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First published online 15 February 2006
doi: 10.1242/dev.02280
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Section of Cell and Developmental Biology, Division of Biological Sciences, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0116, USA.
Author for correspondence (e-mail:
lsmith{at}biomail.ucsd.edu)
Accepted 10 January 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: WAVE/Scar, Arp2/3 complex, HSPC300, BRICK1, Trichomes, Pavement cells
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). In
S. cerevisiae, Arp2/3 complex-dependent actin polymerization drives
the internalization and inward motility of endocytic vesicles
(Engqvist-Goldstein and Drubin,
2003; Chang et al.,
2003). In plants, mutations in genes encoding four different
subunits of a putative ARP2/3 complex (ARP2, ARP3, ARPC2 and ARPC5) cause
epidermal cell shape defects. The most conspicuous defect is a `distorted'
trichome (epidermal hair) shape that has been associated with alterations in
the organization and/or density of F-actin in expanding trichomes
(Le et al., 2003;
Li et al., 2003;
Mathur et al., 2003a;
Mathur et al., 2003b;
El-Assal et al., 2004a;
Saedler et al., 2004).
Activators of the Arp2/3 complex, such as members of the Scar/WAVE family,
greatly increase the efficiency of actin filament nucleation by the Arp2/3
complex in vitro and in vivo (Welch and
Mullins, 2002). In recent years, significant advances have been
made in understanding the regulation of Scar/WAVE activity in animal cells.
WAVE1 and WAVE2 proteins isolated from mammalian cell extracts were both found
in a complex with four additional proteins: PIR121/Sra-1, Nap1, Abi and
HSPC300 (Eden et al., 2002;
Gautreau et al., 2004); WAVE3
has also been shown to associate with Abi and HSPC300 in vivo
(Stovold et al., 2005).
PIR121/Sra-1 and Nap1 have been implicated in responsiveness of the Scar/WAVE
complex to the upstream regulators Rac and Nck, respectively
(Eden et al., 2002). Abi
enhances WAVE activity in vitro (Innocenti
et al., 2004) and also plays a key role in complex assembly
(Gautreau et al., 2004).
Genetic analyses in a variety of animal systems have confirmed crucial roles
for PIR121/Sra-1, Nap1 and Abi in regulation of Scar/WAVE-dependent actin
polymerization in vivo (Blagg and Insall,
2004; Vartiainen and Machesky,
2004).
The significance of HSPC300 in the Scar/WAVE complex is less clear than
that of other subunits. A very small protein that is highly conserved in
multicellular eukaryotes, HSPC300 has been shown to bind directly to WAVE2 in
vitro (Gautreau et al., 2004),
to interact in vivo with the N-terminal Scar homology domains (SHDs) of
Scar/WAVE 1, 2 and 3, and to co-localize with all three WAVE proteins in vivo
(Stovold et al., 2005).
HSPC300 was present in WAVE complexes isolated from HeLa cell extracts, but
unlike all other subunits, the majority of HSPC300 was found in a cytoplasmic
pool not associated with the complex
(Gautreau et al., 2004).
Moreover, mammalian WAVE, PIR121/Sra-1, Nap1 and Abi expressed in insect cells
formed a complex that stimulated Arp2/3-dependent actin polymerization in
vitro, and the activity of this complex was not affected by addition of
HSPC300 (Innocenti et al.,
2004). RNAi-mediated knockdown of HSPC300 in Drosophila
cultured cells resulted in a reduction of cortical F-actin and alterations in
cell morphology that were similar to, but much weaker than those resulting
from RNAi-mediated knockdown of Scar, PIR121/Sra-1, Nap1 and Abi
(Kunda et al., 2003).
Mutations in the maize brick1 gene, which encodes the maize homolog
of HSPC300, cause a complete loss of pavement cell lobe formation, minor
defects in epidermal hair morphology and occasional defects in the
polarization of asymmetrically dividing subsidiary mother cells
(Frank and Smith, 2002;
Gallagher and Smith, 2000).
These defects were associated with loss of localized cortical F-actin
enrichments, pointing to a role for BRK1 in promoting actin polymerization.
However, the relationship of maize BRK1 to the ARP2/3 complex is not
clear.
A family of four proteins distantly related to Scar/WAVE at their N and C
termini (SCAR1-4) has been identified in Arabidopsis
(Deeks et al., 2004;
Brembu et al., 2004).
