Download Mastoparan Alters Subcellular Distribution of

Plant Cell Physiol. 42(9): 912–922 (2001)
JSPP © 2001
Mastoparan Alters Subcellular Distribution of Profilin and Remodels F-Actin
Cytoskeleton in Cells of Maize Root Apices
František Baluška 1, 2, 4, Matthias von Witsch 1, 5, Mechthild Peters 1, Andrej Hlava-ka 3 and Dieter Volkmann 1
1
Botanisches Institut, Rheinische Friedrich-Wilhelms-Universität Bonn, Department of Plant Cell Biology, Kirschallee 1, D-53115 Bonn, Germany
Institute of Botany, Slovak Academy of Sciences, Dúbravská cesta 14, SK-84223 Bratislava, Slovakia
3
Department of Plant Physiology, Comenius University, Mlynská dolina, SK-84512, Bratislava, Slovakia
2
;
Staiger et al. 1997, Gibbon and Staiger 2000). Profilin is
enriched at peripheral cytoplasmic domains of animal cells
which have dynamic AFs that rapidly respond to diverse
signals (Buß et al. 1992, Mayboroda et al. 1997). Moreover,
birch pollen profilin co-localizes with dynamic AFs when
expressed in animal cells (Mayboroda et al. 1997). This suggests that plant and animal profilins obey the same principles
responsible for their subcellular localizations (for animal cells
see, e.g., Hartwig et al. 1989, Buß et al. 1992, Bubb et al.
1998). Importantly, recent genetic studies have revealed that
profilin plays critical roles for basic processes of plant development (Ramachandran et al. 2000, McKinney et al. 2001).
Profilins from non-plant systems are well known to interact with several classes of proteins that contain long continuous stretches of proline residues (Reinhard et al. 1995, Kay et
al. 2000). These interactions are implicated in allowing profilins to be effectively targeted to distinct subcellular domains
(Kang et al. 1997). In addition, data from animal cells show
that profilin can be sequestered from the G-actin-associated
cytoplasmic pool via interactions with phospholipid phosphatidylinositol-4,5-bisphosphate (PIP2) (e.g. Lassing and Lindberg
1985). For yeast cells, it was shown that metabolism of PIP2
plays a crucial role in subcellular localization of profilin
(Ostrander et al. 1995). Since profilin inhibits the diesteratic
cleavage of PIP2 by phospholipase C (for plant cells see
Drøbak et al. 1994), profilin–PIP2 interactions are relevant for
signal transduction cascades implicated in regulation of intracellular cytoplasmic calcium levels (Berridge and Irvine 1989,
for plant cells see Munnik et al. 1998). Interestingly in this
respect, both PI 4-kinase and PI 3-kinase can associate with the
actin cytoskeleton (Xu et al. 1992, Dove et al. 1994, Stevenson
et al. 1998). On the other hand, profilin can stimulate PI 3kinase activity (Singh et al. 1996).
In the present study, we have investigated subcellular distributions of profilin and PIP2 in cells of maize root apices.
Roots are suitable for such a study as their cells are, in contrast
to other plant organs, rich in transcripts for phosphatidylinositol4-phosphate 5-kinase (Mikami et al. 1998), an enzyme which
phosphorylates phosphatidylinositol-4-phosphate to produce
PIP2. We report that maize profilins loosely co-distribute with
PIP2 at cell peripheries as well as in the cytoplasm. Mastoparan, a potent stimulator of PIP2 hydrolysis (for plants see
Indirect immunofluorescence localization of profilin in
cells of maize root apices revealed that this abundant protein was present both in the cytoplasm and within nuclei.
Nucleo-cytoplasmic partitioning of profilin exhibits tissuespecific and developmental features. Mastoparan-mediated
activation of heterotrimeric G-proteins, presumably
through triggering a phosphoinositide-signaling pathway
based on phosphatidylinositol-4,5-bisphosphate (PIP2),
induced relocalization of profilin from nuclei into the cytoplasm of root apex cells. In contrast, PIP2 accumulated
within nuclei of mastoparan-treated root cells. Intriguingly, cytoplasmic accumulation of profilin was associated
with remodeling of F-actin arrays in root apex cells. Specifically, dense F-actin networks were dismantled and distinct
actin patches became associated with the periphery of small
vacuoles. On the other hand, disruption of F-actin with the
G-actin sequestering agent latrunculin B does not affect the
subcellular distribution of profilin or PIP2. These data suggest that nuclear profilin can mediate a stimulus–response
action on the actin cytoskeleton which is somehow linked to
a phosphoinositide-signaling cascade.
Key words: Actin — Maize root — Nucleus — Phosphoinositide signaling — Profilin.
Abbreviations: AFs, Actin filaments; PA, Phosphatidic acid; PI,
Phosphoinositide; PIP2, Phosphatidylinositol-4,5-bisphosphate; PLP,
Poly-L-proline; PRA, Profilin rabbit antibody; ZmPRO3, Zea mays
profilin isoform 3.
Introduction
Profilins are small, ubiquitous and abundant soluble proteins that affect the dynamic behavior of actin filaments (AFs)
in cells of all eukaryotes (e.g. Schlüter et al. 1997, Staiger et al.
1997, Gibbon and Staiger 2000, Staiger 2000). Two contrasting activities of profilin result from its ability to both promote
or inhibit the assembly of AFs depending on the ratio of profilin to G-actin, ionic conditions and on presence/activities of
other actin-binding proteins (Pantaloni and Carlier 1993,
4
5
Corresponding author: Email, [email protected]; Fax, +49-228-739004.
Present address: Purdue University, Department of Biological Sciences, West Lafayette, IN 47907-1392, U.S.A.
912
Mastoparan redistributes nuclear profilin
913
Drøbak and Watkins 1994, Cho et al. 1995, Franklin-Tong et al.
1996, den Hartog et al. 2001), induces relocalization of maize
profilin from the nucleoplasm into cytoplasm. This profilin
redistribution occurs concomitantly with the remodeling of the
F-actin networks in the cytoplasm. On the other hand, disruption of AFs by latrunculin B (for higher plants see e.g. Gibbon
et al. 1999, Baluška et al. 2001a), which transforms the actin
cytoskeleton into high affinity G-actin–latrunculin B complexes (Ayscough et al. 1997, Gibbon et al. 1999), does not
affect subcellular distributions of profilin and PIP2.
Results
Western blots
Western blot analysis (Fig. 1) was accomplished with two
different profilin antibodies: one antibody was raised against
recombinant maize isoform ZmPRO3 (Fig. 1, lanes 1–4) while
another one, called here PRA, was raised against native maize
pollen profilin (Fig. 1, lanes 5–8). Both these profilin antibodies recognized profilins as distinct bands at about 14 kDa in the
soluble protein fraction (Fig. 1, lanes 1, 3, 5 and 7). The respective pre-immune sera did not react with these 14 kDa species
but did faintly recognize several minor protein species of
higher Mr (data not shown). Competitive blocking of both antibodies, using a mixture of recombinant profilins, efficiently
depleted the 14 kDa bands (Fig. 1, lanes 2, 4, 6 and 8) while
the faint non-specific bands were still visible (data not shown).
Mastoparan-treated roots invariably showed stronger profilin
signal than control roots (Fig. 1, compare lanes 1 and 5 for control roots with lanes 3 and 7 for mastoparan-treated roots).
