Journal of Experimental Botany, Vol. 51, No. 344, pp. 521–528, March 2000
Immunolocalization of actin in root statocytes of
Lens culinaris L.
D. Driss-Ecole1,4, J. Vassy2, J. Rembur1, A. Guivarc’h1, M. Prouteau1, W. Dewitte3 and
G. Perbal1
1 Université Pierre et Marie Curie, Laboratoire CEMV, case courrier 150, Bât. N2, 4 place Jussieu, F-75252 Paris
Cedex 05, France
2 Hôpital Saint-Louis, Laboratoire d’Analyse d’Images en pathologie cellulaire, 1 avenue Claude Vellefaux,
F-75475 Paris Cedex 10, France
3 Universiteit Antwerpen, Department of Biology, Universiteitsplein 1, B-2610 Wilrijk, Belgium
Received 2 July 1999; Accepted 25 October 1999
Abstract
Lentil root statocytes show a strict structural polarity
of their organelles with respect to the g vector. These
cells are involved in the perception of gravity and are
responsible for the orientation of the root. Actin filaments take part in the positioning of their organelles
and could also be involved in the transduction of the
gravitropic signal. A pre-embedding immunogold silver
technique was carried out with a monoclonal antibody
in order to study the distribution of actin cytoskeleton
in the statocytes at the electron microscopic level.
Some areas were never labelled (cell wall, vacuole,
nucleoplasm, mitochondria, starch grains of the amyloplasts) or very slightly labelled (stroma of the amyloplasts). The labelling was scattered in the cytoplasm
always close to, or on the nuclear and amyloplast
envelopes and the tonoplast. Associations of 2 to 6
dots in file were observed, but these short files were
not oriented in one preferential direction. They corresponded to a maximum distance of 0.9 mm. This work
demonstrated that each statocyte organelle was
enmeshed in an actin web of short filaments arranged
in different ways. The images obtained by rhodaminephalloidin staining were in accordance with those of
immunogold labelling. The diffuse fluorescence of the
cytoplasm could be explained by the fact that the
meshes of the web should be narrow. The vicinity of
actin and of the amyloplasts envelope could account
for the movement of these organelles that was
observed in spatial microgravity.
Key words: Actin, cytoskeleton, immunolocalization, lentil
root, pre-embedding technique, statocyte.
Introduction
Root statocytes, located in the centre of the cap, are
responsible for gravisensing (Perbal, 1978; Audus, 1979;
Volkmann and Sievers, 1979). These cells show a strict
structural polarity of their organelles with respect to the
g vector (Sievers and Volkmann, 1972; Perbal, 1978;
Hensel and Sievers, 1980; Olsen et al., 1984; Sack and
Kiss, 1989; Perbal and Driss-Ecole, 1993): the nucleus is
positioned near the wall closest to the meristem (proximal
wall ), whereas tubules of endoplasmic reticulum ( ER)
are located near the distal wall. Experiments using cytochalasin B (Hensel, 1985), which inhibits actin filament
elongation (Brenner and Korn, 1979; Flanagan and Lin,
1980; Brown and Spudich, 1981), have shown that these
organelles are maintained in their respective position by
actin filaments. The linkage of the cytoskeleton with the
amyloplasts (or statoliths) is more difficult to demonstrate
since they sediment under the influence of gravity.
4 To whom correspondence should be addressed. Fax: +33 1 44 27 45 82. E-mail: [email protected]
Abbreviations: BB, Blocking buffer; BCIP/NBT, 5-bromo-4-chloro-3-indolyl phosphatase/nitroblue tetrazolium; BSA, bovine serum albumin; CRA, cress
root actin antibody; DMSO, dimethylsulphoxide; EDTA, ethylenediaminetetraacetic acid; EGTA, ethyleneglycol-bis-(b-aminoethylether)-N,N,N∞,N∞tetraacetic acid; ICN, anti-chicken gizzard actin IgG, Biomedicals, Meckenheim/Germany; MBS, m-maleimidobenzoyl N-hydroxysuccinimide ester;
NaN , sodium azide; NBT, ( p-nitroblue tetrazolium chloride); NGS, normal goat serum; PIPES, piperazine-N,N∞-bis(2-ethane-sulphonic acid); PBS,
3
phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; TBS, Tris-buffered saline.
