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The Plant Cell, Vol. 15, 251–269, January 2003, www.plantcell.org © 2002 American Society of Plant Biologists
In Vitro Assays Demonstrate That Pollen Tube Organelles Use
Kinesin-Related Motor Proteins to Move along Microtubules
Silvia Romagnoli,1 Giampiero Cai, and Mauro Cresti
Dipartimento Scienze Ambientali “G. Sarfatti,” Università di Siena, via Mattioli 4, 53100 Siena, Italy
The movement of pollen tube organelles relies on cytoskeletal elements. Although the movement of organelles along
actin filaments in the pollen tube has been studied widely and is becoming progressively clear, it remains unclear what
role microtubules play. Many uncertainties about the role of microtubules in the active transport of pollen tube organelles and/or in the control of this process remain to be resolved. In an effort to determine if organelles are capable
of moving along microtubules in the absence of actin, we extracted organelles from tobacco pollen tubes and analyzed
their ability to move along in vitro–polymerized microtubules under different experimental conditions. Regardless of
their size, the organelles moved at different rates along microtubules in the presence of ATP. Cytochalasin D did not inhibit organelle movement, indicating that actin filaments are not required for organelle transport in our assay. The
movement of organelles was cytosol independent, which suggests that soluble factors are not necessary for the organelle movement to occur and that microtubule-based motor proteins are present on the organelle surface. By washing organelles with KI, it was possible to release proteins capable of gliding carboxylated beads along microtubules.
Several membrane fractions, which were separated by Suc density gradient centrifugation, showed microtubule-based
movement. Proteins were extracted by KI treatment from the most active organelle fraction and then analyzed with an
ATP-sensitive microtubule binding assay. Proteins isolated by the selective binding to microtubules were tested for the
ability to glide microtubules in the in vitro motility assay, for the presence of microtubule-stimulated ATPase activity,
and for cross-reactivity with anti-kinesin antibodies. We identified and characterized a 105-kD organelle-associated
motor protein that is functionally, biochemically, and immunologically related to kinesin. This work provides clear evidence that the movement of pollen tube organelles is not just actin based; rather, they show a microtubule-based motion as well. This unexpected finding suggests new insights into the use of pollen tube microtubules, which could be
used for short-range transport, as actin filaments are in animal cells.
INTRODUCTION
In eukaryotic cells, the transport of organelles and molecules among different cellular regions occurs along the fibrillar cytoskeleton: microtubules and actin filaments (AFs). Organelle transport is essential for cell viability and is well
studied in a variety of cellular processes, such as fast axonal
transport in neurons (Mercer et al., 1994) and the transport
of pigment granules in melanophores (Rogers et al., 1997).
Organelle movement is driven along cytoskeletal filaments
by motor proteins: kinesin and dynein are the two families of
microtubule-based motor proteins (Hirokawa et al., 1998),
whereas myosin is the only family of AF-based motor protein found in eukaryotic cells (Titus, 1993). Motor proteins
anchor to the organelle surface and drive them along cyto-
1 To
whom correspondence should be addressed. E-mail romagnolis@
unisi.it; fax 39-0577-232860.
Online version contains Web-only data.
Article, publication date, and citation information can be found at
www.plantcell.org/cgi/doi/10.1105/tpc.005645.
skeletal filaments by converting into movement the chemical energy released by ATP hydrolysis. In animal cells, microtubules are the primary support for organelle transport;
they drive and regulate the positioning of many organelles,
including the Golgi apparatus, the endoplasmic reticulum,
lysosomes, peroxisomes, and secretory vesicles (Rogers
and Gelfand, 2000). Nevertheless, various pieces of evidence have suggested that microtubule- and AF-based motility systems cooperate functionally during organelle transport, such as in dermal pigment cells (Gross et al., 2002), in
epithelial cells (Fath et al., 1994), during exocytosis (Bi et al.,
1997), and in nerve cells (Coy and Howard, 1994). Cooperation between AF- and microtubule-dependent motility also
extends to lower eukaryotes. In the moss Physcomitrella
patens, chloroplasts use both the microtubule and AF motility systems, which are regulated differently by two types of
photoreceptor (Sato et al., 2001). Therefore, the coordinated
activity of microtubule- and AF-dependent motility is likely
to be a general principle of organelle transport in eukaryotic
cells.
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Studies of cytoplasmic streaming have revealed that organelle transport in plant cells is an AF-dependent process.
Dynamic interactions between AFs and membranes are responsible for vesicle movement during secretion, endocytosis, mitochondrial inheritance, the maintenance of an even
distribution of subcellular components in large cells, and the
asymmetric delivery of membrane material in polarized
growing cells (Shimmen and Yokota, 1994). AFs are the
main scaffold for organelle movement in plant cells, as
shown by studies with AF inhibitors (Williamson, 1972), the
analysis of organelle movement in cells (Nebenführ et al.,
1999), and the association of different myosin classes with
plant organelles (Uchida et al., 1999; Yokota, 2000). Microtubules are not likely to be involved in the transport of plant
organelles; however, they could be involved in other dynamic processes, such as the positioning of nuclei (Lloyd et
al., 1987) and mitochondria (Van Gestel et al., 2002), the
transport of chloroplasts (Sato et al., 2001), and vesicle delivery during cytokinesis (Julie Lee et al., 2001). These few
examples suggest that microtubules could support the
movement of plant cell organelles different from those
transported by AFs; alternatively, microtubules could regulate the movement of organelles that translocate along AFs.
In fungal hyphae and in tip-growing plant cells (root hairs
and pollen tubes), AFs and microtubules participate in the
distribution of organelles according to the cell growth rate.
The relative contributions of AFs and microtubules generally
are understood in fungal hyphae and root hairs, but data are
controversial for pollen tubes (Geitmann and Emons, 2000).
The angiosperm pollen tube acts as a cellular channel for
delivering sperm cells from the pollen grain to the ovary during fertilization. Membrane trafficking in the pollen tube occurs in both directions and determines the even distribution
of organelles and molecules along the tube (except in the
apical and subapical regions) (Pierson et al., 1990). Although
AFs and microtubules are equally abundant in the pollen
tube, current models of organelle transport indicate that AFs
are the main tracks along which organelles are transported.
This hypothesis is based on inhibition studies (Mascarenhas
and Lafountain, 1972; Lancelle and Hepler, 1988), motility
assays with isolated organelles (Kohno and Shimmen,
1988), and the localization of myosins on the organelle surface (Heslop-Harrison and Heslop-Harrison, 1989; Tang et
al., 1989; Miller et al., 1995; Tirlapur et al., 1995). On the
other hand, microtubules most likely correlate with the
movement of both the generative cell and the vegetative nucleus (Astrom et al., 1995; Miyake et al., 1995). Other possible roles of microtubules involve pulsatory growth (Geitmann
et al., 1995) and the accumulation of vacuoles in the pollen
tube tip (He et al., 1995).
The relationships between microtubules and organelle
trafficking in the pollen tube are not clear, because microtubule inhibitors do not produce particular effects on both cytoplasmic streaming and pollen tube growth (Heslop-Harrison
et al., 1988; Geitmann et al., 1995). On the other hand, a series of microtubule motor proteins has been identified and
characterized in the pollen tube. A pollen kinesin homolog
(Tiezzi et al., 1992) and two dynein-related polypeptides
(Moscatelli et al., 1995) have been identified in the vegetative cytoplasm of pollen tubes and localized in association
with unclassified membrane structures (Cai et al., 1993;
Moscatelli et al., 1998). More recently, a 90-kD kinesinrelated protein (90-kD ATP–microtubule-associated protein
[ATP-MAP]) showing motor activity was found in association
with organelles and microtubules in the cortical region of
pollen tubes (Cai et al., 2000), which suggested that the 90kD motor protein translocates organelles along microtubules in tobacco pollen tubes. Microtubule-based motors
identified to date in the pollen tube have different distributions. The pollen kinesin homolog is localized in the apex
and flanks of the tube (Cai et al., 1993), whereas dyneinrelated polypeptides have a more homogenous distribution
(Moscatelli et al., 1998). By contrast, the 90-kD ATP-MAP is
localized in the cortical regions but is absent from the apex
(Cai et al., 2000). The different distribution of microtubulebased motors could imply that these proteins have distinct
functions. Additional evidence for the role of microtubule
motors in Arabidopsis pollen tubes has come from the genetic analysis of double mutants, which suggested that a kinesin-like protein is involved in pollen tube growth and vesicle transport (Krishnakumar and Oppenheimer, 1999).