Immunofluorescence of profilin in control and mastoparantreated root apices
Polyclonal rabbit antibodies PRA and anti-ZmPRO3
showed diffuse and punctate labeling of the cytoplasm which
was typically complemented by distinct enrichment of profilin
within nuclei and at the cell periphery (for PRA see Fig. 2A–L,
for anti-ZmPRO3 see Fig. 2M–P). Importantly, using PRA preimmune serum (not shown), as well as immunodepletion of
both PRA (Fig. 2G) and anti-ZmPRO3 (Fig. 4C) antibodies,
resulted in no visible signals when the same exposure times
were used. The cell periphery-associated labeling is most
prominent in epidermis cells located in the distal part of meristem (Fig. 2A) and in the transition zone (Fig. 2C), as well as
metaxylem cells in the transition zone (Fig. 2H). Epidermis
cells in the basal part of meristem accumulate profilins in
nuclei and only faint signal remains associated with the cell
periphery (see arrow in Fig. 2B). In the cortex, nuclear labeling predominated both in the basal (Fig. 2B) and apical (Fig.
2I, M) parts of meristem. But in the transition zone, some profilin-depleted nuclei (about 15% in the cortex and epidermis)
occurred (white star in Fig. 2F).
Nuclei of epidermal cells near the root cap junction are
only faintly stained (Fig. 2A) when compared with nuclei of
Fig. 1 Immunoblots of maize profilin antibodies anti-ZmPRO3
(lanes 1–4) and PRA (lanes 5–8) on soluble extracts from control
(lanes 1, 2, 5 and 6) and mastoparan-treated (lanes 3, 4, 7 and 8) root
apices. Both antibodies recognized profilins as distinct bands at about
14 kDa in the soluble protein fraction (lanes 1, 3, 5 and 7). Competitive blocking of both antibodies, using a mixture of recombinant profilins (for details see Materials and methods), efficiently depleted the
14 kDa bands (lanes 2, 4, 6 and 8).
developmentally more advanced epidermis cells (Fig. 2C). The
unique staining of the epidermis, when compared with other
root tissues, was more evident in the elongation region (Fig.
2D, E). Here, specialized epidermal cells transform from diffusely growing cells into root hair initiating trichoblasts with
growth (exocytosis) restricted to distinct domains (open stars in
Fig. 2D, E). These domains ultimately transform into tips of
emerging root hairs (not shown). Accumulation of profilin
within the outgrowing bulge is typically associated with progressive depletion of nuclear profilins (black star in Fig. 2D)
and profilin-positive nuclei were never found in trichoblasts
with profilin “caps” at bulging domains. Similarly, about 10%
of nuclei in the root cortex are depleted in profilins (for PRA
antibody see white star in Fig. 2F; the same is true for the
ZmPRO3 antibody, data not shown). This subcellular distribution pattern of PRA-positive profilin was confirmed using a
polyclonal antibody raised against recombinant maize profilin
ZmPRO3 (not shown). Generally, we can conclude that antiZmPRO3 labeling patterns are almost identical to those
obtained with the PRA antibody.
Mastoparan proved to be very effective in altering the subcellular distributions of profilin. In mastoparan treated root apices, intranuclear profilin labelings were depleted and both PRA
(Fig. 2J–L) and anti-ZmPRO3 (Fig. 2N–P) reactive profilin
accumulated in the cytoplasm. This feature was more prominent in cortical cells of the transition zone (Fig. 2K, O) and the
apical part of the elongation region (Fig. 2L, P) than in cortical
cells of the meristem (Fig. 2J, N; for appropriate control cortex
cells see Fig. 2I, M). Mastoparan analog M17 was also effective in this respect (not shown) suggesting that its biological
activity is high for maize roots (for soybean suspension cells
see Schroeder-Taylor and Low 1997).
914
Mastoparan redistributes nuclear profilin
Mastoparan redistributes nuclear profilin
Immunofluorescence localization of PIP2
A commercially available monospecific PIP2 antibody
proved to be highly reactive in maize root cells. The strongest
signal was in meristematic cells having the whole cytoplasm
labeled while nuclei showed almost no signal (Fig. 3A).
Whereas the addition of PIP2 to the antibody inhibited its
immunofluorescence reactivity almost completely (Fig. 3B),
the addition of PA was without any discernible effect (Fig. 3C).
In cortex cells of the transition zone (Fig. 3D) and elongation region (Fig. 3E, F), the most prominent signal was associated with cellular peripheries in a pattern that strongly resembled the plasma membrane-associated profilin labelings. To
substantiate the plasma membrane association of PIP2, we have
applied a monoclonal antibody raised against the maize plasma
membrane H+-ATPase (Jahn et al. 1998). This labeled cellular
peripheries (Fig. 3J) in a fashion similar to the PIP2 antibody
(compare Fig. 3E, F). The nuclear envelopes with associated
cytoplasmic strands (Fig. 3E), as well as nucleoplasm of some
(10%) cortical cells in the transition zone (not shown), were
also PIP2-positive. In order to further assess the punctate cell
periphery labelings, presumably associated with the plasma
membrane, we inspected paradermal sections encompassing
the whole plasma membranes within root sections. Such
images showed that plasma membrane-associated PIP2 is
organized in the form of dots and patches (Fig. 3G, H). In mastoparan treated root apices, anti-PIP2 labeling was depleted in
the cytoplasm and increased within nuclei of transition zone
cells of the root cortex (Fig. 3I).
Mastoparan alters F-actin arrangements
While latrunculin B-mediated disintegration of AFs (Fig.
4F, compare with Fig. 4D, E) had no discernible effects on
either profilin (Fig. 4A) or PIP2 distributions (Fig. 4B), 2 h of
mastoparan treatment altered F-actin distributions in most root
apex cells. In cells of control maize roots, AFs are organized in
the form of networks radiating from nuclear envelopes towards
cellular peripheries (Fig. 4E shows interphase cells of the cortex) or F-actin cables organized between actin-enriched crosswalls (Fig. 4D shows post-mitotic cells of the outer stele and
915
pericycle; for more details on F-actin organization in cells of
maize root apices see Baluška et al. 1997a). Mastoparan dismantled both F-actin networks and bundles in cortical cells of
maize root apices (Fig. 4G corresponds to Fig. 4D) which
transformed into distinct patches associated with peripheries of
small vacuoles (Fig. 4H–J). On the other hand, latrunculin Binduced depolymerization of F-actin was associated with accumulation of actin fluorescence within nuclei (black stars in Fig.
4F).
Discussion
Our study documents that maize root apices are suitable
model object to study basic principles of phosphoinositide (PI)
signaling pathways in plants (for other suitable plant systems
see Munnik et al. 1998, Drøbak et al. 1996) and their putative
links with the actin cytoskeleton (Volkmann and Baluška 1999,
Staiger 2000). Our immunofluorescence data document that, in
addition to the diffuse cytoplasmic labeling, profilin localizes
to distinct subcellular domains distributed at cellular peripheries, cytoplasmic strands and within nuclei of maize root cells.
