Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, BC, Canada V5A 1S6
* Author for correspondence (e-mail: Quarmby{at}sfu.ca )
Accepted 13 January 2002
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Summary |
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Key words: NIMA, Cell cycle, Chlamydomonas, Microtubule severing, Deflagellation
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Introduction |
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Nek2 is of particular interest with respect to the present work. Expression
studies have implicated vertebrate Nek2 in cell cycle progression, but there
is no evidence that it is playing the same roles as NIMA in G2/M transition
and chromosome condensation (Fry et al., 1995;
Fry et al., 1998;
Rhee and Wolgemuth, 1997
;
Tanaka et al., 1997
). Instead,
Nek2 appears to play an essential role in the progression of the centrosomal
cycle. Overexpression of Nek2 results in premature separation of the
replicated centrioles (Fry et al.,
1998
; Mayor et al.,
2000
). Furthermore, biochemical data demonstrates that Nek2B
facilitates assembly of the Xenopus zygotic centrosome
(Fry et al., 2000
). Like Nek2,
the FA2 gene of Chlamydomonas encodes a NIMA family kinase
that has a centrosome/basal-body-associated function.
The FA2 gene was discovered during a genetic screen for
deflagellation-defective mutants of Chlamydomonas reinhardtii
(Finst et al., 1998).
Deflagellation is a highly specific process that involves a Ca2+
signal transduction pathway originating at the plasma membrane and culminating
in the severing of the nine outer-doublet axonemal microtubules at a precise
site distal to the transition zone between the axoneme and the basal bodies
(for a review, see Quarmby,
2000
). The microtubule-severing ATPase katanin has been implicated
in this severing event by its localization at the site of severing, the
inhibition of calcium-induced severing by katanin p60 antibodies and its
demonstrated ability to sever the complex doublet microtubules of the axoneme
(Lohret et al., 1998
). Our
genetic screen identified three additional genes, ADF1, FA1 and
FA2, which are essential for in vivo deflagellation. ADF1
plays a role in signal transduction, whereas, cells with mutations in either
FA1 or FA2 fail to deflagellate because of a defect in
calcium-induced axonemal microtubule severing
(Finst et al., 1998
;
Quarmby, 1996
). Fa1p is a
novel 170 kDa protein with a large coiled coil domain; it localizes to the
base of the flagella (Finst et al.,
2000
).
In this work we have identified a genomic clone that rescues fa2
mutants. Sequence analysis indicates that the FA2 gene encodes a NIMA
kinase. This is the first indication that this family of kinases might play a
role in the regulation of microtubule severing. It is clear that microtubules
break in vivo (e.g. Odde et al.,
1999), and regulated microtubule severing probably plays a role in
the establishment of non-centrosomal microtubules in neurons, myocytes and
epithelial cells (Ahmad et al.,
1999
; Rodionov et al.,
1999
; Waterman-Storer and
Salmon, 1997
; Odde et al.,
1999
). Furthermore, immunolocalization, genetic and biochemical
evidence suggest that katanin is important in mitotic and meiotic cell
division (McNally et al.,
1996
; McNally and Thomas,
1998
; McNally et al.,
2000
; Srayko et al.,
2000
). The role(s) of microtubule severing during the cell cycle,
and the regulation of microtubule severing in general remain unclear. Whether
Fa2p regulates centrosome-associated microtubule severing or, like Nek2, plays
a role in centrosome assembly (or both), it was of interest to discover in the
current work that fa2 mutants are delayed in cell cycle
progression.
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Materials and Methods |
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Genomic library and BAC DNA screening
An insertion of exogenous NIT1 DNA (in pUC119 plasmid) genetically
mapped to the FA2 locus in the fa2-3 strain
(Finst et al., 1998) and was
therefore used as a molecular tag to clone flanking DNA. A lambda Fix II
bacteriophage library of fa2-3 was created according to manufacturers
instructions (Stratagene, La Jolla, CA). The library was screened with
radiolabelled fragments of the transforming DNA (specifically, pUC119 and 0.9
kb of the 3' end of NIT1) as described by Finst and coworkers
(Finst et al., 2000
).
Following identification of positive clones, a 1.5 kb fragment flanking the
inserted DNA was used to probe a wild-type Chlamydomonas genomic BAC
library (Incyte Genomics, St. Louis, MO). To verify specificity of
cross-reactivity, DNA of positive BAC clones was isolated (as described by
Incyte Genomics) and hybridized on Southern blots with the same 1.5 kb
fragment described above.
