Download Motile Apparatus in Vallisneria Leaf Cells. II. Effects of Cytochalasin

CELL STRUCTURE
AND FUNCTION
5, 13-20 (1980)
by Japan Society for Cell Biology
C
Motile Apparatus
in Vallisneria
Leaf Cells.
II. Effects of Cytochalasin
B and Lead Acetate
and Direction of Streaming
on the Rate
Mitsuo Ishigami* and Reiko Nagai**
*Department of Biology
, Faculty of Education, Shiga University, Otsu, Shiga
520, and **Department of Biology, Faculgy of Science, Osaka University,
Toyonaka, Osaka 560, Japan
ABSTRACT.
The
"primary
streaming"
of
streaming"
in
streaming
in
of
the
of cytochalasin
in
Vallisneria
and
the
Cytoplasmic
range
effects
streaming
10-100 ƒÊg/ml.
reverse
direction
observed
before
duration
of
of
its
was
The
cells
and
lead
acetate,
inhibitors
examined
on
"secondary
were
with
reference
reversibly
of
inhibited
cells
removal
increased
reaching
of
CB
depending
50 %
by
showing
after
CB
newly
to
not
apparatus
induce
in
a change
secondary
in
the
the
cessation
on
24 h
total
the
for
streaming
streaming
are
direction.
discussed
on
in the
of
streaming
number
of
concentration
CB
Properties
cells
and
treatment
cessation
however,
this
concentration
established
the
µg/ml.
Lead
acetate
at 50-100
mM caused
the complete
but the streaming
gradually
recovered
after
its removal;
did
to
direction.
ratio
after
treatment
treatment,
cells,
mesophyll
reversal
B (CB)
Characean
at
the
10-100
of streaming,
lead acetate
of
the
motile
basis.
In leaf cells of the higher aquatic plants e.g., Elodea and Vallisneria, rotational
streaming of the cytoplasm is induced by such external stimuli as irradiation with
light or the application of various chemicals. This type of streaming is called "secondary streaming" (4, 6), and that seen in Characean cells is called "primary streaming", as it is steady and ceaseless under natural conditions.
Considerable information of the mechanism of primary streaming has been obtained for the internodal cells of Characeae. The rotational cytoplasmic streaming in the
Characean cells generally is understood to be caused by the unidirectional sliding of
the endoplasmic organelles equipped with myosin-like protein along bundles of
F-actin filaments, all of which are anchored on the stationary chloroplast files. The
direction of the streaming is thought to be determined by the polarity of the F-actin
composing the bundles of microfilaments (7).
Less is known, however, about the structure and function of the motile system for
secondary streaming, though the nature of the photoreceptor involved in light-induced
cytoplasmic streaming and chloroplast movement has been clarified to some extent
(3, 12, 13). Information obtained from investigations of Characean cells should help
us to understand the mechanism of rotational streaming in the leaf cells of aquatic
plants. Forde and Steer (2) looked for microfilament bundles in leaf cells of Elodea,
but failed to find large bundles that might be responsible for the generation of cytoplasmic streaming. We recently have found many bundles of microfilaments in
13
14
M. Ishigami
and R. Nagai
Vallisneria leaf cells which manifested active cytoplasmic streaming (unpublished
results). These bundles, which appear very similar to those in Characean cells, are
localized in the peripheral region of the cell. Their orientation coincides well with the
direction of streaming. Whether these bundles are generated prior to the induction
and establishment of streaming attracted our interest. If so, the mechanism of secondary streaming must be closely related to the stability of these microfilament bundles.
The present study was performed to obtain information on the properties of the
motile apparatus in secondary streaming. Using two drugs, cytochalasin B (CB) and
lead acetate, we examined i) the cessation of streaming induced by their application
and ii) the reversal of the streaming direction after their removal.
As stated above, the endoplasm in Characean cells streams unidirectionally, supposedly due to the polarity of the microfilaments. Reversal of the streaming direction,
therefore, can occur only through the disorganization and reconstruction of the
microfilament bundles. The stability of these bundles in Vallisneria in relation to the
mechanism of secodary streaming is discussed.
