Plant Physiol. (1987) 85, 268-272
0032-0889/87/85/0268/05/$O 1.00/0
Actin of Chara Giant Internodal Cells
A SINGLE ISOFORM IN THE SUBCORTICAL FILAMENT BUNDLES AND A LARGER,
IMMUNOLOGICALLY RELATED PROTEIN IN THE CHLOROPLASTS
Received for publication February 18, 1987 and in revised form May 4, 1987
RICHARD E. WILLIAMSON*, DAVID W. MCCURDY, URSULA A. HURLEY, AND JEAN L. PERKIN
Plant Cell Biology Group, Research School ofBiological Sciences, The Australian National University,
Canberra, ACT 2601, Australia
dles but possessing the other structures that may contain actin;
an immunologically related protein of higher Mr exists inside the
ABSTRACT
Internodal cells of Chara cormllina Klein ex. Wild have been studied
to determine the number of actin isoforms they contain and whether actin
occurs at locations in the cortical cytoplasm outside the filament bundles.
A monoclonal antibody to chicken actin is specific for actin in numerous
animal cells but binds to two Chara proteins after their separation by
two-dimensional polyacrylamide gel electrophoresis. One protein resembles known actins in relative molecular mass (43,000-Mr) and isoelectric
point (5.5) while the other is distinctly different (58,000-Mn isoelectric
point = 4.8). Because it is indetectable in cells whose actin bundles have
been extracted, the 43,000-Mr protein is assigned to the bundles and
concluded to be rare or absent in the remaining cortical cytoplasm. The
58,000-M, protein, in contrast, does not extract with the actin bundles.
It was localized within the chloroplasts by immunofluorescence and by
the dependence of proteolysis on the permeabilization of the chloroplast
envelope.
chloroplasts.
MATERIALS AND METHODS
The following papers describe: the collection and cultivation
Actin has important roles in motility and in the cytoplasmic
architecture of plant (6,30) and animal cells. It is usually encoded
by a multigene family in animals (18) and some, but not all, of
the protein products can be separated by pl' and Mr (4, 7). In
plants, soybean and maize have small families of actin genes (5,
12, 19, 20) although the alga, Dunaliella, may have only one
(10). Only actin from Chlamydomonas flagella has been separated by the techniques usually required to identify isoforms and
only a single form was detected (16).
Internodal cells of Chara show cytoplasmic streaming that
involves subcortical bundles of actin filaments (14, 23). Actin
could, however, also occur elsewhere in the cell. For example:
pea chloroplasts contain 41,000- and 58,000-Mr proteins related
to actin (11); Chara Golgi bodies react with a monoclonal
antibody that binds to Chara actin bundles and can be preabsorbed with muscle F-actin (28); a detergent-insoluble layer
occurs beneath the Chara plasma membrane which could have
similarities to actin-rich meshworks in animal cells (25). However, the protein(s) at those locations in Chara have yet to be
adequately compared to the well-authenticated actin of the subcortical filament bundles.
In this study we have therefore used two-dimensional gel
electrophoresis to demonstrate that: a single resolvable actin
isoform occurs in the subcortical filament bundles; there are
indetectable quantities of this protein in cells lacking actin bun' Abbreviations: pl, isoelectric point; CHAPS, 3([3-cholamidopropyl]
-dimethylammonio)- I -propane sulfonate.
of Chara corallina Klein ex. Wild (26); the technique of vacuolar
perfusion (23); the composition of the solution containing ATP
and 10-7M free-Ca2l that removes tonoplast and endoplasm but
leaves intact the actin bundles (25); the low salt solution that
removes the actin bundles (24); the methods for expelling cell
contents, running one-dimensional SDS gels and staining with
silver (27); the running of two-dimensional gels (11) with CHAPS
(Sigma Chemical Co., St. Louis, Mo.); the transfer of proteins to
nitrocellulose and immunostaining (28) using a monoclonal
antibody to chicken actin (N350; Amersham International,
Bucks, England), biotinylated anti-mouse and streptavidin-peroxidase complexes (Amersham). One-dimensional gels were routinely loaded with the contents of four cells of approximately 50
mm length, two-dimensional gels with the contents of six cells.