Mutant rescue, FA2 genomic and cDNA isolation
To determine whether positive BAC clones could rescue the deflagellation
defect of fa2 mutants, fa2-2 cells (nit)
were co-transformed with NIT1 and BAC DNA. Nit1+
transformants were selected and assayed for deflagellation as previously
described (Finst et al.,
1998). A BAC clone that rescued the deflagellation defect in
fa2-2 mutants was digested with various endonucleases including
EcoRI, SalI, SacI, BamHI, PstI
and ApaI. The products of these digests were co-transformed with
NIT1 into fa2-2 cells to identify enzymes whose recognition
sites did not fall within the FA2 gene. As reported below, one
subclone, containing a 4.6 kb SacI-SalI insert, rescued the
deflagellation defect. To confirm that the 4.6 kb fragment was responsible for
the rescue, it was co-transformed with another selectable marker pARG7.8
(encoding arginosuccinate lyase, linearized by digestion with BamHI)
(Debuchy et al., 1989
) into
fa2-1 /arg7- cells. Arg7+ transformants were selected by
growth on medium lacking arginine and assayed for deflagellation. The 4.6 kb
subclone was sequenced by the Emory University DNA Sequence Facility (Atlanta,
GA) and the University of British Columbia DNA Sequencing Laboratory
(Vancouver, BC, Canada).
In order to identify the cDNA, RNA isolation and RT-PCR were performed as
previously described (Finst et al.,
2000). Polyadenylated RNA was isolated from wild-type strain cells
(strain B214) and reverse transcribed using Superscript II reverse
transcriptase (Life Technologies, Gaithersburg, MD) and
oligo(dT)(12-18) primers. The PCR primers were designed according
to putative exon sequences predicted by the GeneMark algorithm
(Benian et al., 1996
). 3'
RACE was then performed as previously described
(Finst et al., 2000
). PCR
products were separated by agarose gel electrophoresis, purified using an
ultra-MC filter unit (Millipore, Bedford MA) and subcloned into the pGEMT-Easy
vector (Promega, Madison, WI) prior to sequencing.
Sequence analysis
Searches of the GenBank database were performed using the BLAST program
(Altschul et al., 1997). The
top hits from the BLAST search were aligned with the amino-acid sequence of
FA2 using Clustal W (Thompson et
al., 1994
) and Genedoc
(Nicholas et al., 1997
). The
catalytic kinase domain and ATP-binding site were identified by eye using
consensus sequences (Hayashi et al.,
1999
). Phylogenetic analysis was performed using the PHYLIP
package (Felsenstein, 1989
)
and PhyloBLAST (Brinkman et al.,
2001
).
FA2 RNAi construct
The goal for the FA2 RNAi construct was to produce a transgene which, when
transcribed by Chlamydomonas, would produce a double-stranded RNA
structure. In order to achieve efficient expression, we started with the
5' end of the genomic clone, containing upstream sequence and introns
presumed to be important for efficient expression. Specifically, the
FA2 genomic clone was digested with NruI (3108) and
SalI (4657) in order to release the 3' end of the gene (a
fragment of about 1.5 kb from the middle of exon 5 through to the end of the
genomic clone). Similarly, the FA2 cDNA clone was digested with
NruI (1796) and EcoRI (0001), and the fragment containing
the first five and a half exons of FA2 was purified by gel
extraction. The 5' and 3' ends of both the cDNA fragment and
genomic clone (lacking the 3' 1.5 kb) were filled in with T4 DNA
polymerase and dNTPs (Invitrogen, Burlington, ON). The ends of the partial
genomic clone were dephosphorylated with CIAP (Invitrogen) and then the cDNA
fragment was ligated into this genomic clone with T4 DNA ligase (Invitrogen)
and transformed into competent DH5 cells. Ampicillin-resistant
transformants were assayed for insertion, and clones carrying the correct
orientation of the cDNA piece with respect to the genomic piece were
identified by restriction digestion. The desired clone would produce a spliced
RNA transcript that would be a perfect inverted repeat
(Fig. 8A). This primary
structure is expected to form a hairpin secondary structure, which is the most
efficient trigger for RNAi in plants
(Smith et al., 2000
) and has
been shown to be effective in Chlamydomonas (Furhmann et al.,
2001).
|
Molecular characterization of fa2 mutant alleles
To determine the mutation in each fa2 allelle, genomic DNA
isolated from each mutant strain was amplified by PCR with four sets of primer
pairs chosen to span the FA2 gene in four segments of 1.2 kb.