MATERIALS
Cultures.
Vallisneria
cultured
The
in
tap
bottom
Leaves
layers.
first
slip
five
cells,
each
were
was
the
drug
on
and
The
were
streaming
recorded
for
had
been
direction
were
the
per
of
same
cells.
changed
by
one
by
The
the
ratio
drug
the
water.
equipped
between
rates,
condition.
The
passed
the
rate
im-
10-20 ƒÊm/sec.
drug
with
the
of
number
total
The
five
and
directions
of
to
in tap
inspected
the
part.
mesophyll
that
of
were
epidermal
inner
streaming
of the
drug,
river.
streaming
test
the
a range
the
irrigation
the
of
30ƒÊ
a razor.
IMT)
particles
sample
the
a slide
measure
application
cells
and
on
value
of
epidermal
under
within
layers
was
at
with
the
applied
the
usually
before
removal
the
two
cytoplasmic
to
from
region
(Olympus
To
Japan,
lux
occupying
of
examined
of
twenty
the
cells,
mounted
stage.
were
was
collected
cells
and
Otsu,
2,000
basal
microscope
solutions
checked
in about
After
off
normalized
which
was
of
in
about
sand
the
consisting
microscope
were
cells
directions
layers
cut
movement
application,
apparatus.
then
of sheet,
the
River
of
at
mesophyll
a sheet
control
the
piece
timing
in the
to make
off
of
were
and
Seta
muddy
four
a phase-contrast
glass
divisions
of
layers
with
Test
of
them
sheet
a different
streaming
the
of
a razor
10 cm
up
other
the
illumination
cutting
is made
two
with
by
drawing
established
which
from
leaf.
the
leaf
slide
of streaming
stripped
with
the
before
direction
by
apparatus.
and
eyepiece
mediately
with
needed
observed
measured
calibrated
of
were
a drawing
cover
covered
and
from
continuous
was
side
METHODS
biwaensis,
when
pieces
cells
the
under
stripped
Small
Mesophyll
var.
a pail
The
exterior
were
rates
pail
harvested
each
Leaves
with
the
asiatica
in
Microscopy.
on
cell
of
were
Light
cells
water
AND
pieces
recorded
the
of
newly
cells
number
of
in
cells
calculated.
Chemicals.
in
1, 5, 10,
Lead
of
fore
Cytochalasin
dimethyl
sulfoxide
50,
acetate
10,
50,
100
and
(Nakarai
100,
application.
200
B (Imperial
(DMSO),
200 ƒÊg/ml.
Chemicals,
and
500
mM.
then
A
1%
Ltd.)
The
Chemical
diluted
DMSO
was
solution
Industries)
with
tap
water
solution
dissolved
of
lead
to
was
in tap
acetate
was
dissolved
give
final
used
water
was
for
the
to give
prepared
at
20 ƒÊg/ml
concentrations
control
final
of
medium.
concentrations
immediately
be-
Motile
Apparatus
in Vallisneria
15
Cells
RESULTS
Effects
of cytochalasin
centration
of
1, 5,
concentration.
For
illuminated
off
the
the
B.
10,
rates.
irrigation.
50,
about
cells
30
on
the
for
streaming
to
the
drug
Five
rate
the
of
the
stage
(about
was
at
examined
drug
each
rates,
5,500
lux)
was
replaced
a conat
streaming
the
solution
applied
were
monitoring,
test
was
cells
monitoring
of
min,
The
1 h.
microscope
start
60
rate:
for
prior
light
after
treatment
the
200 ƒÊg/m1
min
the
min
On
or
15
with
Then
After
1)
100
we
to
level
applied
by
the
by
control
solution.
Streaming
g/ml.
was
Fig.
drug
application,
tion
and
the
control
10
and
was
the
at
5 ƒÊg/ml
Fig.
1),
1.
CB
control,
was
as
cells.
of
applied
cells
The
CB
at
treated
by
irrigating
application,
1.00;
not
were
removed
before
recover
other
length
which
data
were
of
50
of
the
min.
