing the importance of the tail domain for the assembly of
striated fibers. The removal of a N- or C-terminal domain, or both, had a
severe effect on assembly of certain intermediate filament proteins (for a
review, see Heins and Aebi,
1994). We were not able to heat-depolymerize SFA-GFP fibers in
vitro, suggesting that the disassembly observed in vivo as a reaction to
elevated temperatures was accomplished by cellular factors such as
phosphorylation. In vivo, SFA is phosphorylated with a high turnover
(Lechtreck and Melkonian,
1991
). To test this hypothesis we exchanged Ser275 by either
alanine or glutamic acid. The latter mimics phosphoserine and increased the
solubility of SFA-GFP. In strains expressing the serine-to-alanine mutation we
observed aberrant fiber formation and the mutation conferred heat stability to
the polymers. Thus, phosphorylation of Ser275 could regulate the solubility of
SFA, a mechanism that could be important to maintain a certain size of the SFA
fibers and prevent aberrant fiber formation. Our attempts to express
N-terminal truncated SFA molecules, which in vitro are assembly incompetent
(Lechtreck, 1998
), have failed
up to now, indicating that the 5' UTR or the introns, or both, in the
5' region of the sfa gene are necessary for expression from the
HSP70B/rbcS2 fusion promoter (not shown).
SFA-GFP fibers were largely depolymerized before cells enter mitosis,
indicating that the mitotic processes dissolving the SMAFs also acted on the
GFP-tagged protein. During mitosis SFA-GFP was located in several dot-like
structures, and in vivo observations showed rapid and directional movements of
these structures with velocities of up to 0.4 µm/minute. Previously,
dot-like structures cross-reacting with anti-SFA were observed during cell
division of Chlamydomonas
(Lechtreck and Silflow, 1997).
The exact localization and function of the SFA structures outside of the basal
apparatus needs to be addressed in future. The results show that GFP tagging
is a potent method for the in vivo analysis of cytoskeletal dynamics in
Chlamydomonas.
Overexpression of SFA-GFP in Chlamydomonas caused a decrease in
the amount of the wild-type protein. This effect indicated that SFA-GFP
expression interfered with the metabolism of SFA. Less SFA was also observed
in cells expressing the assembly incompetent tail-less SFA-GFPs. Cells
overexpressing full-length SFA-GFP always contained a pool of soluble SFA-GFP.
Thus, we assume that soluble SFA-GFP or SFAC-GFP downregulates the
expression of SFA. The expression of tubulin is also autoregulated: high
amounts of soluble tubulin dimers provoke the degradation of mRNAs coding for
ß-tubulin (for a review, see
Cleveland, 1989
). In
Chlamydomonas, the control of SFA expression by the soluble protein
could downregulate synthesis before mitosis when SMAFs are depolymerized, or
in early interphase when SMAFs have reached a sufficient length. However, the
assumed feed back of soluble SFA/SFA-GFP on the expression of SFA is
insufficient to explain all effects observed during experiments manipulating
fiber length and composition. After shifting cells to elevated temperatures,
which caused shrinkage of the SFA-GFP fibers, the amounts of soluble SFA-GFP
remained more or less constant, but the amount of SFA in the fibers increased.
One possible explanation for this effect is that the temperature-dependent
disassembly of the fibers dominated by SFA-GFP liberates binding sites for
constitutively expressed SFA. Alternatively, fiber depolymerization would
induce the expression of SFA to ensure SMAFs of a certain minimal length. This
model would need a mechanism by which cells sense the insufficient size,
length or state of the SMAFs. The presence of cycloheximide during a
fiber-destabilizing temperature shift resulted not only in lower amounts of
SFA but also caused higher levels of SFA-GFP and longer polymers. We suppose
that cycloheximide indirectly delayed the degradation of soluble SFA-GFP,
which could cause the assembly of longer SMAFs even at elevated
temperatures because of an equilibrium between soluble and insoluble
forms of SFA-GFP. After the removal of the drug, we observed a boost in the
expression of SFA and SFA-GFP. It has been shown previously that cycloheximide
interferes with stability of mRNAs coding for tubulin
(Pachter et al., 1987
). In
Chlamydomonas the drug prevents the degradation of transcripts coding
for
-tubulin that accumulate after deflagellation
(Baker et al., 1989
). The
effect of cycloheximide on mRNAs coding for SFA is not known. Our data
indicate that the length of the SMAFs depends on a regulatory pathway
controlling SFA synthesis, degradation, and the equilibrium between
polymerization and depolymerization.
Despite the presence of oversized SMAFs, GFP8 cells had no obvious
phenotype (that is, growth, cell size and shape, flagellar position and
number, and swimming behavior as observed by light microscopy were
indistinguishable from controls), qualified by the observation that cultures
of GFP8 maintained at 15°C often contained elevated numbers (2-9%) of
quadriflagellate cells compared with controls (not shown). Furthermore,
SFA
C-GFP did not cause a dominant negative effect as observed for
truncated keratin molecules, for example (for a review, see
Fuchs, 1995
). To explore SFA
function, a partial cDNA of SFA was fused in antisense orientation behind the
corresponding region of the genomic DNA and expressed to induce gene silencing
via the formation of dsRNA, as recently described in Chlamydomonas
(Fuhrmann et al., 2001
).
Indeed, we identified cell lines with severely reduced amounts of SFA that
often had less than two flagella. The observed phenotype of the antisense
strains indicated that SFA or the SMAFs are necessary for flagella assembly or
maintenance, or both. Microtubules serve as track for intracellular transport
and thus the microtubular roots, which terminate near the basal bodies, could
function as supply lines for flagella precursors
(Porter et al., 1999
). This
transport could somehow depend on the SMAFs, which are attached to the
flagellar root microtubules. Furthermore, we observed microtubules terminating
laterally on the oversized SMAFs of GFP8 cells, and SMAFs of the colorless
green alga Polytomella function as microtubule organizing centers in
vitro (Stearns et al., 1976
).
However, antibodies for acetylated tubulin and
-tubulin did not
identify significant structural changes in the microtubular cytoskeleton of
antisense strains as visible by indirect immunofluorescence, but the
distribution of
-tubulin was altered in the strains expressing SFA-GFP
and SFA antisense: GFP8 cells often showed anti-
-tubulin staining along
the large SFA-GFP fibers, whereas in AS8 the
-tubulin signals were
smaller than in wild-type cells (K.-F.L., unpublished). Although many
molecular details remain to be elucidated, reduced expression levels of SFA
might alter the microtubular system near the basal bodies, which could hinder
the transport of flagellar proteins. Similar functions could also be valid for
similar noncontractile rootlets, which are common in flagellate cells over a
broad range of species.
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Acknowledgments |
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Footnotes |
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References |
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