C-terminal domains of SCAR2, SCAR3 and SCAR4 have been shown to activate the
bovine Arp2/3 complex in vitro, indicating that these are bona fide WAVE/Scar
homologs (Frank et al., 2004;
Basu et al., 2005). Homologs of
the other four mammalian WAVE/Scar complex subunits (Nap1, PIR121/Sra-1, Abi
and HSPC300) are also present in Arabidopsis
(Szymanski, 2005). Like the
corresponding mammalian proteins, the SHDs of Arabidopsis SCARs bind
to BRK1/HSPC300 and an Abi-like protein ABIL1
(Frank et al., 2004;
Basu et al., 2005;
Zhang et al., 2005); binding
interactions have also been observed between SRA1 and NAP1
(Basu et al., 2004;
El-Assal et al., 2004b) and
between SRA1 and ABIL1 (Basu et al.,
2005). Thus, although not yet directly demonstrated, it is very
likely that a complex equivalent to the mammalian Scar/WAVE complex exists in
Arabidopsis. Moreover, genetic evidence strongly supports the
conclusion that this complex plays an essential role in activation of the
ARP2/3 complex in Arabidopsis. `Distorted' trichome mutants
gnarled (grl) and pirogi/klunker
(pir/klk), with phenotypes very similar to those of ARP2/3 complex
subunit mutants, are mutations in the Arabidopsis homologs of Nap-1
and PIR121/Sra-1, respectively (El-Assal
et al., 2004b; Basu et al.,
2004; Deeks et al.,
2004; Brembu et al.,
2004; Zimmermann et al.,
2004a; Li et al.,
2004). In addition, dis3/itb1 mutations disrupting
Arabidopsis SCAR2 produce a milder version of the same phenotype
(Basu et al., 2005;
Zhang et al., 2005).
Furthermore, analyses of double mutants lacking both an ARP2/3 complex subunit
and SCAR2 or NAP1 provide strong genetic evidence that both of these proteins
function in a pathway with the ARP2/3 complex
(Deeks et al., 2004;
Basu et al., 2005).
We present an in vivo functional analysis of Arabidopsis BRK1. Our observations indicate that BRK1 functions in a pathway with the ARP2/3 complex and suggest that BRK1 plays an essential role in its function. Further observations demonstrate a requirement for BRK1 in stabilization of SCAR protein in vivo, providing an explanation for the apparent dependence of the ARP2/3 complex on BRK1.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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)
(http://tilling.fhcrc.org:9366/)
identified two premature stop codon mutations at the locations shown in
Fig. 1A in the BRK1
gene (At2g22640) in ABRC stocks CS86554 and CS93199 (Columbia background;
homozygous for erecta mutation er105). To genotype for these
mutations, PCR products amplified with primers BRK1-01 and BRK1-02 (see
Table 1) were digested with
MseI (CS86554=brk1-1) or Hpy188III
(CS93199=brk1-2). brk1-1 mutants used for the experiments
presented in Figs 6 and
7 had been backcrossed twice to
Columbia and were homozygous wild type at the ERECTA locus.
brk1 mutant plants used for other experiments shown had either not
been backcrossed or backcrossed once to Columbia, but the trichome morphology
phenotype was confirmed to remain after two backcrosses. ABRC stocks
SALK_077920 and SALK_123936 (Alonso et al.,
2003) (Columbia background) with T-DNA insertions in ARP2
(WURM) and ARPC5 (CRK) genes, respectively, were
used for crosses to brk1 mutants and all other experiments without
prior backcrossing. For genotyping of brk1-1;arp2 and
brk1-2;arpc5 double mutants, the ARP2 wild-type allele was
amplified with ARP2-01 and ARP2-02, the arp2 mutant allele was
amplified with ARP2-03 and T-DNA-1, the ARPC5 wild-type allele was
amplified with ARPC5-L and ARPC5-R, and the arpc5 mutant allele was
amplified with ARPC5-R and T-DNA-1. Homozygous mutants in stocks SALK_010045,
SALK_038799 and SALK_106757 (Alonso et al.,
2003) (Columbia background) carrying T-DNA insertions in
ARP3 (DIS1), NAP1 (GRL) and SRA1
(PIR/KLK), respectively, were identified from their previously
characterized phenotypes. FLAGdb/FST line 123F07
(Samson et al., 2002) carrying
a T-DNA insertion in the SCAR1 gene was obtained from
INRA-Versailles. The wild-type SCAR1 allele was amplified with
SCAR1-02 and SCAR1-03, and the mutant scar1 allele with T-DNA-2 and
SCAR-02. Sterilized seeds were plated on 1x MS salts with 0.05%
2-(4-morpholino)-ethane sulfonic acid (MES) and 0.8% agar. For the experiments
presented in Figs 3,
4 and Fig. S2 (in the
supplementary material), 2-week-old seedlings were transplanted to soil and
grown for at least 3 additional weeks. Plants were grown on a 16 hour light/8
hour dark cycle at 20-22°C.
