Plant Cell Physiol. 48(4): 585–597 (2007)
doi:10.1093/pcp/pcm030, available online at www.pcp.oxfordjournals.org
ß The Author 2007. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: [email protected]
Wide-Ranging Effects of Eight Cytochalasins and Latrunculin A and B
on Intracellular Motility and Actin Filament Reorganization in Characean
Internodal Cells
Ilse Foissner
1
2
1,
* and Geoffrey O. Wasteneys
2
Department of Cell Biology, University of Salzburg, Salzburg, Austria
Department of Botany, University of British Columbia, Vancouver, Canada
Introduction
Numerous forms of cytochalasins have been identified
and, although they share common biological activity, they may
differ considerably in potency. We investigated the effects of
cytochalasins A, B, C, D, E, H and J and dihydrocytochalasin
B in an ideal experimental system for cell motility, the giant
internodal cells of the characean alga Nitella pseudoflabellata.
Cytochalasins D (60 kM) and H (30 kM) were found to be most
suited for fast and reversible inhibition of actin-based motility,
while cytochalasins A and E arrested streaming at lower
concentrations but irreversibly. We observed no clear correlation between the ability of cytochalasins to inhibit motility and
the actual disruption of the subcortical actin bundle tracks on
which myosin-dependent motility occurs. Indeed, the actin
bundles remained intact at the time of streaming cessation and
disassembled only after one to several days’ treatment. Even
when applied at concentrations lower than that required to
inhibit cytoplasmic streaming, all of the cytochalasins induced
reorganization of the more labile cortical actin filaments into
actin patches, swirling clusters or short rods. Latrunculins
A and B arrested streaming only after disrupting the
subcortical actin bundles, a process requiring relatively high
concentrations (200 kM) and very long treatment periods of
41 d. Latrunculins, however, worked synergistically with
cytochalasins. A 1 h treatment with 15 nM latrunculin A and
4 mM cytochalasin D induced reversible fragmentation of
subcortical actin bundles and arrested cytoplasmic streaming.
Our findings provide insights into the mechanisms by which
cytochalasins and latrunculins interfere with characean actin
to inhibit motility.
Actin-targeted fungal metabolites known as cytochalasins have been used extensively to understand the role of
F-actin in different aspects of cellular function (Petersen
and Mitchison 2002). It is generally acknowledged that
these compounds cap the plus ends, and thereby alter the
dynamic properties of actin filaments. Intracellular motility
in plant cells is predominantly generated by myosindependent movement of organelles along actin filaments
(Staiger et al. 2000). In the giant internodal cells of
characean algae, cytochalasin B (CB) and cytochalasin D
(CD) rapidly inhibit cytoplasmic streaming without disassembling the prominent and highly stable actin bundles
located at the interface of the stationary cortex and the
motile endoplasm (Chen 1973, Bradley 1973, Williamson
1975, Foissner and Wasteneys 2000b). In contrast, the
delicate cortical actin filaments near the plasma membrane
are readily reorganized into short stable rods upon
application of CB and CD (Collings et al. 1995), and
mechanical disruption of the subcortical actin bundles by
wounding allows CD to reorganize them extensively into
thick networks (Foissner and Wasteneys 1999). These and
other findings indicate that cytochalasins have greater
nucleating and bundling properties when actin networks
are relatively labile (e.g. Goddette and Frieden 1986).
Nevertheless, it is still a mystery how cytochalasins inhibit
and arrest organelle motility without affecting the structure
or polymer status of the long, continuous bundles of actin
filaments along which these organelles move. Several
different cytochalasins are commercially available, and
structure–activity relationships have shown that subtle
changes in molecular structure can result in dramatic
differences in specific activities (Walling et al. 1988).
One aim of the present study was to exploit the highly
organized and reproducible cytoplasmic features of characean giant internodal cells to compare and quantify the
effects of different cytochalasins on cytoplasmic streaming
and on the arrangement of the actin cytoskeleton.
Specifically, we wanted to determine if the ability to arrest
Keywords: Cortical actin filaments — Cytoplasmic
streaming — Cytoskeleton — Nitella — Subcortical
actin bundles.
Abbreviations:
ADF,
actin-depolymerizing
factor;
CA, cytochalasin A; CB, cytochalasin B; CC, cytochalasin C;
CD, cytochalasin D; CE, cytochalasin E; CH, cytochalasin H;
CJ, cytochalasin J; DHCB, dihydrocytochalasin B; DMSO,
dimethylsulfoxide; FITC, fluorescein isothiocyanate; EGTA,
ethyleneglycoltetraacetic acid; LatA, latrunculin A; LatB,
latrunculin B; MBS, m-maleimidobenzoyl N-hydroxysuccinimide
ester; PIPES, piperazine-N,N0 -bis (2-ethanesulfonic acid).
*Corresponding author: E-mail, [email protected]; Fax, þ43-662-8044-619.
585
586
Wide-ranging effects of cytochalasins
cytoplasmic streaming is correlated with the ability of a
given cytochalasin to alter the structural organization of
actin filament arrays in an attempt to clarify the role of
actin filament reorganization in streaming inhibition.
We therefore undertook a rigorous evaluation of the effects
of eight different cytochalasins in a broad concentration
range, on both actin filament architecture and the inhibition
and recovery of cytoplasmic streaming.
With the discovery of latrunculins isolated from sea
sponges, other actin-perturbing drugs with a more defined
and less complex action became available (Spector et al.
1983). Latrunculins are monomer sequestering agents and
disassemble the actin cytoskeleton of rapidly growing cells
such as pollen tubes at nanomolar concentrations
(e.g. Gibbon et al. 1999, Vidali et al. 2001, Foissner et al.
2002). During the course of this study, we also investigated
the effects of latrunculins A (LatA) and B (LatB) and
found that, unlike studies with higher plants, they are
not able rapidly and reversibly to inhibit cytoplasmic
streaming in characean internodal cells. Latrunculins,
however, were found to work synergistically with cytochalasins, providing greater insight into the mechanisms by
which cytochalasins and latrunculins interact with actin
filament networks.
