1 Agronomy Department, Purdue University, Lilly Hall, 915 West State Street,
West Lafayette, IN 47907-2054, USA
2 Purdue Motility Group, Purdue University, Lilly Hall, 915 West State Street,
West Lafayette, IN 47907-2054, USA
* Author for correspondence (e-mail: dszyman{at}purdue.edu)
Accepted 14 June 2004
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SUMMARY |
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Key words: Trichome, WAVE, ARP2/3, SRA1, PIR121
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Introduction |
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Arabidopsis leaf trichomes are unicellular structures that have
highly constrained and sequential requirements for the microtubule and actin
filament cytoskeletons (Beilstein and
Szymanski, 2003). Unlike tip-growing root hairs
(Baluska et al., 2000
), the
earliest phases of polarized trichome outgrowth, the initiation of stalk
(stage 2) and branch (stage 3) buds, require an intact microtubule
cytoskeleton (Mathur et al.,
1999
; Szymanski et al.,
1999
). Following branch initiation, microtubules remain important
(Szymanski, 2001
), but the
refinement of the branch tip from a hemispherical dome to a fine point (stage
4) defines the first detectable requirement for an intact actin-cytoskeleton
(Szymanski et al., 1999
).
Mutations in ARP2/3 subunit genes block this morphological transition, and
either disorganization of cytoplasmic actin filaments and/or bundles
(Szymanski et al., 1999
;
El-Assal et al., 2004a
;
Le et al., 2003
) or increased
bundling of actin filaments (Li et al.,
2003
; Mathur et al.,
2003a
; Mathur et al.,
2003b
) is believed to cause cell swelling and reduced branch
elongation.
ARP2/3 is a seven protein complex that binds to the sides of existing actin
filaments, and nucleates `daughter' actin filaments
(Amann and Pollard, 2001;
Blanchoin et al., 2000
).
ARP2/3, along with additional actin-binding proteins, can generate dendritic
actin networks or highly organized bundles
(Svitkina et al., 2003
;
Vignjevic et al., 2003
) that
drive plasma membrane protrusion and organelle motility
(Pollard and Beltzner, 2002
;
Schafer, 2002
). In yeasts
(Winter et al., 1999
), flies
(Stevenson et al., 2002
) and
worms (Sawa et al., 2003
),
ARP2/3 subunit mutations cause severe developmental defects and lethality.
Arabidopsis ARP2/3 subunit genes are expressed ubiquitously, and
plants that carry strong loss-of-function mutations in ARP2/3 subunits have a
reduced fresh weight and widespread cell-cell adhesion defects in the shoot
(El-Assal et al., 2004a
;
Le et al., 2003
;
Li et al., 2003
;
Mathur et al., 2003a
;
Mathur et al., 2003b
).
However, ARP2/3 subunit mutants are viable and overall plant architecture is
not affected.
The actin filament nucleation activity of ARP2/3 requires trans-activators
such as the WASP (Wiscott-Aldrich syndrome protein)/WAVE (WASP family
VERPROLIN-homologous protein) family members
(Welch and Mullins, 2002).
WAVE directly interacts with the ARP2/3 complex
(Machesky and Insall, 1998
)
and potently enhances the actin filament nucleation activity of the complex
(Machesky et al., 1999
). The
activity of WASP is regulated by relief of autoinhibition
(Rohatgi et al., 2000
). WAVE
is an intrinsically active ARP2/3 activator, and the primary function of the
pentameric WAVE complex is to regulate the activity of WAVE
(Eden et al., 2002
). Although
the biochemical details of how the WAVE complex regulates ARP2/3-dependent
actin filament nucleation are unresolved
(Blagg and Insall, 2004
),
recent data from several laboratories indicate that the complex controls the
stability and/or localization of the WAVE subunit
(Blagg et al., 2003
;
Innocenti et al., 2004
;
Kunda et al., 2003
;
Steffen et al., 2004
).