In this respect, our findings are fundamentally different from
previous profilin immunolocalization studies in plant cells
which, however, were done almost exclusively on in vitro
germinating pollen grains and on tip-growing pollen tubes
(Grote et al. 1995, Mittermann et al. 1996, Hess and Valenta
1997, Vidali and Hepler 1997). The only study on subcellular
distribution of plant profilin in non-pollen cells is that of Vidali
et al. (1995) in which tissue-specific distributions of profilin
are described in control and Rhizobium-infected roots of common bean. As profilin interacts specifically with PIP2 which
can sequester profilin from its cytoplasmic pool (for plant cells
see Drøbak et al. 1994), intracellular distributions of PIP2 are
of particular interest. From earlier biochemical work it is
known that a large portion of cellular PIP2 associates with plant
plasma membrane (Wheeler and Boss 1987, Irvine et al. 1989).
Recently we have shown localization of maize profilin at the
plasma membrane of developing pollen of birch and maize
(von Witsch et al. 1998).
Fig. 2 Profilin distributions in cells of control and pharmacologically treated maize root apices as revealed with two antibodies raised against
maize profilins, PRA (A–L) and anti-ZmPRO3 (M–P). (A) Young meristematic cells of epidermis showed abundant profilin labeling with PRA
antibody, especially at cellular peripheries facing the root surface (thick arrow). Note that nuclei (stars) were only faintly labeled at this developmental stage of the epidermis. (B) Depletion of peripheral labeling (thick arrow) and increase in nuclear labeling just a few epidermal cells further, in the proximal part of the meristem. (C) In the transition zone, epidermal peripheries facing root surface showed again profilin
accumulations (thick arrow) and nuclei were strongly labeled with the PRA antibody which also recognized cytoplasmic strands. (D) In the elongation region, root hair initiation in epidermis was accompanied with massive accumulation of profilins within outgrowing bulge (indicated with
open star) and a slight reduction of the nuclear labeling (black star). (E) Finally, the bulge (open star) is the most dominant profilin domain while
nuclei are often not recognizable at all. (F) Similarly in the root cortex, 10% of nuclei in transition zone cells showed no profilin staining (white
star) while the adjacent ones were typically strongly labeled (black star). (G) Blocking of PRA antibody with profilins resulted in no labeling at
all. (H) Prominent cell periphery-associated aggregates (see arrow pointing at a cross wall) were often labeled in prospective metaxylem elements
with the PRA antibody. Metaxylem nucleus is also profilin-positive (star). (I–P) After mastoparan treatment, both PRA (J–L) and ZmPRO3 (N–P)
signal decreased in nuclei and accumulated under side walls in cells of the basal part of cortex meristem (J,N; for appropriate control cells see
I,M). Nuclei in post-mitotic cells of the cortex (K,O) and metaxylem (L,P) did not retain any profilin signal. Nuclei are indicated by stars. Bar =
7 mm.
916
Mastoparan redistributes nuclear profilin
Fig. 3 PIP2 distribution in control and experimentally-treated maize root apices. (A) Typical image from meristem showing strong cytoplasmic
signal and negative nuclei. (B) Blocking with PIP2 resulted in no obvious signal. (C) Blocking with PA had no effect and labeling was strong. (D–
F) Distribution of PIP2 in cells of the transition zone (D) and elongation regions (E,F). Note that PIP2 signal localized in the form of fine dots to
nuclear peripheries (D,E), perinuclear cytoplasmic strands (E) and to the cell periphery (D–F). (G,H) The plasma membrane-associated PIP2-positive dots were recognizeable in cells that were sectioned peripherally, encompassing the plasma membrane within the section. (I) Treatment of
roots with mastoparan reversed subcellular PIP2 distribution in all post-mitotic cortex cells in favor of nuclear labeling which is associated with
depletion of the plasma membrane-associated signal. (J) To support the plasma membrane-associated labeling of PIP2, we applied a monoclonal
antibody raised against the plasma membrane H+-ATPase which labeled the plasma membrane exclusively. Position of nuclei is indicated by stars.
Bar corresponds to 15 mm for (A–C) and 7 mm for (D–J).
Here, we have applied well-characterized monoclonal
antibody raised against PIP2 (Fukami et al. 1988, Hay et al.
1995, Gilmore and Burridge 1996, Bubb et al. 1998, Huang et
al. 1998, Kaznacheyeva et al. 2000, Mayer et al. 2000) on
Steedman’s wax sections taken from maize root apices. This
antibody labels distinct domains at cellular peripheries, cytoplasmic strands and nuclear envelopes. Strong cytoplasmic
labeling in meristematic cells might be attributed to the fact
Mastoparan redistributes nuclear profilin
917
Fig. 4 (A, B) Double labeling with anti-ZmPRO3 (A) and anti-PIP2 (B) antibodies in elongating cortex cells of latrunculin-treated root apices.
(C) Blocking of anti-ZmPRO3 with profilins resulted in no labeling. (D, E) AFs in cells of the stele-cortex interface (D), and in cortex cells (E).
(F) Latrunculin B treatment depolymerizes all AFs resulting in diffuse fluorescence which accumulates within nuclei (stars). (G–J) Mastoparan
(10 mM for 2 h) induced dramatic reorganization of the actin cytoskeleton in cells of maize root apices. Note that all AF networks and bundles
were effectively dismantled (G) and that actin-positive patches were associated preferentially with vacuolar peripheries (arrowheads in H and
asterisks in I, J). (G) Cortex cells at the middle part of meristem; (H) endodermis/pericycle interface at the apical part of transition zone; (I) cortex at the apical part of transition zone; (J) cortex at the apical part of meristem. Nuclei are indicated by stars. Bar = 7 mm (A–I) and 12 mm (J).
that, besides the plasma membrane, cytoplasmic leaflets of all
endocellular membranes are also associated with PIP2 (e.g.
Helms et al. 1991, Hay et al. 1995, Chung et al. 1998, Godi et
al. 1999). PIP2 localizes also to apices of tip growing root hairs
(Braun et al. 1999, Baluška et al. 2000b, Baluška et al. 2000c)
and pollen tubes where it apparently regulate their actin-based
polarized growth in a common pathway with Rho GTPases
(Kost et al. 1999, Molendijk et al. 2001).
The general consensus is that PI signaling cascades also
exist in higher plant cells (reviewed by Drøbak 1993, Drøbak
et al. 1996, Munnik et al. 1998). Several pharmacological
agents are used to dissect PI signaling cascades in plant cells
918
Mastoparan redistributes nuclear profilin
among which mastoparan, a tetracapeptide isolated from wasp
venom, represents one of the best understood agents (van Himbergen et al. 1999). Mastoparan is a potent stimulator of phospholipase A-, C- and D-activities in plants (Drøbak and Watkins 1994, Scherer 1995, Cho et al. 1995, Tucker and Boss
1996, Franklin-Tong et al. 1996, van Himbergen et al. 1999,
den Hartog et al. 2001). Importantly, our data reveal that subcellular distributions of profilin and PIP2 are altered profoundly by mastoparan. In particular, mastoparan redistributed
profilin from nuclei into the cytoplasm. In this respect it is
interesting to note that mastoparan, when imposed on protoplasts isolated from transgenic tobacco plants expressing calcium reporter aequorin, stimulated transient increases of
nuclear free calcium levels independently from those induced
in the cytoplasm (Pauly et al. 2000).
We have recorded similar depletion of nuclear profilin
also using mastoparan analog M17 (van Himbergen et al.
1999), which is known to exert rather lower activities in other
plant systems (e.g. van Himbergen et al. 1999, Frank et al.