Control immunoblots were processed identically except for the
omission of the monoclonal anti-actin. The pH gradient (measured by equilibrating 10-mm segments of duplicate rods with
distilled water) was essentially linear over the ranges shown in
Fig. 1, B and C, which used pH 5-6 and pH 4-6.5 Pharmalytes
(Pharmacia), respectively.
Comparing Immunoblotted and Silver-Stained Two-Dimensional Polyacrylamide Gels. To compare immuno- and silverstained two-dimensional separations, isoelectric focusing rods
were laid on a slab assembly divided vertically by a thin glass
plate. Focused proteins therefore partitioned between two slabs
that could be used for silver and antibody staining, respectively.
The nitrocellulose sheet was cut to the exact size of the pretransfer
gel and silver and immunostained spots compared by x,y coordinates expressed as fractions of the width and length of the
stained gel and replica, respectively.
Immunofluorescence. The methods used for immunofluorescence microscopy of Chara have been described (28). Cell segments or perfused cells were treated before fixation with either
the low salt or the 10-' M Ca2" solutions, fixed in 1% (w/v)
paraformaldehyde for 20 min and, where required, treated subsequently for 7 min with ice-cold acetone. Antibodies to spinach
ribulose bisphosphate carboxylase was the gift of Dr. J. Seeman.
Proteolysis. Proteolysis used thermolysin (3) at 0.5 mg/ml in
high Ca2" perfusion solution (200 mm sucrose, 70 mM KCI, 4
mM MgCl2, 0.5 mM CaCl2, 10 mm Pipes, pH 7.0). Cells were
perfused for 3 min with high Ca2l solution. Samples with intact
chloroplast envelopes were then briefly perfused with protease,
left for 30 min at 4°C, their contents expelled and made 25 mM
268
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ACTIN IN CHARA
269
for EDTA to inhibit the thermolysin. Samples with permeabil- main spot, one had an identical Mr and a pI about 0.1 pH unit
ized chloroplast envelopes were prepared by expelling the cell more acidic, the other a slightly lower pI and a M, of approxicontents after the initial perfusion, making the expelled material mately 38,000.
1% (v/v) for Triton X-100 and 0.5 mg/ml for thermolysin,
Identification of an Actin-Related Protein. When the isoelectric
holding at 4°C for 30 min and then inhibiting proteolysis with focusing gradient was extended to lower pH, a second protein
EDTA. Samples were prepared for one-dimensional SDS-PAGE (58,000-MA, pI = 4.8) reacted with the monoclonal anti-actin
and immunoblotted by the standard methods using Ponceau S (Fig. 1C). This protein was retained in cells depleted of actin
to stain for total protein on the nitrocellulose before immuno- bundles whereas the 43,000-Mr protein was no longer detected
staining ( 17).
by immunostaining (Fig. 2). Since 43,000-Mr actin could be
detected in a sample equivalent to 0.2 whole cells but not in one
RESULTS
equivalent to four cells lacking actin bundles, an approximate
estimate is that less than 5% (0.2 x 100/4) of the cell's 43,000Identification of Actin. After one-dimensional SDS-PAGE, Mr
actin remains after extracting the actin bundles. The intensity
Chara actin migrates with a Mr of 43,000, slightly behind muscle
actin (27) and, based on other actins, would be expected to have of the immunostaining reaction at 43,000Mr was less for pera pl of approximately 5.4 (4, 7). To identify the actin of the fused cells with actin bundles than for whole cells (Fig. 2).
Localization of Actin-Related Proteins. Indirect immunofluofilament bundles, we used two-dimensional PAGE to compare
rescence
was carried out to localize the 43,000- and 58,000-Mr
the proteins of perfused cells having actin bundles with the
proteins of cells from which the actin bundles had been extracted proteins detected with the monoclonal anti-actin. No reaction
with low salt. Only one of several spots in the appropriate region was obtained with the anti-actin using formaldehyde and perfuwas lost (Fig. IA). The identification of the spot as actin and the sion solutions-conditions allowing several monoclonal antiabsence of other actin isoforms was confirmed by immunostain- bodies (28) and one polyclonal antiserum (29) to bind to the
ing with monoclonal anti-actin extracts of whole cells and of actin bundles. When acetone was used in the preparative proceperfused cells containing actin bundles. A single 43,000-Mr spot dure, however, strong binding of the anti-actin to the actin
was stained in both cases (Fig. 1B) and located to within 2 mm bundles and the periphery of the chloroplasts was obtained (Fig.