Arabidopsis does not have an obvious ortholog of WASP or WAVE, but
WAVE-like activity in Arabidopsis is expected given the presence of
SRA1-, NAP125- and HSPC300-like genes in Arabidopsis.
We provide strong evidence that PIR encodes a homolog of the WAVE
complex subunit that has been named SPECIFICALLY RAC1-ASSOCIATED (SRA1)
(Kobayashi et al., 1998) or
PIR121 (p53-121F-induced)
(Saller et al., 1999
). Both
names are used in the literature, and for simplicity in this paper we will
refer to the non-plant homologs collectively as SRA1. SRA1 assembles into the
WAVE complex via a direct interaction with the NAP125 subunit
(Gautreau et al., 2004
;
Innocenti et al., 2004
). SRA1
directly binds to active Rac1 (Kobayashi
et al., 1998
), and imparts Rac1 responsiveness to the WAVE
complex. SRA1 is required for lamellipodia formation in cultured animal cells
(Kunda et al., 2003
;
Rogers et al., 2003
), and in
vitro activation of Rac1 signaling can cause the relocalization of the fully
assembled WAVE complex to the plasma membrane
(Steffen et al., 2004
).
The `distorted group' of trichome mutants provides a useful genetic system with which to study a pathway of functions from SRA1 activation to ARP2/3-dependent morphogenesis in vivo. PIR encodes a homolog of the WAVE complex subunit SRA1. PIR appears to positively regulate ARP2/3, because loss of function mutations in PIR and ARP2/3 subunits cause a similar array of cell shape and actin cytoskeleton phenotypes. PIR is 30% identical to human SRA1, yet in transformation and pir rescue experiments, the human and plant proteins are functionally interchangeable. Despite the high degree of amino acid sequence divergence between the plant and human SRA1 homologs, their cross-kingdom interactions with RHO GTPases and the Arabidopsis homolog of NAP125 (ATNAP125) were indistinguishable.
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Materials and methods |
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Plasmid construction
To create a full length PIR clone, 3849 bp of PIR-coding
region was amplified from pATPOP140 (a gift from C. Staiger) and cloned into
GatewayTM vector, pENTR/D/TOPO (pEN) (Invitrogen, Carlsbad, CA) to
generate pEN-PIR. To generate a full-length human cDNA, 3762 bp of human
SRA1 was amplified by PCR from a full-length cDNA (GenBank Accession
Number AB032994). The PCR product was cloned into pEN vector to generate
pEN-HSPIR. These and all other PCR-generated clones were sequenced on both
strands prior to use. The Arabidopsis and human pEN clones were
recombined into yeast two-hybrid Gal4 DNA-binding (bait) vector pDEST32 to
generate pDS32PIR and pDS32HSPIR, respectively. pEN-PIR and pEN-HSPIR were
also recombined into the GatewayTM compatible binary vector pGWB2 (a gift
from T. Nagawa, Shiman University, Japan), to generate pB2PIR and pB2HSPIR,
respectively. pB2PIR and pB2HSPIR were transformed in Col and pir-3
backgrounds using floral dip protocol
(Clough and Bent, 1998). The
4193 bp of ATNAP125 and 3387 bp of Human NAP125 were
amplified from full-length cDNA (GenBank Accession Numbers AV554904 and
AB011159, respectively) and cloned into pEN generating pENATNAP and pEN-HSNAP,
respectively. These Arabidopsis and human NAP125 clones were then
recombined into the GatewayTM yeast two-hybrid GAL4-activation domain
(prey) vector pDEST22 to generate pDS22NAP and pDS22HSNAP, respectively. For
protein expression in E. coli, pEN-NAP was recombined to
GatewayTM N-terminal 6x-His-tag destination vector pDEST17 to
generate pDS17NAP. The coding regions of ATROP2 and ATROP8 were PCR amplified