The presence of organelle-associated motors and evidence from mutant analysis suggested that pollen tube organelles interact dynamically with and translocate along microtubules, likely through the activity of motor proteins.
Because inhibition studies indicated that the contribution of
microtubules to cytoplasmic streaming and pollen tube
growth is likely to be poor, the result of any dynamic interactions between microtubules and membranes should be
found elsewhere. According to the model of functional cooperation between AF- and microtubule-dependent motility
described in other cells, we can hypothesize that pollen
tube microtubules cooperate with AFs in the fine-tuning of
organelle and vesicle transport or, alternatively, that microtubules could be necessary for the local movement of specific membrane-bound organelles (Cai et al., 2001).
Biochemical, cytological, and genetic analyses have indicated that pollen tube microtubules possess the appropriate
protein machinery to move organelles. However, these data
do not explain the correlations between microtubules, motor
proteins, and membrane-bound organelles and do not clarify the movement of pollen tube organelles along microtubules. In addition, unequivocal evidence of pollen tube organelles that move along microtubules still is missing. In the
present study, we have used the microtubule-organelle motility assay to establish that pollen tube organelles exhibit
clear motor activity along microtubules and to define some
crucial aspects of this movement. The attachment of microtubule tracks to a substrate is a powerful tool with which to
evaluate the motor activity of undefined or purified membrane-bound organelles under defined experimental conditions (Urrutia et al., 1993). The aim of the present work is to
Movement of Pollen Tube Organelles along Microtubules
analyze the conditions under which the movement of pollen
tube organelles occurs along microtubules and to investigate the molecular machinery that supports this activity.
Pollen tube organelles are shown to move along microtubules under different experimental conditions in an ATPdependent manner in both the presence and the absence of
pollen tube cytosol. This finding suggested that the motor
proteins responsible for organelle motility are bound to the
organelle surface. Organelle movement is cytochalasin independent, which suggested that AFs are not required. Because cytosol is not necessary for organelle movement, we
removed peripheral proteins from the organelle surface and
showed that the extracted proteins promote the movement
of latex beads along microtubules. An ATP-dependent microtubule binding assay was used to identify putative motors from the pool of peripheral organelle proteins. We have
isolated a kinesin-related protein that is able to translocate
microtubules in in vitro motility assays and that cofractionates with a microtubule-enhanced ATPase activity. These
results provide clear evidence that organelles from a higher
plant cell (the pollen tube) actively move along microtubules
by means of motor proteins and challenge the notion that
organelle transport in the pollen tube is exclusively an AFdependent process.
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motor proteins (which could invalidate our results), as demonstrated by the absence of motor and ATPase activity and
by negative results with both MMR44 (Marks et al., 1994)
and HYPIR pan-kinesin antibodies in immunoblotting assays
(data not shown). When in vitro–polymerized microtubules
were observed by Allen video enhanced contrast–differential
interference contrast microscopy (Figure 1B), no fields showed
RESULTS
Components of the in Vitro Motility Assay
In this work, we have developed a method for the isolation
of pollen tube organelles that move along microtubules under an in vitro motility assay. Germinated pollen tubes were
homogenized, and then low- and high-speed centrifugation
was used to separate the cytosol from the organelle fraction. The pool of organelles was termed the postnuclear supernatant (PNS) because the generative cell and the vegetative nucleus were likely excluded after the low-speed
centrifugation. The main fractions used in the motility assays were analyzed using 7.5% PAGE. Figure 1A shows the
polypeptide composition of bovine brain tubulin (lane 2), of
the organelle fraction before (lane 3) and after (lane 4) the
second 20% pelleting step, and of cytosol (lane 5) from pollen tubes. The sample shown in lane 3 was used for the motility assay in the presence of cytosol. The second pelleting
step through 20% Suc, which was introduced to clean the
organelle surface of cytosolic proteins, yielded the material
in lane 4, which was used for the assays in the absence of
soluble proteins. Bovine brain tubulin, which was used for
the in vitro polymerization of taxol-stabilized microtubules,
was purchased from Cytoskeleton, Inc. (catalog number
T238), and is 99% pure according to the technical information accompanying the product. The purity level of tubulin
was confirmed by analysis with Fluor-S and Quantity One
software. The tubulin sample did not contain contaminating
Figure 1. Components of the in Vitro Motility Assay.
(A) Silver-stained SDS gel showing the components used for the in
vitro motility assay. Lane 1, molecular mass standards (indicated in kD
at left); lane 2, bovine brain tubulin (5 g) used to prepare in vitro–
polymerized microtubules; lane 3, PNS organelles from pollen tubes
(5 g); lane 4, PNS organelles after the second 20% pelleting step (5
g); lane 5, cytosolic proteins from pollen tubes (5 g). The front of
migration of the electrophoresis is indicated at bottom left (df, dye
front).
(B) Single video frame of in vitro–polymerized microtubules at a concentration (0.4 mg/mL) higher than that used for the motility assay.
Bar 2 m.
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organelles either on the microtubules or in solution, demonstrating that bovine brain tubulin was pure and not contaminated by animal organelles.
Pollen Tube Organelles of Different Size Move
along Microtubules
Different conditions were used during the in vitro motility assays to study the molecular basis of the movement of pollen
tube organelles along microtubules. Table 1 summarizes the
components used in each motility assay. In vitro motility assays were performed by preattaching microtubules to the
cover slips of perfusion chambers, which then were saturated with casein to prevent the aspecific adsorption of organelles. A mixture of organelles, cytosol, and ATP then was
added to the microtubules (the so-called standard condition). Samples were observed by Allen video enhanced contrast–differential interference contrast microscopy, which
allowed the observation of different types of dynamic interactions between microtubules and organelles. Some organelles showed Brownian motions in the medium and then
suddenly attached to microtubules and, either immediately
or soon after, started to move along. The distance covered
by organelles along microtubules was variable but in the
range of 5 to 20 m. Some organelles that moved along microtubules suddenly dissociated, whereas others stopped
moving and remained attached to microtubules. In some instances, binding and subsequent dissociation of organelles
occurred without movement in between. Movements were
smooth and continuous, even for the larger organelles,
whereas recoils or saltatory movements were very infrequent.
When moving organelles reached the intersections of different microtubules, some of them switched to a different
microtubule and resumed their movement. Moving organelles sometimes were observed to pass through unmoving
organelles (Figure 2A, arrowhead) on microtubules without
apparent collision. The size of the organelles that moved
along microtubules was variable, because larger (Figure 2A,
arrowhead) and smaller (Figure 2B, arrowhead) organelles
were observed. The organelle indicated by the arrowhead in
Figure 2A moved with a speed of 178 nm/s, whereas the
smaller organelle in Figure 2B translocated at 133 nm/s. A
typical view field (corresponding to 400 m2) usually
showed 49 21 organelles, of which 35 17 bound to microtubules. In most cases, microtubule-associated organelles retained the ability to move along in most, but not all,
cases. Motionless organelles remained bound to microtubules even when moving organelles approached and
crossed over them (Figure 2A, arrow). The binding of organelles to microtubules appeared to be specific, because
the number of organelles out of microtubules was very low.
The movie sequence of Figure 2A can be viewed in the supplemental data online.
Transport of organelles along in vitro–polymerized microtubules was inhibited by 5 mM AMPPNP (5 -adenylylimidodiphosphate), a nonhydrolyzable ATP analog, which also
caused more organelles to bind to microtubules. Every view
field under these experimental conditions showed no organelles moving along microtubules. Some occasional
Brownian motion of organelles occurred in solution, but we
never observed organelles attaching/dissociating from microtubules or moving along. Furthermore, organelle movement was not observed when nucleotides were omitted
from the microtubule-organelle mixture (data not shown).
