Plant Cell Physiol. 48(2): 345–361 (2007)
doi:10.1093/pcp/pcm001, 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]
Microtubule- and Actin Filament-Dependent Motors are Distributed on Pollen
Tube Mitochondria and Contribute Differently to Their Movement
Silvia Romagnoli 1, Giampiero Cai 1, Claudia Faleri 1, Etsuo Yokota 2, Teruo Shimmen
Mauro Cresti 1, *
1
2
2
and
Dipartimento Scienze Ambientali ‘G. Sarfatti’, Università di Siena, via Mattioli 4, I-53100 Siena, Italy
Department of Life Science, Faculty of Science, University of Hyogo, Harima Science Park City, Hyogo, 678-12 Japan
organelles, which move along actin filaments, as shown by
studies with cytoskeleton inhibitors (Mascarenhas and
Lafountain 1972, Lancelle and Hepler 1988, Gibbon et al.
1999, Vidali et al. 2001). Furthermore, organelles isolated
from Lilium pollen tubes moved along actin filament
bundles of Characeae (Kohno and Shimmen 1988).
Immunocytochemical analysis has shown that myosins,
the actin filament motors, are associated with pollen tube
organelles (Tang et al. 1989, Miller et al. 1995) and one
myosin of 170 kDa has been isolated from Lilium pollen
tubes (Yokota and Shimmen 1994). This myosin is also
present in tobacco plants (Yokota et al. 1999) and belongs
to myosin XI on the basis of the sequence analysis of cDNA
clones encoding heavy chains of tobacco 170 kDa myosin
(Shimmen and Yokota 2004).
Although less well supported, it is nevertheless evident
that microtubules also participate in the regulation of
motility in pollen tubes. Microtubules apparently correlate
with the movement of both the generative cell and the
vegetative nucleus (Astrom et al. 1995, Miyake et al. 1995),
with the pulsed growth (Geitmann et al. 1995), and they
possibly prevent the accumulation of vacuoles in the pollen
tube tip (He et al. 1995). In addition, kinesin-like and
dynein-like proteins have been biochemically identified and
characterized (Tiezzi et al. 1992, Moscatelli et al. 1995, Cai
et al. 2000). These motors show different distributions
inside the pollen tube (Cai et al. 1993, Moscatelli et al.
1998), suggesting that they have distinct functions.
Furthermore, organelles isolated from tobacco pollen
tubes have been shown to move in vitro along microtubules,
and kinesin-related motor proteins are probably involved
in this activity (Romagnoli et al. 2003). The in vitro
velocity of organelles along microtubules is far slower than
that of the streaming induced by actin filament–myosin
in pollen tubes, suggesting that the microtubule-dependent
transport of organelles is overwhelmed in vivo by the
rapid transport generated by the actin filament–myosin
system. To sum up, current data indicate that both actin
filament- and microtubule-based motors contribute,
although differently, to organelle movement in the pollen
tube. However, some issues are unresolved; for example,
it is unclear if the two motor systems cooperate with
The pollen tube exhibits cytoplasmic streaming of
organelles, which is dependent on the actin–myosin system.
Although microtubule-based motors have also been identified
in the pollen tube, many uncertainties exist regarding their
role in organelle transport. As part of our attempt to
understand the role of microtubule-based movement in the
pollen tube of tobacco, we investigated the cooperation
between microtubules and actin filaments in the transport of
mitochondria and Golgi vesicles, which are distributed
differently in the growing pollen tube. The analysis was
performed using in vitro motility assays in which organelles
move along both microtubules and actin filaments. The results
indicated that the movement of mitochondria and Golgi
vesicles is slow and continuous along microtubules but fast
and irregular along actin filaments. In addition, the presence
of microtubules in the motility assays forces organelles to use
lower velocities. Actin- and tubulin-binding tests, immunoblotting and immunogold labeling indicated that different
organelles bind to identical myosins but associate with
specific kinesins. We found that a 90 kDa kinesin
(previously known as 90 kDa ATP-MAP) is associated
with mitochondria but not with Golgi vesicles, whereas a
170 kDa myosin is distributed on mitochondria and other
organelle classes. In vitro and in vivo motility assays indicate
that microtubules and kinesins decrease the speed of
mitochondria, thus contributing to their positioning in the
pollen tube.
Keywords: Kinesin — Microtubule — Mitochondria —
Myosin — Organelle movement — Pollen tube.
Abbreviations: DIC, differential interference contrast;
DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride;
PNS, post-nuclear supernatant.
Introduction
The pollen tube is a highly polarized and rapidly
tip-growing cell used to deliver the male genetic material to
the ovule (Wilhelmi and Preuss 1999). In angiosperms,
pollen tubes show a strong cytoplasmic streaming of
*Corresponding author: E-mail, [email protected]; Fax, þ39-0577-232860.
345
346
Association of motors with pollen mitochondria
each other and how different motors distribute between the
several organelle types.
In other eukaryotic cells (mainly in specialized animal
cells such as neurons and melanophores), the functional
cooperation between microtubules, actin filaments and
molecular motors in organelle trafficking has already been
described (Rogers and Gelfand 2000). Some models
propose that microtubule- and actin filament-dependent
motors perform spatially distinct activities in animal cells,
with the long-range transport of organelles mediated by
microtubules and the short-range local transport provided
by actin filaments (Goode et al. 2000). Alternatively,
it has been proposed that the movement of a single
organelle is the result of the forces exerted simultaneously
on the organelle itself by different motors (Tabb et al. 1998,
Gross et al. 2002).
Although the above-mentioned models derive
essentially from animal cell systems, plants also exhibit
examples of functional cooperation between actin filaments
and microtubules. In non-vascular plants, such as the
alga Chara, both microtubules and actin filaments
are involved in the transport and immobilization of
mitochondria (Foissner 2004). Cooperation between actin
filament- and microtubule-dependent motility also extends
to the transport of chloroplasts in Bryopsis (Menzel and
Schliwa 1986) and in Physcomitrella patens (Sato et al.
2001). In vascular plants, actin filaments are the main tracks
along which organelle transport occurs (Shimmen and
Yokota 2004). Nevertheless, analysis of organelle movement and positioning in some plant species has revealed
that both microtubules and actin filaments mediate the
movement and the anchoring of organelles. In cultured
tobacco cells, for example, the fast directional movement of
mitochondria is dependent on the actin filament–myosin
system, while the positioning of immobile mitochondria in
the cortical cytoplasm is based on both actin filaments
and microtubules (Van Gestel et al. 2002). In the same cells,
the directed movement of the Golgi apparatus depends
on actin filaments and myosins, while microtubules exert
only modest control on organelle streaming (Nebenführ
et al. 1999). In Arabidopsis thaliana leaves, one kinesin
is associated with the Golgi apparatus and is critical for the
dispersal of the Golgi along microtubules, while the
movement of Golgi from the center to the cell cortex
depends on myosin (Lu et al. 2005).
Because current literature suggests that microtubuleand actin filament-dependent motors may cooperate
for organelle movement in plant cells, we analyzed
the motility of individual pollen tube organelles along
microtubules and actin filaments, and assayed these
organelles for the presence of specific motor proteins.
Mitochondria have been chosen as a model organelle,
because information on their movement already exists in
the literature and because they can be easily isolated
from plant cells. Mitochondria are distributed all along
the pollen tube length and move backward and forward
along the pollen tube according to the reverse-fountain
streaming pathway (Parton et al. 2003). However, the
distribution of mitochondria is not uniform as they are
absent or rare in the apical 10 mm domain, and most
abundant behind the apex (Lovy-Wheeler et al. 2006).
Results obtained for mitochondria were compared with
those from Golgi vesicles because the two organelle classes
distribute differently in the tube. Vesicles are generated by
the Golgi bodies, transported along actin filaments
and accumulated in the apical region (Wang et al. 2005).