Results
The cytoplasm of characean internodal cells consists of
a stationary cortex including files of helically arranged
chloroplasts and a streaming endoplasm (Fig. 1A).
A
A
B
C
C
W
EC
EN
Fig. 1 Cytoplasmic and cytoskeletal organization in characean
internodal cells. (A) Schematic longitudinal section showing the
cell wall (W), the stationary ectoplasm (EC) including files of
helically arranged chloroplasts (C) and the streaming endoplasm
(EN). The cortical cytoskeleton (left arrow) consists of actin
filaments (thin lines and circles) and microtubules (thick lines and
circles) near the plasma membrane. Subcortical actin bundles (right
arrow) are present along the inner side of the chloroplasts.
(B) Randomly oriented actin strands and thicker bundles in the
cortex of a non-elongating internode. (C) Continuous subcortical
actin bundles along the chloroplast files. F-actin was visualized by
perfusion of cells with fluorescent phalloidin. Scale bar for b and
c ¼ 10 mm.
The plasma membrane-associated, or cortical, actin cytoskeleton of untreated, non-elongating characean internodes
consists of randomly oriented, delicate, often bent actin
strands, and thicker, straight bundles which are usually
extensions of the subcortical actin bundles located along the
inner side of the stationary chloroplast files (Fig. 1A, B).
These bundles consist of up to 100 single actin filaments and
generate endoplasmic mass streaming through the interaction with myosin associated with the membranes of
endoplasmic reticulum cisternae (Fig. 1A, C; recent reviews
by Foissner and Wasteneys 2000a, Grolig and Pierson 2000,
Shimmen and Yokota 2004).
Inhibition of cytoplasmic streaming and recovery
The effect of cytochalasins on cytoplasmic streaming in
characean internodes is age dependent. Younger cells are
more susceptible and require lower inhibitor concentrations
for streaming arrest than older cells (Collings et al. 1995).
In this study, we used non-elongating internodes of approximately the same age, which were collected from the same
container in order to avoid any differences between culture
batches. We use the terminology from a previous paper
(Foissner and Wasteneys 2000b), in which cytochalasin
concentrations were defined as follows: streaming-arresting
concentration ¼ cytochalasin concentration at or above the
level required to arrest streaming within 1 h; streaminginhibiting concentration ¼ cytochalasin concentration at
which streaming velocity is reduced by 15–80% within 1 h
but not arrested. The streaming velocity in untreated
control cells was about 80 mm s–1 at room temperature.
Fig. 2 shows the time course of streaming inhibition
and recovery of the various cytochalasins used in this study,
and Fig. 3 illustrates the mean relative velocities of cytoplasmic streaming after 1 h recovery from a 1 h treatment at
the predetermined streaming-arresting concentration for
each cytochalasin, maximum recovery velocities and times
required for maximal recovery. The streaming-arresting
concentrations of cytochalasins A, B, D, E, H and
dihydrocytochalasin B (CA, CB, CD, CE, CH and DHCB)
varied between 1 and 200 mM (Fig. 2). Recovery to control
rates of cytoplasmic streaming after 1 h treatment with
streaming-arresting concentrations took about 1 h with CD
and CH, and several hours with CB and DHCB.
Cells treated with CA or CE recovered to 80% of the
original velocities, and these values were reached only after
3 and 5 d, respectively (Fig. 3). Treatment with 200 mM
cytochalasin C (CC) or 200 mM cytochalasin J (CJ) for 1 h
had no significant effect on the velocity of cytoplasmic
streaming (Fig. 2).
All cytochalasins induce the formation of cortical actin rods
with variable length and density
All cytochalasins caused reversible reorganization
of cortical actin filaments at streaming-arresting
Wide-ranging effects of cytochalasins
587
Fig. 2 Time course of cytochalasin-induced streaming inhibition and recovery in internodal cells of N. pseudoflabellata. Cytochalasins
were added immediately after assessing the control rate at 0 min. After 60 min treatment, the inhibitor-containing solution was replaced by
artificial fresh water. Data are means SD.
concentrations, streaming-inhibiting concentrations and
even, in the case of CC and CJ, at concentrations that
had no effect on cytoplasmic streaming. The minimum
concentrations required for cortical actin reorganization
were between 1 and 20% of the concentration required to
arrest streaming. The extent of reorganization varied
considerably between the cytochalasins used, their concentration and the length of treatment. At streaming-inhibiting
concentrations, actin reorganization took longer and
changes were often less pronounced than at streamingarresting concentrations.
CA induced a distinctive swirling pattern of locally
parallel but variably oriented clusters of actin filaments that
frequently appeared frayed or branched (Fig. 4A).
The length of the actin strands or bundles could not be
determined because of their high degree of overlap. Their
density varied along the cell surface (see CE treatments for
more details and figures), but the narrow strips between the
two opposing flows of streaming endoplasm known as
neutral lines were always devoid of F-actin. This curving
actin pattern formed within one to several hours at the
streaming-arresting concentration and persisted for several
hours. The curving pattern was later replaced by short,
isolated actin rods (Fig. 4B) and these were also found at
the neutral line. The cortical rods had a random orientation
typical for non-growing internodes as previously shown for
CD (Collings et al. 1995). Reorganization of cortical F-actin
was reversible even after prolonged incubation in CA
(Fig. 4C).
A similar, transient increase in cortical F-actin was
observed with CB (Fig. 4D, E), but the newly formed F-actin
appeared as short rods from the very beginning and these
were not locally aligned, probably because they were less
dense. DHCB had the smallest effect on the cortical F-actin,
and delicate strands were still present after 1 h treatment at
the streaming-arresting concentration. Only after one to
several days treatment at the streaming-arresting concentration or streaming-inhibiting concentration were actin
588
Wide-ranging effects of cytochalasins
With all cytochalasins tested, cells recovered cortical
actin strands after transfer to inhibitor-free artificial fresh
water (e.g. Fig. 4C). The recovery times were below those
required for recovery of cytoplasmic streaming (not shown).