200
We
the
more
than
min.
duration
10
immediately
150
after
by
the
a
which
applica-
irrigation
with
concentrations
in
noted
10-100
min ƒÊ after
before
min
recovery
of
of
Within
inhibited
But
range
recovery.
rate
velocity.
after
concentration
About
completely
again,
in
concentration
did
Effect
the
cells
of
inhibited
considerable
by
time
depended
on
lag
the
con-
drug.
the
of 1ƒÊg/ml
halt
streaming
was
even
in
and
50 %
normal
observed
not
at the
rotational
to
been
their
drug
inhibition
within
had
complete
was
did
CB
CB
not
of
Streaming
that
the
of
decreased
regained
streaming
by
courses
inhibited
cells
50 ƒÊg/ml
centration
time
completely
solution,
100 ƒÊg/ml
reversibly
the
streaming
was
before
of
inhibited
1 shows
streaming,
was
on
the
streaming
with
of
1 % DMSO
the
cell
mostly
normalized
vertical
with
in
against
bar
is the
min
(-•œ-),
control
of
this
value.
of
the
the
no
cells
treated
detectable
irregularity
or
two
after
the
start
solution.
mean.
or
The
100
pattern.
where
rate
of
CB
at
min
The
track
a stacked
streaming
temperature,
the
(arrow
(-•£-).
(arrow
mean
200
streaming. ƒÊg/ml.
measurements
µg/ml
60
represents
on
cell,
for
at room
point
its
the
of velocity
treatment
10-20µm/sec
Each
of
with
effect
in
areas
(-•¢-)
After
range
S.E.
of
50
(-•›-).
the
the
one
30
10
%
caused
in
rate.
concentrations
was
but
disordered
40
produced
For
2),
the
the
drug
immediately
was
value
regarded
for
five
16
M. Ishigami
and R. Nagai
mass of cytoplasm, like a helmet, containing chloroplasts and other particles was
often formed. This helmet-like mass did not shift position and continued to rotate
there at least for few hours (Fig. 2).
2) On the streaming direction: Bundles of microfilaments in Characean cells are not
affected structurally by cytochalasin B (1, 14). To obtain information of the structural
Fig.
2.
nucleus
with
a)
(N),
5 ƒÊg/ml
plasts.
moved
50ƒÊm, •~
The
Normal
mesophyll
chloroplasts
CB.
nucleus
anticlockwise,
650
The
(C)
arrow
is far
but
cells
and
points
from
the
this
of
other
to
mass.
helmet-like
Vallisneria.
particles
The
shows
a helmet-like
c)
The
mass
thin
mass
same
has
layer
rotational
of
cell
not
(as
moved
of
cytoplasm
streaming.
cytoplasm
in
containing
b)
containing
2b)
after
from
its
10
sec.
original
The
cell
several
The
nucleus
position.
the
treated
chlorohas
Scale,
Motile
Apparatus
stability
of
the
streaming
the
on
the
concentration
average
plateau
reversal
(Fig.
fact
that
suggests
every
cell,
dependent
the
the
and
that
the
With
Vallisneria
drug
on
of
inhibition
investigation;
The
cells
70-80
was
were
%
hibited.
of
toxic.
removal
In
of
Fig.
3.
50
(-•›-).
Each
Percentage
cells
more
the
Effect
(-•œ-),
of
point
cells
examined
than
drug
(-•¢-)
of
at
and
CB
or
on
clear
cells.
30 %
represents
(Ntotai).
the
the
of
streaming
drug,
mean
their
Abscissa:
rate:
Lead
Duration
100,
In
the
and
100
ƒÊ
depending
of
50 (Y.