from pGEX-ROP2 and pGEX-ROP8, while coding regions of HsRac1 and HsCdc42 were
amplified from pGEX-Rac1hs and pGEX-Cdc42hs, respectively. These PCR products
were cloned into pEN to generate pEN-ROP2, pEN-ROP8, pEN-HSRAC1 and
pEN-HSCDC42. To generate yeast two-hybrid constructs, pEN-ROP2, pEN-ROP8,
pEN-HSRAC1 and pEN-HSCDC42 were recombined into pDS22 to generate pDS22ROP2,
pDS22ROP8, pDS22HsRAC1 and pDS22HsCDC42, respectively. Site-directed
mutagenesis was carried out on pEN-ROP2 and pEN-ROP8 using the Quick-change
Site-directed mutagenesis kit (Stratagene, La Jolla, CA) to generate the
clones pEN-ROP2G15V, pEN-ROP2T20N, pEN-ROP2D121A, pEN-ROP8G23V, pEN-ROP8T29N
and pEN-ROP8D133A. The corresponding yeast two-hybrid plasmids were
constructed into pDS22 to generate pDS22ROP2G15V, pDS22ROP2T20N,
pDS22ROP2D121A, pDS22ROP8G23V, pDS22ROP8T29N and pDS22ROP8D133A. The PCR
primers used for plasmid constructions were named according to the name of the
final plasmid produced and are listed in Fig. S4 at
http://dev.biologists.org/cgi/content/full/131/17/4345/DC1.
Protein-protein interaction assays
For yeast two-hybrid assays pairs of bait and prey plasmids defined in the
figures were co-transformed into the S. cerevisiae strain Y190 using
the lithium acetate method. The transformants were selected on
Leu-Trp- medium. Two-hybrid interaction was determined
by colony formation on Leu-Trp-His- medium
using the HIS3 reporter gene. For each two-hybrid plasmid, liquid
ß-galactosidase assays were carried out in triplicate from three
independent colonies using standard protocols
(Ausubel et al., 1994). For
pull-down assays, full-length PIR was expressed as an N-terminal GST fusion
protein. pGEXATPOP140 (PIR) was transformed into E. coli
RossettaTM (DE3) strain (Novagen, Madison, WI), induced at OD600=0.6-0.8
and then grown at 15°C for 40 hours. Soluble protein was purified using
GST beads (Sigma, Steinheim, Germany) using standard protocols. To produce
full-length ATNAP125 protein, pDS17NAP construct was transformed in E.
coli RossettaTM (DE3) strain and induced with 100 µM IPTG at
OD600=0.6-0.7 and grown for 4 hours at 37°C. Soluble HISATNAP125 (GRL)
protein was purified with Ni2+ beads (Novagen, Madison, WI) using
standard protocols. Protein concentrations were determined using the Bradford
method. Bead bound GST-PIR (40 nM) was mixed with 100 nM of HIS-ATNAP125 in
binding buffer containing 50 mM HEPES/KOH (pH 7.6), 20 mM KCl and 5 mM
MgCl2. Binding reactions were incubated at 4°C for 2 hours.
Total, bead-bound and unbound fractions were separated by SDS-PAGE, and
transferred to nitrocellulose membranes. HIS-ATNAP125 was detected with
polyclonal anti-NAP125 rabbit antibody (1:1,000), and blots were quantitated
using densitometry. Anti-NAP125 peptide antibodies were made in rabbits using
amino acids 2-14 (CANSRQYYPSQDES) and 1328-1340 (CSRSGPISYKQHN) of ATNAP125 as
antigens. As a control for nonspecific binding of GST-PIR to large HIS-tagged
proteins, 40 nM bead-bound GST-PIR was mixed with 120 nM of HISAT3G and
analyzed as above. HIS-AT3G was detected with a rabbit polyclonal
antibody.
F-actin localization and quantitation
Whole-mounted seedlings were fixed at room temperature and processed for
F-actin localization and image processing as previously described
(Le et al., 2003).
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Results |
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|
|
We were particularly interested in cloning PIR because it mapped
to an interval on chromosome 5 that did not contain an ARP2/3 subunit-like
gene, and therefore could correspond to a gene with regulatory functions.