Fig. 3 Cytochalasins vary in potency to arrest cytoplasmic
streaming in N. pseudoflabellata internodal cells and in the rate
of recovery after their removal. Concentrations required to arrest
cytoplasmic streaming within 1 h are listed on the x-axis. Mean
relative velocities (left axis) of cytoplasmic streaming after 1 h
recovery from 1 h treatment with streaming-arresting concentrations are shown by white bars, maximum recovery velocities are
shown by gray bars, and times (right axis) required for maximum
recovery are shown by black bars. Data are means SD.
patches and short isolated actin rods found in the cortex
(Fig. 4F, G). CD caused the formation of variably oriented
but locally aligned short actin filaments (Fig. 4H, I),
the density of which differed along the cell surface
(not shown). The initial swirling pattern was, after longer
incubation times, replaced by actin patches and scattered
actin rods (Fig. 4J). CE had similar effects to CA and CD,
and the cells exhibited a characteristic cortical zonation,
where regions with a high density of F-actin abruptly merged
with regions of lower F-actin density (Fig. 4K), both
extending over several hundred micrometers. F-actin was
absent not only from the neutral line but also from circular
areas 10–20 mm across, that were randomly scattered over
the cell surface. The dense swirling pattern was later
replaced by isolated short rods (Fig. 4L). The effects of
CH (Fig. 4M, N) were similar to those observed with
CD and CE. However, after several days’ treatment at
streaming-inhibiting concentrations, the cortex contained
not only isolated short actin rods but also huge clusters of
thick, randomly arranged branching networks of actin
spears that extended into the subcortex (Fig. 4O).
CC (Fig. 4P) and CJ (Fig. 4Q), which both caused only a
slight streaming inhibition after several hours or days of
treatment, induced the formation of isolated, short actin
rods and longer spears without a previous increase in
cortical F-actin, just like the streaming-inhibiting concentrations of the more potent cytochalasins.
Cytochalasin-induced reorganization of subcortical actin
bundles requires several days treatment
The effects of cytochalasins on the subcortical actin
bundles were less immediate than those observed with
cortical actin, and from one to several days’ treatment was
necessary to induce reorganization, if it occurred, even
if cytoplasmic streaming had been arrested. Up to
2 d treatment with CA at streaming-arresting concentrations had no significant effect on the morphology of the
subcortical actin bundles, and 4–7 continuous actin bundles
ran parallel to each cortical chloroplast file, just like in
untreated cells (Fig. 5A). The small percentage of fragmented subcortical actin bundles, i.e. subcortical actin bundles
not continuous over 50 mm in the area investigated, was due
to those bundles extending through the chloroplast files
into the cortical regions, as in control cells (Fig. 6).
Fragmentation of subcortical actin bundles significantly
increased after 2 d treatment at streaming-inhibiting concentrations (Figs. 5B, 6) or following recovery from
treatment with streaming-arresting concentrations that left
the subcortical actin bundles intact (Fig. 5C). Subcortical
actin bundles first disappeared from regions between the
chloroplasts, whereas those parts that had close contact
with the chloroplast surface persisted longer. Some of the
subcortical actin bundles were oblique to the chloroplast
files (Fig. 5B), an orientation rarely found in untreated cells.
CB caused a slight decrease in the mean number of
subcortical actin bundles per chloroplast file after two to
several days treatment at its streaming-inhibiting concentration, but most of the subcortical actin bundles remained
intact (Figs. 5D, 6). DHCB at its streaming-arresting
concentration significantly reduced the mean number of
subcortical actin bundles after 2 d incubation and, interestingly, this effect was even more pronounced at its lower
streaming-inhibiting concentration (Figs. 5E, 6). With the
latter treatment, only about 60% of the subcortical actin
bundles remained continuous over at least 50 mm, and these
subcortical actin bundles were often much thicker than the
short remnants of the fragmented subcortical actin bundles.
CD, CE and CH were most effective in subcortical actin
bundle fragmentation at both their streaming-arresting
concentrations and streaming-inhibiting concentrations
(Figs. 5F–I, 6). With CD and CH, the subcortical actin
bundle fragments were further degraded and, consequently,
their number per chloroplast file declined significantly
(Figs. 5F, I, 6). The thickness of the subcortical actin
bundle fragments varied considerably, and some of
them were oriented obliquely to the chloroplast files.
Wide-ranging effects of cytochalasins
A
CA, SAC
B
CA, SAC
589
C
D
E
CA, recovery
CB, SAC
CB, SAC
F
G
H
I
J
DHCB, SAC
DHCB, SIC
CD, SAC
CD, SIC
CD, SIC
K
L
O
CE, SAC
CE, SAC
CC, SIC
M
N
Q
CH, SAC
CH, SIC
CH, SIC
P
CJ, SIC
Fig. 4 Cortical F-actin can be reorganized in internodal cells of N. pseudoflabellata treated with cytochalasins at concentrations that
inhibit (SIC) or arrest (SAC) cytoplasmic streaming. (A–C) Cytochalasin A: 1 mM, 3 h (A), 1 mM, 1 d (B), 1 mM, 1 d and 2 d recovery in
AFW (C). (D and E) Cytochalasin B: 200 mM, 1 h (D) and 200 mM, 2 h (E). (F and G) Dihydrocytochalasin B: 100 mM, 1 d (F) and 150 mM,
2 d (G). (H–J) Cytochalasin D: 70 mM, 1.5 h (H), 10 mM, 1.5 h (I) and 10 mM, 1 d (J). (K and L) Cytochalasin E: 3 mM, 3 h (K) and 3 mM, 1 d (L).
Note the heterogeneous distribution of F-actin in K. (M–O) Cytochalasin H: 75 mM, 3 h (M), 10 mM, 2 h (N) and 50 mM, 2 d (O). Note local
accumulation of actin spears. (P) Cytochalasin C: 100 mM, 2 h. (Q) Cytochalasin J: 100 mM, 2 h. F-actin was stained by perfusion of cells
with fluorescent phalloidin. Scale bar ¼ 10 mm (O) and 5 mm (all other figures).