We used in vitro motility assays coupled to microtubuleand actin filament-binding analysis, immunochemical and
immunocytological techniques for the characterization of
organelle movement and motor proteins. Our aim was to
define how mitochondria move along microtubules and
actin filaments, to determine the relative contribution of
each motor system to their movement and to understand
whether mitochondria possess specific motor proteins
(kinesin and myosin).
Results
Characterization of the mitochondria and Golgi vesicle
fractions
We used published protocols to purify either
mitochondria or Golgi vesicles from tobacco pollen tubes.
Although the original method for the isolation of
membrane fractions from pollen tubes claims to yield
relatively uncontaminated fractions, we assayed marker
enzymes for mitochondria, Golgi apparatus, endoplasmic
reticulum and plasma membrane to assess the purity of
the isolated organelles (Fig. 1A). As expected, the activities
of these enzymes were equivalent in the post-nuclear
supernatant (PNS). The activity of the marker enzymes
indicated that the mitochondrial and vesicular fractions
were largely free of contamination from other organelles.
To confirm this assessment, mitochondria (Fig. 1B, top)
were also specifically labeled with the dye MitoTracker
Green FM, which showed that almost all 1–2 mm particles
were stained (Fig. 1B, bottom); in contrast, the vesicular
fraction (Fig. 1C, left) was not stained by MitoTracker
(right).
Mitochondria move on both microtubules and actin filaments
under in vitro motility assays
In this work, we examined mitochondria and
Golgi vesicles purified from pollen tubes moving along
microtubules and actin filaments. Both cytoskeletal
filaments were derived from animal sources (tubulin from
bovine brain and actin from rabbit skeletal muscle) but they
Association of motors with pollen mitochondria
A
347
PNS
50
vesicles
mitochondria
µmol/min/mg
40
B
30
20
10
0
CCO
IDPase
CCR
enzyme markers
P-ATPase
C
Fig. 1 Analysis of the organelle fractions used in the motility assays. (A) Analysis of enzyme markers in the PNS, Golgi vesicles and
mitochondria. All enzymatic activities are expressed as mmol min1 mg1. Organelle markers used in the assay were cytochrome c oxidase
activity (CCO) for mitochondria, IDPase activity for Golgi vesicles, cytochrome c reductase activity (CCR) for endoplasmic reticulum and
P-ATPase activity for the plasma membrane. Error bars indicate the standard deviation. (B) An enlarged view of the mitochondrial fraction
observed using DIC microscopy (top panel) and after staining with MitoTracker Green FM (bottom panel). Bar ¼ 4 mm. (C) The vesicle
fraction observed using DIC microscopy (left panel) and after staining with MitoTracker Green (right panel). Bar ¼ 4 mm.
were scarcely contaminated by animal organelles.
In addition, exogenous motor proteins were not detected
in the tubulin or actin samples (as revealed by Western
blotting with antibodies to animal myosin and kinesin;
data not shown).
The movements of mitochondria along microtubules
and actin filaments both were ATP dependent and cytosol
independent, while no movement was recorded either in the
absence of ATP or when non-hydrolyzable ATP analogs or
GTP replaced ATP (data not shown). On microtubules,
mitochondria, initially free in solution, attached and,
after a lag phase, moved slowly while retaining a stable
contact with the microtubule surface (Fig. 2A).
After movement, mitochondria usually detached from the
filaments and became free in solution. Exchange between
different microtubules was uncommon. Only some
microtubule-associated mitochondria exhibited active
movement (Table 1).
The movement of pollen tube mitochondria along
actin filaments (Fig. 2B) was different from that shown
along microtubules. On actin, mitochondria moved rapidly
and irregularly, detaching and then attaching to the same
or different actin filaments (see the mitochondrion in
Fig. 2B). Long-lived contact with the same actin filament
was not rated (Table 1). After movement, organelles again
detached from the filaments. Unlike along microtubule
substrate, most mitochondria moved actively along actin
filaments (Table 1).
When both cytoskeletal elements were present in the
assays, we observed three different conditions. In the
first case (‘absence of simultaneous contact’; Fig. 3A),
mitochondria moved along microtubules or actin filaments
348
Association of motors with pollen mitochondria
mitochondria +
microtubules
A
mitochondria +
actin filaments
0
B
0
MT
AFs
15
3
25
8
35
10
50
13
Fig. 2 In vitro motility assay of mitochondria along microtubules
or actin filaments. (A) Time-lapse video sequences of mitochondria
(arrowheads) moving along in vitro polymerized microtubules (MT)
or (B) in vitro polymerized fluorescent actin filaments (AF).
Numbers on the top right indicate the time in seconds between
each video frame. The black line in the last video frame of B
indicates the route covered by one single mitochondrion in 13 s
along actin filaments. Bar ¼ 5 mm for both sequences.
without interacting with the other cytoskeletal filament.
In the second circumstance (‘consecutive contact’, Fig. 3B),
mitochondria quickly moved along actin filaments,
stopped or decelerated at the intersection between actin
filaments and microtubules, then slowly traveled along the
microtubules. The velocity of mitochondria during each
step was equivalent to the velocity observed under separate
conditions. The third condition (‘simultaneous contact’,
Fig. 3C) occurred when organelles appeared to interact with
accidentally co-aligned microtubules and actin filaments.
In this case, mitochondria alternated between rapid
movements (presumably along actin filaments) and slow
ones (most probably along microtubules); the average speed
was lower than the velocity observed along actin filaments
but higher compared with that along microtubules
(Table 1). The three different conditions were observed in
separate samples; however, their relative incidence was
difficult to evaluate because it depended on the relative
organization of actin filaments and microtubules.
In vitro motility assay of Golgi vesicles along microtubules
and actin filaments
Like mitochondria, Golgi vesicles from tobacco pollen
tubes moved in vitro along microtubules and actin filaments
in an ATP-dependent and cytosol-independent manner. The
movement of Golgi vesicles along microtubules was slow
and continuous; the vesicles did not detach from microtubules. Unlike mitochondria, moving Golgi vesicles
frequently switched to different microtubules and resumed
their movement (Fig. 4A, arrow). The run length of Golgi
vesicles along microtubules was shorter than that of
mitochondria, in agreement with the short time that Golgi
vesicles spent along microtubules (Table 1). On the other
hand, the movement of Golgi vesicles along actin filaments
was similar to the movement of mitochondria on actin
(Fig. 4B), as vesicles moved very rapidly and swapped
frequently from one actin filament to another. When Golgi
vesicles were tested on both cytoskeletal filaments together,
they interacted with both (Fig. 4C) and showed the three
different interaction forms already described for mitochondria. In the most frequent condition, Golgi vesicles bound
to and moved rapidly along actin filaments, with many
saltations; when Golgi vesicles contacted microtubules, they
often stopped or resumed a slow movement along them.
The mean velocity of Golgi vesicles along microtubule–actin
filament matrices was lower that the speed of vesicles along
actin filaments but higher than their velocity along
microtubules (Table 1).
Velocity distribution of mitochondria and Golgi vesicles along
microtubules and actin filaments
The movement of mitochondria and Golgi vesicles
was evaluated statistically. The velocity distribution
of mitochondria and vesicles along microtubules was
Association of motors with pollen mitochondria
Table 1
349
Analysis of mitochondria/vesicle motility along microtubules (MTs) and actin filaments (AFs)
Cytoskeletal
filament
Mitochondria
Golgi vesicles
MTs
AFs
MTs–AFs
MTs
AFs
MTs–AFs
Speed
(mm s1)a
0.17 0.02
1.73 0.73
0.53 0.28
0.22 0.05
1.78 0.80
0.75 0.49
(n ¼ 30)
(n ¼ 45)
(n ¼ 20)
(n ¼ 25)
(n ¼ 45)
(n ¼ 10)d
Distance
covered (mm)
Interaction
time (s)
Moving
organelles (%)
Frequency of
exchange (%)
5–15
20–25
n.d.
5–8
12–15
n.d.
50–60
15–20
n.d.
40–60
5–10
n.d.
20
85
80b
15
80
75b
2
50
10c
10
70
20e
a
Values are expressed as average speed and standard deviation; n is the number of observed samples.