All products resulting from PCR of fa2-2 DNA were of wild-type size
and therefore were sequenced in order to identify the mutation. NIT1
sequence-specific primers were used in combination with FA2
sequence-specific primers to characterize the exogenous DNA insertion in
fa2-1, fa2-3 and fa2-4 because these mutants were generated
by insertional mutagenesis. PCR reactions contained 0.5 µM of each primer,
0.5-2.0 µM MgCl2, 0.3 mM dNTP, 1X Taq DNA polymerase buffer, 2.5
U of Taq DNA polymerase (Qiagen, Valencia, CA) and Q solution (for GC rich
genomes). PCR mixtures were denatured at 94°C for 2 minutes, followed by
35 cycles of 94°C for 1 minutes, 58-68°C for 1 minute, and 72°C
for 1minute followed by a 5 minute extension at 72°C.
Northern analysis
Ten micrograms of polyadenylated RNA was size fractionated on formaldeyhde
gels, transferred to Zeta Probe GT membranes (Bio-Rad, Hercules, CA) using 25
mM sodium phosphate (pH 6.4) and fixed using a Stratalinker UV crosslinker
(Stratagene). Exon-specific probes, 0.4 kb to 0.8 kb, were generated with
[32P]-dATP or [32P]-dCTP (3000 Ci/mmol) by PCR. Products
were purified using the PCR purification kit (Qiagen). Membranes were
hybridized with probes at 106 cpm/ml at 72°C and washed as
described (Virca et al.,
1990
). Imaging was performed with the Storm system (Molecular
Dynamics, Sunnyvale, CA). Before reprobing, blots were stripped using
2x500 mL of 0.5% SDS/0.1x SSC at 94°C for 15 minutes.
Production of FA1 antibodies and western analysis
The C-terminus of FA1 (encoding 545 amino acids) was cloned into
pGEX-6P-2 (Amersham) and pET28a (Novagen) to produce GST-tagged (GST-Fa1pC)
and His-tagged (His-Fa1pC) fusion proteins. Two rabbits were immunized with
purified His-Fa1pC protein (Spring Valley Laboratories, Sykesville, MD).
Polyclonal anti-sera was affinity purified by standard procedures (Harlow and
Lane, 1988), using immobilized GST-Fa1pC (UltraLink Biosupport Medium;
Pierce).
For western analysis, flagella-basal body complex protein was isolated as
previously described (Lohret et al.,
1998). Protein concentration was determined using the Advanced
Protein Assay (Cytoskeleton Inc., Denver, CO). Thirty micrograms of protein
was separated on a 6% SDS-PAGE gel and electroblotted to supported
nitrocellulose (Bio-Rad). To confirm efficient transfer of protein samples,
membranes were stained with Ponceau S (Allied Chemicals, Morristown, NJ) and
then washed with 0.05% Tween 20 in Tris-buffered saline (TBST). The membrane
was blocked in 5% skimmed milk in TBST for 1 hour at room temperature and then
incubated overnight at 4°C with anti-Fa1C antibody (at 1:100). The
membrane was washed with TBST and incubated with horseradish-peroxidase-linked
donkey anti-rabbit Ig (Amersham) at room temperature with rocking for 1 hour.
Immunoreactive proteins were visualized using the ECL chemiluminescent
detection system (Amersham).
Cell size determination
For cell size measurement, aliquots of cells were centrifuged and
resuspended in media containing 2% glutaraldehyde. The sizes of 90-110 cells
in randomly selected fields were determined microscopically at 1000x
magnification by measuring length (l) and width (w) using software supplied by
Motic Images 2000 (Causeway Bay, Hong Kong). Volume was calculated per Umen
and Goodenough (Umen and Goodenough,
2001) on the basis of the approximate prolate ellipsoid shape of
the cells (4/3
[1/2][w/2]2).