2000, den Hartog et al. 2001). Our data, however, indicate that
biological activities of this analog can be high in maize root
apices, and we strongly recommend not to take for granted its
alleged inactivity (see also van Himbergen et al. 1999). High
biological activity of the mastoparan analog M17 was recorded
also for soybean cell suspension cultures (Schroeder-Taylor
and Low 1997).
From other organisms, it is well known that PIP2 is
enriched at specific plasma membrane domains in a tissue- and
development-specific fashion (e.g. Hope and Pike 1996, Glaser et al. 1996). These latter domains may act as putative Factin organizing centers (Baluška et al. 2000b) where, under
the action of external stimuli, PIP2 hydrolysis might lead to dissociation of profilin and other PIP2-actin binding proteins
which then mediate changes in organization of the actin
cytoskeleton (Hartwig et al. 1995). Lipid rafts are preferred
platforms for PIP2-activated actin polymerization (Rozelle et
al. 2000). Moreover, PIP2-based lipid rafts appear to play a
central role in signaling across the plasma membrane (Lisanti
et al. 1994, Kurzchalia and Parton 1999). Numerous data implicate PIP2 and diverse actin-binding proteins as effectors of
extracellular signals (e.g. Machesky and Pollard 1993, Liscovitch and Cantley 1995, Clarke et al. 1998, Stauffer et al. 1998,
Toker 1998), allowing effective perception of extracellular signals at the plasma membrane and its further transduction via
cytoskeletal complexes (Janmey 1998, Machesky and Insall
1999, for plant cells see Volkmann and Baluška 1999, Staiger
2000). Such a scenario was recently reported for the tip growing pollen tubes where compartmentalized PIP2 acts in a common pathway with Rho-family GTP-binding proteins to support
the actin-dependent polar tube growth (Kost et al. 1999). This
may also be true for actively growing root hair tips where apical enrichments of PIP2 colocalize with accumulations of profilin, dense F-actin meshworks (Braun et al. 1999, Baluška et al.
2000a, Baluška et al. 2000b, Baluška et al. 2000c) and Rop
GTPases (Molendijk et al. 2001).
Other authors also reported nuclear localization of plant
profilin using either chemical fixation (Vidali et al. 1995) or
high-pressure freeze fixation technique (Hess and Valenta
1997, Holzinger et al. 1997, Holzinger et al. 2000). Similarly,
nuclear profilin has occassionally been reported for some animal cells (e.g. Mayboroda et al. 1997). Maize profilin localizes
to root cell nuclei in a constitutive fashion. Importantly, however, few post-mitotic cortical nuclei (about 10%) are depleted
in profilin in control cells indicating that this small and soluble
molecule might shuttle between the nuclear and cytoplasmic
compartments, similarly as it was shown for G-actin (Wada et
al. 1998, Rando et al. 2000). Plant cells seem to be different
from other eukaryotic systems, not only because they express
larger number of profilin isoforms (Gibbon and Staiger 2000)
but also because a significant portion of plant profilin constitutively localizes to nuclei. This unique feature of plant profilin
might prove to be relevant for signal transduction cascades
between the plasma membrane and the nucleus as these are
known to be mediated, at least in part, via nuclear shuttling
proteins (Goldfarb 1991, Laskey and Dingwall 1993, Mahanty
et al. 1999). In support of the nucleus–cytoplasm shuttling concept, we show that mastoparan induces redistributions of profilin from nuclei to the cytoplasm, especially in cortical cells of
the transition zone (Baluška et al. 2001c). Intriguingly, this
sudden increase of cytoplasmic profilin was accompanied by a
re-organization of F-actin arrays throughout maize root apices.
A sudden increase of profilin levels within the cytoplasm, after
microinjection of profilin, was shown to rapidly depolymerize
AFs in vivo in living Tradescantia stamen hair cells (Staiger
et al. 1994, Karakesisoglou et al. 1996, Gibbon et al. 1997,
Gibbon et al. 1998). Therefore, putative stimulus-responsive
relocations of nuclear profilin into the cytoplasm would
impinge on organization of actin cytoskeleton around the
nucleus. In fact, our study documents that AF arrays disintegrate and the actin signal accumulates in the form of distinct
actin patches at peripheries of small vacuoles.
Plant profilin might turn out to be a shuttling protein, acting perhaps as a second messenger within the framework of PIbased signaling pathways (Clarke et al. 1998). Similar second
messenger-like behavior was proposed for another actin binding protein, cofilin/ADF, in both animal and plant cells (Samstag et al. 1994, Jiang et al. 1997). By efficient sequestration of
plant profilin within the nuclear compartment and its stimulusresponsive release into the cytoplasm, plant nuclei could
directly remodel the actin cytoskeleton within the cytoplasm.
This feature fits well to our plant “cell body” concept (Baluška
et al. 1997b, Baluška et al. 1998, Baluška et al. 2000a, Baluška
et al. 2001b) which provides a conceptual framework for the
capability of plant nuclei to actively remodel the cytoplasmic
architecture.
Mastoparan redistributes nuclear profilin
Materials and methods
Materials
Maize grains (Zea mays L. cv. Alarik), obtained from Force Limagrain (Darmstadt, Germany), were soaked for 6 h and left to germinate in well-moistened rolls of filter paper for 4 d in darkness at 20°C.
Young seedlings with straight primary roots, 50–70 mm long, were
selected and growing root apices were excised for fixation. For experimental treatments, some seedlings were transferred to containers with
latrunculin B (10 mM, 1 h), mastoparan (10 mM, 2 h) and mastoparan
analog M17 (10 mM, 2 h). With the exception of latrunculin B (Calbiochem, Bad Soden, Germany) and mastoparan analog M17 (Bachem
AG, Heidelberg, Germany), all chemicals were obtained from Sigma
Chemicals.
Sample preparation
Maize root tips were processed for indirect immunofluorescence
as previously described in detail (Baluška et al. 1992, Baluška et al.
1997a). In brief, apical root segments (6–8 mm long) were fixed in
3.7% formaldehyde prepared in stabilizing buffer (SB: 50 mM PIPES
buffer, 5 mM MgSO4, 5 mM EGTA, pH 6.9), for 1 h. Following a rinse
in SB, they were dehydrated in a graded ethanol series diluted with
phosphate-buffered saline (PBS). Subsequently, segments were infiltrated with Steedman’s wax (a mixture of PEG 400 distearate and 1hexadecanol, 9 : 1, v/v) by an over-night incubation in absolute ethanol/
wax (1 : 1, v/v). This was followed by incubation (6 h) in pure wax to
remove residual ethanol from root tissues. Root segments were then
embedded by allowing the wax to polymerize at room temperature.
Immunoblotting of protein extracts from maize roots
Root apices (8 mm long) of 4-day-old Zea mays seedlings, grown
on wet filter paper and submerged in water (2 h) and in mastoparancontaining solution (10 mM, 2 h), were excised and collected into
buffer containing 50 mM Tris-HCl pH 7.4, 300 mM sucrose, 5 mM
KCl, NaCl and EDTA, 2 mM ascorbic acid, 10 mM freshly added
DTT and a cocktail of protease inhibitors (10 mg ml–1 pepstatin A, leupeptin, aprotinin, benzamidin, 1 mg ml–1 phenanthrolin). Afterwards,
all segments were mechanically homogenized using mortar on dry ice.
