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Annals of Botany 85 (Supplement A): 69-77, 2000
doi:10.1006/anbo.1999.1038, available online at http://www.idealibrary.com on
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Cytoskeletal Basis of Organelle Trafficking in the Angiosperm Pollen Tube
G. CAI*, C. DEL CASINO and M. CRESTI
Dipartimento Scienze Ambientali, Universitdt degli Studi di Siena, Via Mattioli 4, 53100 Siena, Italy
Received: 21 July 1999
Returned for revision: 25 August 1999
Accepted: 22 October 1999
The pollen tube is a cell with unusual features that plays a fundamental role during reproduction in higher plants,
transporting the generative cell and sperm cells from the pollen grain to the ovary. Pollen tubes elongate by apical
growth, which is, in turn, elicited by the fusion of secretory vesicles carrying plasma membrane and cell wall
components. Organelle trafficking in the pollen tube, which occurs bi-directionally along the longitudinal axis, has
been the focus of much research for many years. The movement of organelles along the pollen tube relies on the
cytoskeleton, which consists of microtubules, actin filaments and associated proteins. Motor proteins are a special
subset of cytoskeleton proteins that induce organelles to move by using the energy of ATP hydrolysis. The pollen
tube, like other plant cells, uses actin-myosin interactions as a basis for the intracellular movement of organelles. The
role of microtubules during organelle movement is not yet defined, despite the fact that microtubule motor proteins
have been identified. The differential distribution of organelles and vesicles, which is observable along the main axis
of the pollen tube, is essential for the proper growth of the pollen tube. How this level of organization is preserved is
not known. However, other mechanisms of regulation are being identified in the pollen tube which might also be
important in modulating the activity and structure of the cytoskeleton elements.
© 2000 Annals of Botany Company
Key words: Pollen tube, actin filament, microtubule, cytoskeleton, organelle movement, apical growth, cytoplasmic
streaming, molecular motors, kinesin, dynein, myosin, secretory vesicles.
INTRODUCTION
Of all the stages of sexual reproduction in higher plants,
growth of the pollen tube, a cellular extension generated by
the pollen grain following activation on the stigma of a
receptive flower, is probably the most intensively studied.
The pollen tube is a cell with unusual characteristics as it
accomplishes the transport of other cells-the generative
cell (GC) and sperm cells (SCs) from the pollen grain to
the ovary. The growth of the pollen tube occurs in the
apical region, where secretory vesicles provide plasma
membrane and cell wall elements (Mascarenhas, 1993;
Derksen et al., 1995; Li et al., 1997; Taylor and Hepler,
1997). Bi-directional movement of organelles takes place
along the longitudinal axis of the tube (Pierson and Cresti,
1992; Cai et al., 1997) and probably facilitates the distribution of solutes during tube growth. Organelle trafficking
in the pollen tube has received considerable attention for
many years, mainly because of the importance of the pollen
tube during sexual reproduction in plants and because very
long distances are covered by the organelles. Besides, the
pollen tube is an interesting cellular model because it can be
easily cultured and manipulated under in vitro experimental
conditions. The contribution of many investigators has
been invaluable for our understanding of the biology of
organelle movement in the pollen tube. The scientific
community owes much to the work of Professor Jack
Heslop-Harrison (1920 98) who contributed considerably
* For correspondence. Fax +39-0577-232860, e-mail cai(qunisi.it
0305-7364/00/0A0069 + 09 $35.00/00
to our understanding of the role of the cytoskeleton in
organelle movement and pollen tube growth.
In eukaryotic cells, organelle transport relies upon the
cytoskeleton, mainly microtubules (MTs) and actin filaments (AFs). Both systems take advantage of a particular
group of associated proteins, called motor proteins, that use
the energy of ATP hydrolysis to transport cargo (Howard,
1997). Similar motor machinery characterizes the plant
cells, but the relative contribution of the two main cytoskeleton structures to organelle movement is reversed
(Asada and Collings, 1997) as AFs are the main scaffolds
for organelle transportation (Williamson, 1993). This is also
likely to take place in plant cells with apical growth
(e.g. root hairs, fungal hyphae and pollen tubes), where, in
most cases, both AFs and MTs are oriented approximately
in an axial direction (Kropf et al., 1998). In the pollen
tube, as in other eukaryotic cells, the cytoskeleton is the
molecular apparatus that supplies the force driving
organelle movement. This activity is achieved through the
dynamic interactions between motor proteins and cytoskeleton filaments (Cai et al., 1997). The movement of
organelles and the specific role of cytoskeleton proteins in
the pollen tube are known in general terms, but many
points remain to be clarified. For example, except for the
movements of specific organelles observable through the
optical microscope, little information exists on other types
of intracytoplasmic movement in the pollen tube and on
their relationship with the cytoskeleton. Furthermore, we
have little information on the movement of vesicles between
different endocellular compartments and how this movement is produced or controlled. Nor do we know anything
( 2000 Annals of Botany Company
Cai et al.-Pollen Tube Cytoskeleton
70
about vesicle trafficking between dictyosomes and the
endoplasmic reticulum, or about the endocytotic process.