© Oxford University Press 2000
522 Driss-Ecole et al.
In addition to their role in the positioning of the
statocyte organelles, actin filaments could play a role in
the transduction mechanism of the gravitropic signal
(Sievers et al., 1989, 1991). Another interesting feature
was observed in statocytes of roots grown in microgravity,
in space: a displacement of the amyloplasts occurred in
microgravity (Lorenzi and Perbal, 1990; Volkmann et al.,
1991). The kinetics of this movement was studied in lentil
root statocytes grown on a 1 g centrifuge and placed
during various periods in microgravity (Driss-Ecole et al.,
2000). This movement could be due to actin filaments
and motor proteins.
Evidence for the presence of actin in many plant cells
(Staiger and Schliwa, 1987; Sievers et al., 1989; Tewinkel
et al., 1989; Tang et al., 1989; Wada et al., 1998) has
been provided using rhodamine-phalloidin staining.
Nevertheless, these studies dealt with rhizoids (Chara),
protonema (Funaria, Adiantum) or pollen tubes
(Nicotiana) where, most of the time, actin microfilaments
are organized into bundles. The images concerning actin
in root statocytes are less numerous (Hensel, 1989; White
and Sack, 1990; Perbal and Driss-Ecole, 1993; Baluska
and Hasenstein, 1997) due to the fact that the staining is
weak and diffuse.
Other attempts have been made using immunofluorescence (Hensel, 1986; 1989; Koropp and Volkmann, 1994).
Koropp and Volkmann used either a new monoclonal
antibody (CRA) directed against the actin antigen of
cress roots or a commercially available antibody (ICN ).
The staining was obtained using fluorescein isothiocyanate-conjugated secondary antibody. In both cases, as with
rhodamine-phalloidin staining, a diffuse labelling in the
statocyte cytoplasm was observed except that the fluorescence intensity with CRA seemed to be stronger than
with the ICN antibody.
A new sectioning method suitable for the analysis of
microtubules has been developed (Brown et al., 1989)
and modified ( Vitha et al., 1997) to visualize F-actin
arrays (immunofluorescence on low melting point wax
sections). However, concerning the actin microfilaments
of statocytes (Baluska et al., 1997; Baluska and
Hasenstein, 1997), this staining provided almost the same
results as those obtained by rhodamine-phalloidin (Perbal
and Driss-Ecole, 1993) or by a more conventional
immunofluorescence technique ( Koropp and Volkmann,
1994).
The authors have chosen to adapt the pre-embedding
immunogold silver technique (Holgate et al., 1983;
Danscher and Rytter Nörgaard, 1983; Merdes and De
Mey, 1990; Vandenbosch, 1991; Burry et al., 1992) to the
material in order to visualize actin in the statocytes at
the electron microscopic level. This technique permitted
the location of G and F actin. Secondary antibody was
coupled with 10 nm gold particles. A pretreatment with
MBS has also been included (O’Sullivan et al., 1978;
Sutoh, 1984) which is effective in reducing the risk of
observing artificially bundled actin filaments (Sonobe and
Shibaoka, 1989).
Materials and methods
Dry seeds of Lens culinaris L. cv. Verte du Puy were placed on
cellulose sponges in culture chambers and hydrated. The
growing period was 28 h in darkness at 22 °C.
Immunoblotting
Soluble proteins from root tips (5 mm length) were prepared
according to the following procedure. 100 mg of root tips were
homogenized in liquid nitrogen with a mortar and a pestle.