100 ƒÊg/ml,
the
observed
in
are
the
control,
the
50
completely
of
the
by
CB
regenerated
in-
regenerated
acetate
micro-
100
five
of
the
or
500
the
mM
streaming
was
mM
was
were
samples
the
for
for
1 h.
was
this
not
When
observed
completely
(i.e.,
200
and
was
not
resumed
applied
treated
of
after
drug
though
is useful
in-
500
mM)
after
long.
drug
direction
a reversible
examined.
streaming
cells
has
cells,
action
streaming
before
The
also
its
200
of
cells,
died
for
first
50,
than
Control
value
h.
of
of
was
cells,
direction.
streaming
24
internodal
cessation
treated
cells
(-•£-).
the
reversed
probability
10,
other
the
and
bundles
the
higher
the
the
reversibility
of
In
of
most
drug
inhibition
of
50
was
of
50
a plateau
of
disorganized
Characean
The
a concentration
100 ƒÊg/ml
which
in
(5).
of
10,
50 %.
streaming
10 mM
the
the
streaming
concentrations
treated
acetate
within
are
of
is
the
mode
at
with
the
On
reaching
direction
because
polarity
the
finally
10 ƒÊg/ml
removal
reversal
of
concentrations
streaming
polarity,
is not
applied
at
the
of 1 % DMSO.
increased
gradually
treatment,
at
checked
concentrations
microfilaments
cytoplasmic
treated
Lead
the
the
1)
therefor,
drug
of
and
of
after
acetate.
effect
mechanism
of
we
at
in a solution
cells
examined
treatment
bundles
cells,
treatment
duration
reversed
of lead
inhibitory
total
the
3).
original
having
Effects
was
and
reversal
that
of
filaments
in
by
was
reached
within
3 h,
remained
a few
percent.
The
the
in
caused
As a control,
cells
were
immersed
ratio
of reversed
cells
to the total
on
in
17
Cells
microfilaments
direction
g/ml.
The
cells
in Vallisneria
about
the
treatment.
at
with
twenty
removal
concentrations
1 %
cells
of the
DMSO
each.
drug
of
10
solution
Ordinate:
(Nx)O to
the
18
M. Ishigami
and R. Nagai
Reversible inhibition
was observed with treatments
at 50 and 100 mM. Fig. 4 shows
the time courses of the inhibition
of streaming
and its recovery.
Streaming
in every
treated cell was inhibited completely
within 30 min at 100 mM, and within 45 min at
50 mM. Disturbances
in the streaming track and the stack of cytoplasm,
observed with
CB treatment,
did not occur with lead acetate. Cells inhibited
completely
at 50 mM
resumed their normal velocity within 90 min after the drug had been washed out. The
recovery in cells that had been treated at 100 mM was slightly delayed.
We noted that streaming
started without a time lag after removal of the drug as
was observed
with CB treatment.
Fig. 4.4. Effect
Fig.
Effect of
of lead
lead acetate
acetate
on the
on
the streaming
streaming
rate.
rate.
As in
As
in the
the
case
case
of CB,
of
CB,
lead
lead
acetate
acetate
was applied
was
applied
at the
at
the concentrations
concentrations
of 50
of
50 (-0—)
(-•œ-)
or 100
or
100 mM
mM
(-•¢-).
(—A—).
Tap
Tap
water
water
was
was
used
used
as the
as
the
control
control
medium
medium
(-0—). Each
(-•›-).
Each
point
point
represents
represents
the mean
the
mean
value
value
for five
for
five cells.
cells.
The
The
length
length
of the
of
the vertical
vertical
bar
bar
is the
is
the S.E.
S.E.
of the
of
the mean.
mean.
TABLE
1.
EFFECT OF LEAD ACETATE ON THE STREAMING
*1 Number of cells which reversed
*2 Total number of cells examined
streaming .
in one sample .
DIRECTION
Motile
Apparatus
in Vallisneria
19
Cells
2) On the streaming direction: Effects of the drug at 50 and 100 mM were examined
as described above. The treatment periods were set at 1 and 3 h because treating cells
for a longer period (such as 1 day) killed even those immersed in rather dilute solutions
(i.e., 50 mM). Results are shown in Table 1. Lead acetate produced no reversal of
the streaming direction in almost all the cells. This differs sharply from the results
obtained for CB treatment, and suggests that the microfilament bundles must be
structurally stable during the cessation of cytoplasmic streaming caused by lead
acetate.