PIR mapped near KLUNKER
(Schwab et al., 2003), but
reciprocal complementation tests proved that PIR and KLUNKER
were different genes. The mapping interval did contain one gene,
ATSRA1 (AT5G18410), which shared a high degree of amino acid sequence
identity with the SRA1 subunit of the WAVE complex
(Fig. 2A). To determine if
PIR corresponded to this gene, we assayed the trichome phenotype of
plants that were homozygous for the SALK_106757 T-DNA insertion
(Alonso et al., 2003
). The
insertion was located in the first intron of the 5' UTR of
ATSRA1 (Fig. 2B), and
lines that were homozygous for the insertion displayed a clear distorted
trichome phenotype. Complementation tests indicated that SALK_106757 was a
pir allele. To confirm the identity of PIR, we sequenced the
ATSRA1 gene in each of the four pir backgrounds. In each
case we found point mutations and deletions that affected ATSRA1
coding (Fig. 2B, Fig. S1 at
http://dev.biologists.org/cgi/content/full/131/17/4345/DC1).
The pir-3 fast neutron allele was a null because we failed to detect
an ATSRA1 transcript after two successive rounds of RT-PCR. The
pir-3 RNA sample was intact because the control gene GAPC
was detected easily in both rounds of RT-PCR (data not shown). As final proof
of gene identity, we transformed pir-3 plants with a transgene that
used the viral 35S promoter to overexpress the full-length PIR cDNA.
All 12 primary transformants had trichome shape
(Fig. 2C) and cotyledon
pavement cell-adhesion phenotypes (data not shown) that were indistinguishable
from the wild type. For the rest of this manuscript we will refer to
ATSRA1 as PIR.
|
Based on the animal literature, Arabidopsis PIR is expected to
function as a regulator of the ARP2/3 complex. As an initial test of this
idea, we wanted to determine if PIR, ATNAP125 and ARP2/3 subunit-like
genes had similar levels and patterns of gene expression. Homologs of each of
the seven ARP2/3 subunits share a similar level of expression throughout the
plant, with some differences among subunit-like genes such as DIS2
(ARPC2) and ATARPC4, in their relative expression levels in
leaves and stems (El-Assal et al.,
2004a; Le et al.,
2003
; Li et al.,
2003
). PIR, ATNAP125 and DIS2 (ARPC2)
were expressed at similar levels in the major organs, several of which lacked
trichomes (Fig. 3).
|
|
If PIR and human SRA1 functions are truly interchangeable, one would expect
to detect conserved interactions with RHO-family GTPases. SRA1 interacts
specifically with the small GTPase Rac1
(Kobayashi et al., 1998), and
we were able to use the two-hybrid assay to confirm this interaction
(Fig. 5A). We next tested the
ability of PIR to interact with Arabidopsis RHO-family GTPases.
Arabidopsis encodes 11 RHO-family GTPases that are members of a plant
specific family of small GTPases termed ROPs (Rho of plants)
(Christensen et al., 2003
;
Vernoud et al., 2003
).
ATROP2 is expressed in leaves and overexpression of dominant mutant
forms of this isoform causes epidermal shape defects
(Fu et al., 2002
). Therefore,
we tested whether or not Arabidopsis PIR could interact with ATROP2,
ATROP8 as a control, human RAC1 and human CDC42 in a two-hybrid assay. In
these experiments, each of the small GTPases were expressed at similar levels
and did not activate the HIS3 or ß-Gal reporter genes when
tested in isolation (data not shown). However, when ATROP2 was co-expressed
with PIR a strong interaction was detected
(Fig. 5A). No interactions were
detected between PIR and ATROP8 or human CDC42. In another set of
inter-kingdom two-hybrid assays, PIR displayed a strong interaction with RAC1
and human SRA1 interacted with ATROP2 (Fig.