Subsequently, the powder was suspensed in 9 ml of extraction
buffer (0.7 M saccharose, 0.5 M TRIS-HCl pH 7.5, 50 mM
EDTA, 0.1 M KCl, 2% b-mercaptoethanol (v/v), and 2 mM
PMSF ) for 10 min at 4 °C according to the phenol procedure
devised earlier (Schuster and Davies, 1983). The protein pellet
was washed three times with 0.1 M ammonium acetate in
methanol and once with acetone (100%) and then air dried.
For electrophoresis, soluble proteins were suspended in buffer
(Laemmli, 1970) and boiled for 3 min. 20–60 mg of protein
were loaded to a 10% SDS-polyacrylamide gel. The low
molecular range of proteins from Bio-Rad was used as a
standard. Protein content was determinated using the Bio-Rad
Laboratory kit. After electrophoresis (2 h at 20 mA), the
proteins were transfered to nitrocellulose sheets at 50 V for 3 h
at 4 °C using a Transblot cell (Bio-Rad, München, Germany).
Then the part of the membrane with standard proteins was
stained with Ponceau S (0.2% in 5% acetic acid ) and the other
part of the membrane subjected to immunostaining. The blots
were soaked in blocking solution overnight at 4 °C (TBS, 5%
dry milk, 0.02% NaN ), rinsed three times in TBS with 0.05%
3
Tween-20 and then incubated for 3 h at room temperature with
a mouse monoclonal antibody (Amersham N.350; anti-chicken
gizzard actin; dilution 151000).The antibody was diluted in
TBS, 0.05% Tween-20 and 5% dry milk. After washing three
times (in TBS with 0.05% Tween-20) the blots was incubated
in the secondary antibody for 1.5 h at room temperature (antimouse IgG-alkaline phosphatase Fab fragments; Boehringer
No. 1198661; dilution: 2.5 U ml−1). After three 10 min washing
periods in TBS-Tween-20 the blot was incubated in a development buffer (TRIS-MgCl pH 9.5) and then in a BCIP/NBT
2
colour development solution following the Bio-Rad procedure.
Controls omitted the primary antibody. No band was detected.
Pre-embedding immunogold labelling
At the end of the growing period, the seedlings were treated
for 10 min with 100 mM MBS (stock solution: 100 mM in
DMSO), washed in stabilizing buffer (SB: 50 mM PIPES, 5 mM
EGTA, 2 mM MgSO , pH 6.8) and were fixed in the vertical
4
position for 3 h at 4 °C with 3% paraformaldehyde plus 0.5%
glutaraldehyde in SB (according to Sonobe and Shibaoka,
1989). The roots were rinsed in SB and sectioned longitudinally
and axially in two parts under a stereomicroscope with a razor
blade. The two half-sections of the roots were treated for 5 min
by Triton X-100 0.5% in SB, washed in SB and then treated
twice with sodium borohydride (1 mg ml−1 in SB; according to
Merdes et al., 1991). Then the sections were treated for 30 min
in a blocking buffer (BB: 2% BSA, 1% fish gelatin, 1% NGS,
0.1% NaN , and 0.05% Tween-20) and then in a Tween solution
3
(0.025% in PBS). The sections were incubated overnight at 4 °C
with the primary anti-actin antibody (see previous section:
N-350 dilution 1=20 in BB, filtered through Whatman filter
device, 0.2 mm pore size).
Immunolocalization of actin in root statocyte
523
The sections, washed twice in filtered PBS and once in BB,
were incubated with the secondary antibody gold (10 nm)
conjugated goat anti-mouse IgG Sigma G-7652; dilution 1520
in BB). Following several washing periods with PBS and one
with BB the sections were post-fixed with 1.25% glutaraldehyde
in PBS for 15 min and rinsed in excess bi-distilled water.
Silver amplification were performed in the dark for 10 min
with SEM kit (Amersham). The sections were then washed in
excess bi-distilled water and stained en bloc with 0.5% uranyl
acetate in the dark for 10 min. Half-sections of the roots were
dehydrated in ethanol and embedded in araldite. Ultra-thin
sections were made for observation in electron microscopy.