Cell division and flow cytometry
Synchronization of cells was carried out as described by Umen and
Goodenough (Umen and Goodenough,
2001) with modification. Cultures of wild-type B214 and
fa2 mutant strains were grown in M-media
(Harris, 1989
) at room
temperature with shaking. Flasks were bubbled with 5% CO2 and grown
asynchronously to a density of
5x106 cells/ml in the
light, then placed in the dark at 1x106 cells/ml for 24
hours. Cultures were then moved back to the light, and aliquots were sampled
over the next 24 hours. For each sample, we examined 300 fixed cells
microscopically for cleavage furrows in order to determine the fraction of
cells that had entered M phase. In Chlamydomonas, incipient cleavage
furrows form at preprophase and are visible throughout mitosis and cytokinesis
(Kirk, 1998
). To analyse DNA
content, cells were harvested at various times after return to light,
collected by centrifugation, resuspended and incubated in 1 volume of 70%
ethanol for 1 hour at room temperature. Cells were then prepared for FACS
analysis according to a protocol developed by A. Shutz and S. Dutcher
(Washington University) as follows. The cells were washed with 1 volume of
FACS buffer (0.2M Tris-Cl, pH 7.5, 20 mM EDTA, 5 mM sodium azide) and then
resuspended in 0.5 volumes of FACS buffer. 106 of these cells were
pelleted by centrifugation, resuspended in 100 µL FACS buffer with 1 mg/ml
RNase A and incubated for 3 hours at 37°C. Cells were washed with 1mL PBS
and then incubated overnight in the dark with 100 µL of PI solution (PBS
supplemented with 50 ug/mL propidium iodide; Sigma). To each sample 900 µL
of PBS was added. The samples were then analyzed by flow cytometry at the UBC
Multiuser FACS Facility (University of British Columbia, Vancouver,
Canada).
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Results |
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BAC clone 38g23 was digested with various restriction enzymes to identify
the smallest fragment of DNA that contained the FA2 gene.
Transformation of fa2 mutants with these fragments lead to the
identification of a 4.6 kb SacI-SalI fragment that rescued
the deflagellation defect in fa2-1 and fa2-2 (21 rescues out
of 1108 transformants assayed; 2%). Southern analysis showed that the 4.6
kb insert was present in wild-type cells and transformation-rescued
fa2-1 cells but not in fa2-1 mutant cells (data not shown).
Because the 4.6 kb fragment rescues the deflagellation defect, and because it
physically maps to the FA2 locus, we concluded that it contains the
FA2 gene. The rescuing clone was sequenced, and seven putative exons
were identified by the Genemark gene prediction algorithm. This prediction was
used to design primers for RT-PCR and 3' RACE of polyadenylated RNA
isolated from wild-type cells. DNA sequencing and subsequent alignment of the
cDNA clone with the genomic sequence confirmed the seven exons and defined the
5' and 3' untranslated regions. The cDNA sequence was used by C.
Silflow and M. LaVoie (University of Minnesota) to map the FA2 gene
to linkage group VII (Vysotskaia et al.,
2001
).
FA2 encodes a Nek kinase
The FA2 cDNA encodes 618 amino acids with a predicted molecular
mass of 68 kDa (Fig. 1).
Searches of public databases identified many proteins in the NIMA family of
expressed kinases (Neks), with high amino acid identity (35-40%) to
FA2 in the N-terminal kinase domain
(Fig. 2). The motif (IKSAN) in
the catalytic domain suggests that Fa2p is a serine/threonine kinase
(Fig. 2) (Hanks et al., 1991).
Little or no sequence similarity exists in the C-terminal regions of these
proteins, but, like several other Neks, the C-terminal region of Fa2p is basic
(pI 9.6) (Wang et al., 1998).
Phylogenetic analysis using the complete Fa2p amino-acid sequence or
N-terminal domain showed that Fa2p is a NIMA kinase member but failed to
tightly associate Fa2p with a specific subfamily of NIMA kinases (data not
shown).
|
|
Characterizing the fa2 mutant alleles
PCR analysis and sequencing of the three mutant alleles generated by
NIT1 insertion revealed an insertion in the fourth intron of
fa2-1 and an insertion in the fourth exon of fa2-4
(Fig. 3A.). By Southern
analysis, FA2 is completely deleted in fa2-3 (data not
shown). The UV-generated allele, fa2-2, has a C to T transition at
position 861bp, causing a codon change from glutamine to a stop codon
(Fig. 3A).
|
Northern analysis showed a wild-type transcript of 3.0 kb, which was
not detected in fa2-1 or fa2-3 cells, demonstrating that FA2
is not an essential gene (Fig.