5A).
|
Depending on the cell type, loss of SRA1 function can lead to
either increased levels of actin filaments and gain-of-function actin-based
phenotypes (Blagg et al.,
2003), or to reduced amounts of cortical actin at the leading edge
of migrating cells and a failure to generate normal lamellipodia
(Kunda et al., 2003
;
Rogers et al., 2003
).
Therefore, we wanted to determine if the actin cytoskeleton in stage 4
pir trichomes differed from that of the wild-type and dis2
(aprc2). Because changes in branch morphology during stage 4 is an early
and obvious indicator of actin and ARP2/3 function, we concentrated our actin
localization efforts on this stage of development. Stage 4 wild-type
(Fig. 6A), dis2
(aprc2) (Fig.
6D) and pir (Fig.
6G) trichomes had an intricate, interconnected actin cytoskeleton
that was present throughout the stalks and developing branches. Wild-type
(Fig. 6B), dis2
(Fig. 6E) and pir
(Fig. 6H) stage 4 branches have
a dense reticulate network of cortical actin filaments. Given the complexity
of cortical actin organization and the lack of a specific marker for ARP2/3
subunits, we have not detected clear phenotypes in this region of the cell.
Wild-type stage 4 trichomes contain a dense population of core bundles, and in
all observations (n=10), many of the core bundles terminated within 1
µm of the apical plasma membrane. Fig.
6C contains a representative image. Fifty-three percent of
pir early stage 4 branches (n=15) contained a dense
population of core cytoplasmic bundles that were aligned with the long axis of
the branch (Fig. 6I). In 29% of
the stage 4 pir branches analyzed (n=15), the bundles
terminated at least 2 µm distal to the apical plasma membrane
(Fig. 6I). At the same time,
the apical surface of the pir branches tended to be prematurely
refined to a sharp tip. Therefore, we do not know the cause-and-effect
relationships between the core bundles and the branch tip morphology in
pir trichomes. Ninety-one percent (n=11) of dis2
trichomes lacked a population of core bundles
(Fig. 6F).
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Discussion |
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The only known function of vertebrate SRA1 is to regulate ARP2/3-dependent cell motility. Several facts support the idea that PIR and ARP2/3 subunit genes have related functions. First, mutation of PIR and ARP2/3 subunit-like genes causes identical trichome distortion, epidermal cell-cell adhesion and reduced fresh weight phenotypes. Second, although the actin bundle phenotypes of pir and ARP2/3 subunit mutants are distinct at early stages, in both mutant classes there is a failure to maintain core actin bundles in elongating branches (Figs 6, 7). Third, PIR interacts with ATNAP125 and active forms of ATROP2 (Figs 4, 5); these binding interactions are expected for a SRA1 family member. Fourth, overexpression of human SRA1 completely rescues the pir trichome phenotypes. The simplest explanation of the pir rescue result is that Arabidopsis and human SRA1 function interchangeably by linking small GTPase inputs to altered ARP2/3 activity. Last, PIR, ATNAP125 and ARP2/3 subunit-like genes such as DIS2 (ARPC2A) are expressed at similar relative levels in all of the major organs tested (Fig. 3).
If PIR directly regulates Arabidopsis ARP2/3, then the
similar cell shape and actin phenotypes of PIR and ARP2/3 subunit
mutants suggest that PIR positively regulates ARP2/3. These results
are consistent with those obtained by using RNA interference of SRA1
in cultured insect cells (Kunda et al.,
2003; Rogers et al.,
2003
). A positive role for SRA1 in signaling to ARP2/3 is also
suggested by the ability of activated Rac1 to relocalize fully assembled and
active WAVE complex to the leading of stimulated cells
(Innocenti et al., 2004
;
Steffen et al., 2004
). Other
biochemical (Eden et al.,
2002
) and genetic (Blagg et
al., 2003
) data suggest that SRA1 negatively regulates ARP2/3. The
reasons for these discrepancies are not known, but they could, in part, be
explained if cell types differ, either with respect to functional redundancy
at the level of F-actin nucleation, or in their ability to accumulate or
localize WAVE-like proteins in a SRA1-independent manner.
