Actin-Bundling Protein Isolated from Pollen Tubes of Lily
: Biochemical and Immunocytochemical Characterization |
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Authors: | Etsuo Yokota and Kei-ichiro Takahara and Teruo Shimmen |
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Institution: | Department of Life Science, Faculty of Science, Himeji Institute of Technology, Harima Science Park City, Hyogo 678–12, Japan |
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Abstract: | A 135-kD actin-bundling protein was
purified from pollen tubes of lily (Lilium longiflorum)
using its affinity to F-actin. From a crude extract of the pollen
tubes, this protein was coprecipitated with exogenously added F-actin
and then dissociated from F-actin by treating it with
high-ionic-strength solution. The protein was further purified
sequentially by chromatography on a hydroxylapatite column, a
gel-filtration column, and a diethylaminoethyl-cellulose ion-exchange
column. In the present study, this protein is tentatively referred to
as P-135-ABP (Plant 135-kD
Actin-Bundling Protein). By the
elution position from a gel-filtration column, we estimated the native
molecular mass of purified P-135-ABP to be 260 kD, indicating that it
existed in a dimeric form under physiological conditions. This protein
bound to and bundled F-actin prepared from chicken breast muscle in a
Ca2+-independent manner. The binding of 135-P-ABP to actin
was saturated at an approximate stoichiometry of 26 actin monomers to 1
dimer of P-135-ABP. By transmission electron microscopy of thin
sections, we observed cross-bridges between F-actins with a
longitudinal periodicity of 31 nm. Immunofluorescence microscopy using
rhodamine-phalloidin and antibodies against the 135-kD polypeptide
showed that P-135-ABP was colocalized with bundles of actin filaments
in lily pollen tubes, leading us to conclude that it is the factor
responsible for bundling the filaments.Actin filaments, one of the major components of the cytoskeleton,
are organized into a highly ordered architecture and are involved in
various kinds of cell motility. Their architecture is regulated by
several kinds of actin-binding proteins, including cross-linking
proteins, severing proteins, end-capping proteins, and
monomer-sequestering proteins in animal, protozoan, and yeast cells
(Stossel et al., 1985; Pollard and Cooper, 1986; Vandekerckhove
and Vancompernolle, 1992). In plant cells the organization of the actin
cytoskeleton also changes remarkably during the cell cycle or during
developmental processes, and it is suggested that actin-binding
proteins are involved in their dynamic change. However, little is known
about actin-binding proteins in plant cells.Only a low-Mr actin-binding and -depolymerizing
protein, profilin, in white birch (Betula verrucosa;
Valenta et al., 1991), maize (Zea mays; Staiger
et al., 1993; Ruhlandt et al., 1994), bean (Phaseolus
vulgaris; Vidali et al., 1995), tobacco (Nicotiana
tabacum; Mittermann et al., 1995), tomato (Lycopersicon
esculentum; Darnowski et al., 1996), Arabidopsis
(Arabidopsis thaliana; Huang et al., 1996), and lily
(Lilium longiflorum; Vidali and Hepler, 1997), and an ADF in
lily (Kim et al., 1993), rapeseed (Brassica napus; Kim
et al., 1993), and maize (Rozycka et al., 1995; Lopez et al., 1996),
have been identified by biochemical or molecular biological means.The native and recombinant forms of these proteins are capable of
binding to animal or plant actin (Valenta et al., 1993; Giehl et al.,
1994; Ruhlandt et al., 1994; Lopez et al., 1996; Perelroizen et al.,
1996; Carlier et al., 1997). Plant profilin expressed in mammalian
BHK-21 cells (Rothkegel et al., 1996) or profilin-deficient Dictyostelium discoideum cells (Karakesisoglou et al., 1996) was
able to functionally substitute for endogenous profilin in these cells.
The introduction of plant profilin into living stamen hair cells by
microinjection caused the rapid reduction of the number of actin
filaments (Staiger et al., 1994; Karakesisoglou et al., 1996; Ren et
al., 1997). These results indicate that plant profilin and ADF share
many functional similarities with other eukaryote profilins and
ADFs.It is well known that the actin cytoskeleton undergoes dynamic changes
in organization during hydration and activation of the vegetative cells
of pollen grains (Pierson and Cresti, 1992). Before hydration actin
filaments exist as fusiform or spiculate structures (a storage form),
but they are rearranged to form a network upon hydration
(Heslop-Harrison et al., 1986; Tiwari and Polito, 1988). In the growing
pollen tube there are strands or bundles of actin filaments parallel to
the long axis (Perdue et al., 1985; Pierson et al., 1986; Miller et
al., 1996) that are involved in cytoplasmic streaming (Franke et al.,
1972; Mascarenhas and Lafountain, 1972) and transport of vegetative
nuclei and generative cells to the growing tip (Heslop-Harrison et al.,
1988; Heslop-Harrison and Heslop-Harrison, 1989). Characterization of
the function of actin-binding proteins is essential to understanding
the regulation of actin organization during the developmental process
of pollen. Since only a small number of vacuoles containing proteases
develop in pollen grains and pollen tubes at a younger stage, pollen
tubes are suitable materials for isolating and biochemically studying
actin-binding proteins responsible for organizing actin filaments into
various forms.In a previous paper we reported that several components in a crude
extract prepared from lily pollen tubes, including a 170-kD myosin
heavy chain and 175-, 135-, and 110-kD polypeptides, could be
coprecipitated with exogenously added F-actin (Yokota and Shimmen,
1994). We also found that rhodamine-labeled F-actin was tightly bound
to the glass surface treated with the fraction containing the 135- and
110-kD polypeptides (Yokota and Shimmen, 1994). These results suggested
that either one or both of the 135- and 110-kD polypeptides possesses
an F-actin-binding activity. In the present study, we purified the
135-kD polypeptide from lily pollen tubes by biochemical procedures and
then characterized its F-actin-binding properties and distribution in
the pollen tubes. This protein was able to bundle F-actin isolated from
chicken breast muscle and colocalized with actin-filament bundles in
pollen tubes. We refer to this protein as P-135-ABP (Plant
135-kD Actin-Bundling
Protein). |
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