Mechanisms that control these movements (including those
of larger organelles) are almost unknown as well. Moreover,
the role of motor proteins identified in pollen tubes is far
from clear. These few examples suggest that many aspects
of the cytoskeleton-based organelle trafficking in the pollen
tube have still to be elucidated. The aim of this review is to
summarize current knowledge of organelle movement in the
pollen tube, highlighting the role of cytoskeleton motor
proteins.
ROLE OF ACTIN FILAMENTS AND
MICROTUBULES IN ORGANELLE
TRANSPORT
The movement of organelles in the pollen tube can be
described as a continuous stream of membrane material
along the main axis of the tube, with the exception of the
apical zone, which does not contain larger organelles
(Heslop-Harrison and Heslop-Harrison, 1990). Videomicroscopy analyses have underlined different types of
movement, showing that organelles are independently
transported both towards the tip and in the direction of
the pollen grain (Pierson et al., 1990). In the proximity of
the apical region, the transport orientation of organelles
changes and they move in the opposite direction (HeslopHarrison and Heslop-Harrison, 1990). The involvement of
AFs in organelle transport has been established by several
studies which helped elucidate the organization, structure
and biochemical properties of AFs [for reviews on AF
characterization see Pierson and Cresti (1992) and Li et al.
(1997)]. According to the current model of organelle
transport in the pollen tube, AFs act as tracks along
which organelles are transported by means of myosin
molecules (Cai et al., 1997). Organelles move from the grain
to the apex and vice versa. This suggests that AFs in the
pollen tube do not have the same polarity, but some are
oriented with the plus end towards the apex and the
remainder with the plus end in the direction of the grain.
This assumption is not based on concrete evidence relating
to the orientation of AFs (which is still missing), but merely
on the fact that the myosins so far identified move towards
the plus end of AFs (Spudich et al., 1985). This organization requires that diametric bundles of AFs are distinctly
organized in different cellular regions. How this arrangement could be produced and maintained is not known.
Recently, mathematical analyses have allowed the description of movement of single organelles during tube growth
(de Win et al., 1997), supporting the model of bipolar
organization of AFs (de Win et al., 1998, 1999).
Myosins are the main motor proteins responsible
for organelle movement in the pollen tube (Kohno and
Shimmen, 1988; Kohno et al., 1990). Different subclasses of
myosin have been identified in the pollen tube on the basis of
heterologous antibodies (Heslop-Harrison and HeslopHarrison, 1989; Tang et al., 1989; Tirlapur et al., 1995)
(Table 1) and localized in association with specific organelle
categories (Miller et al., 1995). This suggests that different
myosin subclasses may possibly be involved in the transport
of definite organelles, hence contributing to the control of
organelle trafficking. The specific interaction between
myosin subclasses and organelles could also explain the
finding that organelles moving along the same cytoskeleton
tracks can display different speeds (de Win et al., 1999). This
result could also be the consequence of steric as well as
hindrance effects. Myosins have also been localized in the
apical region of the pollen tube, but their role in the tip
growth process is not known (Tang et al., 1989; Miller et al.,
1995; Tirlapur et al., 1995; Yokota et al., 1995). Myosins
may be used for the AF-mediated accumulation of secretory
vesicles in the apex [as suggested by immunofluorescence
observations (see previous references)], but this hypothesis
does not exclude the involvement of these motor proteins in
TABLE 1. Myosin polypeptides identified in the pollen tube
Molecular
Method of
weight (kD)
identification
Purification
Associated
proteins
Myosin-II related
185
Heterologous
antibodies
No
Myosin-II related
175
Heterologous
antibody
Myosin-l A,B
125
Myosin-II
Sequence
Localization
Citations
NI
NA
Organelles,
vesicles, GC
Tang et al.. 1989
No
NI
NA
Organelles,
vesicles, GC
Heslop-Harrison and
Heslop-Harrison,
1989*; Tirlapur
et al., 1995*
Heterologous
antibody
No
NI
NA
Organelles,
GC, VN
Miller et al., 1995
205
Heterologous
antibody
No
NI
NA
Larger organelles
Miller et al., 1995
Myosin-V
190
Heterologous
antibody
No
NI
NA
Smaller organelles
Miller et al., 1995
170-kD myosin
170
Functional assays
Yes
CaM
NA
Vesicles?
Yokota et al., 1994,
1995, 1999
Type
*The same antibody has been used in both manuscripts. NA, Not available; NI, not identified; GC, generative cell; VN, vegetative nucleus.