Controls were run by omitting the primary antibody from the
first incubation: no labelling was detected on the sections.
Rhodamine-phalloidin staining
Seedlings were treated by MBS and washed in SB as described
above. Then they were fixed in the vertical position for 3 h at
4 °C by 3% paraformaldehyde in SB and rinsed in SB. The
roots were sectioned longitudinally and axially in two parts and
the sections were placed for 3 h in the dark at room temperature
in 0.22 mM rhodamine-phalloidin (Molecular Probes, Inc).
After a brief wash the sections were mounted in Mowiol
(Calbiochem) for examination by confocal microscopy
(MRC-600, Bio-Rad ).
Results
Rhodamine-phalloidin staining
By using confocal microscopy it is possible optically to
section the statocytes ( Fig. 1A). The section goes through
Fig. 2. Western immunoblotting of lentil root extract with a monoclonal
anti-actin antibody (Amersham, N 350): only one band at 43 kDa can
be seen. Numbers indicate the molecular weights of standard proteins
in kDa.
the amyloplasts and makes it possible to see that the
internal part of these organelles is not labelled. A diffuse
fluorescence is observed in the cytoplasm located around
the amyloplasts. At the proximal part of the statocyte the
optical section goes tangentially to the nucleus where a
bright and diffuse fluorescence can be detected as well as
some short and bright small strands (small group of
filaments; Fig. 1A, black arrows).
The most external cells of the root tip ( Fig. 1B) show
large vacuoles crossed by thin cytoplasmic trabeculae
( Fig. 1B, white arrow) which are labelled by rhodaminphalloidin demonstrating that actin is present at this level.
Some bright small strands can be seen in the cytoplasm
( Fig. 1B, black arrows) and around the amyloplasts. It
must be noted that typical bundles of microfilaments are
not observed in these cells as in Lepidium secretory cells
(Baluska et al., 1997).
Immunoblotting and immunogold-silver staining
Fig. 1. (A, B) Lentil root statocytes (A) and cell of the external part
of the root tip (B) stained by rhodamine-phalloidin and visualized by
confocal microscopy. (A) The focal plane is going through the
amyloplasts (a) and a diffuse fluorescence is observed in the cytoplasm
(cy) around these organelles. A tangential view of the nuclei (N ) at the
proximal pole of the statocytes shows a bright zone of actin which
surrounds these organelles. The vicinity of the nucleus display some
fluorescent small strands (black arrows). (B) Bright small strands
(black arrows) can be observed around the amyloplasts (a), and in the
transvacuolar trabeculae of cytoplasm (white arrow); v, vacuole. In
controls prepared without primary antibody fluorescence is not detected
in the cells.
The monoclonal anti-actin antibody (Fig. 2) recognizes
one polypeptide since only one band located at 43 kDa
can be revealed. This 43 kDa polypeptide corresponds
to actin.
Figure 3A–D shows a whole lentil root statocyte and
details of the nucleus, amyloplasts and ER of other
statocytes treated by the monoclonal anti actin antibody.
The gold–silver particles are spread all over the cytoplasm
( Fig. 3A) but the cell walls, the starch grains in the
amyloplasts ( Fig. 3A, C ), the mitochondria (Fig. 3A–D),
the nucleoplasm (Fig. 3A, B) do not show any particles.