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) with primers BRK1-03 and BRK1-04
(Table 1). Northern blot
analysis was carried out as previously described
(Smith et al., 2001) using an
RT-PCR product amplified with primers BRK1-05 and BRK1-06 as a probe to detect
BRK1 mRNA. To detect ß-tubulin RNA, a 1.2 kb
BamHI/EcoRI fragment of a ß-tubulin cDNA clone isolated
by Marks et al. (Marks et al.,
1987) was used as a probe. Radioactive hybridization signals were
digitized using a Molecular Dynamics Phosphorimager, and signals quantitated
using ImageQuant version 1.2 software.
Production of BRK1p::BRK1-T7 transgenic plants
A fragment comprising the BRK1-coding region minus the stop codon
and 1.4 kb upstream sequence was amplified from Columbia genomic DNA with
primers BRK1-07 and BRK1-08, and cloned into pET28a (Novagen) upstream of the
T7 tag. BRK1p::BRK1-T7 was then amplified from this construct with
primers BRK1-08 and T7 (Table
1), and cloned into pEZRK-LCY (a gift of David Ehrhardt, Carnegie
Institution, Department of Plant Biology), replacing the CaMV 35S promoter and
YFP-coding region with BRK1p::BRK1-T7. The 3'UTR was then
amplified from genomic DNA with primers UTR-01 and UTR-02, and cloned
downstream of the T7 tag. This construct was introduced into Columbia wild
type and brk1-2 mutants via Agrobacterium-mediated
transformation using the floral dip method
(Clough and Bent, 1998).
Visualization and analysis of F-actin
Immunofluorescent labeling of F-actin in expanding trichomes was carried
out on intact 10- to 14-day-old seedlings as described by Zhang et al.
(Zhang et al., 2005), except
that cell wall permeabilization was achieved by incubating in 0.5% Pectolyase
Y23 (ICN Biomedicals) for 80 minutes, and Alexa Fluor 488-conjugated
anti-mouse Ig (Molecular Probes) was used to visualize antibody labeling. Leaf
primordia were excised and mounted in Vectashield (Vector Laboratories) for
confocal microscopy. Fluorescence was excited with a 488 nM line from an argon
laser and visualized using a Nikon TE-200U microscope equipped with a
60x 1.2 NA water immersion objective, a Yokogawa Nipkow spinning disk
confocal head, a Chroma HQ525/50 band pass emission filter, and a Coolsnap HQ
cooled CCD camera controlled by MetaMorph software (Universal Imaging).
For imaging of F-actin in expanding cotyledon pavement cells, 3- to
4-day-old seedlings grown on MS-agar plates were bombarded with 1 mg of 1
µm gold particles (Bio-Rad) coated with 200 ng of 35S-GFP-mTALIN plasmid
described by Fu et al. (Fu et al.,
2001). Particle coating and bombardment using a Bio-Rad PDS-1000
helium biolistic system was carried out according to the manufacturer's
instructions at a helium pressure of 1100 psi. Eighteen to 24 hours later,
cotyledon pavement cells exhibiting intermediate levels of GFP-mTALIN
fluorescence located in the upper two-thirds of the cotyledon (excluding the
extreme edges) with areas between 900 and 8000 µm2 were imaged
by confocal microscopy as described above except that a 20x objective
was used. For quantitative analysis of cortical F-actin distribution, the
linescan tool of Metamorph was used to measure fluorescence intensities around
the entire periphery of each cell and also around the peripheries of emerging
lobes (defined here as areas of the cell surface exhibiting outward
curvature). Linescan data were transferred to Excel files and used to
calculate the proportion of the cell periphery exhibiting fluorescence
intensities in the lowest quarter (`dim'), next to lowest quarter
(`intermediate') and upper half (`bright') of the fluorescence intensity range
for each cell. In addition, the proportion of `bright' peripheral fluorescence
associated with lobes was calculated.
Trichome and pavement cell shape analyses
To visualize trichome shapes, mature rosette leaves were attached to stubs
and imaged without further processing using an FEI Quanta 600 Environmental
Scanning Electron Microscope at a low pressure setting of 133 Pa. For trichome
branch length analysis, rosette leaves were fixed and cleared with chloral
hydrate as described previously (Hamada et
al., 2000) and visualized using DIC optics on a Nikon E600
microscope with a 4x objective. Images were captured using NIH ImageJ
version 1.32j software with a DAGE MTI CCD72 camera coupled to a Scion LG-3
framegrabber. Branch lengths were then measured using ImageJ.
For imaging of cotyledon pavement cell shapes, fully expanded cotyledons
were removed from 12-14 day old seedlings grown aseptically on MS-agar plates.
Cotyledons were mounted under a coverslip in 0.01% Triton X-100 and 10
µg/ml propidium iodide, and adaxial surfaces imaged by confocal microscopy
as described earlier except that a Chroma HQ610/75 band pass emission filter
and 10X objective were used. For each cotyledon analyzed, a set of images was
collected spanning the entire width of the cotyledon along a straight line
perpendicular to the tip-base axis approximately halfway from base to tip, but
excluding the extreme edges where cells are more lobed than they are elsewhere
in the cotyledon. The freehand selections tool in ImageJ version 1.32j was
used measure the perimeter and area of every cell in each image collected. For
cells with areas from 10,000 to 30,000 µm2 (the majority) a
`form factor' was calculated as (area2)/(4xperimeter) - a
unitless number reflecting the degree of convolution of the cell periphery
(Russ, 2002).