Both fragmentation and a decline in subcortical actin
bundle number by CD and CH were reversible, and cells
formed continuous, nearly evenly thick subcortical
actin bundles parallel to the chloroplast files after 2 d
recovery in artificial fresh water (Figs. 5G, 6). With CE,
however, all subcortical actin bundles became irreversibly
fragmented after 2 d treatment at a streaming-arresting or
streaming-inhibiting concentration, and the fragments
persisted along the inner chloroplast surface (Figs. 5H, 6).
Subcortical actin bundles also became fragmented after 2 d
recovery from 1 h treatment at the streaming-arresting
concentration although the subcortical actin bundles were
intact when cells were transferred to artificial fresh water
(not shown, compare CA). CC did not cause reorganization
590
Wide-ranging effects of cytochalasins
A
CA, SAC
F
CD, SAC
B
C
CA, SIC
G
CD, recovery
D
CA, recovery
E
I
H
CE, recovery
DHCB, SIC
CB, SIC
J
CH, SAC
CC, SIC
Fig. 5 Subcortical actin bundles in internodal cells of N. pseudoflabellata treated with or recovering from cytochalasins applied at
concentrations that inhibit (SIC) or arrest (SAC) cytoplasmic streaming. (A–C) Cytochalasin A: 1 mM, 1 d (A), 0.05 mM, 3 d (B), 1 mM, 3 h and
several days recovery in artificial fresh water (C). (E) Cytochalasin B: 60 mM, 2 d. (E) Dihydrocytochalasin B: 30 mM, 2 d. (F and G)
Cytochalasin D: 50 mM, 1 d (F), 50 mM, 1 d and 2 d recovery in artificial fresh water (G). (H) Cytochalasin E: 1 mM, 2 d and 2 d recovery in
artificial fresh water. Subcortical actin bundles are still fragmented. (I) Cytochalasin H: 50 mM, 2 d. (J) Cytochalasin C: 200 mM, 2 d. F-actin
was stained with fluorescent phalloidin. Scale bar ¼ 10 mm (C) and 5 mm (all other figures).
of subcortical actin bundles (Figs. 5J, 6) and CJ had a weak
effect (Fig. 6).
With all cytochalasins tested, the decrease in subcortical actin bundle number and extent of fragmentation was
more pronounced with the lower streaming-inhibiting
concentrations than with streaming-arresting concentrations (Fig. 6). In the non-elongating internodes used in this
study, additional endoplasmic actin, as described for CD
treatment of younger cells (Collings et al. 1995), was not
generated.
Cytochalasin applied by perfusion does not interact with actin
filaments or induce actin reorganization
Cytochalasins are readily cell permeant, and it seems
unlikely that their mode of action is limited by the ability to
enter cells. CD introduced by perfusion, however, had no
effect on the organization of actin filaments, even at
concentrations up to 70 mM and incubation times up to
1.5 h, a treatment that caused extensive reorganization of
cortical F-actin when applied from the outside of intact
cells (Fig. 4H). The inability of cytochalasins to reorganize
actin filament structures when applied by transcellular
perfusion may relate to their inability to bind actin under
these semi-in vitro conditions. CD conjugated with a
Bodipy fluorophore is non-permeant and was therefore
introduced by transcellular perfusion. CD–Bodipy at
concentrations of up to 3.5 mM failed to label cortical
actin strands or the subcortical bundles but did stain
organelles which, according to their dimension and location, corresponded to mitochondria and cisternae of the
endoplasmic reticulum (not shown). Similar staining results
were obtained with other Bodipy-conjugated proteins, e.g.
the microtubule-stabilizing paclitaxel and the actin-stabilizing phalloidin. Therefore, labeling is probably due to nonspecific adsorption of the dye molecule.
Latrunculins fail on their own but work synergistically with
cytochalasins to arrest intracellular motility
Latrunculins are generally more effective disruptors
of actin filaments than cytochalasins and work at
Wide-ranging effects of cytochalasins
591
B
A
LatA, 200 mM
D
LatA, rec
E
LatA + CD
G
LatA, rec
F
LatA, 15 mM
H
LatA + CD
C
LatA, 15 mM
CD, 4 mM
I
CD, 4 mM
Fig. 6 Effects of cytochalasins on the arrangement of subcortical
actin bundles of N. pseudoflabellata internodal cells after 2 d
treatment with streaming-arresting concentrations (SAC 2 d), 2 d
treatment with streaming-arresting concentrations followed by
2 d recovery in artificial fresh water (SAC 2d, AFW 2d; CD, CE
and CH only) and after 2 d treatment with streaming-inhibiting
concentrations (SIC 2d). White bars indicate the number of
subcortical actin bundles per chloroplast file (left axis); gray bars
show the extent of fragmentation (right axis). Streaming-arresting
concentrations for CA, CB, DHCB, CD, CE and CH were 1, 220,
150, 60, 1 and 30 mM. Streaming-inhibiting concentrations for CA,
CB, DHCB, CD, CE, CH, CC and CJ were 0.5, 40, 75, 30, 0.5, 25,
200 and 200 mM. Data are means SD.
Fig. 7 Actin cytoskeleton of N. pseudoflabellata internodal cells
after treatment with LatA and CD. (A) One day treatment with
200 mM LatA is required for streaming cessation and correlates with
the disassembly of subcortical actin bundles. (B) Continuous
subcortical bundles but only a few cortical actin filaments
(C) have regenerated after 3 d recovery in artificial fresh water.
(D) Combined treatment of 15 mM LatA with 4 mM CD arrests
streaming within 1 h and disassembles the subcortical bundles.