3B). The fa2-4 strain produces a message of
8.7 kb,
consistent with the insertion of NIT1
(Fig. 3B). The UV-generated
mutant, fa2-2, expressed a transcript of the correct size, but at a
lower level than that of wild-type cells. fa2-2 cells rescued for
deflagellation by transformation with the 4.6 kb subclone express wild-type
levels of FA2 message (Fig.
3B).
Patterns of FA2 expression
By northern analysis, we discovered that FA2 mRNA levels increase
approximately 3.5-fold 30 minutes post deflagellation compared with CBL, a
constitutively expressed message (Schloss,
1990), and return to normal expression levels by 60 minutes
(Fig. 3C,D). The peak of
FA2 expression occurs more rapidly than peak expression of the highly
up-regulated tubulin (Fig. 3C)
(Schloss et al., 1984
).
Fa1p is localized to the flagellar transition zone in fa2
mutants
We have previously used isolated, de-membranated, flagellar-basal body
complexes (FBBCs) for the in vitro assay of axonemal microtubule severing
(Lohret et al., 1999).
Therefore, to test whether Fa2p might affect the targeting of proteins to the
axonemal severing complex, the localization of Fa1p to FBBCs was examined in
fa2 cells. Fa1p is essential for axonemal microtubule severing and
localizes to the basal body/flagellar transition region
(Finst et al., 2000
). This
localization appears to be unaltered in the fa2 mutants
(Fig. 4). Therefore, it is
likely that Fa2p is doing something other than facilitate localization of the
axonemal microtubule severing complex to the flagellar transition zone.
|
Some fa2 mutant cells are larger than wild-type cells
We noticed that fa2 cells were larger than wild type
(Fig. 5A). This was quantified
by measuring volumes of wild-type, fa2 mutant and
fa2-2-rescue strains. Two independent examinations of asynchronous
populations of cells confirmed that there is a difference in mean volume of
wild-type (151 µm3) and fa2 mutant cells
(fa2-2, 287 µm3; fa2-3, 257µm3;
fa2-4, 214 µm3). The fa2-2-rescue strain
regained a wild-type cell volume (152 µm3). The size
distribution of mutants and wild-type cells was also strikingly different
(Fig. 5B). This difference in
cell size distribution could reflect faster rates of fa2 cell growth
relative to wild type or a delay in cell cycle progression, providing
fa2 cells more time to grow before cell division. To distinguish
these possibilities, we examined cell growth in synchronized populations.
|
Chlamydomonas uses a multiple fission mechanism of cell division
(Pickett-Heaps, 1975).
Vegetative cells can grow to many times their original size during a prolonged
G1 phase. There is a point during G1, called `commitment', when cells that
have achieved a minimal cell volume will commit to at least one round of cell
division even in the absence of further growth (larger cells will undertake
multiple rounds of division)
(Pickett-Heaps, 1975
;
Spudich and Sager, 1980
).
Subsequently, the cells undergo multiple rounds of rapidly alternating S and M
phases, producing 2, 4, 8 or 16 daughter cells of equal size
(Coleman, 1982
;
Craigie and Cavalier-Smith,
1982
; Donnan and John,
1983
).
In asynchronous populations grown in continuous light, fa2 cells
are larger than wild-type cells (Fig.
5). When these same cultures are placed in the dark for 24 hours
there is no further cell growth, and cells that were larger than commitment
minimum when placed in the dark will divide to produce daughter cells (which
then join the population of cells below the threshold size for division).
After 24 hours in the dark, all cells larger than the commitment threshold are
expected to have divided. Thus the maximum cell size after 24 hours in the
dark should correspond approximately to the commitment threshold. Wild-type
and fa2 mutant cells showed no difference in the maximum cell size
after 24 hours in the dark (Fig.
6A). Furthermore, the size threshold was comparable to that
previously reported for wild-type cells (178 µm3)
(Umen and Goodenough, 2001).
This indicates that fa2 cells are not defective in the coupling of
cell size to commitment and provides an opportunity to assess rates of cell
growth after return to the light. Fig.
6A shows that 10 hours after a shift into the light, both
populations of cells increased in mean volume; fa2 cells did not grow
faster than wild-type cells. In contrast to the disparate size distributions
observed for asynchronous cultures of fa2 and wild-type cells,
populations within the first 10 hours of return to growth conditions show
similar distributions (compare Fig.