The homogenate was centrifuged at 23,000´g for 20 min at 4°C to
remove cellular debris, nuclei and other large organelles. The pellet
was discarded and the supernatant containing soluble and microsomal
proteins was subjected to discontinuous SDS-PAGE using 15% mini
slab gels at 15 mg of protein per lane. Gels were wet-blotted onto nitrocellulose and membranes were then used for immunoblotting. This
was accomplished with rabbit polyclonal profilin antibodies raised
against recombinant ZmPRO3 maize profilin isoform (anti-ZmPRO3;
Karakesisoglou et al. 1996) and maize native pollen profilins (PRA;
von Witsch et al. 1998), as well as with corresponding pre-immune
sera, all in a dilution of 1 : 200 in TTBS. Preincubations of antiZmPRO3 and PRA with recombinant profilins were carried out by
incubating 1 ml of diluted antibody with 60 mg of ZmPRO3 and 20 mg
of ZmPRO1, 2 and 4 (Gibbon et al. 1997, Gibbon et al. 1998) for 1 h
at room temperature. For protein visualization, alkaline phosphatasecoupled secondary antibodies (Promega, WI, U.S.A.) were employed
and visualized with Fast-Red kit of Sigma Chemicals.
Indirect immunofluorescence
Median longitudinal 7 mm thick Steedman’s wax sections were
placed on slides coated with glycerol-albumen (Serva, Heidelberg,
Germany), allowing them to expand on drops of distilled water. In
order to facilitate penetration of antibodies, the sections were dewaxed
in ethanol, rehydrated in an ethanol/PBS series, and allowed to stand
919
in SB for 45 min. After a 10 min rinse with absolute methanol at –
20°C, they were transferred to SB for 30 min at room temperature.
They were then incubated with rabbit polyclonal profilin antibodies
PRA and anti-ZmPRO3, raised against native maize pollen profilins
and recombinant maize profilin isoform ZmPRO3, respectively. Both
antibodies and their respective pre-immune sera were applied at
1 : 100 dilution, made up in PBS, for 90 min at room temperature.
Antibodies raised against maize H+-ATPase (provided by W. Michalke,
University of Freiburg, Germany), actin (monoclonal, clone C4, ICN
Biomedicals, Costa Mesa, CA, U.S.A.) and PIP2 (PerSeptive Biosystems, Inc., Framingham, MA, U.S.A.) were applied on sections in
1 : 200 dilutions. After another rinse in SB, the root sections were
stained with FITC-conjugated anti-rabbit (PRA, ZmPRO3) and antimouse IgG (PIP2, actin, H+-ATPase) raised in goat (Sigma Chemical
Co., St. Louis, MO, U.S.A.) diluted 1 : 200 in PBS for 90 min at room
temperature. For double labelings, Alexa dyes (Alexa 488-FITC and
Alexa 546-TRITC) were used (Molecular Probes Europe, Leiden, The
Netherlands). A further rinse in PBS (10 min) preceded 10 min in
0.01% Toluidine Blue (in PBS), which diminished the natural autofluorescence of root tissues (Baluška et al. 1992).
Using anti-fade mountant containing p-phenylenediamine, root
sections were mounted under a coverslip. Fluorescence was examined
with an Axiovert 405M inverted light microscope (Zeiss, Oberkochen,
Germany) equipped with epifluorescence and standard FITC exciter
and barrier filters (BP 450–490, LP 520). Photographs were taken on
Kodak T-Max films.
Competitive inhibition and other controls for profilin and PIP2
antibodies
Immunoblocking of profilin antibodies was accomplished with a
mixture of recombinant maize pollen profilins. In particular, 1 ml of
diluted PRA or anti-ZmPRO3 was incubated with 60 mg of ZmPRO3
and 20 mg of ZmPRO1, 2 and 4 (Karakesisoglou et al. 1996, Gibbon et
al. 1997, Gibbon et al. 1998). PIP2 antibody used in the present study
(PerSeptive Biosystems, Inc., Framingham, MA, U.S.A.) is in use for
several years now (Fukami et al. 1988, Hay et al. 1995, Gilmore and
Burridge 1996, Bubb et al. 1998, Huang et al. 1998, Kaznacheyeva et
al. 2000, Mayer et al. 2000). Importanly, its specificity has been confirmed here for maize roots: 10 ml of PIP2 antibody was incubated with
20 mg of native PIP2 and this immuno-coupling negated any signal in
immunofluorescence. According to the manufacturer (PerSeptive Biosystems), the PIP2 antibody may also recognize (<5%) phosphatidic
acid (PA). We have tested this for maize roots using native PA (Sigma
Chemicals). In contrast to PIP2, addition of 20 mg of PA to 10 ml of
PIP2 antibody did not influence the labeling pattern confirming the
specificity of this antibody for PIP2 in maize roots.
Additional controls included omitting the first antibody or using
the appropriate pre-immune sera. No labeling was detected in these
cases. Moreover, applying another rabbit polyclonal antibody raised
against maize calreticulin using the same labeling procedure, resulted
in completely different labeling patterns (Baluška et al. 1999).
Acknowledgements
We would like to thank C.J. Staiger (Purdue University, U.S.A.)
for critical reading of our manuscript and for providing us with recombinant profilin and polyclonal ZmPRO3 antibody. Financial support by
Deutsches Zentrum für Luft- und Raumfahrt (DLR, Köln) is gratefully acknowledged. F.B. is partially supported by the Grant Agency
VEGA (Slovak Academy of Sciences, project No. 6030).
920
Mastoparan redistributes nuclear profilin
References
Ayscough, K.R., Stryker, J., Pokala, N., Sanders, M., Crews, P. and Drubin, D.G.
(1997) High rates of actin filament turnover in budding yeast and roles for
actin in establishment and maintenance of cell polarity revealed using the
actin inhibitor latrunculin-A. J. Cell Biol. 137: 399–416.
Baluška, F., Barlow, P.W. and Volkmann, D. (2000c) Actin and myosin VIII in
developing root cells. In Actin: A Dynamic Framework for Multiple Plant
Cell Functions. Edited by Staiger, C.J., Baluška, F., Volkmann, D. and Barlow, P.W. pp. 457–476. Kluwer Academic Publishers, Dordrecht, The Netherlands.
Baluška, F., Jasik, J., Edelmann, H.G., Salajová, T. and Volkmann, D. (2001a)
Latrunculin B-induced plant dwarfism: plant cell elongation is F-actin
dependent. Dev. Biol. 231: 113–124.
Baluška, F., Lichtscheidl, I.K., Volkmann, D. and Barlow, P.W. (1998) The plant
cell body: a cytoskeletal tool for cellular development and morphogenesis.
Protoplasma 202: 1–10.
Baluška, F., Parker, J.S. and Barlow, P.W. (1992) Specific patterns of cortical
and endoplasmic microtubules associated with cell growth and tissue differentiation in roots of maize (Zea mays L.). J. Cell Sci. 103: 91–200.
Baluška, F., Salaj, J., Mathur, J., Braun, M., Jasper, F., Šamaj, J., Chua, N.-H.,
Barlow, P.W. and Volkmann, D. (2000b) Root hair formation: F-actindependent tip growth is initiated by local assembly of profilin-supporterd Factin meshworks accumulated within expansin-enriched bulges. Dev. Biol.
227: 618–632.