(E and F) When LatA (E) or CD (F) are applied separately,
cytoplasmic streaming continues and subcortical bundles remain
intact. (G–I) The cortex of LatA-treated cells [in combination with
CD (G) or alone, (H)] is devoid of F-actin, whereas delicate actin
rods are seen in cells treated with CD (I). F-actin was stained with
fluorescent phalloidin. Scale bar ¼ 10 mm.
submicromolar concentrations. In the characean internodal
cell, however, LatA and LatB proved to be relatively
ineffective at inhibiting cytoplasmic streaming. At the
highest concentration tested (200 mM) and after 1 h treatment, LatA and LatB decreased the mean streaming rates
by only 56 5 and 62 12%, respectively. At these
concentrations, cortical F-actin either was no longer visible
or consisted of scattered short actin rods. Subcortical actin
bundles, although faintly stained, remained intact.
Prolonged exposure to LatB failed to reduce streaming
rates any further, but streaming could be completely
arrested after 1 d treatment with 200 mM LatA. These
cells contained faintly stained cortical actin patches
(not shown) and fragmented subcortical actin bundles of
uneven thickness, which were occasionally obliquely
oriented (Fig. 7A), suggesting that latrunculins also have
reorganizing properties at concentrations insufficient for
complete disassembly of F-actin (compare Gibbon et al.
1999, Vidali et al. 2001, Hörmanseder et al. 2005). Both
streaming cessation and staining results (Fig. 7B, C) were
reversible, but recovery in artificial fresh water took
several days.
592
Wide-ranging effects of cytochalasins
Table 1 Effects of simultaneous and separate treatment with LatA and CD on cytoplasmic streaming and the actin
cytoskeleton of N. pseudoflabellata internodal cells
Velocity of cytoplasmic streaming (% of control)
Fragmentation of subcortical actin bundles (%)
No. of subcortical actin bundles per chloroplast file
Velocity of cytoplasmic streaming after 1 h recovery
from 1 h treatment (% of control)
Control
15 mM LatA,
4 mM CD; 1 h
15 mM LatA;
1h
4 mM CD;
1h
100 8.6
3.4 1.4
5.4 0.8
00
100 0
3.5 1.3
40.9 13.3
36.0 14.4
4.4 1.1
4.6 1.2
47.6 15.4
73.7 17.6
3.7 3.5
5.7 0.6
91.3 14.8
Data are means SD.
Combined treatment with latrunculins and cytochalasins, however, rapidly arrested cytoplasmic streaming even
at concentrations which, when applied separately, had only
mild effects on the streaming rate. The combination of
15 mM LatA and 4 mM CD arrested cytoplasmic streaming
and disrupted subcortical actin bundles into short fragments within 60 min (Fig. 7D; Table 1). Cells recovered
continuous bundles and about 40% of the control streaming rate after 1 h in artificial fresh water (Table 1). Recovery
to near control rates took several days. When applied
separately, 15 mM LatA and 4 mM CD decreased the
streaming rate by about 64 and 26%, respectively, and did
not affect the appearance of the subcortical bundles
(Figs 7E, F; Table 1). The cortex of cells treated with
15 mM LatA (with or without CD) was completely devoid of
F-actin (Fig. 7G, H). Cells treated with 4 mM CD contained
delicate actin rods (Fig. 7I).
Discussion
Cytochalasins D and H are best suited for reversible
streaming inhibition in characean internodal cells
Characean internodal cells are one of the best studied
model systems for understanding myosin-based motility.
In this study, we found that latrunculins, which have
previously been shown to be potent disruptors of actinbased processes in higher plants (e.g. Gibbon et al. 1999),
have relatively mild effects on streaming in characean
internodal cells. Similarly, jasplakinolide (Sawitzky et al.
1999) and the jasplakinolide-related chondramides (own
unpublished data; Holzinger and Lütz-Meindl 2001) are
unable rapidly and reversibly to arrest streaming in Nitella
internodes. It seems plausible that the extraordinary
bundling within the actin cables and the protective effect
of actin-binding proteins renders them very stable towards
the action of these drugs. Cytochalasins thus remain the
only actin-perturbing drugs suited for inhibition of cytoplasmic streaming for these cells.
Ideal inhibitors are effective at low concentrations, are
highly soluble and have actions that are readily reversible.
The relative potencies of cytochalasins on cytoplasmic
streaming and actin reorganization are summarized in
Table 2. Those inhibitors with the lowest streaming-arresting concentration (1 mM) and most potent in rapidly
arresting streaming, CA and CE, however, also had the
lowest capacity for recovery. These cytochalasins presumably have a strong affinity for characean F-actin that
greatly slows recovery. DHCB and CB reversibly arrested
cytoplasmic streaming, but the required streaming-arresting
concentrations were relatively high (150 and 200 mM,
respectively). CC and CJ failed to arrest streaming even at
the highest concentrations tested.
Complete and rapid recovery within 1 h was only
possible with CD and CH, and the streaming-arresting
concentrations were 60 and 30 mM, respectively. These
cytochalasins are thus best suited for rapid and reversible
streaming arrest in internodal cells of N. pseudoflabellata.
In Chara corallina, a concentration of 8 mM CD was
sufficient to arrest cytoplasmic streaming (Foissner and
Wasteneys 2000b), which suggests that sensitivity to cytochalasins may vary between taxa within the Characeae,
although relative sensitivities to different cytochalasins are
probably conserved. Interestingly, CD and CH are also the
cytochalasins that induced the most pronounced and yet
most reversible changes in the arrangement of subcortical
actin bundles (Table 2, see below).
Streaming arrest by latrunculins but not by cytochalasins is
due to the disassembly of subcortical actin bundles
The subcortical actin bundles of untreated characean
internodes consist of up to 100 single filaments (Nagai and
Hayama 1979) and are arranged in groups of 3–7 located
along and parallel to the helically arranged chloroplast files
(Fig. 1A). Some extend into the cortex, but most of them
appear continuous over huge distances, eventually encircling the whole cell end to end (Wasteneys et al. 1996).
This continuity implies that individual actin filaments are
very long, suggesting that the number of exposed actin
filament plus ends, the preferred targets of cytochalasins
(e.g. Bonder and Mooseker 1986, Cooper 1987), is low.