These values mainly refer to organelles moving along actin filaments, as they are the majority of moving objects.
c
Mitochondria that move from actin filaments to microtubules.
d
Calculation of the mean velocity takes into account the distance covered by a vesicle within a given time, during which it interacted
repeatedly with both actin filaments and microtubules.
e
Vesicles that exchange from actin filaments to microtubules.
n.d., not determined.
b
mitochondria + microtubules + actin filaments
A
AF
0
0
B
0
C
MT
MT
AF
MT
AF
11
7
2
30
14
5
47
45
15
Fig. 3 In vitro motility assay of mitochondria along microtubules and actin filaments. (A) Time-lapse video sequences of one
mitochondrion (arrow) that moves in vitro along a single microtubule (MT). One actin filament (AF) is aligned with the microtubule but is
relatively distant. Numbers on the top right indicate the time in seconds. Bar ¼ 4 mm. (B) Time-lapse video sequences of the sequential
movement of one mitochondrion (arrow) that firstly moves along actin filaments (AF), then switches to and moves on one microtubule
(MT). The black line in the last video frame of B shows the course covered by the mitochondrion. Bar ¼ 4 mm. (C) Time-lapse video
sequences showing one microtubule (MT) and one actin filament (AF) that align closely; one single mitochondrion (arrow) moves while
simultaneously interacting with both cytoskeletal filaments. The black line in the last video frame of C shows the route covered by the
mitochondrion. Bar ¼ 4 mm. Numbers in all frames indicate the time in seconds.
350
Association of motors with pollen mitochondria
Vesicles + MTs
A
Vesicles + AFs
0
MT
B
Vesicles + MTs/AFs
0
C
0
AF
AF
15
1
2
25
3
10
35
4
15
50
5
25
Fig. 4 In vitro motility assay of Golgi vesicles along microtubules and/or actin filaments. (A) One vesicle (arrow) moves along and
switches between two different microtubules (MT). Bar ¼ 2 mm. (B) One vesicle (arrow) moves in vitro along fluorescent actin filaments
(AF). The long black line in the last video frame of B indicates the route covered by the vesicle. Bar ¼ 2 mm. (C) In vitro motility assay of
vesicles along both microtubules (MT) and actin filaments (AF). One vesicle (arrow) firstly travels along one single actin filament, then
switches to and moves slowly along microtubules. A second vesicle (arrowhead) initially moves along microtubules and then switches to
actin filaments. The black line in the last video frame of C shows the route covered by the first vesicle. Bar ¼ 1.5 mm. Numbers on the top
right of each frame indicate the time in seconds. The dotted line in the first video frame indicates the microtubule.
Association of motors with pollen mitochondria
12
A
10
The results observed in vitro compared closely with those
observed in in vivo conditions (Fig. 6E, F). Given the
absence of Golgi vesicle-specific dyes, we focused our
attention on mitochondria. In control conditions
(when both microtubules and actin filaments are present,
Fig. 6E), the lowest velocity ranges of mitochondria are
substantially represented, whereas the highest ones (from
2.4 to 3.0 mm s1) are scarcely present. In the case of
oryzalin-treated pollen tubes (when only actin filaments are
supposed to be present, Fig. 6F), the lowest ranges
almost disappeared while the highest ranges significantly
increased. The two velocity distributions differed significantly according to the Kolmogorov–Smirnov test
(P50.004). The results were similar when using oryzalin
at 2 or 10 mM. Treatment with cytochalasin D inhibited the
movement of mitochondria within a few minutes following
drug application (data not shown).
Mitochondria on
microtubules
8
6
Frequency
4
2
0
7
6
351
B Golgi vesicles on
microtubules
5
4
3
2
>0.3
0.28-0.3
0.26-0.28
0.24-0.26
0.2-0.22
0.22-0.24
0.18-0.2
0.16-0.18
0.14-0.16
0.1-0.12
0.12-0.14
0.08-0.1
0.06-0.08
0.04-0.06
0-0.02
0
0.02-0.04
1
velocity distribution (µm/sec)
Fig. 5 Velocity distribution of mitochondria and Golgi vesicles
along microtubules. The velocity distribution of mitochondria
(A) and Golgi vesicles (B) along in vitro polymerized microtubules
was analyzed using the software Retrac; both organelles use
velocity ranges that fit a normal distribution (according to the
Shapiro–Wilk test; P50.002 and P50.001, respectively).
indistinguishable from a normal distribution (Fig. 5A, B).
However, the distribution of organelle velocity on actin
differed significantly from a normal distribution, being
spread out rather evenly among represented velocities
(Fig. 6A, B). The statistical analysis of mitochondria and
Golgi vesicles on the combined actin and microtubule
system was performed taking into account the conditions of
consecutive and simultaneous contact (which are supposed
to occur in the pollen tube). The velocity distribution of
Golgi vesicles (Fig. 6C) was again spread throughout
different values, but the frequency of lower velocity
ranges significantly increased whereas the highest values
(43 mm s1) were absent. Likewise for mitochondria
(Fig. 6D), there was a notable reduction of high velocity
movement and an increase in low velocity movement. The
velocity distributions of both organelles moving in the
combined system could be distinguished statistically from
distributions for movement on actin alone (compare
Fig. 6A and B with C and D).
To extend these results, we obtained velocity distributions for organelles moving in living pollen tubes.
Amyosin of 170 kDa is present on the surface of mitochondria
and Golgi vesicles
As mitochondria and Golgi vesicles from tobacco
pollen tubes moved along actin filaments in an ATPdependent manner, we investigated the presence of myosins
associated with pollen tube organelles using actin filament
binding assays coupled to immunoblotting. We used an
anti-myosin antibody that cross-reacts with a single
170 kDa polypeptide in the crude sample of lily pollen
tube proteins (Yokota and Shimmen 1994) and with
170 kDa polypeptides in pollen tubes of tobacco and
Tradescantia virginiana, and in suspension cultured cells of
tobacco and A. thaliana (Yokota et al. 1995). In our assays,
the anti-myosin antibody cross-reacted with a 170 kDa
polypeptide in the PNS proteins (Fig. 7A, lane 2; gel and
blot); a band of similar molecular mass was identified in
both the mitochondria and vesicle fractions (gel and blot,
lanes 3 and 4). As shown by the binding assay, the PNS
fraction contained membrane proteins that bind to actin
filaments in the absence of ATP (gel, pellet in lane 5).
The 170 kDa myosin was detected in this sample (blot in
lane 5) and in the PNS proteins that are released from actin
filaments by addition of ATP (supernatant in lanes 6,
gel and blot).
The 170 kDa pollen tube myosin belongs to the myosin
XI class, which is represented by different isoforms.
To determine whether mitochondria and Golgi vesicles
use different 170 kDa myosin isoforms, both fractions were
analysed by two-dimensional electrophoresis and immunoblotting. Both organelle classes contained a similar pattern
of cross-reacting polypeptides (gel and blot panels of
Fig. 7B, C), consisting of three spots at 170 kDa and a pI
of around 4.8. The relative intensity of the three spots
was comparable in both cases, with the exception of the
more acidic spot, which was less intense in the Golgi
352
Association of motors with pollen mitochondria
8
7
A
Vesicles on actin filaments
B
Mitochondria on actin filaments
6
5
4
3
2
1
0
16
14
C
Vesicles on actin filamentsmicrotubules
Mitochondria on actin
filaments-microtubules
D
frequency
12
10
8
6
4
2
Mitochondria in
control pollen tubes
F Mitochondria in
oryzalin-treated
pollen tubes
0-0.2
0.2-0.4
0.4-0.6
0.6-0.8
0.8-.1
0.1-1.2
1.2-1.4
1.4-1.6
1.6-1.8
1.8-2
2-2.2
2.2-2.4
2.4-2.6
2.6-2.8
2.8-3
>3
E
0-0.2
0.2-0.4
0.4-0.6
0.6-0.8
0.8-.1
0.1-1.2
1.2-1.4
1.4-1.6
1.6-1.8
1.8-2
2-2.2
2.2-2.4
2.4-2.6
2.6-2.8
2.8-3
>3
0
100
90
80
70
60
50
40
30
20
10
0
velocity distribution (µm/sec)
vesicle preparation (compare the magnified insets in
Fig. 7B, C).