5 with Fig. 6A). In
asynchronous populations fa2 cells are on average larger than
wild-type cells, but the cells seem to grow at the same rate and have a
similar size threshold for commitment to divide. These data lead us to predict
that the larger fa2 cells would, on average, undergo more rounds of
fission per division cycle than wild-type cells. As shown in
Fig. 6B, this is what we
observed. In other words, the larger fa2 cells are dividing into more
daughter cells, thus producing a spectrum of cell size comparable to wild type
after 24 hours in the dark, when all cells above the threshold will have
divided. On the basis of these data, we hypothesized that fa2 cells
are slow to transit through the cell cycle. This would provide more time for
growth, allowing the cells to grow larger, followed by an increased number of
fission events, producing daughter cells of wild-type size.
|
fa2 mutant cells are slow to progress through the cell
cycle
In order to compare the rate of progression of fa2 and wildtype
cells through the cell cycle, we measured DNA content and assessed the
division status of cells returned to the light after 24 hours in the dark.
Mitotic figures and the mitotic spindle are difficult to visualize in
Chlamydomonas, thus we choose to use the readily visible cleavage
furrows to identify cells in M phase. Cleavage furrows are first visible in
preprophase and persist through cytokinesis, they therefore provide a good
indication of cells in mitosis (Kirk,
1998).
We sampled cells immediately after return to light (0 hours of continuous light) and then again after 10 hours and 21 hours in continuous light. After 24 hours in the dark, wild-type and fa2 cells have similar distributions of DNA per cell. Ten hours after return to the light, both populations increased their DNA content to the same extent, indicating that S phase is not delayed in the fa2 mutant. By 21 hours in the light, a significant fraction of the wild-type population has undergone cytokinesis and hatched from the mother cell wall, yielding cells of 1N. In contrast, the fa2 cells had not hatched at the 21 hour time point (Fig. 7). Apparently, the fa2 cells take longer to transit from G2 through mitosis and cytokinesis to hatch as individual cells of 1N.
|
A count of cells with cleavage furrows indicates that fa2 cells are slower to enter M phase (Fig. 7E). Fig. 7A and 7C shows that fa2 cells complete S phase in synchrony with wildtype cells, yet Fig. 7E shows that they are slower to enter mitosis. We conclude from this that fa2 mutant cells are delayed at the G2/M transition. But this is clearly not the only point in the cell cycle where fa2 cells are slower than wild-type cells. A comparison of Fig. 7B and 7D illustrates that fa2 cells are also slow to return to 1N. Microscopic examination of these samples revealed that the delay in return to 1N was in large part caused by failure of the daughter cells to hatch from the mother cell wall (daughter cells, held together by the mother cell wall, were counted as single particles by the FACS machine). Flagella-less cells often have difficulty hatching from the mother cell wall; therefore, we digested the mother walls from these cells. We discovered that the daughter cells had not yet assembled flagella (data not shown). We have also observed that in rich (TAP) media, rapidly growing asynchronous populations of fa2 cells accumulate clusters of flagella-less daughter cells. We conclude that fa2 mutant cells are delayed at the G2/M transition and in the assembly of flagella after exit from mitosis.
RNAi of FA2 mimics deflagellation but not cell size profile
of fa2 mutants
RNA interference (RNAi) is a mechanism of gene silencing that has been
shown to be effective in a wide range of organisms
(Smith et al., 2000). One
approach to RNAi involves introduction of a gene construct which, following
transcription in vivo, produces a hairpin dsRNA. Recently, this method has
been shown to be an effective way of reducing the function of a target gene in
Chlamydomonas (Furhmann et al., 2001). Because all of our
fa2 alleles are probably null
(Fig. 3A,B), RNAi was used to
generate strains of Chlamydomonas with reduced expression of
FA2. To generate a DNA construct that would produce a hairpin dsRNA,
exons 1 to 5 of the cDNA were inverted and ligated to
3 kb of the
FA2 genomic fragment representing the same exons
(Fig. 8A). This construct was
cotransformed with the selectable marker Ble into wildtype cells.