Wide-ranging effects of cytochalasins
593
Table 2 Relative potencies of cytochalasins on cytoplasmic streaming, reorganization of the actin cytoskeleton and
recovery in internodal cells of N. pseudoflabellata in decreasing order (based on data presented in Figs. 3 and 6 and starting
with the lowest streaming-arresting concentration, most severe actin reorganization, fastest and most effective recovery)
Cytoplasmic streaming
Recovery of cytoplasmic streaming after
1 h treatment with streaming-arresting
concentration
Fragmentation of subcortical actin
bundles after 2 d treatment with
streaming-arresting concentration
Fragmentation of subcortical actin
bundles after 2 d treatment with
streaming-inhibiting concentration
Reduction of subcortical actin bundles
number after 2 d treatment with
streaming-arresting concentration
Reduction of subcortical actin bundles
number after 2 d treatment with
streaming-inhibiting concentration
Recovery of continuous subcortical actin
bundles after 2 d treatment with
streaming-arresting concentration
Reorganization of cortical actin
CA
CD
¼CE 4CH
¼CH 4CB
4CD
4DHCB 4CB
4DHCB 4CA
44CE
CH
¼CD 4CE
4DHCB 4CB
CE
¼CD
4CA
¼CH
44CJ 44CC
CA
4DHCB 4CJ
4CB
CC
4CE
¼CC
CD 4CH
DHCB 4CE
CA
CB
CD 4CH
4DHCB
4CJ
4CB
44CA
¼CH
4CB
4DHCB 44CJ 44CC
CH
¼CD 44CE
CA
¼CD
This and the presence of actin bundling protein(s) that may
inhibit binding of cytochalasins explains why subcortical
actin bundles are less susceptible to reorganization than the
shorter cortical actin strands, whose ends are more exposed.
A protective effect of actin-binding proteins would also
explain the high concentrations of cytochalasins and
latrunculins needed for the disassembly of subcortical
actin bundles and the ineffectiveness of jasplakinolide
(Sawitzky et al. 1999).
With LatA, fragmentation of subcortical actin bundles
clearly coincided with streaming arrest after 1 d treatment at
200 mM, but the loss of motility achieved with cytochalasins
was never correlated with disruption of the subcortical actin
bundles. Indeed, subcortical actin bundles remained intact
when exposed to the streaming-arresting concentration of
CA for several days. This suggests that inhibition of
streaming by cytochalasins is not caused by the disassembly
of F-actin or even its reorganization through plus end
capping, but rather it results from lateral binding, which
somehow inhibits interaction with the motor protein
myosin. In agreement with this hypothesis, Nothnagel
et al. (1981) found that the staining of subcortical actin
bundles by fluorescent heavy meromyosin was diminished
in the presence of CB. The experiments of Urbanik and
Ware (1989) indicate that there is more than one
cytochalasin-binding site on the actin molecule, and this is
consistent with a mechanism by which cytochalasins may
¼CE
bind along the length of, rather than only at the ends of,
actin filaments.
CD has been reported to depolymerize F-actin
via dephosphorylation of and activation of actindepolymerizing factor (ADF)/cofilin (Rückschloß and
Isenberg 2001). In this case, CD-induced fragmentation of
subcortical actin bundles should be prevented by phosphatase inhibitors that cause hyperphosphorylation of
ADF/cofilin. We found that the combined treatment with
CD and the phosphatase type II inhibitor calyculin A did
not ameliorate the fragmentation observed in the presence
of CD alone (results not shown). Thus, the effect of
cytochalasins on characean F-actin is probably more direct.
In agreement with this, Selden et al. (2001) found that CD
and profilin bind competitively to Mg-ATP-actin isolated
from vertebrate non-muscle cells.
Comparison of cytochalasin-dependent disruption of
subcortical actin bundles with in vitro experiments
The subcortical actin bundle fragmentation activities of
cytochalasins after 2 d treatment at streaming-arresting
concentrations were in the order of CH ¼ CD4CE4
DHCB4CB CA (Table 2). This correlates with the
order of activity found in in vitro experiments, where CH,
CD and CE were most effective in cleaving filaments and
inhibiting filament elongation and steady-state assembly
594
Wide-ranging effects of cytochalasins
(Walling et al. 1988). CD and CH were also very active in
further disassembly and reorganization of subcortical actin
bundles, which was reflected in the reduced number of
subcortical actin bundle fragments per chloroplast file and
their uneven thickness (Fig. 5 F, I). In contrast, CE did not
degrade further the remnant subcortical actin bundles
found in close contact with the chloroplast envelope.
The changes to the organization of subcortical actin bundles
induced by CH and CD, whose structures differ only by one
peripheral oxygen (Walling et al. 1988), were readily
reversible, just as was the arrest of cytoplasmic streaming
(Table 1). Paradoxically, CE had less effect on the
subcortical actin bundles but recovery took up to 2 weeks.
The different effects of CD/CH and CE could reflect the
fact that, in contrast to CD and CH, CE does not accelerate
microfilament assembly under certain in vitro conditions
(Walling et al. 1988). The structural integrity of subcortical
actin bundles in cells treated with CA or CB was barely
disturbed, consistent with the absence of cleavage and a low
filament shortening activity (Walling et al. 1988).
Also seemingly paradoxical is the fact that the
disruption of the subcortical actin cytoskeleton was more
pronounced at the lower concentrations that slowed but did
not arrest streaming (cf. Foissner and Wasteneys 2000b).
Similarly, disruption was observed only after transfer of
cells into artificial fresh water to allow the drug levels to
drop, and not during treatment with CA and CE.
This suggests that repeated binding and detachment of
cytochalasins enhances their capacity to reorganize the actin
cytoskeleton, whereas treatment at the higher streamingarresting concentration is more likely to stabilize existing
bundles (Williamson 1978, Williamson and Hurley 1986).
Unfortunately, fluorescent CD did not label or induce actin
reorganization and, therefore, we still do not know whether
cytochalasins are a major component of the actin rods and
bundles formed during treatment.