Baluška, F., Šamaj, J., Napier, R. and Volkmann, D. (1999) Maize calreticulin
localizes preferentially to plasmodesmata. Plant J. 19: 481–488.
Baluška, F., Vitha, S., Barlow, P.W. and Volkmann, D. (1997a) Re-arrangements
of F-actin arrays in growing cells of intact maize root apex tissue: a major
developmental switch occurs in the postmitotic transition region. Eur. J. Cell
Biol. 72: 113–121.
Baluška, F., Volkmann, D. and Barlow, P.W. (1997b) Nuclear components with
microtubule-organizing properties in multicellular eukaryotes: functional and
evolutionary considerations. Int. Rev. Cytol. 175: 91–135.
Baluška, F., Volkmann, D. and Barlow, P.W. (2000a) Actin-based domains of the
‘cell periphery complex’ and their associations with polarized ‘cell bodies’ in
higher plants. Plant Biol. 2: 253–267.
Baluška, F., Volkmann, D. and Barlow, P.W. (2001b) Motile plant cell body: a
‘bug’ within a ‘cage’. Trends Plant Sci. 6: 104–111.
Baluška, F., Volkmann, D. and Barlow, P.W. (2001c) A polarity crossroads in the
transition growth zone of maize root apices: cytoskeletal and developmental
implications. J. Plant Growth Regul. 20: (in press).
Berridge, M.J. and Irvine, R.F. (1989) Inositol phosphates and cell signalling.
Nature 341: 197–205.
Braun, M., Baluška, F., von Witsch, M. and Menzel, D. (1999) Redistribution of
actin, profilin and phosphatidylinositol-4,5-bisphosphate (PIP2) in growing
and maturing root hairs. Planta 209: 435–443.
Bubb, M.R., Baines, I.C. and Korn, E.D. (1998) Localization of actobindin, profilin I, profilin II, and phosphatidylinositol-4, 5-bisphosphate (PIP2) in Acanthamoeba casrellanii. Cell Motil. Cytoskel. 39: 134–146.
Buß, F., Temm-Grove, C., Henning, S. and Jockusch, B.M. (1992) Distribution
of profilin in fibroblasts correlates with the presence of highly dynamic actin
filaments. Cell Motil. Cytoskel. 22: 51–61.
Cho, M.H., Tan, Z., Erneux, C., Shears, S.B. and Boss, W.F. (1995) The effects
of mastoparan on the carrot cell plasma membrane polyphosphoinositide
phospholipase C. Plant Physiol. 107: 845–856.
Chung, S.H., Song, W.J., Kim, K., Bednarski, J.J., Chen, J., Prestwich, G.D. and
Holz, R.W. (1998) The C2 domains of Rabphilin3A specifically bind phosphatidylinositol 4,5-bisphosphate containing vesicles in a Ca2+-dependent
manner — in vitro characteristics and possible significance. J. Biol. Chem.
273: 10240–10248.
Clarke, S.R., Staiger, C.J., Gibbon, B.C. and Franklin-Tong, V.E. (1998) A
potential signaling role for profilin in pollen of Papaver rhoeas. Plant Cell
10: 967–979.
den Hartog, M., Musgrave, A. and Munnik, T. (2001) Nof factor-induced phosphatidic acid and diacylglycerol pyrophosphate formation: a role for phospholipase C and D in root hair deformation. Plant J. 25: 55–65.
Dove, S.K., Lloyd, C.W. and Drøbak, B.K. (1994) Identification of a phosphatidylinositol 3-hydroxy kinase in plant cells: association with the cytoskeleton.
Biochem. J. 303: 347–350.
Drøbak, B.K. (1993) Plant phosphoinositides and intracellular signaling. Plant
Physiol. 102: 705–709.
Drøbak, B.K., Dove, S.K. and Staiger, C.J. (1996) Inositides and plant signalling. In Plant Membrane Biology. Edited by Møller, I.M. and Brodelius, P. pp.
29–50. Clarendon Press, Oxford, U.K.
Drøbak, B.K. and Watkins, P.A.C. (1994) Inositol (1,4,5) trisphosphate production in plant cells: stimulation by the venom peptides, melitin and mastoparan. Biochem. Biophys. Res. Commun. 205: 739–745.
Drøbak, B.K., Watkins, P.A.C., Valenta, R., Dove, S.K., Lloyd, C.W. and
Staiger, C.J. (1994) Inhibition of a plant plasma membrane phosphoinositide
phospholipase C by the actin-binding protein, profilin. Plant J. 6: 389–400.
Frank, W., Munnik, T., Kerkmann, K., Salamini, F. and Bartels, D. (2000) Water
deficit triggers phospholipase D activity in the resurrection plant Craterostigma plantagineum. Plant Cell 12: 111–123.
Franklin-Tong, V.E., Drøbak, B.K., Allan, A.C., Watkins, P.A.C. and Trewavas,
A.J. (1996) Growth of pollen tubes of Papaver rhoeas is regulated by a slowmoving calcium wave propagated by inositol 1, 4, 5-trisphosphate. Plant Cell
8: 1305–1321.
Fukami, K., Matsuoka, K., Naganishi, O., Yamakawa, A., Kawai, S. and Takenawa, T. (1988) Antibody to phosphatidylinositol 4, 5-bisphosphate inhibits
oncogene-induced mitogenesis. Proc. Natl. Acad. Sci. USA 85: 9057–9061.
Gibbon, B.C., Kovar, D.R. and Staiger, C.J. (1999) Latrunculin B has different
effects on pollen germination and tube growth. Plant Cell 11: 2349–2364.
Gibbon, B.C., Ren, H. and Staiger, C.J. (1997) Characterization of maize (Zea
mays) pollen profilin function in vitro and in live cells. Biochem. J. 3: 909–
915.
Gibbon, B.C. and Staiger, C.J. (2000) Profilin. In Actin: A Dynamic Framework for Multiple Plant Cell Functions. Edited by Staiger, C.J., Baluška, F.,
Volkmann, D. and Barlow, P.W. pp. 45–65. Kluwer Academic Publishers,
Dordrecht, The Netherlands.
Gibbon, B.C., Zonia, L.E., Kovar, D.R., Hussey, P.J. and Staiger, C.J. (1998)
Pollen profilin function depends on interaction with proline-rich motifs. Plant
Cell 10: 981–993.
Gilmore, A.P. and Burridge, K. (1996) Regulation of vinculin binding to talin
and actin by phosphatidylinositol-4-5bisphosphate. Nature 381: 531–535.
Glaser, M., Wanaski, S., Buser, C.A., Boguslavsky, V., Rashidzada, W., Morris,
A., Rebecchi, M., Scarlata, S.F., Runnels, L.W. and Presttwich, G.D. (1996)
Myristoylated alanine-rich C kinase substrate (MARCKS) produces reversible inhibition of phospholipase C by sequestering phosphatidylinositol 4,5bisphosphate in lateral domains. J. Biol. Chem. 271: 26187–26193.
Godi, A., Pertile, P., Meyers, R., Marra, D., Di Tullio, G., Iurisci, C., Luini, A.,
Corda, D. and De Matteis, M.A. (1999) ARF mediates recruitment of PtdIns4-OH kinase-> and stimulates synthesis of PtdIns (4,5)P2 on the Golgi complex. Nat. Cell Biol. 1: 280–287.
Goldfarb, D.S. (1991) Shuttling proteins go both ways. Curr. Biol. 1: 212–214.