In Fig. 3B (arrow) a longitudinal section of the statocyte
shows labelling in a cytoplasmic area surrounded by the
nucleoplasm. The density of the cytoplasm decreases
substantially upon a short extraction by Triton X-100
( Fig. 3B, C ), but gold–silver particles remain located
close to or on the nuclear envelope (Fig. 3B) or close to,
or on the amyloplast envelope ( Fig. 3A, C ). A few times,
dots are observed in the stroma of the amyloplasts
524 Driss-Ecole et al.
Fig. 3. Pre-embedding immunogold-silver labelling of actin on lentil root statocytes. The nucleoplasm N (A, B), the longitudinal, proximal and
distal walls (A–D), the mitochondria (A–D) and the starch grains in the amyloplasts (A, C ) are never labelled. Very few dots are localized in the
stroma of the amyloplasts (C ). The labelling is scattered in the cytoplasm (A) and in close vicinity to the nucleus envelope (B) or amyloplasts
envelope (C ). ER displays numerous dots (A, D) sometimes associated with the ER tubules (A). The gold-silver particles can be arranged in files
by two, three (B and D, arrowheads) or by four to six (D, arrow). (B) The arrow shows a hole in the nucleus through which the labelling of actin
in the cytoplasm can be seen. (C ) The arrow shows a thin trabeculae of cytoplasm associated with gold-silver particles demonstrating that actin is
present at this level. a, Amyloplast; d, dictyosome; dw, distal wall; ER, endoplasmic reticulum; lw, longitudinal wall; m, mitochondria; n, nucleolus;
N, nucleus; pw, proximal wall; s, stroma of the amyloplast; st, starch grain.
Immunolocalization of actin in root statocyte
525
Fig. 4. Pre-embedding immunogold-silver labelling of actin on cells located in the external part of the lentil root tip. As in the statocytes, the
nucleoplasm N (A), the longitudinal wall and the distal wall (A, B), the mitochondria and the starch grains in the amyloplasts (B) are not labelled.
There are no dots inside the vacuoles (A, B) but the tonoplast (A) and the transvacuolar trabeculae (A, B, arrows) are labelled. a, Amyloplast;
dw, distal wall; lw, longitudinal wall; N, nucleus; t, tonoplast; v, vacuole.
(Fig. 3C ). The remaining parts of cytoplasm are associated with labelled particles and sometimes the residues
look like filaments on which the dots are lined up (Fig. 3C,
arrow). Numerous dots are observed in the distal cytoplasm occupied by the ER (Fig. 3D) and the dots can be
linked to the ER tubules (Fig. 3A, ER). Frequently the
gold–silver particles are associated by two or three
(Fig. 3B, D, arrowheads) or by four to six dots ( Fig. 3D,
arrow) which correspond to a distance of 0.35–0.9 mm. It
must be stressed that these files of dots are not oriented
in a preferential direction.
Figure 4A and B show cells of the external layer of the
root tip identical to the cell observed in confocal microscopy (Fig. 1B). As in the statocytes, the particles are
spread over the cytoplasm, and the cell walls, the nucleoplasm (Fig. 4A), the mitochondria and the starch grains
in the amyloplasts ( Fig. 4B) are not labelled. No dots
can be detected in the vacuoles (Fig. 4A, B) but the
tonoplast and the transvacuolar trabeculae of cytoplasm
are labelled (Fig. 4A, B, arrow) demonstrating that actin
is present at this level.
Discussion
Taking account of the involvement of actin in the polarity
of lentil root statocytes and of the putative role of this
element of the cytoskeleton in the transduction of the
gravitropic signal, it was interesting to adapt a method
for the vizualization of actin in these cells at the electron
microscopic level. As suggested previously (Sonobe and
Shibaoka, 1989) MBS was used prior to fixation in order
to prevent the formation of artificially bundled actin
filaments and the pre-embedding immunogold silver technique was adjusted to the material. Immunocytochemistry
was carried out on root tips prior to their embedment in
order to increase antibody access to the antigen in the
absence of resin ( Vandenbosch, 1991). A good ultrastructural preservation and a maximum retention of antigenicity was insured by the use of a monoaldehyde
(paraformaldehyde) in addition to a small amount of
glutaraldehyde (Sonobe and Shibaoka, 1989; Merdes and
De Mey 1990; Merdes et al., 1991; Vandenbosch, 1991;
Koropp and Volkmann, 1994). After a phenol extraction
of the proteins of the root, only one band (at 43 kDa)
was obtained on the Western blot which demonstrated
the specificity of the antibody.