The 2-D gel analysis was also performed on the PNS
and on the mixture of mitochondria and Golgi vesicles. In
the PNS, the number of spots was higher (at least five),
although the three spots previously discussed were the most
evident (data not shown). When mitochondria and vesicles
were mixed, the number and position of cross-reacting spots
do not change, suggesting that the myosin XI isoforms
were the same in both samples (magnified blot in Fig. 7D).
The 90 kDa ATP-MAP is associated with pollen tube
mitochondria
Because mitochondria and Golgi vesicles moved
in vitro along microtubules, we examined the presence of
kinesin-related proteins in both organelle classes by
immunoblotting with two different commercially available
anti-kinesin antibodies (AKIN01 and AKIN02). AKIN01
cross-reacted with one polypeptide of 90 kDa (Fig. 8A,
Fig. 6 Velocity distribution of mitochondria
and Golgi vesicles along microtubule–actin
filaments and in living pollen tubes.
(A) Analysis of vesicle movement along
in vitro polymerized actin filaments; the
distribution is non-normal (in accordance
with the Shapiro–Wilk test; P40.2).
(B) Velocity distribution of mitochondria
along actin filaments; the distribution is
non-normal (consistent with the Shapiro–
Wilk test; P40.1). (C) Golgi vesicles analyzed
in the presence of microtubules and actin
filaments lack the highest velocities while the
frequency of the lowest velocities increases
(compared with A). (D) Movement of mitochondria along microtubules/actin filaments;
the velocity distribution increases in the lowest
values but disappears in the uppermost ones
(compared with B). (E) Velocity distribution
of mitochondria in living pollen tubes.
The lowest velocity ranges are recognizable.
(F) Velocity distribution of mitochondria in
living pollen tubes treated with oryzalin.
The lowest ranges disappear and the frequency of the highest velocities increases.
The velocity distribution of organelles was
determined using Retrac software. The distributions of velocities in A–C, B–D and E–F
are considered different according to the
Kolmogorov–Smirnov test (P50.001 in all
cases).
lanes 1–4). However, cross-reactivity was fainter in the
cytosolic fraction (lane 2) and absent in the vesicle fraction
(lane 5). The AKIN02 antibody showed almost the same
pattern of cross-reactivity (Fig. 8A, lanes 6–10); reaction
was hardly visible in the cytosolic fraction (lane 7) and
absent in the vesicle fraction (lane 10).
To characterize the 90 kDa polypeptide, we assessed its
ability to bind to microtubules in an ATP-dependent
manner using the PNS as the starting material (Fig. 8B,
top panel). Except for a weaker polypeptide of 105 kDa
(dot), the final sample (lane 8) contained one main
polypeptide of 90 kDa (arrow), the latter being recognized
by AKIN01 in most of the protein fractions but particularly
in the final ATP supernatant (bottom panel, lane 8, arrow).
Comparable results were also obtained with AKIN02
(data not shown).
Since the polypeptide identified by AKIN01 in the
mitochondrial fraction showed the same molecular mass
as the 90 kDa ATP-MAP from tobacco pollen tubes
B
353
7
4
250
S+ATP
P-ATP
PNS
proteins
Vesicles
gel
PNS
Markers
A
Mitochondria
Association of motors with pollen mitochondria
150
100
250
75
150
100
50
75
50
37
1
2
3
4
5
6
blot
C
7
4
250
D
150
100
75
50
Fig. 7 Assay of binding to actin filaments and characterization of pollen tube myosin. (A, gel) Binding assay of PNS proteins to actin
filaments. Lane 1, molecular mass standards. Lane 2, PNS proteins (20 mg). Lane 3, pollen tube mitochondria (5 mg). Lane 4, pollen tube
vesicles (3 mg). Lane 5, PNS proteins that bound to the actin pellet (arrowhead) in the absence of ATP. Lane 6, PNS proteins released from
the actin pellet by ATP wash. Equal volumes were loaded in lanes 4–7. All lanes are from the same gel. (A, blot) Immunoblotting with the
anti-myosin antibody on the same samples shown in the gel. A single band of 170 kDa is labeled in all fractions (arrow). All lanes are from
the same blot. (B) Top panel, 2-D electrophoresis of mitochondrial proteins separated by 4–7 pH gradients. Bottom panel, immunoblotting
with the anti-myosin antibody on the same sample. The antibody cross-reacts with three spots (large view in the inset) with an average pI of
4.8. (C) Left panel, 2-D electrophoresis of vesicle proteins separated as described for mitochondria. Right panel, immunoblotting with the
anti-myosin antibody, which cross-reacts with three spots (enlarged view in the inset). (D) Magnified inset of 2-D immunoblotting of mixed
mitochondria and vesicle proteins, showing the correspondence of the myosin spots.
(Cai et al. 2000), we also tested the cross-reactivity of
AKIN01 on the ATP-MAP fraction (Fig. 8C). The 90 kDa
ATP-MAP is definitely recognized by AKIN01 in the final
ATP-MAP sample (lane 2, gel; lane 4, blot, arrowhead),
while no significant cross-reactivity was detected in the
microtubule protein pellet after ATP wash (lane 1, gel;
lane 3, blot). The results indicated that the 90 kDa
mitochondrial protein and the 90 kDa ATP-MAP represent
the same molecule.
Immunolocalization of 90 kDa ATP-MAP and 170 kDa
myosin on pollen tube mitochondria
To study the distribution of both the 90 kDa
kinesin and the 170 kDa myosin in tobacco pollen
tubes, we used the antibodies immunocytochemically.
Efforts to compare the localization of the AKIN01
antibody with the mitochondrial dye MitoTracker
were unsuccessful (Supplementary Fig. S1). Therefore,
the distribution patterns of the 90 kDa kinesin and
354
Association of motors with pollen mitochondria
A
B
150
200
100
116
97
75
1
2
3
4
5
6
7
AKIN01
8
9
10
AKIN02
66
T
45
1
C
2
3
4
5
6
7
8
250
200
150
100
90 kD
116
97
75
66
50
45
37
1
2
3
4
Fig. 8 Identification of the 90 kDa kinesin in different organelle fractions. (A) Immunoblotting with AKIN01 and AKIN02 on pollen tube
organelle fractions; lanes 1 and 6, total pollen extract; lanes 2 and 7, cytosolic proteins; lanes 3 and 8, PNS proteins; lanes 4 and 9, pollen
tube mitochondria; lanes 5 and 10, pollen tube vesicles. A 5 mg aliquot of proteins was loaded in each lane. (B, top panel) Microtubule
binding assay of PNS proteins extracted with KI. Lane 1, molecular weight standards. Lane 2, PNS proteins (10 mg). Lane 3, KI-extracted
proteins (10 mg). Lane 4, desalted proteins (15 mg). Lane 5, proteins binding to microtubules in the presence of AMP-PNP. Lane 6, proteins
that do not bind to microtubules (lanes 5 and 6 are equivalent volumes). Lane 7, microtubule pellet after ATP wash. Lane 8, ATP-released
proteins (lanes 7 and 8 are equally loaded). A major protein of 90 kDa is recovered (arrow) along with a second weaker band of 105 kDa
(dot). Tubulin (T) is indicated. Bottom panel: immunoblotting with AKIN01 on the gel fractions. (C) Left: final fractions obtained in the
preparation of ATP-MAPs; lane 1, pellet after incubation with ATP; lane 2, corresponding supernatant (ATP-MAPs). Identical volumes were
loaded in both lanes. Right: immunoblotting on the same samples with AKIN01, which cross-reacts with the 90 kDa ATP-MAP
(arrowhead).
mitochondria were compared using AKIN01 and immunogold electron microscopy. In these preparations, the signal
was essentially found in association with the surface of
pollen tube mitochondria (M) (Fig. 9A, B, arrows).