Cytochalasin D Does Not Affect Organelle Movement
Short AFs could be present in the cytosol or in membrane
fractions from pollen tubes. To establish that the in vitro
movement of pollen tube organelles was dependent on microtubules but not on AFs, we added cytochalasin D (an actin-depolymerizing factor) to the organelle-cytosol-ATP mixture. The inhibitor was applied at concentrations (5 M) that
usually inhibit cytoplasmic streaming in the pollen tube. Under these experimental conditions, we still observed or-
Table 1. Different Experimental Conditions of the Microtubule-Organelle Motility Assays
Condition
Cytosol
Nucleotide(s)
Other
Standard condition
Without cytosol
Cytochalasin plus cytosol
Cytochalasin without cytosol
AMPPNP
GTP
No nucleotide
6,5 g/L
–
6,5 g/L
–
6,5 g/L
6,5 L/mL
6,5 L/mL
5 mM ATP
5 mM ATP
5 mM ATP
5 mM ATP
5 mM ATP and 5 mM AMPPNP
5 mM GTP
–
–a
Buffera
5 M cytochalasin D
5 M cytochalasin D and bufferb
–
–
Bufferb
Each test contained in vitro–polymerized microtubules (0.1 mg/mL), the PNS at 0.15 g/mL, 20 M taxol, and other components as indicated.
a –, no data.
b Buffer (BRB25 buffer containing 20 M taxol and 1 mM DTT) was added to adjust the assay volume to 200 L.
Movement of Pollen Tube Organelles along Microtubules
255
Figure 2. Motility Assay under Standard Conditions.
Time-lapse video sequences of PNS organelles moving along microtubules under standard conditions (a mixture of pollen tube organelles, pollen tube cytosol, and ATP was added to preattached microtubules, the latter at 0.1 mg/mL). Both larger (arrowhead in [A]) and smaller (arrowhead in [B]) organelles were observed to translocate along microtubules. The arrow in (A) indicates a motionless organelle whose position is unaffected by collision with the moving organelle. Numbers in each frame indicate the time in seconds. The movie sequence of (A) can be viewed
in the supplemental data online. Bars 2 m.
ganelles moving along microtubules. The percentage of
moving organelles was relatively the same compared with
that in the standard conditions. Figure 3 shows images from
two different time-lapse video sequences of organelles
moving along microtubules in the presence of cytochalasin
D. In Figure 3A, two to three organelles were observed to
move simultaneously in the same direction along a single
microtubule. Two organelles (arrowheads outlined in black
and white) covered a longer distance without interruption at
velocities of 285 nm/s. The organelle indicated by the
white arrowhead without outline detached from the microtubule or disappeared from the focal plane when it collided
with the unmoving organelle marked by the arrow (the movie
sequence of Figure 3A can be viewed in the supplemental
data online). Figure 3B shows images from a time-lapse
video sequence of a pollen tube organelle (arrowhead) that
switched between three different microtubules. This specific
assay was performed in the absence of cytosol.
Cytosol Is Not Critical for Organelle Movement
To evaluate the importance of cytosolic proteins in promoting
the transport of pollen tube organelles along microtubules,
the organelle fraction was assayed for motility in the absence
of cytosol. The high-speed pellet was centrifuged again on a
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Figure 3. Motility Assay in the Presence of Cytochalasin D.
(A) Three different organelles (arrowheads) move continuously along the same microtubule in the presence of 5 M cytochalasin D and cytosol.
The organelle indicated by the white arrowhead without an outline detached from the microtubule or disappeared from the focal plane when it
collided with the unmoving organelle marked by the arrow. Numbers in each frame indicate the time in seconds. The movie sequence can be
viewed in the supplemental data online. Bar 2 m.
(B) One pollen tube organelle (arrowhead) switches between three different microtubules in the presence of 5 M cytochalasin D but in the absence of cytosol. Numbers in each frame indicate the time in seconds. Bar 2 m.
20% Suc cushion to further separate the organelle fraction
from cytosolic proteins. A mixture of organelles and ATP
(but not cytosol) was added to microtubules in the perfusion
chamber. Figure 4 shows two examples of pollen tube organelles that moved along microtubules in the absence of
cytosol with velocities of 208 and 156 nm/s (A and B, respectively; the movie sequence of Figure 4B can be viewed
in the supplemental data online). Dynamic interactions between organelles and microtubules under these experimental conditions were the same as those described for the
standard conditions. Organelles were observed to move
continuously along microtubules even when they approached motionless organelles and crossed over them.
These results indicate that cytosolic factors are not necessary for the movement of pollen tube organelles along microtubules.
Organelle Proteins Move Latex Beads
As further evidence for the presence of microtubule-based
motors on the surface of pollen tube organelles, peripheral
Movement of Pollen Tube Organelles along Microtubules
proteins were extracted from organelles and assayed for
their ability to move latex beads by in vitro motility assay.
Peripheral membrane proteins were removed from pollen
tube organelles by treatment with KI. The sample was centrifuged to separate the KI-washed organelles (Figure 5A,
lane 2) from extracted proteins. The supernatant (Figure 5A,
lane 3) was desalted to remove KI from extracted proteins
and then used to coat latex beads. As a control, we performed in vitro motility assays with a commercial kinesin
from Cytoskeleton, Inc. (Figure 5A, lane 4) that was attached
to beads under exactly the same experimental conditions
as were used for pollen proteins. In this case, many beads
257
moved at high speed (400 nm/s) in the same direction, as
shown by the two video frames in Figure 5B. When organelle protein–coated beads were introduced into the perfusion chambers, they stuck to the microtubule surface just
as pollen tube organelles did. In most cases, the beads
moved along microtubules (Figures 5C and 5D). The velocity
of beads was equivalent to that of unextracted organelles
(183 44 nm/s for beads versus 171 58 nm/s for organelles). Beads showed unidirectional and continuous
movement. As an additional control, KI-washed organelles
were centrifuged, resuspended in KI-free buffer, and assayed for the ability to translocate along microtubules by in
Figure 4. Motility Assay of Pollen Tube Organelles in the Absence of Cytosol.
Two time-lapse video sequences of pollen tube organelles (arrowheads) that move along a microtubule in the absence of cytosol. Velocities of
the organelles were 208 and 156 nm/s. Numbers in each frame indicate the time in seconds. The movie sequence of (B) can be viewed in the
supplemental data online. Bars 2 m.
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Figure 5. Motility Assay of Carboxylated Beads along Microtubules.
(A) Silver-stained SDS gel showing the main components used in the motility assay. Lane 1, molecular mass standards (indicated in kD at left);
lane 2, pellet of KI-washed PNS organelles (5 g); lane 3, supernatant containing KI-stripped proteins from PNS organelles (10 g); lane 4, commercial kinesin (1 g). The front of migration of the electrophoresis is indicated at bottom left (df, dye front).
(B) In vitro motility assay of carboxylated beads coated with commercial kinesin. All beads moved in the same direction at 400 nm/s. The arrow in the time-0 frame indicates the direction of bead movement. Numbers in each frame indicate the time in seconds. Bar 2 m.
(C) and (D) Two examples from in vitro motility assays of carboxylated beads coated with proteins extracted from pollen tube organelles. Beads
moved along in vitro–polymerized microtubules at 183 44 nm/s. Numbers in each frame indicate the time in seconds. Bars 2 m.
vitro motility assay. In this case, we observed no organelles
showing active movement along microtubules (data not
shown). Uncoated beads never moved along microtubules
(data not shown).
Organelle Velocities under Different Conditions
Organelle velocity was calculated with the Speed command
of the Argus 20 image processor, taking into account all
moving organelles regardless of their size. Figure 6 shows
the average velocities of pollen tube organelles in different
experimental conditions. Under standard conditions (organelles, ATP, and cytosol), we determined an average speed of
171 58 nm/s. Both the average speed and the standard deviation increased in the absence of cytosol (309 205 nm/s)
compared with the presence of soluble factors. The addition
of cytochalasin D to the standard conditions increased the
average speed to 360 67 nm/s. The effect of cytochalasin
D was analyzed in the absence of cytosol as well. In this
case, the average velocity was comparable to that observed
in the presence of cytosol and cytochalasin D, and the standard deviation was higher. Organelle protein–coated beads
moved along microtubules with an average speed of 183 44 nm/s.