of the extractable spot on a duplicate silver-stained gel. (This 3A). Immunofluorescence carried out on cells from which low
divergence was comparable to that seen between the correspond- salt perfusion had extracted the 43,000- but not the 58,000-Mr
ing spots on duplicate silver-stained gels.) There were no indica- immunoreactive protein (Fig. 2), showed that the monoclonal
tions that either the silver or the immunostained spot could be anti-actin still bound to the chloroplasts but not to the actin
resolved further, even though the focusing gradient was as shal- bundles (Fig. 3B). (See Ref. 24 for the rapidity and lack of visible
low as 0.01 pH unit/mm. A small minority of immunoblots chloroplast damage associated with the low salt extraction of
showed two very minor spots (data not shown). Relative to the bundles.) A similar pattern of predominantly peripheral labeling
!
6.0
4.5
Imil;;L
...:
S
-
-
4
**
*
~.
..1
Is
A
4
5.9
43.5.5
B
6.1
58, 4.8
5.0
@*
*
FIG. 1. Chara proteins separated by two-dimensional
PAGE and stained with silver (A, A') or with anti-actin (B,
C). The pH gradient measured on duplicate rods is shown
at the top of each panel. A, Silver-stained gel showing the
proteins of six perfused cells that contained actin bundles.
The inset (A') shows part of a gel on which proteins from
six perfused cells without actin bundles had been separated.
Compared to the equivalent area of the main gel (dotted
line), a single spot (arrow; 43,000-M, pl = 5.5) is lost when
the bundles are removed. While differing in abundance
between the two gels, all the major proteins around the
missing spot can be detected on the two gels (e.g. the two
proteins just above and the three proteins just below the
putative actin). B, Among the proteins from cells containing actin bundles that are resolved on a pH 5-6 gradient
and transferred to nitrocellulose, only a single spot (arrow)
with the same fractional coordinates as that identified in
(A) reacts with anti-actin. C, A second spot (arrow: 58,000Mr, pl = 4.8) stained with anti-actin when the gradient was
extended to more acidic pH. A small amount of 43,000Mr actin failed to enter the pH gradient (arrowhead). The
major 43,000-M, isoform is to the right of this and is well
resolved from the 58,000-M, spot.
43, 5.5
C
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Copyright © 1987 American Society of Plant Biologists. All rights reserved.
270
WILLIAMSON ET AL.
2
1
Plant Physiol. Vol. 85, 1987
3
94--
NT.
7-
O N
.
..
.J..
*
43 - -mw
W Nm
_
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-
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~~~~~~~~~-/
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30-
20FIG. 2. Nitrocellulose sheets carrying proteins transferred from a onedimensional SDS polyacrylamide gel and stained with monoclonal antiactin. Lane 1, proteins from whole Chara cells; lane 2, perfused cells
containing actin bundles; lane 3, perfused cells lacking actin bundles.
The 43,000-Mr immunostained band is depleted by perfusion (lane 2
versus lane 1) and fully extracted by low salt (lane 3). The 58,000-M,
immunostained band is not reliably depleted by either treatment.
of acetone-permeabilized chloroplasts was obtained using an
antibody to ribulose bisphosphate carboxylase (data not shown).
To decide whether the 58,000-Mr chloroplast protein was
exposed on the chloroplast surface or protected by the chloroplast
envelope, proteolysis of Chara proteins with thermolysin was
performed under conditions in which an intact chloroplast envelope would protect intrachloroplastic proteins and after permeabilizing the envelope to expose the intrachloroplastic proteins. The validity of the conditions was demonstrated by the
behaviour of a known stromal polypeptide, the large subunit of
ribulose bisphosphate carboxylase (Fig. 4A). This was degraded
only when the chloroplast envelope had been permeabilized. The
58,000-Mr immunoreactive protein behaved identically (Fig. 4B),
whereas the 43,000-Mr immunoreactive protein was degraded in
both the permeabilized and unpermeabilized preparations (Fig.
4B).