The mouse monoclonal anti-kinesin AKIN02 and the
rabbit polyclonal anti-myosin antibody were used in double
labeling experiments to determine the relative distribution
of both motor proteins (Fig. 9C, D). Although the
anti-myosin antibody labeled different organelles in the
cell, double labeling was specifically found on
mitochondria (M), suggesting that the 90 kDa kinesin
(10 nm gold particles, arrows) and the 170 kDa myosin
(15 nm gold particles, arrowheads) are associated with the
surface of mitochondria.
Quantification analysis of the immunogold labeling
experiment with AKIN01 indicates that 480% of examined
mitochondria were labeled (Fig. 9E). The number of gold
particles on mitochondria corresponded to the majority of
the total particles in examined areas, suggesting that the
90 kDa kinesin is specifically associated with pollen tube
mitochondria. The quantitative analysis of the immunogold
labeling with both AKIN02 and anti-myosin antibody
(Fig. 9F) indicates that a small number of mitochondria
were individually labeled by AKIN02 or anti-myosin,
whereas both antibodies simultaneously labeled most of
the mitochondria. Although the anti-myosin antibody
labeled other organelle classes, labeling with the anti-kinesin
antibody on any of these organelles was not statistically
significant. Therefore, the analysis indicated that myosin is
not specifically associated with mitochondria, unlike the
90 kDa kinesin.
Discussion
In this work, we provide evidence that mitochondria
from tobacco pollen tubes move slowly along microtubules
Association of motors with pollen mitochondria
A
355
B
M
M
M
C
D
M
M
100.0
100.0
Control 2
Control
0.0
≥2 Particle
0.0
1 Particle
20.0
Particles on
mitochondria
20.0
Control 1
40.0
Kinesin particles
40.0
60.0
Myosin particles
60.0
80.0
Double-labelled
80.0
Analysis of double-labelled samples
Myosin-labelled
Percentage
F 120.0
Kinesin-labelled
Analysis of kinesin-labelled samples
120.0
Labelled
mitochondria
Percentage
E
Fig. 9 Immunolocalization of the 90 kDa kinesin and myosin in tobacco pollen tube. (A, B) Immunogold labeling with the AKIN01
antibody. The 90 kDa kinesin is mainly detected in association with mitochondria (arrows). Bar ¼ 300 nm for A; 200 nm for B. (C and D)
Double labeling with the monoclonal AKIN02 and the polyclonal anti-myosin antibodies. The 90 kDa kinesin (10 nm gold particles) was
detected on the surface of mitochondria (arrows). Myosin (15 nm gold particles) was also found in association with mitochondria
(arrowheads). Bar ¼ 150 nm. (E) Histogram of mitochondria labeled with AKIN01. The graph shows the percentage of labeled
mitochondria, of gold particles on mitochondria (compared with the cytoplasm) and the percentage of mitochondria labeled with one or
with two (or more) gold particles. ‘Control’ bar: percentage of labeled mitochondria without the primary antibody. Error bars indicate the
standard deviation. (F) Histogram of mitochondria labeled with AKIN02 and anti-myosin. The graph shows the percentage of mitochondria
labeled by kinesin or myosin only, the percentage of mitochondria labeled by both antibodies, and the percentage of kinesin
(‘kinesin particles’ bar) or myosin (‘myosin particles’ bar) on mitochondria compared with other cell structures. ‘Control 1’ and ‘control 2’:
percentage of gold particles on mitochondria without the primary anti-myosin or anti-kinesin antibodies. Error bars indicate the standard
deviation.
356
Association of motors with pollen mitochondria
but more rapidly along actin filaments, and that mitochondria are associated with a specific microtubule-dependent
motor. A 170 kDa myosin is bound to mitochondria and
Golgi vesicles, while a 90 kDa kinesin is associated with
mitochondria but not with vesicles. The different distribution of the two proteins suggests that the set of motor
proteins associated with mitochondria and Golgi vesicles is
distinct and may comprise similar myosins but different
kinesins. In vitro and in vivo motility assays indicate
that microtubule-dependent motors influence the overall
velocity of mitochondria, suggesting that kinesin and
myosin cooperate for the positioning of mitochondria in
the pollen tube.
Functional cooperation between motor proteins has
been studied in different cell types (fungal, plant and
animal) using a variety of technical approaches, including
in vitro motility assays. In this study, we investigated the
transport of pollen tube mitochondria and Golgi vesicles
mediated by both kinesin and myosin. The two classes of
organelles interact and move along both microtubules and
actin filaments, occasionally switching from one filament to
the other or interacting simultaneously with both.
Nevertheless, some differences in the motility pattern
along microtubules and actin filaments may be observed.
The average speed of mitochondria (1.73 0.73 mm s1)
and Golgi vesicles (1.78 0.80 mm s1) along actin filaments
is comparable with the speed of organelles observed
within the growing pollen tube of tobacco (2.13 mm s1)
(de Win et al. 1999), rye (2.58 mm s1) and iris (2.47 mm s1)
(Heslop-Harrison and Heslop-Harrison 1987, HeslopHarrison and Heslop-Harrison 1988, Heslop-Harrison
and Heslop-Harrison 1990). In contrast, the speed of
organelles along microtubules (0.22 0.05 mm s1 for
Golgi vesicles and 0.17 0.02 mm s1 for mitochondria)
is at least 10 times slower than the corresponding velocity
along actin filaments. A remarkable difference is in the
distribution of velocity. Mitochondria and Golgi vesicles
have normally distributed velocities while moving along
microtubules, and movement is continuous; whereas, for
organelles on actin, the distributions of velocity are flat,
and movements are irregular. When actin filaments and
microtubules are present simultaneously, the higher velocity
ranges are not used while the frequency of lower ranges
substantially increases. The in vitro analysis of mitochondria is comparable with that observed in living pollen tubes
and also agrees with the evidence that the oryzalin
treatment does not negatively interfere with the mitochondrial distribution in lily pollen tubes (Lovy-Wheeler et al.
2006). Based on the similarity between the in vitro
and in vivo movement of mitochondria, we suggest that
the role of microtubule-dependent motors is to control the
overall velocity of mitochondria rather than their general
distribution in the pollen tube.
Characterization of the mitochondria-associated
kinesin was achieved by immunological techniques.
Among the anti-kinesin antibodies known to cross-react
with pollen tube proteins, k71s23 (Tiezzi et al. 1992) and
MMR44 (Marks et al. 1994, Cai et al. 2000) are no longer
available. The K1005 antibody has already been shown
to cross-react with different organelle classes (Romagnoli
et al. 2003). In the current work, we have extended the
immunological analysis using two additional antibodies,
the polyclonal AKIN01 and the monoclonal AKIN02.
Both antibodies cross-reacted with one polypeptide of
90 kDa, which was found exclusively associated with
mitochondria but not with vesicles. Consequently, vesicles
presumably use a different kinesin to move along
microtubules. Since AKIN01 and AKIN02 cross-react
with the 90 kDa mitochondrial microtubule-binding protein
and with the 90 kDa ATP-MAP (Cai et al. 2000), we assume
that the two molecules are the same (hereafter simply
referred to as 90 kDa kinesin) and that the protein is
associated with mitochondria. The absence of signal in the
apical region of pollen tubes and the immunoblotting
results suggest that the 90 kDa kinesin is not associated with
Golgi vesicles. The velocity of purified 90 kDa kinesin
(0.040 0.008 mm s1, tested using in vitro microtubule
gliding assays) (Cai et al. 2000) is different from the velocity
of isolated mitochondria moving along microtubules
(0.17 0.02 mm s1). However, this discrepancy may reflect
damage, insofar as the rate found in vivo is rather low for
kinesin motors.
We found that the 170 kDa myosin, which is
distributed among all the purified organelle fractions,
binds to actin filaments in the absence of ATP and is
released after addition of ATP; furthermore, the motor
protein shows a similar cross-reactivity of the 170 kDa
myosin heavy chain from lily pollen tubes and cultured
tobacco cells (Yokota et al. 1999). Therefore, the 170 kDa
myosin is a candidate to promote the cytoplasmic streaming
in tobacco pollen tubes, as it supposedly does in lily pollen
tubes and in cultured tobacco cells. This model is also
supported by the diffuse localization of the protein in the
entire pollen tube of tobacco (Yokota et al. 1995).