Organelles of Different Fractions Move
along Microtubules
To demonstrate that microtubule-based motility is not restricted to a particular class of pollen tube organelles, we re-
Movement of Pollen Tube Organelles along Microtubules
quired fractionation of the initial PNS. A discontinuous Suc
gradient was used to separate pollen tube organelles into
five distinct fractions, whose polypeptide profiles are shown
in Figure 7A (lanes 4 to 8). When tested by in vitro motility
assay, the organelles of each fraction were observed to
move along microtubules in both the presence and the absence of cytosol (data not shown). This finding suggested
that most pollen tube organelles are able to move along microtubules. We found that organelles of the 50/57 fraction
(lane 7) showed a higher level of motor activity than the
other organelle fractions. In this context, higher motor activity means that moving organelles in the 50/57 fraction were
greater in number and covered longer distances. Figure 7B
shows one organelle from the 50/57 fraction that moved
along a microtubule. The movement of 50/57 organelles
was smooth and continuous without recoils or saltatory activities and was similar to that of PNS organelles under the
same experimental conditions.
The organelle fractions were assayed for the presence of
specific organelle marker proteins to provide indications of
the organelle composition of each fraction. We selected four
organelle markers: cytochrome c oxidase activity for mitochondria, inosine 5-diphosphatase activity for the Golgi apparatus and secretory vesicles, P-ATPase activity for the
Figure 6. Velocities of Pollen Tube Organelles and Beads under Different Experimental Conditions.
Velocities of organelles (expressed as nm/s) under the standard conditions increased when the cytosol was not added to the mixture.
Higher velocities also were obtained in the presence of cytochalasin D
(in both the presence and the absence of cytosol). The velocity of
beads coated with proteins from pollen tube organelles was comparable to the velocity of organelles under standard conditions. Approximately 20 organelles (or beads) were tested to calculate the speed
and standard deviation for each experimental condition.
259
plasma membrane, and cytochrome c reductase activity for
rough and smooth endoplasmic reticulum. The results are
expressed in Figure 7C as relative percentages of enzymatic
activity. We found that the 50/57 organelle fraction did not
contain peaks of relative enzymatic activity. However, this
fraction was quite enriched in cytochrome c oxidase activity; therefore, it could be ascribed as a mitochondrial membrane fraction. More specifically, the S33 fraction contained
the highest level of inosine 5-diphosphatase activity (it was
enriched in the Golgi apparatus and/or secretory vesicles).
The 33/44 fraction had the highest intensity of both cytochrome c reductase and P-ATPase activity (which suggested enrichment in endoplasmic reticulum and plasma
membrane, respectively). The highest level of cytochrome c
oxidase activity was found in the 57/65 fraction, which likely
contains mitochondria.
Proteins from the 50/57 Organelle Fraction Bind to and
Glide Microtubules in Vitro
Because the 50/57 organelles exhibited higher motor activity, this organelle fraction was investigated for the presence
of microtubule-based motors. Proteins from the 50/57 organelle fraction (Figure 8A, lane 3) were extracted with KI.
After desalting, KI-extracted proteins were assayed for the
ability to bind to microtubules in an ATP-sensitive manner.
The pool of KI-extracted proteins (Figure 8A, lane 4) was incubated with microtubules in the presence of AMPPNP. Microtubules were pelleted by centrifugation. The AMPPNPmicrotubule pellet (lane 6) was washed with ATP and KCl at
a low concentration (200 mM). Centrifugation was used to
separate the ATP/KCl-released proteins (S1-ATP; lane 7)
from microtubule-bound proteins. The S1-ATP contained
essentially polypeptides at 105 kD (arrowhead) and 74 kD
(dot). A 53-kD band represented tubulin, as indicated by
positive blots with a monoclonal antibody to tubulin (data
not shown). The microtubule pellet was washed further with
ATP and KCl at a higher concentration (500 mM) and then
centrifuged to yield a second ATP/KCl-released sample (S2ATP; lane 8) and the final microtubule pellet (lane 10). The
second ATP supernatant contained predominantly two
bands at 183 kD (lane 8, arrow) and 53 kD. The presence of
polypeptides at 105 kD was not detected, showing that they
had been released quantitatively in the first ATP/KCl wash.
The ATP-dependent microtubule binding assay also was
performed on proteins extracted from the PNS by KI treatment. In this case, the final ATP supernatant contained additional bands but still included polypeptides at 183 and 105
kD (lane 9, arrow and arrowhead, respectively).
A key feature of motor proteins is the ability to glide microtubules in an in vitro motility assay. The motor activity of
the S1-ATP fraction was determined by gliding assay of microtubules on S1-ATP proteins bound to cover slips. Single
microtubules bound to cover slips coated with S1-ATP proteins and moved in the presence of ATP with a velocity of
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Figure 7. Motility Assay of the 50/57 Organelle Fraction.
(A) Silver-stained SDS gel showing the organelle fractions obtained after separation of PNS on a discontinuous Suc gradient. Lane 1, molecular
mass standards (indicated in kD at left); lane 2, cytosolic proteins (5 g); lane 3, PNS organelles; lane 4, the S33 fraction (the organelle fraction
just above the 33% Suc layer); lane 5, the 33/44 fraction; lane 6, the 44/50 fraction; lane 7, the 50/57 fraction; lane 8, the 57/65 fraction. Lanes 3
to 8 contained 10 g of protein. The front of migration of the electrophoresis is indicated at bottom left (df, dye front).
(B) In vitro motility assay of the 50/57 organelle fraction. The arrowhead indicates one organelle that moved along in vitro–polymerized microtubules. Numbers in each frame indicate the time in seconds. Bar 2 m.
(C) Detection of organelle marker proteins in the discontinuous Suc fractions. The specific activity of each marker is expressed as the relative percentage of enzymatic activity. The results are averages of three different experiments. Cytochrome c reductase, P-ATPase, inosine 5-diphosphatase, and
cytochrome C oxidase activities are markers of endoplasmic reticulum, plasma membrane, the Golgi apparatus, and mitochondria, respectively.
133 47 nm/s. Figure 8B shows an example of three to four
microtubules that moved on S1-ATP proteins. The motor
activity of S2-ATP was not investigated.
Immunoblot Analysis of the 105-kD Polypeptide
To determine if any S1-ATP proteins belonged to the kinesin
superfamily, we probed different anti-kinesin antibodies.
Specifically, we used the polyclonal HYPIR antibody, which
was raised against highly conserved amino acid sequences
of the kinesin superfamily (Sawin et al., 1992); the polyclonal
AKIN01, raised against bovine brain kinesin; the monoclonal
AKIN02, which corresponds to SUK4 (Ingold et al., 1988)
and was raised originally against the head of sea urchin kinesin; and the polyclonal K1005, which corresponds to the
HD antibody (Rodionov et al., 1993) and was raised originally against the motor domain of Drosophila kinesin. Al-
Movement of Pollen Tube Organelles along Microtubules
though the first three antibodies failed to give a positive reaction, the polyclonal K1005 cross-reacted with the 105-kD
polypeptide (Figure 9A). The K1005 anti-kinesin antibody
was probed on the protein fractions shown in Figure 8A to
analyze the distribution of the 105-kD polypeptide during
the isolation of microtubule binding proteins. As shown in
Figure 9A, the 105-kD polypeptide was detected throughout
the purification steps (arrowhead). In the PNS (lane 2), the
105-kD polypeptide was not the only band detected by the
K1005 antibody. The protein was detected in the 50/57 organelle fraction (lane 3) and in the KI-extracted protein sample (lane 4). At this stage, most of the contaminating bands
were lost. The 105-kD polypeptide bound preferentially to
microtubules in the presence of AMPPNP (lane 6) and was
released after washing with ATP/KCl (lane 7). The band was
almost undetectable in the S2-ATP sample (lane 8), and only
a small amount was found in the microtubule pellet (lane 10).