DISCUSSION
Internodal cells of Chara have a single 43,000-M, protein (pl
= 5.5) identifiable as actin by two criteria. It is specifically
extracted when cells are perfused with a low salt solution that
removes the subcortical actin bundles (24) and it is stained by
the monoclonal antibody to chicken actin. In addition, a band
from one-dimensional gels with the same Mr and extraction
properties gives fragments resembling those from rabbit muscle
actin when cleaved at Trp residues (27). A very minor spot
having the same Mr and a pl about 0.1 pH unit more acidic than
the major spot was occasionally immunostained. It has the
FIG. 3. Immunofluorescent localization of 43,000- and 58,000-M,
actin-related proteins using monoclonal anti-actin. A, Binding of monoclonal anti-actin to Chara actin bundles and chloroplasts treated with
acetone. B, Cell perfused with low salt before fixation so that it contained
58,000 but not 43,000-Mr protein (see Fig. 2). Labeling is confined to
the periphery of the chloroplasts. Bar represents 30 ,m.
A. Ponceau S
An
t
LS
123
FIG. 4. Proteolysis experiment localizing the 58,000OMr protein inside
the chloroplasts and the 43,000-Mr protein outside the chloroplasts.
Proteins were resolved by one-dimensional SDS-PAGE, tnsferred to
nitrocellulose and stained with Ponceau S (panel A) and monoclonal
anti-actin (panel B). The lanes contain extracts of perfused cells with: 1,
intact chloroplasts without thermolysin; 2, intact chloroplasts with thermolysin; 3, permeabilized chloroplasts with thermolysin; 4, permeabilized chloroplasts without thermolysin. The 43,000-Mr immunostained
protein is degraded without chloroplast permeabilization (panel B, lane
2). In contrast, the 58,000Mr immunostained protein (panel B, lane 3)
resembles a known stromal protein, ribulose bisphosphate carboxylase
large subunit (LS, panel A, lane 3), in being degraded only after chloroplast permeabilization.
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ACTIN IN CHARA
characteristics of a charge heterogeneity artifact (4, 11, 13). The
well documented actin isoforms of mammals differ by less than
the single charge likely to be involved in this case (4). The very
occasionally detected spot at 38,000-M may result from slight
proteolysis; actins readily cleave to a fragment of this size that is
resistant to several proteases and commonly contaminates actin
preparations (15). Neither of these artifactual spots could be
detected on a duplicate silver stained gel, emphasizing the ability
of immunostaining to recognize trace quantities of other isoforms. Given that the monoclonal anti-actin reacts with all actin
isoforms from diverse animal species (8, 9, 22), it seems unlikely
that an isoform lacking the relevant epitope would occur in
Chara. The ability of the present techniques to recognize isoforms is shown by similar experiments with seedlings of Arabidopsis thaliana where five immunoreactive isoforms of 43,000Mr actin are resolved (RE Williamson, DW McCurdy, UA
Hurley, unpublished results). We conclude, therefore, that mature Chara internodal cells have a single major electrophoretic
isoform of 43,000-Mr actin. However, because Acanthamoeba
has an isoform representing only 1.5% of its total actin (7), the
existence of such minor species in Chara cannot yet be excluded.
Judged by the intensity of staining with monoclonal anti-actin,
whole cells have considerably more 43,000-Mr actin than perfused cells (Fig. 2). This is consistent with: the same protein
occurring in the endoplasm that is extracted by perfusion (23);
part of the actin in the subcortical bundles being extracted by
perfusion; actin extracting from another (unknown) site in the
cortex. By analogy with nonmuscle animal cells (1), substantial
amounts of unpolymerized (and therefore extractable) actin are
likely to exist in Chara and account for much, perhaps all, of
the actin lost on perfusion. When the actin bundles have been
extracted by low salt, no 43,000OMr actin is detected on either
immuno- or silver-stained gels. As a first approximation we
estimated that immunostaining would have detected actin if 5%
of the actin present in unperfused cells was retained in low-saltextracted cells. While this estimate cannot be considered highly
accurate it gives an approximate upper limit to the quantity of
43,000-M, actin that could exist on Golgi bodies and inside
chloroplasts, both of which survive the low-salt extraction that
removes the actin bundles (24, 28). Organelle anchorage via the
cortical gel and a visible detergent-insoluble layer beneath the
plasma membrane also survive low-salt extraction (25). It seems
unlikely, however, that such small quantities of actin (less than
5% of the original actin) would allow this protein to be a major
structural component of the subplasmalemmal region. We therefore consider that there is currently no evidence for 43,000-M,
actin occurring in perfused cells at sites other than the actin
bundles.