SCAR1 protein analysis
Polyclonal chicken antibodies were raised and affinity purified against an
internal fragment of SCAR1 corresponding to amino acids 302-463 at GenWay
Biotech (San Diego, CA). Shoot tips from 3-week-old seedlings were homogenized
in extraction buffer [TBS with 5 mM 2-mercaptoethanol, 0.05% Tween, 10%
sucrose and 1/100 plant protease inhibitor cocktail (Sigma)] using an Omni TH
homogenizer at 4°C. Following centrifugation at 700 g then
16,000 g, 1 µg of anti-SCAR1 was added to the supernatant.
Following incubation at 4°C for 30 minutes with end-over-end rotation, 10
µl of goat anti-chicken IgY antibody-conjugated microbeads (Genway) were
added, and samples were incubated with rotation at 4°C for another hour.
After two washes in extraction buffer, bead-bound proteins were removed by
boiling in SDS loading buffer and electrophoresed on NuPage Novex Bis-Tris
4-12% acrylamide gels (Invitrogen) using MES-SDS running buffer, and
transferred to polyvinyldifluoride membrane (Millipore). Western blotting was
carried out as described by Harlow and Lane
(Harlow and Lane, 1998) using
anti-SCAR1 antibody diluted to 0.1 µg/ml and horseradish
peroxidase-conjugated goat anti-chicken antibody (GenWay) diluted
1:10,000.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), Arabidopsis BRK1 is widely expressed,
suggesting a global function. Two mutations introducing premature stop codons
in the first exon of Arabidopsis BRK1 were identified by TILLING
(Colbert et al., 2001); these
mutations truncate the BRK1 protein at the locations shown in
Fig. 1A. All aspects of the
phenotype reported here were observed in plants homozygous for either of these
mutations. Moreover, a transgene comprising the Arabidopsis BRK1 gene
expressed from its native promoter was able to rescue all the epidermal cell
morphology defects in brk1-2/brk1-2 mutants described below. Thus, we
can conclude that these phenotypes resulted from mutations in the
BRK1 gene.
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;
El-Assal et al.,, 2004b), we
found that sra1 and nap1 trichome branches are longer than
those of brk1, arp2 and arp3 mutants, and are similar to
those of arpc5 mutants (Fig.
3I,J). Thus, with respect to trichome branch length, the phenotype
of brk1 mutants is equivalent to that of the most severe ARP2/3
complex subunit mutants examined, and is more severe than that of other SCAR
complex subunit mutants.
Like trichome branch elongation, tip growth of root hairs and pollen tubes
is well known to depend crucially on actin polymerization
(Hepler et al., 2001).
Nevertheless, tip growth has been reported to be affected very little
(Mathur et al., 2003a;
Mathur et al., 2003b;
Li et al., 2003) or not at all
(e.g. Le et al., 2003;
Brembu et al., 2004;
El-Assal et al., 2004a) in
ARP2/3 complex and SCAR complex subunit mutants. When brk1-2
heterozygotes were self pollinated, the frequency of brk1-2 mutants
in the next generation was found not to be significantly different from 25%
(79 brk mutants/360 progeny, 0.1<P<0.5 by
2 analysis). Moreover, no difference was observed between root
hairs of wild type, brk1-1, arp2, arp3, arpc5, nap1 and sra1
(see Fig. S1 in the supplementary material). Thus, tip growth of both root
hairs and pollen tubes appears to be unaffected in Arabidopsis brk1
mutants, as previously reported for maize brk mutants
(Frank and Smith, 2002;
Frank et al., 2003).
Apparently, other actin nucleators besides the ARP2/3 complex are sufficient
to stimulate the actin polymerization that is essential for tip growth.
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1.4 kb of 5' and 200 bp
of 3' sequence was introduced into the Columbia wild-type background.
Surprisingly, in eight out of 16 independent T2 transgenic lines analyzed,
some plants exhibited trichome morphology defects similar to those of
brk1 loss-of-function mutants
(Fig. 4A, white arrowheads),
although abnormal trichomes were always accompanied by normal trichomes
(Fig. 4A, black arrowhead).