The number and type of putative myosin isoforms were
similar on mitochondria and Golgi vesicles, suggesting that
both organelle classes use relatively the same myosin. This is
in apparent contrast to results from A. thaliana, where
MYA2 (a myosin XI isoform) was localized in association
with peroxisomes (Hashimoto et al. 2005). However, other
observations suggest that MYA2 may be involved in the
movement of both vesicles and larger organelles (presumably the endoplasmic reticulum) (Holweg and Nick 2004).
It is consequently possible that different organelle classes
will use a broad repertoire of myosin motors during
cytoplasmic streaming. In addition, an antiserum against
Association of motors with pollen mitochondria
a 175 kDa myosin cross-reacted with different plant tissues
but failed to detect bands in germinating tobacco pollen
(Yokota et al. 1999), suggesting that pollen tubes may use a
restricted number of myosins for organelle movement.
Cooperation between myosin and kinesin in the
transport of mitochondria is becoming a general trait of
cell biology. In animal cells, the long-range movement of
mitochondria occurs on microtubules while the short-range
movement depends on actin filaments (Hollenbeck and
Saxton 2005). Although plants are relatively distant
from animal and fungal cells, examples of functional
cooperation have also been reported, as in characean
internodal cells (Foissner 2004). Association of kinesin
with mitochondria has also been shown in A. thaliana root
cells (Ni et al. 2005), suggesting that microtubule-dependent
motors may also have a role in the movement of
mitochondria in flowering plant cells. In the pollen tube,
the movement of organelles (including mitochondria) is
described as vectorial in the base domain (de Win et al.
1999) and is supported by the actin filament bundles
(Lovy-Wheeler et al. 2005). Results from our in vitro
motility assays suggest that actin filaments and the 170 kDa
myosin promote the irregular but fast movement of
mitochondria in the pollen tube. In contrast, the 90 kDa
kinesin may promote slow movements of mitochondria,
similar to the processive motility of animal kinesin-1
(Howard et al. 1989), along the microtubule bundles in
the base domain (Del Casino et al. 1993). This activity
may delay the trafficking of mitochondria or immobilize
them at specific cell areas. This is in agreement with our
observations that mitochondria move rapidly in vitro along
actin filaments and stop at the intersection between actin
filaments and microtubules. This combined activity
may consequently regulate the positioning of mitochondria
in the pollen tube, allowing a precise distribution in
accordance with the growth rate. In this context, similar
myosin machinery may be used by different organelles,
whereas specific kinesins may be required for the movement
of distinct organelle classes.
According to the data presented, the 90 kDa kinesin is
absent from the Golgi vesicle fraction. In addition, the
motility of vesicles along microtubules is different from
that shown by mitochondria, as vesicles interact for
shorter times with microtubules and frequently exchange
to different filaments. Consequently, this fraction must
contain some other kinesin(s). The so-called pollen kinesin
homolog of 100–105 kDa (Tiezzi et al. 1992) was originally
identified as an ATP-dependent microtubule-binding
protein with microtubule-enhanced ATPase activity.
The protein localized in the pollen tube apex and showed
a distribution consistent with its binding to Golgi vesicles.
In addition, one immunological homolog of 100 kDa was
found in hazel pollen in association with Golgi vesicles
357
(Liu et al. 1994). A second 105 kDa polypeptide with
kinesin-like properties was identified in tobacco pollen
tubes and shown to be associated with different organelle
fractions (Romagnoli et al. 2003). The 105 kDa kinesin(s)
hypothetically represents the microtubule-dependent
motor(s) responsible for the movement of Golgi vesicles
along microtubules.
Materials and Methods
Chemicals and antibodies
Reagents for electrophoresis and molecular mass standards
(unstained or pre-stained) were purchased from Bio-Rad
(Hercules, CA, USA). Blotting membranes, secondary antibodies
and enhanced chemiluminescence reagents were from GE
Healthcare (Uppsala, Sweden). Buffer reagents and nucleotides
were purchased from Sigma-Aldrich (St Louis, MO, USA).
Tubulin, actin (fluorescent and non-fluorescent), heavy meromyosin, the anti-kinesin antibodies AKIN01 (rabbit polyclonal) and
AKIN02 (mouse monoclonal), and the antifade reagent were
purchased from Cytoskeleton, Inc. (Denver, CO, USA). The antimyosin antibody (rabbit polyclonal) was raised against the heavy
chain of 170 kDa myosin isolated from germinating Lilium pollen
(Yokota and Shimmen 1994) and it also cross-reacts with the
170 kDa myosin from tobacco pollen (NtMY-170: accession No.
AB180675). Secondary antibodies for fluorescence microscopy,
rhodamine–phalloidin, MitoTracker Red and Green FM were
from Molecular Probes (Invitrogen Corporation, Carlsbad, CA,
USA), while secondary antibodies for electron microscopy were
from British Biocell (BBInternational Ltd, Cardiff, UK).
Pollen culture, preparation of post-nuclear supernatant and cytosol
fractions from tobacco pollen tubes
Pollen of Nicotiana tabacum was collected from plants
grown in the Botanical Garden of Siena University and germinated
in BK medium (Brewbaker and Kwack 1963). Culture of
tobacco pollen tubes and preparation of both PNS and cytosol
fractions have already been described (Romagnoli et al. 2003).
Purification of pollen tube mitochondria
Mitochondria were isolated from pollen tubes following
the protocol outlined in Hajek et al. (2004). After germination,
the pollen was washed twice with BRB25 buffer (25 mM HEPES
pH 7.5, 2 mM EGTA, 2 mM MgCl2) plus 15% sucrose. The pollen
was resuspended in 1 vol. of lysis buffer [BRB25 containing 2 mM
dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride
(PMSF), 10 ml ml1 protease inhibitor cocktail (Sigma-Aldrich),
1 mM NaN3 and 10% mannitol]. After lysis on ice with a motordriven Potter–Elvehjem homogenizer, the sample was centrifuged
at 1,000g for 5 min at 48C to remove large cellular debris.
The supernatant was centrifuged again at 6,000g for 10 min
at 48C. The resulting supernatant was layered on a Percoll cushion
(0.3 M sucrose, 10 mM MOPS-KOH, pH 7.2, 1 mM EDTA, 28%
Percoll) and centrifuged at 40,000g for 60 min at 48C.
The centrifugation yielded two closely related bands in the
middle of the centrifuge tube. Mitochondria were recovered in
the lighter band. The second denser layer (which is contaminated
by nuclear material according to the original method)
was discarded. The mitochondria-containing band was diluted
10 times with washing buffer (0.3 M mannitol, 10 mM
MOPS-KOH, pH 7.5, 1 mM DTT) and centrifuged at 11,000g
358
Association of motors with pollen mitochondria
for 15 min at 48C. Mitochondria were resuspended in washing
buffer and used for the biochemical and in vitro motility assays.
Purification of pollen tube Golgi vesicles
Golgi vesicles were purified from the PNS using a step sucrose
gradient according to the method described in Helsper et al. (1977),
which is based on the protocol of Engels (1974). The PNS was
layered on a step sucrose gradient composed of (from bottom
to top) 2, 1.5, 1 and 0.5 M sucrose in BRB25. After centrifugation
at 100,000g for 60 min at 48C, the sample at the 0.5/1 M interface
was removed and diluted 2-fold with BRB25. The diluted sample
was layered on a step sucrose gradient composed of (from bottom
to top) 1.1, 0.9 and 0.7 M sucrose in BRB25 solution and
centrifuged at 100,000g for 60 min at 48C. Golgi vesicles were
removed from the 0.7/0.9 M sucrose interface and used for the
biochemical and in vitro motility assays.