We also checked the distribution of the 105-kD polypeptide in the organelle fractions obtained after discontinuous
Suc gradients (shown in Figure 7A). The K1005 antibody
cross-reacted with all of the organelle fractions, but the 105-kD
polypeptide was found mostly in the 50/57 and 57/65 fractions (Figure 9B). Densitometric analysis was performed in
all lanes with Quantity One software to calculate the relative
intensity of the blots. Assuming that the signal in the 50/57
fraction is 100% and that equal protein amounts were
loaded in each lane, the 105-kD polypeptide was detected
with a relative percentage of 13.4% in S33, 39.5% in 33/44,
32.8% in 44/50, and 86.1% in 57/65.
ATPase Activity on S1-ATP Proteins
Almost all microtubule-based motor proteins have microtubule-enhanced ATPase activity. To establish whether the
S1-ATP proteins share this feature, we separated the S1ATP polypeptides by Suc gradient centrifugation. Figure
10A shows a typical SDS gel representing the separation of
S1-ATP polypeptides. The main proteins were eluted in distinct fractions: the 74-kD polypeptide at 4 Svedberg units
and the 105-kD protein at 9 S. The ATPase activity of each
fraction was determined in both the presence and the absence of microtubules. We found evidence for microtubuleenhanced ATPase activity (Figure 10B) that peaked in fraction 11 (from 0.08 to 3.7 nmol Pi/mL) and that corresponded
exactly with the elution pattern of the 105-kD polypeptide.
Analysis of the other fractions showed lower ATPase activity
for the 74-kD polypeptide, which did not require the presence of microtubules.
DISCUSSION
Here, we present evidence that membrane-bound organelles of pollen tubes move along microtubules in in vitro
261
motility assays. In vitro organelle movement occurs in the
absence of cytosol, indicating that motor proteins are attached firmly to the organelle surface and that no cytosolic
factors are required. The motility of organelles along microtubules is continuous, not saltatory, and is dependent on
ATP. Motor protein(s), which can be extracted from the organelle surface, are able to move polystyrene beads along
microtubules. The ATP-dependent microtubule binding of
peripheral organelle proteins allowed the isolation of a protein fraction that glides microtubules and that contains a
105-kD protein showing microtubule-enhanced ATPase activity and cross-reactivity with anti-kinesin antibodies. These
data constitute evidence that pollen tube organelles move
along microtubules under in vitro conditions and that microtubule-dependent organelle movements are driven by motor
proteins. A rational extension of these findings is that pollen
tube organelles can move along microtubules under in vivo
conditions as well.
We used a reconstituted system to examine the movement of pollen tube organelles along microtubules. The in
vitro motility assay using video-enhanced techniques is a
powerful tool with which to study the movement of membrane-bound organelles under defined experimental conditions. This technique was used to study the motility of organelles from a wide variety of different cells and to
characterize the proteins involved in the transport of organelles along microtubules (Schroer and Kelly, 1985). We
used bovine brain tubulin in our motility assays mainly because of the poor quantity of tubulin that can be obtained
from tobacco pollen tubes. Although it would be preferable
to use the native track (i.e., pollen tube microtubules), extensive evidence indicates that animal tubulin can efficiently
replace plant tubulin: the binding of plant MAPs to neuronal
or plant tubulin is the same (Schellenbaum et al., 1993), microinjected brain tubulin incorporates into plant microtubules (Wasteneys et al., 1993), and sea urchin dynein translocates efficiently along plant microtubules (Yokota et al.,
1995). Under standard conditions (i.e., in the presence of
cytosol), we found that pollen tube organelles of different
size move along microtubules. We have not yet investigated
the polarity of organelle movement along microtubules.
Organelles can switch from different microtubules and
can pass each other without colliding, as described in previous studies (Schnapp et al., 1985). Organelles move only after the addition of ATP, which implies the presence of ATPdriven motor proteins on the organelle surface. The mean
velocity of such organelle movement (171 58 nm/s) is very
low compared with the velocity of pollen tube organelles involved in cytoplasmic streaming ( 1 m/s). Other authors
have reported that isolated vesicles can translocate along
microtubules in in vitro motility assays with velocities close
to the speed that vesicles attain within cells (Schnapp et al.,
1985). This equivalence was not found when isolated pollen
tube organelles were assayed for the ability to slide along
actin bundles in characean cell models (Kohno and Shimmen,
1988). In that case, the velocity of organelle movement was
262
The Plant Cell
Figure 8. Preparation of Proteins from the 50/57 Organelle Fraction
That Bind to Microtubules in an ATP-Sensitive Manner and That Are
Capable of Gliding Microtubules.
(A) Silver-stained SDS gel showing the intermediate fractions obtained during the preparation of S1-ATP and S2-ATP from KI-extracted
proteins of the 50/57 organelle fraction. Lane 1, molecular mass
standards (indicated in kD at left); lane 2, PNS organelles; lane 3, the
50/57 organelle fraction; lane 4, desalted KI-extracted proteins from
the 50/57 organelle fraction; lane 5, supernatant after incubation of
proteins from lane 4 with microtubules and AMPPNP; lane 6, the
corresponding pellet; lane 7, S1-ATP obtained by incubating the
sample from lane 6 with 10 mM ATP and 200 mM KCl; lane 8, S2-ATP
obtained by further incubation with 10 mM ATP and 500 mM KCl;
lane 9, S-ATP obtained from the pool of PNS organelles; lane 10, the
corresponding pellet of S1-ATP. The arrowheads and the dot indicate the polypeptides at 105 and 74 kD that are released specifically
greater than that of the cytoplasmic streaming in pollen
tubes, indicating that the potential velocity of pollen tube organelles is moderated within the cell. Nevertheless, these
examples suggest that organelle speed in an in vitro motility
assay is comparable to or greater than the in vivo organelle
velocity. Consequently, evidence that the in vitro velocity of
pollen tube organelles along microtubules is considerably
less than that in vivo suggests that either (1) we have been
unable to reproduce the in vivo conditions (which may be
more stringent for pollen tubes), or (2) microtubules are not
involved in the primary route of cytoplasmic streaming in the
pollen tube. Organelle movement along microtubules does
not occur in the presence of nucleotides other than ATP
and is sensitive to AMPPNP, as shown for animal kinesins
(Urrutia et al., 1991) and for pollen tube myosin (Kohno et al.,
1990).
Whether organelle speed is dependent on size is not
known; it appeared that larger organelles exhibited a lower
velocity, but statistical analysis is needed to confirm this observation. Movement of pollen tube organelles was not saltatory, as was observed in other cases (Vale et al., 1985a).
Organelle movement in our assay was not actin dependent.
The addition of cytochalasin D did not prevent organelle
movement but, surprisingly, increased organelle speed to
360 67 nm/s (possibly by removing networks of AFs that
could prevent the free movement of organelles along microtubules). In addition, no AFs were visualized in our assay
samples after staining with rhodamine-phalloidin.
The in vitro movement of pollen tube organelles occurred
in both the presence and the absence of cytosol. This result
indicated that organelles do not require the presence of soluble motor proteins to move along microtubules and, consequently, that motor proteins are present on the surface of
isolated organelles. The association of microtubule motor
proteins with membrane-bound organelles has been shown
for different cells (Allan and Schroer, 1999), leading to the
conclusion that functional motor proteins are attached stably to the organelle surface and that the cytosol contains a
nonfunctional pool of motor proteins (Reilein et al., 2001).
The requirement of soluble components other than motor
proteins to activate or regulate organelle-associated motor
proteins in eukaryotic cells still is under debate. For example, the first evidence that soluble factors other than kinesin
are essential for organelle motility (Schroer et al., 1988) was
not confirmed by subsequent studies, in which purified kinesin was shown to promote vesicle motility (Urrutia et al.,
in S1-ATP. The arrows indicate the 183-kD polypeptide released in
S2-ATP. The front of migration of the electrophoresis is indicated at
bottom left (df, dye front).