Northern blot analysis showed a minor increase in BRK1 mRNA levels
(endogenous and transgenic combined) in BRK1p::BRK1-T7 plants with
abnormal trichomes compared with wild-type plants
(Fig. 4B), ruling out
co-suppression as the cause of the phenotype. Moreover, our observation that
the same construct was able to complement the phenotypes of brk1-2
homozygous mutants demonstrates that the C-terminal T7 tag does not interfere
with BRK1 function. Together, these data suggest that the occasional
appearance of aberrantly shaped trichomes in wild type plants carrying the
BRK1p::BRK1-T7 transgene is due to slightly elevated levels of
functionally normal BRK1 protein. Presumably, the transgene restores wild-type
trichome morphology in a brk1 mutant background because transgenic
BRK1 protein levels in this background do not significantly exceed the levels
of endogenous BRK1 protein normally produced in wild-type plants.
Similar alterations in F-actin organization are observed in expanding trichomes of Arabidopsis brk1 and arp2 mutants
If BRK1 functions in ARP2/3 complex activation, then brk1 and
ARP2/3 complex subunit mutations would be expected to have similar effects on
the F-actin cytoskeleton. We directly compared F-actin in expanding trichomes
of wild-type plants to that in arp2 and brk1 mutant
trichomes following formaldehyde fixation and immunofluorescent labeling of
F-actin. As previously reported in studies where aldehyde fixation and
phalloidin or antibody labeling were used to visualize F-actin (e.g.
Szymanski et al., 1999), we
observed arrays of cytoplasmic F-actin bundles at all stages of wild type
trichome expansion examined that were of relatively uniform thickness and were
fairly well aligned with each other along the long axes of the trichome
branches (Fig. 5A-C). As
previously shown (Zhang et al.,
2005), close examination of the surfaces of elongating wild-type
trichome branches also revealed a very fine network of cortical F-actin
filaments that are aligned transversely to the axis of the trichome branch,
perpendicular to the overall alignment of cytoplasmic F-actin bundles (gray
arrowheads, Fig. 5D).
In expanding trichomes of both arp2 and brk1-1 mutants,
we observed alterations in F-actin organization similar to those previously
described for arp2 mutants when aldehyde fixation and antibody
labeling were used to visualize F-actin
(Le et al., 2003). At all
stages of arp2 and brk1-1 mutant trichome expansion
examined, actin filament bundles permeated the cytoplasm of branches and
elongating stalks, and no striking reduction was observed in the overall
density of cytoplasmic F-actin compared with wild type
(Fig. 5E-J). Shortly after
initiation of trichome branches, misaligned actin filament bundles were
observed in arp2 (white arrowheads,
Fig. 5E,F) and brk1
mutants (white arrowheads, Fig.
5H,I), although defects in actin organization were subtle at these
early stages. As arp2 and brk1-1 mutant trichomes continued
to expand, alterations in F-actin organization became increasingly pronounced,
such that relatively little overall alignment was observed within the network
of cytoplasmic F-actin bundles by the stage illustrated in
Fig. 5G (arp2) and 5J
(brk1). Thus, an overall loss of alignment within the network of
cytoplasmic F-actin bundles was associated with the swelling of mutant
trichomes. Notably, the fine network of transversely aligned cortical F-actin
observed in wild type was also present in aberrantly expanding trichome
branches of brk1-1 and arp2 mutants (gray arrowheads,
Fig. 5K,L, respectively).
|
;
Li et al., 2003;
Mathur et al., 2003a;
Mathur et al., 2003b;
Brembu et al., 2004).
Therefore, we were interested to determine the effects of Arabidopsis
brk1 mutations on pavement cell lobe formation. Pavement cells of wild
type cotyledons and rosette leaves have irregularly-shaped but well-defined
lobes that interlock with those of neighboring cells
(Fig. 6A and see Fig. S2A in
the supplementary material, respectively). Reduced pavement cell lobing was
observed in brk1 mutant cotyledons, as well as in ARP2/3 complex and
other SCAR complex subunit mutants (Fig.
6 and data not shown). To quantify these phenotypes, we calculated
for each genotype a mean `form factor', which measures the relationship
between cell perimeter and area with higher numbers indicating a higher degree
of lobing (Russ, 2002) (see
Materials and methods for details). As shown in
Fig. 6H, cotyledon pavement
cell form factors were significantly decreased (P<0.01) in
brk1, arp2, arp3, arpc5, sra1 and nap1 mutants relative to
wild type (Fig. 6H). As shown
earlier for trichome branch lengths (Fig.
3I), form factors are reduced less in arpc5 mutants than
in arp2 or arp3 mutants
(Fig. 6H). Moreover, as
described previously for rosette leaves
(Brembu et al., 2004), we found
a greater reduction in pavement cell form factors for nap1 cotyledons
than sra1 cotyledons (Fig.