All cytochalasins induce spatio-temporal reorganization of
cortical F-actin
It has been described previously (Collings et al. 1995,
Foissner and Wasteneys 2000b) that CB and CD reorganize
the delicate cortical actin filaments into short, relatively
stable rods. Here we show that actin rod formation
occurred with all cytochalasins tested. A hitherto unknown
fact is, however, that formation of actin rods is often
preceded by a spectacular increase in more delicate, nonrigid cortical actin strands. This effect was most pronounced with CA, CD, CE and CH. All of these drugs
transiently increased the number of cortical actin filaments,
which became organized into dense, swirling patterns.
Transient, dense patterns of shorter, rod-like cortical
F-actin were also observed in cells treated with CB. Only
with DHCB, CC and CJ were cortical actin strands directly
replaced by sparingly distributed actin rods, although we
cannot fully exclude the possibility of a very short period
with an enhanced cortical F-actin content. As yet, we have
no explanation for this transient increase in nucleation and
elongation activity of most cytochalasins because, contrary
to our expectation, CA and CE are the two cytochalasins
that do not accelerate assembly of F-actin in vitro (Walling
et al. 1988).
The increase in number and length of cortical actin
filaments prior to the formation of rods suggests that,
similar to pollen tubes, a considerable fraction of the actin
pool in the cortex of characean internodes is present in the
form of G-actin (Collings et al. 1995, Yokota et al. 2005).
The heterogeneous distribution of cytochalasin-induced
cortical actin may result from the well-known pH banding
phenomenon of characean internodal cells (Shimmen et al.
2003, Babourina et al. 2004, and references therein).
One previous study has shown that cortical microtubules
are less abundant in the alkaline bands of C. corallina
internodal cells than they are in the acid bands where net
Hþ efflux occurs (Wasteneys and Williamson 1992). In the
case of cortical F-actin, the activity of Hþ-ATPases and the
resulting changes in the ion content of the cortical
cytoplasm may regulate the G-actin pools and/or the actin
dissociation and polymerization rates (Sampath and Pollard
1991). The fact that perfusion of cytochalasins did not cause
an increase in cortical F-actin probably reflects their
inability to bind actin under these semi-in vitro conditions
or the depletion of G-actin in perfused cells.
Cytochalasins potentiate the effects of latrunculins
Long treatment times and high concentrations of LatA
or LatB were required to arrest cytoplasmic streaming in
our experiments with Nitella internodal cells, even though
cortical F-actin was rapidly affected. Since latrunculins are
monomer-sequestering drugs, these findings probably indicate a low turnover of the subcortical actin bundles. At far
lower concentrations, however, LatA rapidly arrested
cytoplasmic streaming when applied together with low
concentrations of CD. As with latrunculins alone, streaming cessation correlated with the fragmentation of subcortical actin bundles, suggesting that CD potentiated the
destructive effects of LatA. One possible explanation is that
during the combined treatment, CD may bind not only
laterally but also to the free filament ends created by LatA
by complexing G-actin required for regeneration of actin
filaments and bundles. The capping of actin filament plus
ends might then enhance the severing activities of CD
(Cooper 1987, Urbanik and Ware 1989) and cause further
disassembly of actin bundles.
Cytochalasins as well as latrunculins affected cortical
F-actin at concentrations below those required to arrest
cytoplasmic streaming. Low concentrations of these drugs
Wide-ranging effects of cytochalasins
can therefore be used to study F-actin-dependent processes
in the cortex without indirect effects caused by cessation of
endoplasmic streaming. Latrunculins are better suited for
this purpose because they disassembled cortical actin
filaments completely, whereas cytochalasins reorganized
cortical actin filaments into cortical patches, branching
clusters and rods, which may have unpredictable effects on
cortical physiology and even morphology.
It is possible that the high concentrations of latrunculins required for actin depolymerization in characean algae
are due to very low plasma membrane permeability.
Variations in plasma membrane permeability could also
account for the quantitative differences observed with the
different cytochalasins. In order to address these questions
and to shed more light on the mechanism of actindisturbing drugs, we plan to perform perfusion experiments
which allow the introduction of inhibitors under controlled
conditions and independently of a plasma membrane
barrier.
Indirect effects of cytochalasins and actin–microtubule
interactions
The variable responses observed with the different
cytochalasins, cytochalasin concentrations and organisms
(e.g. Yahara et al. 1982, Zackroff and Hufnagel 1998,
Spector et al. 1999) imply that both the impact on
cytoplasmic streaming and the morphology of the
actin cytoskeleton can be cell specific. Furthermore, some
cytochalasin effects may also be indirectly related to the
actin cytoskeleton. It has been reported that cytochalasins
and other actin-disturbing drugs inhibit membrane transport and ion currents (Bray 1992, Rückschloss and Isenberg
2001). Newly formed actin aggregates in cytochalasintreated animal cells associate with receptors, markers and
proteins, suggesting a disturbance of cell signaling and
endocytosis (Mortensen and Larsson 2003). CD leaves
characean microtubules intact (Foissner and Wasteneys
1999), but in vitro experiments have shown that CA inhibits
the depolymerization not only of muscle actin but also that
of brain tubulin (Himes 1976). Therefore, CA may affect
actin filaments and cytoplasmic streaming not only directly
but also indirectly via depolymerization of microtubules.
Earlier studies have indeed shown that depolymerization of
microtubules makes cytoplasmic streaming in characean
internodal cells more sensitive to cytochalasin treatment
(Collings et al. 1995). In another study, we found that
subcortical actin bundle disruption by cytochalasin is
considerably enhanced in the presence of microtubuledepolymerizing drugs, and speculated that the release of
microtubule-bound proteins and their subsequent interaction with the actin cytoskeleton could enhance the
effects of cytochalasin (Foissner and Wasteneys 2000b).
Recent studies in Arabidopsis thaliana demonstrate that
595
mutation-dependent microtubule disruption can generate
hypersensitivity to cytochalasins and latrunculins (Collings
et al. 2006). The similar synergistic effect of LatA on
cytochalasins suggests that the as yet unidentified microtubule-binding factor implicated in the microtubule disruption studies may have G-actin-complexing properties.