Cells that acquired resistance to Zeocin were assayed for a deflagellation
phenotype. We found that 18/121 Zeocinresistant strains were defective for
deflagellation. Specifically, they were defective in calcium-induced axonemal
severing assayed as previously described
(Fig. 8D)
(Finst et al., 2000
). Two of
these strains were examined by northern analysis and both showed two-fold
reductions in levels of FA2 mRNA
(Fig. 8B,C). Surprisingly, the
cell size distribution of the RNAi strains was the same as wild type
(Fig. 8E). From these data we
infer that the deflagellation phenotype is sensitive to FA2
expression levels, whereas the cell size phenotype is less sensitive. However,
when grown asynchronously in rich media, the RNAi cells, like the fa2
mutants, are slow to form flagella and hatch from the mother cell wall. The
high sensitivity of deflagellation to FA2 expression levels is
consistent with the low rates of co-transformation that we achieve in
deflagellation transformation rescue experiments with FA2 (1-3%
compared to 10-15% for FA1)
(Finst et al., 2000
).
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Discussion |
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As we observed previously for the FA1 gene
(Finst et al., 2000) and for
katanin p60 (T. A. Lohret and L.M.Q., unpublished), FA2 mRNA levels
are modestly increased during flagellar regeneration
(Fig. 3C,D). These data suggest
that katanin, Fa1p and Fa2p are components of the flagella or play a role in
the assembly of new flagella. We previously showed that Fa1p is localized to
the basal body/flagellar transition zone
(Finst et al., 2000
), and we
show here that this localization is not disrupted in fa2 mutants
(Fig. 4). This would indicate
that Fa2p is not essential for the localization of Fa1p.
In addition to the axonemal-severing defect, we have discovered that fa2 mutants have subtle cell cycle progression defects. Populations of asynchronously growing fa2 cells have a cell volume distribution that encompasses the wild-type spectrum but also extends to include cells that are almost twice as large as any seen in the wild-type populations (Fig. 5). When the populations are partially synchronized, fa2 cells grow at approximately the same rate as wild-type cells (Fig. 6A), suggesting that fa2 cells are probably larger because they grow for a longer time before completing cell division. We suggest that this may be because of a delay in transit through the cell cycle.
Analyses of DNA content and cell division support the idea that fa2 cells are delayed in transit through at least two points in the cell cycle: (1) the G2/M transition and (2) in the assembly of flagella after exit from mitosis. However, the role of Fa2p at each of these points must be non-essential because fa2 mutant cells do transit the cell cycle, albeit more slowly than wild-type cells. It is possible that other Nek family members in Chlamydomonas compensate for the cell cycle functions of Fa2p in the fa2 mutants. We have identified two related proteins in the Chlamydomonas EST database.
Our discovery that FA2 encodes a Nek kinase is the first
indication that this family might play a role in the regulation of microtubule
severing. It is also possible that the axonemal severing defect of
fa2 mutants is a secondary consequence of defective centrosome/basal
body assembly. During cell division in Chlamydomonas, the flagella
are resorbed and the basal bodies function as centrioles, providing foci for
the spindle poles (for a review, see Kirk,
1998). When division is complete the centrioles reposition as
basal bodies and new flagella are assembled. On the basis of our observation
that flagellar assembly is delayed in fa2 mutants, and the report
that Nek2 facilitates centrosome assembly
(Fry et al., 2000
), we
speculate that Fa2p might play a role in the centriole cycle in
Chlamydomonas. For example, a delay in the differentiation of
centrioles into basal bodies would translate as a delay in flagellar assembly,
which in turn would cause a delay in hatching from the mother cell wall.
Furthermore, the calcium-induced axonemal microtubule-severing defect of
fa2 mutants is associated with mature basal bodies and therefore this
too may be a consequence of defective centriole differentiation. One piece of
evidence that argues against a role for Fa2p in assembly of specific
centrosome/basal-body-associated complexes is the observation that Fa1p, whose
only known function is its essential role in calcium-induced axonemal
microtubule severing, retains its localization to the basal body/transition
zone in fa2 mutant.
Fa2p function, as it relates to both deflagellation and cell cycle
progression, might be directly related to microtubule severing associated with
the basal body/centriole. Microtubule severing has been implicated in cell
cycle progression (for a review, see
Quarmby, 2000), and it is
possible that both the deflagellation and cell cycle phenotypes of
fa2 mutants are related to defects in microtubule severing, directly
or indirectly. Future experiments will discriminate whether the cell cycle
progression defect of fa2 cells is a consequence of a microtubule
severing defect or a hampered centrosomal cycle. Most intriguing is the
possibility that microtubule severing plays a role in the centrosomal
cycle.
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Acknowledgments |
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