6H). Although sra1 and nap1 trichome branch
lengths are similar to those of arpc5 mutants
(Fig. 3I), pavement cell form
factors for sra1 and nap1 are comparable with those of the
more severe ARP2/3 complex subunit mutants arp2 and arp3
(Fig. 6H). Interestingly, lobe
formation was reduced significantly more in brk1 mutants than in any
of the other mutants (P<0.01). This phenotype could be clearly
attributed to loss of BRK1 function as it was observed in both
brk1-1 and brk1-2 mutants, and was fully complemented by the
BRK1p::BRK1 transgene (Fig.
6H). Analysis of rosette leaves also showed a significantly
greater reduction (P<0.01) of pavement cell form factors in
brk1 mutants than in arp2 or arpc5 mutants (see
Fig. S2 in the supplementary material).
Examination of cotyledons also revealed occasional intercellular gaps in
brk1 mutants as well as in all other mutants examined
(Table 2, Fig. 6). These gaps were most
often adjacent to stomata (white arrowheads,
Fig. 6C,E,G) but were sometimes
seen in other locations (white arrows, Fig.
6E). Intercellular gaps in cotyledons and other tissues have been
observed previously in ARP2/3 complex and SCAR complex subunit mutants (e.g.
Le et al., 2003;
Mathur et al., 2003a;
Mathur et al., 2003b;
El-Assal et al., 2004a;
El-Assal et al., 2004b;
Basu et al., 2004;
Basu et al., 2005;
Zhang et al., 2005), and have
in most cases been interpreted to reflect a reduction in intercellular
adhesion.
|
|
). In our hands, cortical F-actin in expanding pavement
cells was not consistently well preserved by fixation. Moreover, when all
pavement cells are labeled, it is difficult to distinguish the cortical actin
of one cell from that of its neighbors. Therefore, we visualized actin
filaments in individual, living pavement cells from 4-5 day old cotyledons
transiently expressing GFP fused to an actin binding fragment of mouse talin
following particle bombardment (Fu et al.,
2002; Fu et al.,
2005). GFP-talin labeled F-actin was examined in cotyledon
pavement cells with areas of 900 to 8000 µm2, spanning a
growth phase in which lobes are forming
(Fig. 7). In wild-type cells,
we observed cytoplasmic and subcortical F-actin cables along with fine
cortical F-actin, with areas of bright cortical actin restricted to a
relatively small proportion of the cell surface
(Fig. 7A-C). Quantitative
analysis (see Materials and methods for details) showed that in wild-type
cells, on average, 8% of the cell periphery exhibited `bright'
fluorescence (values in the upper half of the intensity range), 25% exhibited
`intermediate' fluorescence (values from 25 to 50% of the intensity range) and
66% exhibited `dim' fluorescence (values in the bottom 25% of the intensity
range) (Fig. 7G). As previously
described for expanding rosette leaf pavement cells expressing GFP-talin
(Fu et al., 2002;
Fu et al., 2005), areas of the
cell surface with bright cortical F-actin often
(Fig. 7A-C, white arrowheads),
though not always (Fig. 7C,
gray arrowheads), corresponded to sites where lobes appeared to be emerging.
When fluorescence intensities for the entire cell periphery were compared with
those at sites on the cell surface exhibiting outward curvature, 60% of
`bright' cortical F-actin was found to be associated with apparent sites of
lobe outgrowth (Fig. 7G).
Although the majority of lobes did not exhibit bright cortical F-actin, these
may have been lobes that were not actively growing at the time of
observation.
Expanding brk1-1 cotyledon pavement cells were generally less
lobed than wild-type cells of similar size, but their actin cytoskeletons
appeared similar to those of wild-type cells, with no obvious reduction in
F-actin density either in the cytoplasm or cortex
(Fig. 7D-F). However, cortical
F-actin in brk1 cells appeared to be somewhat more broadly
distributed. Quantitative analysis showed that this difference was small but
statistically significant (P<0.05), with areas occupied by bright
and intermediate fluorescence increased to 12% and 30% of the cell periphery,
respectively, and areas occupied by dim fluorescence decreased to 58%
(Fig. 7G). Notably, the
proportion of bright cortical F-actin located at sites of apparent lobe
outgrowth was reduced from 60% in wild type to 38% in brk1 cotyledons
(Fig. 7G). Similar results were
obtained when F-actin in expanding rosette leaf pavement cells was labeled via
transient expression of GFP fused to an actin-binding domain of
Arabidopsis fimbrin (Wang et al.,
2004) (see Fig. S3 in the supplementary material). Thus, similar
to previous findings for arp2 and arp3 mutants
(Li et al., 2003), we found
that cortical F-actin was more broadly distributed and local F-actin
enrichments were less likely to be associated with emerging lobes in both
cotyledon and rosette leaf pavement cells of brk1 mutants compared
with wild type.