Grote, M., Swoboda, I., Meagher, R.B. and Valenta, R. (1995) Localization of
profilin- and actin-like immunoreactivity in in vitro-germinated tobacco pollen tubes by electron microscopy after special water-free fixation techniques.
Sex. Plant Reprod. 8: 180–186.
Hartwig, J.H., Bokoch, G.M., Carpenter, C.L., Janmey, P.A., Taylor, L.A., Toker,
A. and Stossel, T.P. (1995) Thrombin receptor ligation and activated Rac
uncap actin filament barbed ends through phosphoinositide synthesis in permeabilized human platelets. Cell 82: 643–653.
Hartwig, J.H., Chambers, K.A., Hopcia, K.L. and Kwiatkowski, D.J. (1989)
Association of profilin with filament-free regions of human leukocyte and
platelet membranes and reversible membrane binding during platelet activation. J. Cell Biol. 109: 1571–1579.
Hay, J.C., Fisette, P.L., Jenkins, G.H., Fukami, K., Takenawa, T., Anderson,
R.A. and Martin, T.F.J. (1995) ATP-dependent inositide phosphorylation
required for Ca2+-activated secretion. Nature 374: 173–177.
Helms, J.B., de Vries, K.J. and Wirtz, K.W.A. (1991) Synthesis of phosphatidylinositol 4, 5-bisphosphate in the endoplasmic reticulum of chinese hamster
ovary cells. J. Biol. Chem. 266: 21368–21374.
Hess, M.W. and Valenta, R. (1997) Profilin revealed in pollen nuclei: immunoelectron microscopy of high-pressure frozen Ledebouria socialis Roth (Hyacinthaceae). Sex. Plant Reprod. 10: 283–287.
Holzinger, A., Mittermann, I., Laffer, S., Valenta, R. and Meindl, U. (1997)
Microinjection of profilins from different sources into the green alga Micrasterias causes transient inhibition of cell growth. Protoplasma 199: 124–134.
Holzinger, A., Valenta, R. and Lütz-Meindl, U. (2000) Profilin is localized in
Mastoparan redistributes nuclear profilin
the nucleus-associated microtubule and actin system and is evenly distributed in the cytoplasm of the green alga Micrasterias denticulata. Protoplasma 212: 197–205.
Hope, H.R. and Pike, L.J. (1996) Phosphoinositides and phosphoinositide utilizing enzymes in detergent-insoluble lipid domains. Mol. Biol. Cell 7: 843–851.
Huang, C.-L., Feng, S. and Hilgemann, D.W. (1998) Direct activation of inward
rectifier potassium channels by PIP2 and its stabilization by G>C. Nature 391:
803–806.
Irvine, R.F., Letcher, A.J., Lander, D.J., Drøbak, B.K., Dawson, A.P. and Musgrave, A. (1989) Phosphatidylinositol (4,5)bisphosphate and phosphatidyl
(4)phosphate in plant tissues. Plant Physiol. 89: 888–892.
Jahn, T., Baluška, F., Michalke, W., Harper, J. and Volkmann, D. (1998) Plasma
membrane H+-ATPase in the root apex: evidence for strong expression in
xylem parenchyma and asymmetric localization within cortical and epidermal cells. Physiol. Plant. 104: 311–316.
Janmey, P.A. (1998) The cytoskeleton and cell signaling — component localization and mechanical coupling. Physiol. Rev. 78: 763–781.
Jiang, C.-J., Weeds, A.G. and Hussey, P.J. (1997) The maize actin-depolymerizing
factor, ZmADF3, redistributes to the growing tip of elongating root hairs and
can be induced to translocate into the nucleus with actin. Plant J. 12: 1035–
1043.
Kang, F., Laine, R., Bubb, M., Southwick, F. and Purich, D. (1997) Profilin
interacts with the Gly-Pro-Pro-Pro-Pro-Pro sequences of vasodilatorstimulated phosphoprotein (VASP): implications for actin-based Listeria
motility. Biochemistry 36: 8384–8392.
Karakesisoglou, I., Schleicher, M., Gibbon, B.C. and Staiger, C.J. (1996) Plant
profilins rescue the aberrant phenotype of profilin-deficient Dictyostelium
cells. Cell Motil. Cytoskel. 34: 36–47.
Kay, B.K., Williamson, M.P. and Sudol, M. (2000) The importance of being
proline: the interaction of proline-rich motifs in signaling proteins with their
cognate domains. FASEB J. 14: 231–241.
Kaznacheyeva, E., Zubov, A., Nikolaev, A., Alexeenko, V., Bezprozvanny, I.
and Mozhayeva, G.N. (2000) Plasma membrane calcium channels in human
carcinoma A431 cells are functionally coupled to inositol 1,4,5-trisphosphate
receptor-phosphatidylinositol 4,5-bisphosphate complexes. J. Biol. Chem.
275: 4561–4564.
Kost, B., Lemichez, E., Spielhofer, P., Hong, Y., Tolias, K., Carpenter, C. and
Chua, N.-H. (1999) Rac homologues and compartmentalized phosphatidylinositol 4,5-bisphosphate act in a common pathway to regulate polar pollen
tube growth. J. Cell Biol. 145: 317–330.
Kurzchalia, T.V. and Parton, R.G. (1999) Membrane microdomains and caveolae. Curr. Opin. Cell Biol. 11: 424–431.
Laskey, R.A. and Dingwall, C. (1993) Nuclear shuttling: the default pathway for
nuclear protein? Cell 74: 585–586.
Lassing, I. and Lindberg, U. (1985) Specific interaction between phosphatidylinositol 4,5-bisphosphate and profilactin. Nature 314: 472–474.
Lisanti, M.P., Scherer, P.E., Tang, Z. and Sargiacomo, M. (1994) Caveole, caveolin and caveolin-rich membrane domains: a signalling hypothesis. Trends
Cell Biol. 4: 231–235.
Liscovitch, M. and Cantley, L.C. (1995) Signal transduction and membrane traffic: the PITP/phosphoinositide connection. Cell 81: 659–662.
Machesky, L.M. and Insall, R.H. (1999) Signaling to actin dynamics. J. Cell
Biol. 146: 267–272.
Machesky, L.M. and Pollard, T.D. (1993) Profilin as a potential mediator of
membrane-cytoskeleton communication. Trends Cell Biol. 3: 14027–14034.
Mahanty, S.K., Wang, Y., Farley, F.W. and Elion, E.A. (1999) Nuclear shuttling
of yeast scaffold Ste5 is required for its recruitment to the plasma membrane
and activation of the mating MAPK cascade. Cell 98: 501–512.
Mayboroda, O., Schlüter, K. and Jockusch, B.M. (1997) Differential colocalization of profilin with microfilaments in PtK2 cells. Cell Motil. Cytoskel. 37:
166–177.
Mayer, A., Scheglmann, D., Dove, S., Glatz, A., Wickner, W. and Haas, A.
(2000) Phosphatidylinositol 4,5-bisphosphate regulates two steps of homotypic vacuole fusion. Mol. Cell Biol. 11: 807–817.
McKinney, E.C., Kandasamy, M.K. and Meagher, R.B. (2001) Small changes in
the regulation of one Arabidopsis profilin isovariant, PRF1, alter seedling
development. Plant Cell 13: 1179–1191.