As with the rhodamine-phalloidin staining, the ultrathin sections of the root cap cells observed after immunoreaction showed that some areas were never labelled: the
cell wall, the nucleoplasm, the vacuoles, and the amyloplasts. The two methods permitted the observation that
actin surrounded the nucleus as had been demonstrated
526 Driss-Ecole et al.
in isolated cells previously (Seagull et al., 1987). In the
statocytes the nucleus was enclosed in a basket of actin
and was maintained at the proximal pole at least by these
elements of the cytoskeleton.
The immunogold technique had the advantage of
detecting labelling at the level of the organelles or in
some part of them. Many dots of labelling were located
in the region occupied by the ER and sometimes the dots
were on the ER tubules. In plants cells (epidermal cells
of onion bulb or Drosera tentacles, Chara cells, leaf cells
of Nicotiana) it has been established that actin was in
close association with ER (Quader et al., 1987; Kachar
and Reese, 1988; Lichtscheidl et al., 1990; Hepler et al.,
1990; Boevink et al., 1998) and that these structural
interactions were essential for intracellular movements.
In statocytes from cress roots, microfilaments could play
a role in the translocation of the ER from the proximal
to the distal part of these cells (Hensel, 1989). In lentil
root statocytes actin filaments would be necessary to
maintain the internal cohesion of the ER complex and
these elements of the cytoskeleton could be involved in
the movement of the ER tubules from the proximal to
the distal part during the differentiation of the statocytes
and in the stabilization of the complex near the distal
wall in mature statocytes. The interconnection between
the ER tubules and the amyloplasts via actin cytoskeleton
could account for a role of the ER in gravitropic process.
At the microscopic level the stroma of the amyloplasts
sometimes showed some particles, but the amyloplast
envelope was always associated with the gold-silver particles. The starch grains were never labelled. No dots
were observed in the inner part of the mitochondria, but
the labelling was close to the external membrane of this
organelle. The dots were also localized on the tonoplast.
Such linkage between the periphery of the vacuoles and
microfilaments was reported in human epithelial carcinoma cells (Henics and Wheatley, 1997) and could account
for the positioning of these organelles or for their movement in Saccharomyces cerevisiae as suggested previously
(Hill et al., 1996). The association between actin microfilaments and vacuoles was also reported in pollen tubes
of Pyrus communis ( Tiwari and Polito, 1988).
The dots of labelling were scattered in the cytoplasm
of the statocytes and could be linked to G actin since the
molecule could be detected by immunogold–silver staining
procedure. Nevertheless, the observation of the images
obtained by transmission electron microscopy allowed
the detection of associations of 2–6 particles in file
corresponding to 0.08–0.9 mm. These short files always
had various orientations within the cells and could be
associated with actin microfilaments (F actin). This observation could fit with a randomly interconnected network
of short units (according to the model proposed by
Forgacs, 1995). The bright small strands which were
observed with rhodamine-phalloidin should correspond
to some of these interconnected fibrous units.
Taking into account that the immunogold particles
were located very close to or on the membrane or on the
envelope of the statocyte organelles it could be considered
that these organelles were connected to the actin network.
This linkage allowed the understanding that each organelle movement could be controlled. It was proposed that
the disturbance of cytoskeletal microfilaments could generate the transduction step in the gravitropic phenomenon
(Sievers et al., 1989, 1991). From an experiment performed in space microgravity, a very short presentation
time for the lentil root (26–27 s) was obtained which was
consistent with the fact that actin filaments could transduce the gravitropic signal (Perbal and Driss-Ecole,
1994). This conception could be in agreement with the
more recent theories about intracellular signalling
(Ingber, 1991; Forgacs, 1995; Baluska et al., 1998). In
lentil root statocytes, the actin network, in association
with motor proteins, could also be responsible for the
movement of the amyloplasts observed in roots grown in
microgravity (Driss-Ecole et al., 2000).
Acknowledgements
This work was supported by the Centre National des Etudes
Spatiales (CNES). TEM observations were performed at the
Centre Interuniversitaire de Microscopie Electronique
(CIME ), Paris.
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