Analysis of double mutants indicates that BRK1 functions in a pathway with the ARP2/3 complex
If BRK1 functions to activate the ARP2/3 complex, then brk1
mutations would be expected to have no phenotypic effects in the absence of
ARP2/3 complex function. That is, the phenotypes of brk1;arp2 and
brk1;arpc5 double mutants would be no more severe than those of
arp2, arpc5 and brk1 single mutants. We found the
morphologies of 2-week-old brk1;arp2 and brk1;arpc5 double
mutant seedlings to be very similar to those of brk1, arp2 and
arpc5 single mutants (Fig.
2). Moreover, the appearance and the branch lengths of
brk1;arp2 and brk1;arpc5 double mutant trichomes were not
significantly different from those of the single mutants
(Fig. 3). As described earlier,
pavement cell lobing was reduced more in brk1 mutants than in
arp2 or arpc5 mutants, but the degree of lobing in
brk1;arp2 and brk1;arpc5 double mutants was not
significantly different from that of brk1 single mutants
(Fig. 6F,G,H for cotyledons;
see Fig. S2E,F,G in the supplementary material for rosette leaves). Thus,
analysis of brk1;arp2 and brk1;arpc5 double mutants
indicates that BRK1 acts in a pathway with the ARP2/3 complex.
BRK1 protects SCAR1 from degradation in vivo
In Dictyostelium, cells lacking PIR121/Sra-1 function
(Blagg et al., 2003) as well as
in Drosophila cultured cells with RNAi-mediated inhibition of
PIR121/Sra-1, Nap1 or Abi function (Kunda
et al., 2003; Rogers et al.,
2003), Scar/WAVE protein levels are greatly reduced compared with
wild type. Thus, in addition to the roles these proteins play in complex
assembly and reception of regulatory input, they also protect Scar/WAVE from
degradation in vivo. As shown in Fig.
8 (lane 1, arrowhead), an antibody raised against SCAR1
immunoprecipitates a protein from Arabidopsis seedling extracts that
is close to the expected molecular mass of SCAR1 (92 kDa). This protein
co-migrates with SCAR1 protein produced via in vitro transcription/translation
(not shown), and is undetectable in plants homozygous for a T-DNA insertion in
the SCAR1 gene (lane 2), confirming its identity as SCAR1. As shown
in Fig. 8 (lane 3), levels of
this protein are dramatically reduced in extracts from brk1-1
mutants. Thus, as previously described for other Scar/WAVE complex subunits in
animal cells, BRK1 appears to protect SCAR1 from degradation in vivo.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The
mammalian homolog of BRK1, HSPC300, is a component of a complex regulating the
activity of the Arp2/3 complex activator, Scar/WAVE
(Blagg and Insall, 2004).
Homologs of all components of the mammalian Scar/WAVE complex are present in
Arabidopsis and play essential roles in actin-dependent aspects of
epidermal cell morphogenesis (Szymanski,
2005). Together, these and other findings suggest that BRK1, along
with other components of a SCAR complex, function to stimulate ARP2/3
complex-dependent actin polymerization in plant cells. In this study, we have
taken a genetic approach to investigate this hypothesis, taking advantage of
mutations in the Arabidopsis BRK1 gene and genes encoding ARP2/3
complex and other SCAR complex subunits. Our findings support the conclusion
that BRK1 is essential for activation of the ARP2/3 complex in vivo, and shed
new light on its role.
Consistent with the idea that BRK1 plays an essential role in activation of
the ARP2/3 complex, Arabidopsis brk1 mutants display distorted
trichome shapes, reduced pavement cell lobing, and other phenotypes
characteristic of ARP2/3 complex and other SCAR complex subunit mutants.
Surprisingly, however, lobe formation is reduced more in brk1 mutants
than in any of these other mutants. This finding raises the possibility that
BRK1 may have ARP2/3 complex-independent function(s) that contribute to
pavement cell lobing. Alternatively, it is possible that ARP2/3 complex
function is not completely eliminated in any of the ARP2/3 complex subunit
mutants we analyzed, but is completely eliminated in brk1 mutants.
Indeed, although the arp2, arp3 and arpc5 alleles used in
our study are all RNA null alleles (Le et
al., 2003; Li et al.,
2003), analysis of yeast arp2, arp3 and arpc5
null mutants in S. cerevisiae showed that in some genetic backgrounds
and under some growth conditions, partially assembled Arp2/3 complexes
remained and Arp2/3 complex function was not completely abolished
(Winter et al., 1999). Strong
genetic evidence that BRK1 acts in a pathway with the putative ARP2/3 complex
is provided by the finding that trichome and pavement cell morphology defects
are no more severe in brk1;arp2 and brk1;arpc5 double
mutants than they are in the corresponding single mutants.
|
;
Zhang et al., 2005).
Similarly, HSPC300 binds to WAVE proteins and may also bind directly to Abi
(Gautreau et al., 2004;
Stovold et al., 2005). Thus,
binding of excess BRK1 to Arabidopsis SCARs and/or Abi-like proteins
may make them unavailable for formation of functional SCAR complexes, thereby
reducing the levels of active SCAR. Alternatively, disruption of trichome
morphogenesis by excess BRK1 protein might be due to an ARP2/3
complex-independent function of BRK1, if it has such a function.