Although plant and human SRA1 homologs are functionally
interchangeable, plant development is less sensitive to removal of its
function. The subtle PIR-null phenotypes differ greatly from the
embryonic lethality and severe morphological defects that are caused by null
SRA1 alleles in Drosophila
(Schenck et al., 2001) and
C. elegans (Soto et al.,
2002
). Mutations in several Arabidopsis ARP2/3
subunit-like genes have similar mild-effects on whole plant development. These
results suggest that ARP2/3-dependent nucleation has important but
non-essential functions in Arabidopsis. It is generally accepted that
plants cells use turgor force to push the plasma membrane against the rigid
cell wall and drive cell expansion: therefore, the mechanical energy of
ARP2/3-dependent nucleation may be used for other functions (see
Vidali and Hepler, 2001
;
Wasteneys and Galway, 2003
).
In plant cells, the dynamics of the tonoplast membrane is cytochalasin
D-sensitive (Uemura et al.,
2002
); in S. cerevisiae, vacuole biogenesis is
ARP2/3-dependent (Eitzen et al.,
2002
). The vacuole morphology
(Mathur et al., 2003a
) and
positioning defects in ARP2/3 subunit mutants
(El-Assal et al., 2004a
;
Le et al., 2003
) and
pir (Fig. 6J and
Fig. 7C) imply a role for
Arabidopsis ARP2/3 in vacuole-based functions. More-detailed
information on ARP2/3 function in the context of membrane trafficking and
organelle motility is needed to define the cellular function of ARP2/3 in
plants and to understand why there is so much variability in the extent to
which different cell types rely on ARP2/3 function. For example, is the
apparent unimportance of PIR and ARP2/3 for the tip growth of pollen
tubes, which has a strict actin-dependence
(Gibbon et al., 1999
), owing
to cell-type specific strategies for actin-dependent growth or to differences
in the extent to which pathways leading to actin filament nucleation are
functionally redundant? Perhaps different plant cell types use combinations of
FORMIN (Cvrckova, 2000
;
Deeks et al., 2002
) and ARP2/3
activities to fine tune actin filament nucleation and morphogenesis.
At a molecular level, the protein-protein interactions of the human and
plant SRA1 homologs are remarkably similar. In non-plant cells, the molecular
function of SRA1 is to link Rac1 signaling with WAVE-dependent regulation of
ARP2/3 (Steffen et al., 2004).
PIR interacts with RHO-family GTPases, and binds to ATROP2 with some degree of
isoform and nucleotide selectivity (Fig.
5A,B). NAP125 is an essential subunit of the WAVE complex
(Bogdan and Klambt, 2003
) that
directly interacts with SRA1 (Soto et al.,
2002
). Using full-length recombinant proteins, we detected a
direct interaction between PIR and ATNAP125 in both the yeast two-hybrid and
GST-pull down assays (Fig. 4).
Therefore PIR contains the functional domains that are known to mediate WAVE
complex signaling. Small N- or C-terminal truncations of C. elegans
GEX-2 (SRA1) eliminate its ability to bind GEX-3 (NAP125)
(Soto et al., 2002
). If the
PIR-ATNAP125 interaction is similarly constrained, the similar phenotypes of
the pir null alleles and those that encode C-terminal truncations
(Fig. 2B) could reflect the
inability of any of the mutant proteins to bind ATNAP125.
The interchangeable RHO-GTPase binding and in vivo functions of
Arabidopsis and human SRA1 homologs suggest that during multicellular
development there have been extreme structural constraints on this particular
RHO-GTPase switch. There are substantial in vivo data that support a
Rac1-dependent pathway of SRA1 signaling to ARP2/3. RNA interference of three
Rac1-like functions in cultured insect cells cause phenotypes that are
identical to those of SRA1 loss of function
(Kunda et al., 2003).