Analysis of mitochondria and Golgi vesicles
Isolated mitochondria were allowed to adhere for 15 min to
perfusion chambers pre-coated with 1 mg ml1 poly-L-lysine.
Chambers were washed with washing buffer (25 mM PIPES
pH 6.8, 2 mM EGTA, 2 mM MgCl2, 1 mM DTT, 55% sucrose)
and then incubated with MitoTracker Green FM diluted to
100 nM in washing buffer. The Green FM version of MitoTracker
was chosen because the dye is essentially non-fluorescent in
aqueous solutions but becomes fluorescent as it accumulates in
the mitochondrial membranes. After 15 min, samples were washed
with washing buffer and then observed with both fluorescence
and differential interference contrast (DIC) microscopy.
Golgi vesicles were treated likewise (the washing buffer contained
0.8 M sucrose). Labeling with MitoTracker was performed as a
control to confirm the absence of mitochondria.
Mitochondria and vesicles were assayed for the presence
of characteristic organelle markers: IDPase activity for Golgi
vesicles and cytochrome c oxidase activity for mitochondria. In
addition, cytochrome c reductase activity (for the endoplasmic
reticulum) and P-ATPase activity (for the plasma membrane) were
also assayed as controls. Results were referred to the protein
concentration of samples and expressed as specific enzymatic
activities. The starting PNS sample was also assayed. Protocols
were performed exactly as described in the literature (Robinson
and Hinz 2001), with the exception of mitochondria that were
assayed using the Cytochrome c Oxidase Assay Kit from Sigma.
All assay reactions were performed using spectrophotometer
cuvettes and started by addition of each membrane sample.
The molar concentration of inorganic phosphate was measured
using the PhosFree phosphate assay Biochem Kit from
Cytoskeleton Inc.
Preparation of fluorescent actin filaments for in vitro motility assay
Fluorescent actin filaments for in vitro motility assay
were polymerized using both monomeric fluorescent and nonfluorescent actin following the protocol supplied by Cytoskeleton
Inc. Fluorescent and non-fluorescent actins were separately
diluted in A buffer (5 mM Tris–HCl pH 8.0, 0.2 mM CaCl2,
0.5 mM DTT and 1 mM ATP) to a final concentration of
0.4 mg ml1 and kept on ice for 4 h. In the meantime, 50 APB
buffer (0.5 M KCl, 20 mM MgCl2, 1 mM ATP) was kept at 378C
for 15 min, and then left at room temperature until use. One part
of a 50 stock solution of APB was mixed with four parts of A
buffer to make the PB buffer, and this was kept on ice. Fluorescent
and non-fluorescent actins were mixed equally, supplemented
with PB buffer in the ratio of 1 : 10 and then incubated at room
temperature for 2 h. During the incubation, 1 vol. of PB buffer was
mixed with 9 vols. of A buffer and 6.6 mM rhodamine–phalloidin
to make the stabilization buffer (SB). Rhodamine–phalloidin
was used to increase the stability of actin filaments and to enhance
their fluorescence. Actin filaments were mixed with SB in a ratio of
1 : 20 and then used.
Preparation of taxol-stabilized microtubules
Microtubules were polymerized from monomeric tubulin
(10 mg ml1 in 80 mM PIPES, pH 6.8, 1 mM MgCl2, 1 mM EGTA)
in the presence of 1 mM GTP and 10% glycerol at 358C for 20 min
according to the protocol supplied by the Cytoskeleton Inc.
The microtubule sample was diluted 1 : 25 in microtubule
resuspension buffer (80 mM PIPES, pH 6.8, 1 mM MgCl2, 1 mM
EGTA, 1 mM DTT, 20 mM taxol), placed at room temperature
and used for several motility assays.
In vitro motility assays
The movement of pollen tube organelles along microtubules
and actin filaments was analyzed by in vitro motility assays using
perfusion chambers assembled as described (Romagnoli et al.
2003). For the motility assay along microtubules, 10 ml of
microtubule solution (0.1 mg ml1) was incubated for 10 min in
the perfusion chamber. Mitochondria or Golgi vesicles were
diluted 1 : 10 in 20 mM taxol, 5 mM ATP and 6.5 mg ml1 cytosol
(which was replaced by buffer as control), and then introduced into
the chamber. The movement of mitochondria or vesicles
along microtubules was visualized as already described
(Romagnoli et al. 2003). The motility assay of organelles along
fluorescent actin filaments was performed according to the
literature (Kuznetsov et al. 1994). Briefly, 10 ml of the fluorescent
actin filament solution (0.04 mg ml1) were incubated for 10 min in
the perfusion chamber. Mitochondria or vesicles (diluted 1 : 10 in
5 mM ATP, 6.5 mg ml1 cytosol or buffer, and antifade solution)
were then added. We took care that the distribution of actin
filaments did not change over several minutes. DIC and
fluorescence microscopy were performed with the same type of
video camera (C2400-75i charge-coupled device from
Hamamatsu Photonics, Hamamatsu City, Japan) using the
Argus-20 to enhance the fluorescence signal (Frint command).
The video camera was plugged into a Zeiss Axiophot microscope
(Oberkochen, Germany) equipped with a 100 oil immersion
objective. For monitoring the movement of organelles along actin
filaments, two different video sequences were separately captured.
Organelles were first observed using DIC microscopy and their
movement was recorded for several seconds or minutes into an
AVI file according to the PAL standard (720 576 pixels, 25
frames s1, each frame corresponding to approximately 400 mm2).
Then, the same video frame was observed using fluorescence
microscopy in the rhodamine channel (without changing the
focus), and the distribution of actin filaments was captured as a
separate BMP image. Overlay of the AVI file with the actin
filament image was achieved using the free software VirtualDub
(www.virtualdub.org) and its Logo filter adjusted in order that
both organelles and actin filaments were clearly observed. The
same digital overlay was performed to pick up the movement of
organelles along matrices of microtubules and actin filaments. In
this case, 10 ml of the microtubule solution and 10 ml of the actin
filament solution were mixed and incubated for 10 min in the
perfusion chamber. Mitochondria or vesicles (diluted 1 : 10 in
5 mM ATP, 6.5 mg ml1 cytosol or buffer, and antifade solution)
were added to the microtubule–actin filament matrix. The number
of actin filament bundles in each frame (corresponding to
Association of motors with pollen mitochondria
approximately 400 mm2) was reasonably constant, allowing the
selection of video frames only based on the number of
microtubules (preferably 45).
Analysis of the organelle velocity
The velocity distribution of mitochondria and Golgi vesicles
under different conditions was calculated using the free software
Retrac, which is available from Dr. N. Carter, Marie Curie
Research Institute, Molecular Motors Group, Oxted, Surrey,
UK (http://mc11.mcri.ac.uk/retrac/). Single video frames were
extracted at given times from each video clip and saved as TIFF
images, which were imported into Retrac and analyzed with
the tracking option. Results were evaluated statistically using
Microsoft Excel (Frequency function). Analysis of distribution was
done using the software Statistica (StatSoft, http://www.statsoft.
com). To follow the organelle pathway accurately along
microtubules and actin filaments, we again used the tracking
option of Retrac; at the end of each analysis, the last video
frame was saved overlaying the track line. Consequently, the path
of each organelle is representative of the movement. For the
analysis of organelles moving along microtubules, we considered
ranges of 0.02 mm s1, while in the case of actin filaments
the velocity range was necessarily higher (0.2 mm s1). In the case
of double motility, we considered the higher range in order to
understand how microtubules affect the motility along actin
filaments.
Drug treatments of living pollen tubes
Stock concentrations of 10 mM oryzalin (Sigma Aldrich)
and 1 mM cytochalasin D (Sigma Aldrich) were made in
dimethylsulfoxide (DMSO). Appropriate amounts of stock solutions were dissolved in BK medium to reach the final concentrations of 10 mM oryzalin and 5 mM cytochalasin D. Oryzalin was
also used at 2 mM according to Lovy-Wheeler (2006). Pollen
tubes were observed after 10 min incubation, according to
Geitmann et al (1995). Mitochondria were stained with 100 nM
MitoTracker Green FM for 30 min prior to drug application.