Mikami, K., Katagiri, T., Iuchi, S., Yamaguchi-Shinozaki, K. and Shinozaki, K.
(1998) A gene encoding phosphatidyl-4-phosphate 5-kinase is induced by
water stress and abscisic acid in Arabidopsis thaliana. Plant J. 15: 563–568.
921
Mittermann, I., Heiss, S., Kraft, D., Valenta, R. and Heberle-Bors, E. (1996)
Molecular characterization of profilin isoforms from tobacco (Nicotiana tabacum) pollen. Sex. Plant Reprod. 9: 133–139.
Molendijk, A.J., Bischoff, F., Rajendrakumar, C.S.V., Friml, J., Braun, M.,
Gilroy, S. and Palme, K. (2001) Arabidopsis thaliana Rop GTPases are localized to tips of root hairs and control polar growth. EMBO J. 20: 2779–2788.
Munnik, T., Irvine, R.F. and Musgrave, A. (1998) Phospholipid signalling in
plants. Biochim. Biophys. Acta 1389: 222–272.
Ostrander, D.B., Gorman, J.A. and Carman, G.M. (1995) Regulation of profilin
localization in Saccharomyces cerevisiae by phosphoinositide metabolism. J.
Biol. Chem. 270: 27045–27050.
Pantaloni, D. and Carlier, M.F. (1993) How profilin promotes actin filament
assembly in the presence of thymosin >4. Cell 75: 1007–1014.
Pauly, N., Knight, M.R., Thuleau, P., van der Liut, A.H., Moreau, M., Trewavas, A.J., Ranjeva, R. and Mazars, C. (2000) Control of free calcium in plant
nuclei. Nature 405: 754–755.
Ramachandran, S., Christensen, H.E.M., Ishimaru, Y., Dong, C.–H., ChaoMing, W., Cleary, A.L. and Chua, N.–H. (2000) Profilin plays a role in cell
elongation, cell shape maintenance, and flowering in Arabidopsis. Plant
Physiol. 124: 1637–1647.
Rando, O.J., Zhao, K. and Crabtree, G.R. (2000) Searching for a function for
nuclear actin. Trends Cell Biol. 10: 92–97.
Reinhard, M., Giehl, K., Abel, K., Haffner, C., Jarchau, T., Hoppe, V., Jockusch, B.M. and Walter, U. (1995) The proline-rich focal adhesion and microfilament protein VASP is a ligand for profilins. EMBO J. 14: 1583–1589.
Rozelle, A.L., Machesky, L.M., Yamamoto, M., Driessens, M.H.E., Insall, R.H.,
Roth, M.G., Luby-Phelps, K., Marriott, G., Hall, A. and Yin, H.L. (2000)
Phosphatidylinositol 4,5-bisphosphate induces actin-based movement of raftenriched vesicles through WASP-Arp2/3. Curr. Biol. 10: 311–320.
Samstag, Y., Eckerskorn, C., Wesselborg, S., Henning, S., Wallich, R. and
Meuer, S.C. (1994) Costimulatory signals for human T-cell activation induce
nuclear translocation of pp19/cofilin. Proc. Natl. Acad. Sci. USA 91: 4494–
4498.
Scherer, G.F.E. (1995) Activation of phospholipase A2 by auxin and mastoparan
in hypocotyl segments from zucchini and sunflower. J. Plant Physiol. 145:
483–490.
Schlüter, K., Jockusch, B.M. and Rothkegel, M. (1997) Profilins as regulators of
actin dynamics. Biochim. Biophys. Acta 1359: 97–109.
Schroeder-Taylor, A.T. and Low, P.S. (1997) Phospholipase D involvement in
the plant oxidative burst. Biochem. Biophys. Res. Commun. 237: 10–15.
Singh, S.S., Chauhan, A., Murakami, N. and Chauhan, V.P.S. (1996) Profilin
and gelsolin stimulate phosphatidylinositol 3-kinase activity. Biochemistry
35: 16544–16549.
Staiger, C.J. (2000) Signaling to the actin cytoskeleton in plants. Annu. Rev.
Plant Physiol. Plant Mol. Biol. 51: 257–288.
Staiger, C.J., Gibbon, B.C., Kovar, D.R. and Zonia, L.E. (1997) Profilin and
actin-depolymerizing factor: modulators of actin organization in plants.
Trends Plant Sci. 2: 275–281.
Staiger, C.J., Yuan, M., Valenta, R., Shaw, P.J., Warn, R.M. and Lloyd, C.W.
(1994) Microinjected profilin affects cytoplasmic streaming in plant cells by
rapidly depolymerizing actin microfilaments. Curr. Biol. 4: 215–219.
Stauffer, T.P., Ahn, S. and Meyer, T. (1998) Receptor-induced transient reduction in plasma membrane PtdIns (4, 5)P2 concentration monitored in living
cells. Curr. Biol. 8: 343–346.
Stevenson, J.M., Perera, I.Y. and Boss, W.F. (1998) A phosphatidylinositol 4kinase pleckstrin homology domain that binds phosphatidylinositol 4-monophosphate. J. Biol. Chem. 273: 22761–22767.
Toker, A. (1998) The synthesis and cellular roles of phosphatidylinositol 4,5bisphosphate. Curr. Opin. Cell Biol. 10: 254–261.
Tucker, E.B. and Boss, W.F. (1996) Mastoparan-induced intracellular Ca2+
fluxes may regulate cell-to-cell communication in plants. Plant Physiol. 111:
459–467.
van Himbergen, J.A.J., ter Riet, B., Meijer, H.J.G., van den Ende, H., Musgrave,
A. and Munnik, T. (1999) Mastoparan analogues stimulate phospholipase Cand phospholipase D-activity in Chlamydomonas: a comparative study. J.
Exp. Bot. 50: 1735–1742.
Vidali, L. and Hepler, P.K. (1997) Characterization and localization of profilin
in pollen grains and tubes of Lilium longiflorum. Cell Motil. Cytoskel. 36:
323–338.
Vidali, L., Pérez, H.E., López, V.V., Noguez, R., Zamudio, F. and Sánchez, F.
922
Mastoparan redistributes nuclear profilin
(1995) Purification, characterization, and cDNA cloning of profilin from
Phaseolus vulgaris. Plant Physiol. 108: 115–123.
Volkmann, D. and Baluška, F. (1999) The actin cytoskeleton in plants: from
transport networks to signaling networks. Microsc. Res. Tech. 47: 135–154.
von Witsch, M., Baluška, F., Staiger, C.J. and Volkmann, D. (1998) Plasma
membrane-associated profilin isoforms in pollen grains of Zea mays and
microspores of Betula pumila. Eur. J. Cell Biol. 77: 303–312.
Wada, A., Fukuda, M., Mishima, M. and Nishida, E. (1998) Nuclear export of
actin: a novel mechanism regulating the subcellular localization of a major
cytoskeletal protein. EMBO J. 17: 1635–1641.
Wheeler, J.J. and Boss, W.F. (1987) Polyposphoinositides are present in plasma
membranes isolated from fusogenic carrot cells. Plant Physiol. 85: 389–392.
Xu, P., Lloyd, C.W., Staiger, C.J. and Drøbak, B.K. (1992) Association of phosphatidylinositol 4-kinase with the plant cytoskeleton. Plant Cell 4: 941–951.
(Received September 29, 2000; Accepted June 11, 2001)