DISCUSSION
The
present
g/ml,
but
ing locally
ceased
due
of
treated
the
quickly
the
presence
cessation
of
that
peripheral
its
and
served
when
integrity
the
by
Lead
the
the
of
Vallisneria
the
Based
not
must
integrity
does
on
the
to
is not
that
particles
directed
had
cells
3)
the
caused
CB
occurred
Lead
in
acetate
in
of
the
at
10-100
about
at
same
integrity
CB
reversal
of
check
the
cytoplasmic
further
of
the
data
extent
50 %
50-100
mM
direction
the
acetate
was
is strong
Characean
are
cells.
in
seems
microfilament
bundles
14).
However,
not
to
be
be
the
Our
next
bundles
in
is prestructural
maintained
these
the
to
bundles
Characean
which
This
localized
there
direction.
to
for
as
investigated.
in
CB
rigidly
labile
kept
their
during
step
will
are
broken
be
streaming
the
the
conclusions
microfilaments
the
simple
along
those
1).
in
cessation
were
the
of
are
no
reversion
of
obtained
simple
this
of
cessation
the
inhibition
important
is
evidence
of
streaming
in
Vallisneria
the
bundles
Characean
of
experiment
when
darkness
are
we
microfilament
as
preliminary
disorganized
stability
with
mechanism
However,
bundles;
constructed
in
Vallisneria
The
for
disorganization.
treatment,
that
of our
become
in
is removed.
to be
closely
related
partly
or completely
governs
tentative
(1,
streaming
streaming
drug
microfilament
result
be
so
the
be
not
rotational
as
the
is assumed
around
were
with
along
finally
the
the
The
microfilament
motile
lead
to
their
disinte-
the microfilaments
in darkness.
When
light,
newly
united
of
concerned,
walls
that
kept
cells
However,
would
to regeneration
of
disordered
in darkness.
the
anticline
cells.
streaming
suggested
cells
are
irradiated
in their
pericline
walls
usually
moved
cytoplasmic
tracks.
These
streamlets
factor
of
of
treatment
the
lead
in
However,
stability
Vallisneria
and
that
results).
structural
during
CB
microfilaments
(unpublished
structural
to
by
to
bundles
the
in
of
been
processes
seem
which
become
far
many
The
after
gration.
One
result
have
a tendency
to
Our
direction
is homologous
between
inhibits
seem
integrity
as
its
drug.
by
of rotational
streamƒÊ
2) When
streaming
had
drug.
and
hours
what
inhibited
track
streaming
Vallisneria
that
ceases
direction
clear
fact
microscope
acetate
about
by
because
streaming
in
microfilaments
electron
down
steady
the
removal,
mechanism
streaming
of
use
streaming
Vallisneria.
treatment
cells
of
of
its
completely
5 ƒÊg/ml
broke
the
of the cytoplasm.
reversion
removal
After
difference
Characeae
do
CB,
the
motile
region
a considerable
few
was
CB at
movement
of
after
streaming.
is supported
not
Streaming
restored.
The
to
: 1)
reversibility.
irregular
cells
halted
evidence
CB
showed
good
induced
to
gradually
also
study
with
and
cell
the
cytoplasmic
formed,
and
(cf.
12,
microfilament
We must
now
diversely
established
13).
These
bundles
determine
bundles.
mechanism
is homologous
of
to
secondary
primary
streaming,
streaming
once
a
20
M. Ishigami
and R. Nagai
rotational streaming is established. 2). The motile system of secondary streaming
differs in the structural stability of the microfilament bundles from that of primary
streaming.
Cytoplasmic organelles equipped with myosin-like protein need to be investigated
further. Myosin-like protein already has been extracted from Elodea and partially
purified (11).
REFERENCES
1. BRADLEY,M.O. Microfilaments and cytoplasmic streaming: inhibition of streaming with
cytochalasin. J. Cell Sci. 12, 327-343, 1973
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(Received for publication, November 6, 1979)