Video clips of mitochondrial movements were recorded after drug
treatment and in control samples using the Axiophot microscope
(100 oil immersion objective) and the C2400-75i charge-coupled
device. Single video frames were saved as TIFF files every 0.5 s,
and analyzed using the software Retrac. For statistical evaluation,
the position of about 100 arbitrary mitochondria per test was
monitored in random selected regions of different pollen tubes
(length up to 200 mm) and data were processed with Microsoft
Excel using the Frequency function.
ATP-dependent microtubule binding assay
Proteins were extracted from the PNS of pollen tubes by
washing with 0.6 M KI for 30 min on ice (Schroer et al. 1988) and
then by centrifuging at 100,000g for 90 min at 48C. The resulting
supernatant was desalted using a Hi-Trap desalting column
(GE Healthcare) equilibrated in BRB25 containing 1 mM DTT
and 1 mM PMSF. After desalting, KI-extracted proteins were
mixed with taxol-stabilized microtubules (prepared as described for
the motility assay, 0.4 mg ml1 of tubulin), 20 mM taxol and 10 mM
AMPPNP. The mixture was incubated for 30 min at room
temperature, and then centrifuged at 40,000g for 30 min at
258C. The pellet was washed for 10 min at room temperature with
1 ml of EDTA buffer (25 mM HEPES, pH 7.5, 3 mM EGTA, 1 mM
AMPPNP, 20 mM taxol, 1 mM DTT and 10 mM EDTA) and then
centrifuged at 40,000g for 30 min at 258C. The pellet was
incubated overnight with 0.5 ml of release buffer (25 mM HEPES,
359
pH 7.5, 3 mM EGTA, 2 mM MgCl2, 1 mM DTT, 200 mM KCl,
20 mM taxol and 10 mM ATP). After centrifugation at 40,000g
for 30 min (258C), the supernatant was collected as the ATP
supernatant.
The ATP-MAPs protein sample was isolated from a highspeed supernatant of pollen tubes by the co-sedimentation assay
with taxol-stabilized microtubules (Cai et al. 2000).
ATP-dependent actin filament binding assay
To polymerize actin filaments for the binding assay, 10 ml
of non-fluorescent actin (10 mg ml1) were mixed with 240 ml of
A buffer and kept on ice for 2 h. In the meantime, APB buffer was
kept at 378C for 15 min, and then left at room temperature until
use. Then, 5 ml of APB (50) were added to 250 ml of actin in A
buffer, kept for 1 h at room temperature and then used for the
binding assay. Proteins were removed from the pollen tube PNS
using the carbonate buffer (15 mM Na2CO3, 35 mM NaHCO3 at
pH 11.4) as described by Evans et al. (1998). Carbonate-extracted
proteins were incubated with in vitro polymerized actin filaments
(0.1 mg ml1) for 30 min on ice in the absence of ATP.
After incubation, the sample was centrifuged at 95,000g
for 30 min at 48C. The pellet was resuspended in EMP buffer
(5 mM EGTA, 6 mM MgCl2, 0.5 mM PMSF, 10 ml ml1 protease
inhibitor cocktail, 1 mM DTT, 30 mM PIPES-KOH pH 7) and
kept on ice for 30 min. The resuspended pellet was centrifuged at
95,000g for 30 min at 48C. To release proteins from actin
filaments, the resulting pellet (P-ATP) was resuspended in EMP
buffer containing 10 mM ATP and 5 mM K2HPO4, and kept on ice
for 30 min. The sample was centrifuged again at 95,000g for
30 min at 48C. The resulting supernatant was referred to as the
SþATP sample.
Immunoelectron microscopy
Immunogold labeling of tobacco pollen tubes was performed
as already described (Li et al. 1995). The AKIN01 antibody
was used at the dilution of 1 : 20, whileAKIN02 was used at 1 : 10.
The anti-myosin antibody was used at a dilution of 1 : 100. The
secondary goat anti-rabbit Ig 15 nm gold-conjugated and goat antimouse Ig 10 nm gold-conjugated antibodies were used at
the dilution of 1 : 20. Electron micrographs were taken with a
Philips Morgagni 268D-transmission electron microscope operated
at 80 kV and equipped with a MegaView II charge-coupled device
camera (Philips Electronics, Eindhoven, The Netherlands).
For quantitative analysis, gold particles were counted on randomly
selected micrographs (30 micrographs from three different experiments, each of approximately 10 mm2). Particles were considered
associated with organelles if they were located on their surface
or within 20 nm from it. Particle analysis was done using the
software Scion Image, which is based on NIH Image and is freely
available at www.scioncorp.com; counting of particles was done
using the Particle Macro and the Threshold Option. Graphs
were made using Microsoft Excel.
Electrophoresis of proteins and immunoblotting
SDS–PAGE analysis was performed using 7.5% acrylamide
(Laemmli 1970) on a mini-gel apparatus (Bio-Rad). Gels were
stained with Coomassie brilliant blue or silver according to the
protocol and kit provided by GE Healthcare. Images of gels were
captured using the Quantity One software and the Fluor-S
Multimager apparatus from Bio-Rad. Immunoblotting analysis
(Towbin et al. 1979) was performed on a Mini Trans-Blot
Cell (Bio-Rad). Primary antibodies were used for 1 h diluted as
follows: AKIN01 and AKIN02 at 1 : 1,000 and 1 : 250, respectively,
360
Association of motors with pollen mitochondria
and the anti-myosin antibody at 1 : 1000. Secondary antibodies
were horseradish peroxidase-conjugated goat anti-mouse and goat
anti-rabbit antibodies from GE Healthcare, used at 1 : 3,000 for
1 h. Blots were developed with the enhanced chemiluminescence
kit from GE Healthcare and captured with the Fluor-S apparatus.
Images of pre-stained molecular mass standards (of broad and
narrow range) were captured with the Fluor-S apparatus and
overlaid on the blot results.
For 2-D electrophoresis, samples were prepared using the
ReadyPrep Soluble/Insoluble kit and concentrated using the
ReadyPrep 2-D Cleanup Kit (both from Bio-Rad). Samples were
directly applied to the rehydration buffer and initially separated
using 7 cm long Immobiline DryStrip (GE Healthcare) with a
pH gradient of 3–10. After the first screening, DryStrip gels with a
pH of 4–7 were used for a sharper separation. Proteins were
separated by isoelectric focusing with a Multiphor II apparatus
(GE Healthcare) at 200 V (2 mA, 5 W) for 1 min, 3,500 V for 1.5 h,
and 3,500 V for a further 1.5 h. After the first dimension, gels were
equilibrated in the equilibration buffer (prepared as indicated in
the manufacturer’s protocol) for 15 min or, alternatively,
frozen and stored immediately. Proteins were separated in
the second dimension by SDS gel electrophoresis on a Bio-Rad
Mini-Protean II, using 1.0 mm thick 7.5% acrylamide gels. At least
three gels for each protein fraction were run. Parallel unstained gels
were transferred onto nitrocellulose membranes and probed with
the anti-myosin antibodies as described.
Protein concentration
The protein concentration was determined by the 2-D Quant
Kit from GE HealthCare using bovine serum albumin (BSA) as
standard.
Supplementary material
Supplementary material mentioned in the article is available
to
online
subscribers
at
the
journal
website
www.pcp.oxfordjournals.org.
Acknowledgments
We thank Professor Peter K. Hepler (Biology Department,
University of Massachusetts. Amherst, MA, USA) for critically
reading the manuscript and for constructive suggestions. We are
grateful to Dr. Stefano Loppi and Professor Carlo Gaggi
(Dipartimento Scienze Ambientali, University of Siena) for their
kind assistance in the statistical analysis of data. We also thank the
employees of the Botanical Garden of Siena University for the kind
assistance in the culture of tobacco plants. This work is partially
supported by a grant from the University of Siena in the
framework of the University Research Programme (PAR 2005).
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(Received November 24, 2006; Accepted December 30, 2006)