(B) Microtubules gliding on cover slips coated with S1-ATP. Microtubules (arrows and arrowheads) were observed to move in the
focus plane with velocities of 133 47 nm/s. Numbers in each
frame indicate the time in seconds. Bar 2 m.
Movement of Pollen Tube Organelles along Microtubules
Figure 9. Immunoblot Analysis of the 105-kD Polypeptide.
(A) Immunoblot analysis with the polyclonal K1005 antibody on fractions obtained during the preparation of S1-ATP (lanes 2 to 10 are
the same as in Figure 8A). The antibody cross-reacted with the 105-kD
polypeptide in all lanes (arrowhead). Other contaminating cross-reacting bands were lost during the procedure; as a result, the 105-kD
polypeptide is the only band that binds to and is released from the
microtubules. Lane 1, Coomassie blue–prestained molecular mass
markers. The first three bands correspond to molecular masses of
205, 115, and 93 kD, respectively.
(B) Cross-reactivity of the K1005 antibody with the organelle fractions obtained by discontinuous Suc gradients. Lane 1 to 6, PNS,
S33, 33/44, 44/50, 50/57, and 57/65 fractions, respectively. The
105-kD polypeptide was detected in all fractions, but more strongly
in the 50/57 and 57/65 fractions (lanes 5 and 6, respectively). Equal
protein amounts were loaded in each lane.
1991). On the other hand, cytoplasmic events, such as the
phosphorylation of both kinesin and dynein (Dillman and
Pfister, 1994; McIlvain et al., 1994), are used by cells to regulate the binding of motors to organelles. In our case, the
motility of pollen tube organelles along microtubules occurred in the absence of cytosol, suggesting that the essential motor machinery is located on the organelle surface and
that soluble proteins are not required. The analysis of organelle speed under different experimental conditions suggested additional hypotheses. Evidence that the motility
rate along microtubules is greater when cytosol-free organelle proteins are used indicated that the pollen tube cytosol contains undefined factors that negatively modulate
organelle velocity. These factors could be regulatory proteins, microtubule binding proteins that hinder the movement of organelles along microtubules, or other motors that
counteract the activity of the organelle-associated motor
proteins. Because the addition of cytochalasin D increased
263
the average velocity of pollen tube organelles, AFs could be
involved in this mechanism of control.
The presence of microtubule-based motors on the surface of pollen tube organelles is not unexpected. Microtubule-based motors other than the 105-kD polypeptide are
present in the pollen tube and are found in association with
membrane-bound organelles. The pollen kinesin homolog
was identified initially as a soluble protein that interacted
with microtubules in an ATP-dependent manner (Tiezzi et
al., 1992); however, the protein was immunolocalized at the
tip and on the flanks of pollen tubes with a spot-like pattern
resembling the distribution of organelles or vesicles (Cai et
al., 1993). Furthermore, an immunorelated pollen kinesin homolog was detected in association with the surface of isolated 80-nm membranous structures from hazel pollen (Liu
et al., 1994). Dynein-related polypeptides were discovered
as soluble proteins as well (Moscatelli et al., 1995); however,
they also were found in association with membranous structures (Moscatelli et al., 1998). The 90-kD ATP-MAP was isolated as a soluble protein; however, the MMR44 antibody
detected the 90-kD ATP-MAP in association with organelles
isolated from pollen tubes (Cai et al., 2000). At present, pollen kinesin homolog, dynein-related polypeptides, and 90-kD
ATP-MAP have not been shown to translocate organelles
along microtubules, even though the 90-kD ATP-MAP has
motor activity and interacts with organelles. Nevertheless,
these data suggest that the pollen tube contains two distinct populations of microtubule-based motors—soluble and
membrane associated—whose relationships are not known.
The present evidence on the movement of isolated pollen
tube organelles along microtubules in the absence of soluble proteins confirms previous findings and suggests that
microtubule-based motors are bound to the surface of pollen tube organelles as in other cells.
Motor proteins have been extracted from different organelles by treatment with KI (Schroer et al., 1988) or NaOH
(Lacey and Haimo, 1992), but kinesin was shown to bind to
membranes after strong treatment conditions, such as 1 M
KI or 1 M NaCl (Schnapp et al., 1992). By contrast, pollen
tube myosin was shown to be released from isolated organelles after treatment with 0.5 M KCl (Kohno et al., 1990).
In our hands, 0.6 M KI was sufficient to extract motor proteins from the surface of pollen tube organelles, because
KI-washed organelles did not show motility along microtubules. Presumably, most but not all microtubule-based
motors were extracted from the surface of pollen tube organelles after KI treatment. However, extracted proteins
succeeded in moving polystyrene beads, which suggested
that functionally active motors are detached from the organelle surface by KI treatment. The average velocity of
beads is lower compared with that of organelles in the absence of cytosol, which suggests a partial denaturation of
motor proteins by KI treatment or the requirement of receptor proteins on the cargo.
The ATP-dependent binding of KI-extracted proteins to
microtubules could highlight most of the microtubule-based
264
The Plant Cell
Figure 10. Suc Gradient Centrifugation of S1-ATP and Quantitation of Mg-ATPase Activity.
(A) The S1-ATP fraction was fractionated on a 5 to 25% Suc gradient, and fractions of 0.25 mL were collected. Black arrowheads indicate standard proteins in Svedberg units: thyroglobulin (19.1 S), catalase (11.3 S), and BSA (4.4 S). The positions of both the 105- and 74-kD polypeptides are indicated by white arrowheads. Lane M, molecular mass standards; lane L, protein sample loaded onto the Suc gradient (S1-ATP);
lanes 2 to 19, fractions from the Suc gradient. The front of migration of the electrophoresis is indicated at bottom left (df, dye front).
(B) Mg-ATPase activity with and without microtubules (MT) was analyzed in the Suc gradient fractions shown in (A). Activity is expressed as
nmol/mL inorganic phosphate.
motors associated with pollen tube organelles. When the
assay was performed using proteins extracted from the
PNS, the final ATP supernatant revealed many polypeptides, which likely correspond to putative microtubulebased motors. However, we concentrated our efforts on the
50/57 organelle fraction because it showed a higher motility
rate. The 183-, 105-, and 74-kD polypeptides are likely candidates for motor proteins. We did not investigate the 183-kD
polypeptide, which is eluted only when KCl concentration is
500 mM. In addition, the in vitro microtubule gliding assay
on S2-ATP–coated cover slips was not performed. The
S1-ATP sample, which contained the 105- and 74-kD polypeptides in addition to tubulin, was able to glide microtubules at a speed of 133 47 nm/s. This value is comparable to the velocity of kinesin (Saxton et al., 1988) but is lower
than the velocity of microtubules gliding on dynein-coated
slides (Sakakibara and Nakayama, 1998).
Motor proteins are machines that convert the energy of
ATP hydrolysis in mechanical work; therefore, microtubule
gliding correlates well with the enzymatic ability to hydrolyze
ATP (Oosawa, 1995). In our case, the motor activity of S1-ATP
was related to the 105-kD polypeptide, because it exhibited
exclusively microtubule-dependent ATPase activity. Therefore, the 105-kD polypeptide is a potential candidate for
moving pollen tube organelles along microtubules. This protein is extracted from pollen tube organelles, is able to bind
to microtubules in an ATP-sensitive manner, is the main
band of a protein fraction (S1-ATP) that glides microtubules,
and is the only protein that shows microtubule-dependent
ATPase activity. In addition, the protein cross-reacts with
the anti-kinesin antibody K1005, formerly known as HD,
which was raised against the motor domain of Drosophila kinesin (Rodionov et al., 1993). These findings indicate that the
105-kD polypeptide is related functionally, biochemically, and
Movement of Pollen Tube Organelles along Microtubules
immunologically to kinesin. The 105-kD polypeptide was not
the only band that cross-reacted with the K1005 antibody;
however, it was the only polypeptide that bound to and was
released from microtubules. Therefore, the other cross-reacting bands are likely to be nonmotor proteins that share common epitopes. Because the K1005 antibody stained bands
other than the 105-kD polypeptide and cross-reacted with all
organelle fractions, it could not be used for immunocytochemistry analysis of pollen tubes.