This interpretation, however, remains speculative in view
of the complex interactions of these drugs with G- and
F-actin, and their competition with the cell’s own actinbinding proteins (Selden et al. 2001).
Materials and Methods
Plant material and culture conditions
Shoot tips of Nitella pseudoflabellata A. Br., em. R.D.W. were
planted in a soil–peat–sand mixture covering the bottom of 10 liter
aquaria filled with tap water. The temperature in the culture room
was about 208C, and fluorescent lamps (Gro-lux; Silvana,
Erlangen, Germany) provided a photoperiod of 16 h light and
8 h dark.
The fourth upper internode of each stem was used for this
study. These cells were no longer growing or elongating and were
harvested at least 2 d prior to experiments, trimmed of neighboring
internodal cells and left in artificial fresh water (1 mM NaCl,
0.1 mM KCl, 0.1 mM CaCl2).
Perfusion and staining of the actin cytoskeleton
Perfusion of internodal cells was as described (Williamson
et al. 1989). Briefly, an internodal cell was placed on the cover slip
bottom of a perfusion chamber and pressed lightly into vacuum
grease lines positioned several millilmeters away from the cell ends.
Small reservoirs with grooves were carefully placed over the grease
lines and pressed down firmly without damaging the cells.
The central portion of the cell between the reservoirs was then
covered with silicon fluid (Wacker, Burghausen, Germany) in
order to prevent evaporation. The ends of the cells within the
reservoirs were bathed in isotonic perfusion solution: 200 mM
sucrose, 70 mM KCl, 4.5 mM MgCl2, 5 mM ethyleneglycoltetraacetic acid (EGTA), 1.48 mM CaCl2, 10 mM piperazine-N,N0 -bis
(2-ethanesulfonic acid) (PIPES, pH 7.0). Following reduction of
turgor, cells were cut with small scissors, and a small difference in
solution levels between the two reservoirs ensured a gentle flow of
perfusion solution through the cell. After 1 min, actin filaments
were stained by replacing the perfusion solution with perfusion
solution containing Alexa phalloidin (Molecular Probes, Leiden,
The Netherlands; 6.6 mM in methanol) at a concentration of
0.16 mM. Cells were ready for microscopic examination after
20 min. Prior stabilization of actin filaments with m-maleimidobenzoyl N-hydroxysuccinimide ester (MBS; Sigma, Deisenhofen,
Germany) (Sonobe and Shibaoka 1989) was not required. This
method yielded excellent images of the cortical actin filaments but
the fluorescent signal of subcortical actin bundles was occasionally
attenuated by the intense autofluorescence of the chloroplasts and/
or their starch grains. Subcortical actin bundles were therefore also
visualized in cells fixed in glutaraldehyde (1% in cytoskeletonstabilizing buffer: 10 mM EGTA, 5 mM MgSO4, 100 mM PIPES/
KOH, pH 6.9; Traas et al. 1987) for 10 min and stained in Alexa
phalloidin (0.16 mM in buffer) for at least 20 min after a brief wash
in buffer. Cylindrical fragments of these internodes were opened
596
Wide-ranging effects of cytochalasins
out to produce single layer preparations with the endoplasmic side
face up, and mounted in the staining solution.
To rule out any possibility that the disassembly and
reconstruction of cortical actin filaments was influenced by the
preservation and labeling method, cells were also processed for
indirect actin immunofluorescence as described (Foissner et al.
1996)
using
the
N350
actin
antibody
(Amersham,
Buckinghamshire, UK) and a fluorescein isothiocyanate (FITC)conjugated anti-mouse secondary antibody (Sigma). This method
produced images equivalent to the more rapid phalloidin staining
(not shown).
Inhibitor treatments
Stock solutions of CA, CB, CC, CD, CE, CH, CJ, DHCB
(Sigma; 10 mM) and LatA and LatB (Calbiochem, Darmstadt,
Germany; 10 mM) were prepared in dimethylsulfoxide (DMSO)
and diluted with artificial fresh water. Controls containing DMSO
up to a concentration of 2% affected neither cytoplasmic streaming
nor the organization of the actin cytoskeleton. Fluorescent CD
(Bodipy FL conjugate) for transcellular perfusion was purchased
from Molecular Probes (Leiden, The Netherlands). The 10 mM
stock solution in DMSO was diluted with perfusion solution up to
a concentration of 3.5 mM.
Microscopy
The microscope used for visualizing fluorescently labeled
actin and autofluorescent chloroplasts was a Zeiss Axiovert 100M
inverted microscope equipped with a confocal laser scanner (Zeiss
LSM 510, Oberkochen, Germany) including an argon ion and an
HeNe laser. Projections of series of optical sections (Z-series) and
3D anaglyphs were generated using the Zeiss LSM 510 software
and further processed with Adobe Photoshop.
Statistical evaluation
Cytoplasmic streaming velocity was determined in at least
seven cells by measuring the distance traveled by detached
chloroplasts within a given time period. Movement of organelles
was also studied by analyzing slow-motion or frame-by-frame
playbacks of recorded time series collected in the confocal
microscope’s transmission mode or from video images.
The number of subcortical actin bundles per chloroplast file
was determined from Z-series obtained from 50 50 mm subcortical regions between the chloroplast-free zones known as neutral
lines, which mark the borders between the upward and downward
flows of cytoplasm. A minimum of three cells and three Z-series
per cell were investigated. The extent of fragmentation was
determined from the same regions by calculating the percentage
of interrupted subcortical actin bundles as assessed by gaps in the
fluorescent signal. Further fragmentation within a discontinuous
bundle was not considered. Mean values are given together with
their standard deviation. Data were evaluated using one-way
analysis of variance (ANOVA) and considered to be significant
when P 0.01.
Acknowledgments
I.F. gratefully acknowledges financial support from the
Stiftungs- und Förderungsgesellschaft der Universität Salzburg.
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(Received November 27, 2006; Accepted February 21, 2007)