Just as brk1 and ARP2/3 complex subunit mutations produce similar
changes in cell morphology, these mutations also have similar effects on the
F-actin cytoskeletons of expanding cells. Similar to previous findings for
ARP2/3 complex and SCAR complex subunit mutants when aldehyde fixation and
phalloidin or antibody labeling were used to visualize F-actin
(Szymanski, 2005), we observed
that aberrant trichome expansion in brk1 and arp2 mutants
was associated with a loss of alignment within the network of cytoplasmic
F-actin cables. Given the well-established role of cytoplasmic F-actin in
intracellular transport of Golgi stacks
(Nebenführ et al., 1999)
and secretory vesicles (at least in tip growing cells)
(Hepler et al., 2001),
aberrant morphogenesis of brk1 and arp2 mutant trichomes
could be due to mistargeting of Golgi and/or secretory vesicles by
disorganized actin cables. Indeed, Golgi motility was shown to be impaired in
expanding trichomes of arpc5 (crk) mutants
(Mathur et al., 2003b).
However, a variety of alternative explanations are also possible
(Szymanski, 2005;
Smith and Oppenheimer, 2005).
Similar to previous results for arp2 and arp3 mutants
(Li et al., 2003), we also
found that in expanding brk1 pavement cells, cortical F-actin was
more broadly distributed and F-actin enrichments were less likely to be
associated with apparent sites of lobe outgrowth. Localized cortical F-actin
polymerization has been proposed to promote lobe outgrowth in both maize and
Arabidopsis pavement cells by an unknown mechanism
(Frank and Smith, 2002;
Fu et al., 2002;
Fu et al., 2005). Thus, the
altered distribution of cortical F-actin observed in expanding brk1
pavement cells could potentially explain the reduced lobe formation seen in
these mutants. Determining the intracellular sites of ARP2/3 complex-dependent
actin polymerization in expanding trichomes and pavement cells would most
probably lead to a better understanding of its role in promoting normal
patterns of epidermal cell expansion.
|
;
Frank et al., 2003) and as the
Arp2/3 complex does in S. cerevisiae and migrating animal cells
(Pollard and Borisy, 2003).
Loss of ARP2/3 complex activity in brk1 mutants may somehow alter the
activity or distribution of other actin nucleators to cause the changes in
F-actin organization/distribution observed in brk1 mutant cells, but
such nucleators remain to be identified. In expanding pavement cells of
Arabidopsis, cortical F-actin is markedly depleted in the absence of
Rho-family GTPases ROP2 and ROP4 (Fu et
al., 2002; Fu et al.,
2005). ROP2 binds to Arabidopsis SRA1 (PIR)
(Basu et al., 2004) and may
contribute to the regulation of the putative SCAR complex. However, another
ROP-binding protein, RIC4, appears to play a more crucial role in mediating
ROP-dependent cortical actin polymerization in expanding pavement cells
(Fu et al., 2005). RIC4 may
itself be a novel actin nucleator, or may activate another nucleator such as a
member of the formin family.
|
;
Rogers et al., 2003;
Kunda et al., 2003).
Presumably, degradation of Scar/WAVE is due to reduced stability of the
complex in the absence of any of these three subunits. Similarly, we found
that SCAR1 protein levels are dramatically reduced in brk1 mutants,
indicating that BRK1 functions to protect SCAR1 from degradation in vivo and
providing further indirect evidence for the existence of a complex containing
both SCAR1 and BRK1. If all four Arabidopsis SCAR proteins are
degraded in brk1 mutants [including SCAR2, which unlike SCAR1 has
been shown to play a role in trichome morphogenesis
(Basu et al., 2005;
Zhang et al., 2005)], this
would explain why BRK1 appears to be essential for ARP2/3 complex activity in
vivo. Indeed, based on our findings, it may be that the only function of BRK1
is stabilization of the putative SCAR complex in vivo. Alternatively, like
PIR121/Sra-1, Nap1 and Abi, BRK1 may have additional functions in regulation
of complex assembly or activity, but further work will be required to
determine what these functions might be. |
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/6/1091/DC1
* Present address: Section of Neurobiology, Division of Biological Sciences,
University of California San Diego, 9500 Gilman Drive, La Jolla, CA
92093-0116, USA 
Present address: Department of Microbiology and Molecular Genetics, College
of Medicine, University of California Irvine, Irvine, CA 92697-4025, USA
Present address: Crop Genetics Research and Development, Pioneer Hi-Bred
International, A Dupont Company, 7300 N.W. 62nd Avenue, Johnston, IA
50131-1004, USA
|
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