Activation of Rac1-signaling pathways in cultured cells relocalizes SRA1 and
other WAVE complex subunits (Steffen et
al., 2004
) to locations in the cell that are defined by
ARP2/3-dependent nucleation (Svitkina and
Borisy, 1999
). A similar regulatory pathway in
Arabidopsis may include PIR as an ATROP2 effector. PIR specifically
binds ATROP2 both with isoform specificity and with selectivity for active
forms of the small GTPase (Fig.
5). Some degree of redundancy at the level of PIR-ROP interaction
is expected in Arabidopsis, because overexpression of
dominant-negative or constitutively active forms of ATROP2 within the strong
viral 355-promoter does not cause trichome distortion
(Fu et al., 2002
).
The protein-protein interaction data and the interchangeable functions of
human and Arabidopsis SRA1 homologs are consistent with the idea that
PIR encodes a subunit of the WAVE complex. However, the composition
of the putative Arabidopsis WAVE complex is unknown. Of the five
known WAVE subunits (Eden et al.,
2002), only SRA1-, NAP125- and HSPC300-like genes are easily
identified in sequence databases. With respect to ATNAP125, we recently
learned the `distorted group' gene GNARLED corresponds to this gene,
and that human NAP125 functions interchangeably with the plant homolog
(El-Assal et al., 2004b
).
Arabidopsis encodes a homolog of human HSPC300 that we termed
ATBRK1. ATBRK1 is expressed in Arabidopsis, but has no known
function. In maize, BRK1 is required for the crenulation of leaf
epidermal cells (Frank and Smith,
2002
). The plant protein sequence database does not encode an
obvious ortholog of the ARP2/3 activator WAVE or the WAVE complex subunit ABI2
(ABL-Interaction 2) (Eden et al.,
2002
). The N-terminal domains of WAVE and ABI2 may mediate
assembly of both subunits into the WAVE complex
(Innocenti et al., 2004
).
Arabidopsis encodes small gene families with homology that is
restricted to the corresponding N-terminal domains of WAVE and ABI2 (D.B.S.,
unpublished). Arabidopsis WAVE- and ABI2-like subunits may have
retained the domains needed for the assembly of an analogous plant
complex.
Conclusion
The `distorted group' of trichome mutants provide a powerful genetic entry
point into signaling to ARP2/3, morphogenesis and multi-cellular development.
Specifically, the apex of stage 4 branches will be a useful experimental
testing ground to understand how cortical microtubules and actin filaments
control the dynamics of cytoplasmic organization and polarized growth. The
`distorted group' appears to define a class of genes that specifically affect
the activity and assembly of ARP2/3. Given the diversity of the cell shape and
adhesion defects of the distorted mutants and the known functions of ARP2/3 in
membrane protrusion, motility and endocytosis, we expect `distorted group'
genes to have multiple functions in the cell. It has been proposed that
Arabidopsis ARP2/3 generates actin filament networks that are
required for organelle and vesicle transport
(Li et al., 2003;
Mathur et al., 2003a
;
Mathur et al., 2003b
). In our
hands, the clearest actin phenotype of the ARP2/3 subunit mutants is the
failure to populate the branch cytoplasm with aligned actin filaments and
bundles. In this context, potential functions of PIR, ARP2/3 and core actin
bundles include membrane trafficking to the vacuole and/or the physical
interactions with expanding central vacuole. Actin filament and/or bundle
interactions with the vacuole may serve the purpose of mechanically excluding
the expanding central vacuole from regions of the cell that grow persistently
or to alter the dynamics of the tonoplast. If true, one expects the apical
dome of stage 4 branches to be a busy site for ROP signaling, PIR-dependent
activation of ARP2/3 and the generation of aligned actin bundles. It will be
interesting to find out how directly the rules of PIR signaling and
morphogenesis transfer from trichomes to other cell types and species.
Note added in proof
While this work was under review, related work on the cloning of
GNARLED was published online (2 July 2004)
(El-Assal et al., 2004b).
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ACKNOWLEDGMENTS |
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Footnotes |
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