Studies with cytoskeletal inhibitors have indicated that cytoplasmic streaming of organelles in the pollen tube depends essentially on AFs (Heslop-Harrison et al., 1988). By
contrast, our data from motility assays indicate that pollen
tube organelles are competent to move along microtubules.
Furthermore, we found that all organelle fractions obtained
by Suc gradient centrifugation showed microtubule-dependent motility and that the 50/57 organelle fraction had a
higher motility rate. These findings suggest two possible hypotheses. The first hypothesis suggests that microtubulebased transport is a common feature of pollen tube organelles. This hypothesis, which is confirmed indirectly by
the presence of the 105-kD motor protein in all organelle
fractions, proposes that microtubules play a general role in
the positioning or movement of pollen tube organelles. Because the long-range transport of organelles occurs along
AFs, microtubules could be important for the localization of
organelles at specific sites (perhaps temporarily) or for the
short-range movement of organelles and vesicles in the pollen tube. These assumptions are supported by data showing that the positioning and transport of membranous structures in eukaryotic cells depend on the coordinated activity
of AFs, microtubules, and related motors (Goode et al.,
2000). In plant cells, organelle transport relies mainly on AFs
and myosin, as shown for the Golgi apparatus (Boevink et
al., 1998) and peroxisomes (Mathur et al., 2002). However,
recent evidence has shown that microtubules also could
play a role in the relocation and positioning of other plant organelles, such as chloroplasts (Sato et al., 2001) and mitochondria (Van Gestel et al., 2002).
The second hypothesis suggests that a few classes of
pollen tube organelles move more actively along microtubules. Specific enrichment of the 105-kD polypeptide in the
most active organelle fraction indicates that this protein is
one of the microtubule-based motors that drives organelles
or vesicles of the 50/57 fraction. The 50/57 fraction is more
enriched in cytochrome c oxidase activity than in other
markers, suggesting that mitochondria may be the relevant
organelle class moving along microtubules in the 50/57 fraction. Furthermore, the appearance of membranes is very
similar to that of mitochondria moving in vitro along microtubules in other cell systems (Nangaku et al., 1994). To reinforce these data, we found that the 57/65 organelle fraction
(which contained the peak mitochondrial marker) has motor
activity lower but comparable to that of the 50/57 fraction
and is correspondingly enriched in the 105-kD motor
polypeptide. Although we cannot exclude the presence of
265
other mobile membranes in the 50/57 fraction, these findings suggest that moving organelles in the 50/57 fraction are
enriched in mitochondria and that the 105-kD polypeptide
participates in their positioning or transport along microtubules, as shown for both animal (Nangaku et al., 1994) and
plant (Van Gestel et al., 2002) cells.
Evidence has been reported that the microtubule cytoskeleton is involved in the transport of both the generative
cell and the vegetative nucleus in the pollen tube (Astrom et
al., 1995). The precise role of microtubules during generative cell and vegetative nucleus movement is unknown, because the drug-dependent disorganization of microtubules
did not prevent but markedly reduced the transport efficiency and speed of these larger particles. No microtubulebased motors have been localized on the surface of or close
to the generative cell and the vegetative nucleus. Furthermore, no in vitro motility assays with isolated generative
cells have been reported. Therefore, the involvement of microtubule motors in the transport of the generative cell and
the vegetative nucleus is far from established. We hypothesize that the role of microtubules is to organize the pollen
tube cytoplasm (including the actin cytoskeleton) during the
movement of the generative cell and the vegetative nucleus.
The motor protein and the motility events we have demonstrated here could take part in this process.
METHODS
Chemicals and Antibodies
Chemical reagents for electrophoresis, including molecular mass
standards (unstained or prestained with Coomassie Brilliant Blue
R250), were purchased from Bio-Rad (Hercules, CA). Blotting membranes, secondary antibodies, and enhanced chemiluminescence
reagents were from Amersham Pharmacia Biotech (Uppsala, Sweden). General reagents used in the preparation of buffers (including
nucleotides) were purchased from Sigma-Aldrich (St. Louis, MO).
Chromatographic columns were from Amersham Pharmacia Biotech. Polystyrene beads for motility assays were obtained from
Sigma-Aldrich. Purified tubulin and recombinant kinesin were purchased from Cytoskeleton, Inc. (Denver, CO). Anti-kinesin antibodies
were obtained as follows: the polyclonal HYPIR antibody was a generous gift from K.E. Sawin (Institute of Cell and Molecular Biology,
University of Edinburgh, UK) (Sawin et al., 1992); polyclonal AKIN01
and monoclonal AKIN02 (SUK4) were purchased from Cytoskeleton,
Inc.; and polyclonal K1005 was obtained from Sigma-Aldrich.
Preparation of Taxol-Stabilized Microtubules
Microtubules were polymerized from monomeric tubulin (10 mg/mL in
80 mM Pipes, pH 6.8, 1 mM MgCl2, and 1 mM EGTA) in the presence
of 1 mM GTP and 10% glycerol at 35C for 20 min. 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, and 20 M taxol),
placed at room temperature, and used for several motility assays.
266
The Plant Cell
Pollen Culture
Anthers of tobacco (Nicotiana tabacum) were collected from plants
grown in the Botanical Garden of Siena University. After dehiscence,
pollen was collected, dried on silica gels, and stored at 20C. Before use, the pollen was acclimatized progressively at room temperature and then hydrated in a moist chamber. The pollen was germinated in BK medium [1.62 mM H3BO3, 1.25 mM Ca(NO3)2·4H2O, 2.97
mM KNO3, and 1.65 mM MgSO4·7H2O] containing 15% (w/v) Suc
(Brewbaker and Kwack, 1963). Germination was performed for 2 to 3 h
in a rotary incubator set at 24C. Germinated pollen then was used
for protein preparation.
Preparation of Organelle and Cytosol Fractions from Tobacco
Pollen Tubes
After germination in BK medium containing 15% (w/v) Suc, the pollen
was washed twice with 25 mL of BRB25 buffer (25 mM Hepes, pH
7.5, 2 mM EGTA, and 2 mM MgCl2) plus 15% Suc. The pollen was resuspended in 1 volume of lysis buffer (BRB25 buffer containing 2 mM
DTT, 1 mM phenylmethylsulfonyl fluoride [PMSF], 1 L/mL protease
inhibitor cocktail [Sigma-Aldrich], 1 mM NaN3, and 10% mannitol).
After lysis on ice with a motor-driven Potter-Elvehjem homogenizer
(Millville, NJ), the sample was centrifuged at 10,000g for 10 min at
4C to remove large cellular debris. The low-speed supernatant was
centrifuged again at 100,000g for 45 min at 4C on a 20% (w/v) Suc
cushion. The supernatant (i.e., the pollen tube cytosol) was supplied
with 1 mM DTT. The pellet (representing the pollen tube organelles)
was resuspended with 200 L of resuspension buffer (BRB25 buffer,
10% mannitol, 1 mM DTT, and 1 mM PMSF) for 50 mg of germinated
pollen. The sample of tobacco pollen tube organelles was named the
postnuclear supernatant.
Organelle Motility Assay
The movement of organelles along microtubules was analyzed by in
vitro motility assay (Waterman-Storer, 2001). Perfusion chambers
were assembled by attaching two pieces of double-sided scotch
tape on clean microscope slides, and then clean cover slips were
placed on top of them to form a perfusion chamber of 10 to 15 L. All
steps were performed in a moist chamber. Ten microliters of microtubule solution (0.1 mg/mL) was incubated for 5 min in the perfusion
chamber. The chamber was washed repeatedly with casein (5 mg/mL)
in BRB25 buffer containing 20 M taxol and 1 mM DTT. The assay
sample used in the motility assay consisted of pollen tube organelles
(1:100 final dilution) mixed with 20 M taxol, 5 mM ATP, and 6.5 g/L
cytosol. This combination generally is referred to as the standard
condition. Variations to the standard condition included the type of
nucleotide used (5 mM 5-adenylylimidodiphosphate or GTP), the
absence of cytosol, or the addition of cytoskeletal inhibitors (5 M
cytochalasin D). Chambers were perfused with 10 L of the assay
sample and placed onto the optical microscope to observe the motility of pollen tube organelles along microtubules.