A 58,0004Mr protein in a pea chloroplast preparation binds
the monoclonal anti-actin used in this study (1 1). A 58,000-M,
protein was also detected by one-dimensional immunoblots of
Chara proteins ( 11) and we have now shown that it migrates as
a single spot with a pl of 4.8. Unlike 43,000-Mr actin (this report)
and two other components of the actin bundles (27), the 58,000Mr protein is retained in cells lacking actin bundles. It therefore
cannot be co-polymerized with actin in the filament bundles in
the way proposed for arthrin, a 55,000Mr form of actin found
in insect flight muscle (2). Since the monoclonal anti-actin can
detect only the 58,000-Mr protein in Chara cells lacking filament
bundles, antibody binding to the chloroplasts of such cells associates the 58,000-M, protein with those organelles. A similar
location was deduced for the 58,000Mr pea protein (1 1). Because
antibodies localize the major stromal protein ribulose biphosphate carboxylase in a similar, largely peripheral pattern, we
suspect that antibodies do not have access to the whole stromal
compartment. The peripheral labeling with anti-actin may therefore underestimate the distribution of the 58,000-M, protein
271
within the chloroplasts.
Acetone treatment was required for the monoclonal anti-actin
to immunofluorescently label the actin bundles and chloroplasts.
This precluded carrying out immunofluorescence under conditions in which the chloroplast envelope would be intact to
determine whether the 58,000-M, chloroplast-associated protein
was accessible on the surface of the chloroplast envelope or
protected by being intrachloroplastic. (The acetone is probably
required to denature the proteins of the actin bundles rather than
to remove any permeability barrier since the bundles are already
accessible to many antibodies [28, 29], colloidal gold particles
[28] and myosin-coated plastic beads [21]. Moreover, detergent
treatment [1% (v/v) Triton X-100] cannot substitute for the
acetone.) This difficulty was circumvented by comparing proteolysis of Chara proteins under conditions where the envelope
membranes were intact and after permeabilizing them with
detergent (see Ref. 3 regarding the general validity of the method
and the advantages of thermolysin). The 58,000-Mr protein was
degraded only after permeabilizing the chloroplast envelope,
indicating that it is not exposed on the cytoplasmic surface of
the outer envelope membrane and is therefore intrachloroplastic.
The behavior of two proteins whose locations are known support
the validity of this procedure used for Chara: a stromal protein,
the large subunit of ribulose bisphosphate carboxylase, was degraded only after envelope permeabilization, whereas the 43,000Mr actin of the bundles on the envelope surface (14) was degraded
both with intact and permeabilized chloroplast envelopes. (The
presence of other cortical organelles besides the chloroplasts does
not compromise this experiment, since immunofluorescence
shows that they lack proteins reacting with the anti-actin.)
The behavior of the Chara 43,000-Mr protein contrasts with
that shown by a 41,000-M, actin-related protein associated with
pea chloroplasts ( 11); the latter was degraded by thermolysin
only after rupturing the chloroplasts. It remains to establish why
such an actin-related protein occurs inside pea but not (in
detectable quantities) inside Chara chloroplasts. While both pea
(1 1) and Chara chloroplasts therefore contain 58,000-Mr proteins
that react with the monoclonal anti-actin, the extent of the actinrelated sequences in those proteins remains to be determined.
The pea protein does, however, resemble actin in being retained
by a DNase I column (1 1).
In conclusion, Chara internodal cells have a single 43,000-M,
actin isoform in their actin bundles and a 58,000-Mr protein in
their chloroplasts that shares some sequence homology with
actin.
Acknowledgments-We thank Dr. R. Teliam for purified muscle actin, Dr. J.
Seeman for antibodies to ribulose bisphosphate carboxylase, and Dr. P. Jablonski
for comments on the manuscript.
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