The instrumentation for the in vitro motility assay consisted of a
Zeiss Axiophot microscope (Oberkochen, Germany) equipped with a
100 Planapos 1.3 objective and the differential interference contrast filter set. A Hamamatsu C2400-75i charge-coupled device camera (Hamamatsu Photonics, Hamamatsu City, Japan) connected to
Argus 20 (Hamamatsu) was used to visualize microtubules and organelles. The Background Subtraction and Average commands of
Argus 20 were used to enhance the image quality. Video sequences
were recorded onto U-matic tapes (Sony, Tokyo, Japan). For printing
of single video frames, individual frames were captured using ATI TVPlayer software (ATI Technologies, Markham, Canada) running on a
personal computer equipped with the ATI Radeon All-In-Wonder
video capture card connected to the video recorder. To evaluate organelle velocity, single organelles were tracked with the mouse cursor and their velocity was calculated with the Speed command of Argus 20. To visualize the presence of actin filaments, samples were
counterstained with 1 M rhodamine-phalloidin, which was added
directly to the organelle-cytosol mixture.
Extraction of Peripheral Proteins from Pollen Tube Organelles
Peripheral proteins from pollen tube organelles were extracted by incubating organelles for 30 min on ice with 0.6 M KI (Schroer et al.,
1988). KI-washed organelles were centrifuged at 100,000g for 90 min
at 4C. The pellet was resuspended with an identical volume of
BRB25 buffer containing 10% mannitol, 1 mM DTT, and 1 mM
PMSF. Stripped organelles were used for in vitro motility assays. The
supernatant obtained after centrifugation was desalted using the HiTrap desalting column (Amersham Pharmacia Biotech) in BRB25
buffer containing 1 mM DTT and 1 mM PMSF. Desalted KI-extracted
proteins were used for in vitro motility assay with carboxylated latex
beads.
Motility Assay with Carboxylated Latex Beads
For the in vitro motility assay of carboxylated latex beads, KIextracted proteins after desalting were mixed with carboxylated latex
beads (1:1500 final dilution) and incubated for 5 min on ice (Vale et
al., 1985b). After the addition of 20 M taxol and 5 mM ATP, the mixture was used for the in vitro motility assay, which was performed like
the organelle motility assay.
Fractionation of Pollen Tube Organelles by Suc
Gradient Centrifugation
Pollen tube organelles were separated into five different classes by a
discontinuous Suc gradient (Robinson and Hinz, 2001). Suc solutions at different concentrations (33, 44, 50, 57, and 65% [w/v]) were
layered into a centrifuge tube (0.8 mL of each solution). Postnuclear
supernatant organelles (500 L) were loaded directly onto 4.0 mL
of the discontinuous Suc gradient and centrifuged at 200,000g for 60
min at 4C. The five organelle fractions obtained were named S33 (for
the organelle layer just above the 33% Suc layer), 33/44, 44/50, 50/57
and 57/65 (for the interfaces between each Suc concentration).
Organelle fractions were assayed for the presence of marker proteins to provide indications of their composition. We measured the following specific enzyme activities: cytochrome c reductase for rough
and smooth endoplasmic reticulum, P-ATPase for the plasma membrane, inosine 5-diphosphatase for the Golgi apparatus and secretory
vesicles, and cytochrome c oxidase for mitochondria. Enzyme activities were determined by standard colorimetric methods (Robinson and
Hinz, 2001).
Movement of Pollen Tube Organelles along Microtubules
ATP-Dependent Microtubule Binding Assay
Peripheral organelle proteins were extracted with KI and desalted
with the HiTrap desalting column into BRB25 buffer containing 1 mM
DTT and 1 mM PMSF. Microtubules used in the binding assay were
prepared as described for the motility assay. Microtubules (0.66 mL
for 1 mL of protein solution), 20 M taxol, and 10 mM AMPPNP were
added to the protein solution. The mixture was incubated for 30 min
at room temperature, and the sample was centrifuged at 40,000g for
30 min at 25C. 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 M taxol, 1 mM DTT, and 10 mM EDTA). The sample was centrifuged at 40,000g for 30 min at 25C. The pellet was incubated overnight with 0.5 mL of release buffer 1 (25 mM Hepes, pH
7.5, 3 mM EGTA, 2 mM MgCl2, 1 mM DTT, 200 mM KCl, 20 M taxol,
and 10 mM ATP). After centrifugation at 40,000g for 30 min at 25C,
the supernatant was named S1-ATP. The pellet was washed for 60
min with 0.5 mL of release buffer 2 (25 mM Hepes, pH 7.5, 3 mM
EGTA, 2 mM MgCl2, 1 mM DTT, 500 mM KCl, 20 M taxol, and 10
mM ATP). The sample was centrifuged at 40,000g for 30 min at 25C,
and the resulting supernatant was named S2-ATP.
Microtubule Motility Assays
The in vitro motility assay of microtubules was performed exactly as
described previously (Cai et al., 2000).
267
1:3000 for 1 h. Blots were developed with the enhanced chemiluminescence kit from Amersham Pharmacia Biotech and captured with
the Fluor-S apparatus. Images of molecular mass standards, which
were prestained with Coomassie Brilliant Blue R250, were captured
with the Fluor-S apparatus and superimposed to the blot results.
Protein Concentration
The protein concentration was determined by the colorimetric
method (Bradford, 1976) using BSA as a standard.
ATPase Activity
The ATPase activity of protein fractions was measured as follows. A
total of 80 L of protein samples was supplemented with 10 L of
ATP solution (20 mM Hepes, pH 7.5, 20 mM ATP, 20 mM MgCl2, and
10 mM DTT). For analysis in the presence of microtubules, 10 L of
microtubule solution was added to the mixture. In the control sample, 10 L of BRB25 buffer was added to the mixture in place of microtubules. Samples were incubated at 25C for 45 min. The release
of Pi was measured according to the PhosFree phosphate assay kit
purchased from Cytoskeleton, Inc. A linear working curve was made
from 4.5 to 90 M K-phosphate and used to calculate the nanomoles
of phosphate produced by the protein fractions.
Upon request, all novel materials described in this article will be
made available in a timely manner for noncommercial research purposes.
Velocity Sedimentation
ACKNOWLEDGMENTS
Centrifugation through continuous Suc gradients was used to separate the proteins extracted from the organelle fractions and that
bound to microtubules in an ATP-sensitive manner. Samples of S1ATP (300 L) were loaded onto 4.5 mL of 5 to 25% Suc gradients
prepared using a gradient former from Amersham Pharmacia Biotech and a peristaltic pump from Bio-Rad. Suc was dissolved in
BRB25 buffer containing 1 mM PMSF and 2 mM DTT. Thyroglobulin
(19.1 Svedberg units), catalase (11.3 Svedberg units), and BSA
(4.4 Svedberg units) were used as standards to calculate the sedimentation coefficient (Martin and Ames, 1961). Samples were centrifuged at 80,000g for 15 h at 4C. Fractions of 250 L each were
collected from the top of the centrifuge tubes using a peristaltic
pump.
We thank Kenneth E. Sawin (Institute of Cell and Molecular Biology,
University of Edinburgh, UK) for kindly donating the HYPIR antibody.
We also thank Ronald D. Vale (Department of Cellular and Molecular
Pharmacology, University of California, San Francisco), Clive Lloyd
(Department of Cell Biology, The John Innes Centre, Colney, Norwich, UK), and Antonio Tiezzi (Dipartimento Scienze Ambientali, Università della Tuscia, Viterbo, Italy) for helpful and critical discussions.
Received June 25, 2002; accepted October 8, 2002.
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In Vitro Assays Demonstrate That Pollen Tube Organelles Use Kinesin-Related Motor Proteins to
Move along Microtubules
Silvia Romagnoli, Giampiero Cai and Mauro Cresti
Plant Cell 2003;15;251-269; originally published online December 13, 2002;
DOI 10.1105/tpc.005645
This information is current as of June 14, 2017
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