Cai et al.-Pollen Tube Cytoskeleton
TABLE
2. Microtubule motors described in the pollen tube
Light
chains
Sequence
Localization
Citation
Yes
NI
NA
Vesicles in the
apex?
Cai et al., 1993
Heterologous
antibody
No
NI
NA
Golgi vesicles?
Liu et al., 1994
400-500
Anti-peptide
antibody
No
NI
NA
Vacuoles? Golgi
bodies?
Moscatelli et al.,
1995
120
Anti-peptide
antibody
No
NI
NA
GC, cytoplasm
Liu et al., 1996
Molecular
weight (kD)
Method of
identification
Pollen kinesin homologue
100
Heterologous
antibody
Kinesin-related
100
Type
Dynein-like
Kinesin-like protein
71
Purification
NA, Not available; NI, not identified; GC, generative cell.
the alignment and distribution of new AFs in the growth
region. This hypothesis will be discussed below.
In general, MTs have not been considered to be involved
in organelle movement along the pollen tube. Inhibition of
AF organization is sufficient to halt the movement of
organelles, while treatment with MT inhibitors does not
affect cytoplasmic streaming (Heslop-Harrison et al., 1988).
Studies with more specific inhibitors suggested that MTs
may have a role in controlling the cellular organization of
the pollen tube, but no specific effects on organelle
movement have been observed (Joos et al., 1994, 1995).
Although MTs seem to be related to the translocation of
both the GC and vegetative nucleus (VN) (Astr6m et al.,
1995), it is not possible to completely exclude the involvement of MTs in the positioning or in the translocation of
organelles. On the other hand, cytological, biochemical and
molecular data supporting this hypothesis are scarce. In
some cases, MTs are observed in intimate contact with
vesicular structures, such as elements of the cortical
endoplasmic reticulum (Hepler et al., 1990), but it is not
possible to establish if these motionless interactions hide a
possible dynamic relation. Furthermore, the movement of
endoplasmic tubules in plant cells is more dependent upon
the actomyosin system, while MTs do not seem to be
involved (Knebel et al., 1990). MTs have been found to
have a role in the pulsed growth of the pollen tube
(Geitmann et al., 1995), but how this process relates to
organelle movement is far from understood. The pollen
tube contains motor proteins related to kinesin and dynein
(Table 2), whose specific function is still unknown,
although an interaction with membrane structures seems
feasible. The 'pollen kinesin homologue' (PKH) (Tiezzi
et al., 1992) is mainly located in the apical and cortical
region of the tube (Cai et al., 1993). This particular
distribution suggests the involvement of PKH in regulating
the accumulation of secretory vesicles. A connection
between PKH and vesicles has not yet been shown,
although immunologically related polypeptides have been
located in association with Golgi vesicles (Liu et al., 1994).
The biochemical properties of dynein heavy chain-like
polypeptides suggest that these proteins are bound to
membrane structures, the nature of which is not yet
established (Moscatelli et al., 1995, 1998). The distribution
of these dynein-like polypeptides in the middle and older
parts of the tube suggests their involvement in cytoplasmic
organization, but not in the process of tip growth. More
information on the cellular functions of PKH and dyneinrelated polypeptides should be available after the membrane structures to which they bind are clearly identified. A
further MT-motor has been identified in the pollen tube by
means of a peptide antibody (Liu and Palevitz, 1996) but,
as this protein is mainly distributed within the GC and its
localization in the vegetative cytoplasm is still uncertain, it
will not be discussed here.
The fact that specific classes of organelles can bind
different types of myosin (Miller et al., 1995) suggests that
motor proteins should be targeted from the site of synthesis
to particular types of organelles. Mechanisms through
which motor proteins bind to single organelles are almost
unknown. Establishing a convincing hypothesis on this
topic is an interesting challenge because it is the basis for
understanding how the movement of organelles is
regulated. Proteins related to kinectin (Yu et al., 1995)
and dynactin (Waterman-Storer et al., 1997), that act as
kinesin and dynein 'receptors' on organelles, respectively,
are not yet characterized in pollen tubes (or in plants in
general). It is consequently difficult to understand how
single motor proteins are allocated to specific classes of
organelles and how these 'motor-binding proteins' contribute to the control of intracytoplasmic motility. Proteins that
are able to allocate different classes of myosin to specific
organelles are currently unknown.
CONCERTED ACTIONS BETWEEN
MICROTUBULES AND ACTIN FILAMENTS
While the role of AFs appears to be clear, many
uncertainties have still to be resolved about the role of
MTs during organelle movement in the pollen tube. The
problem is whether pollen tube MTs are required for the
active translocation of organelles along the tube and/or in
the control of this process. Furthermore, we must address
the question of whether a concerted action between the
two motor systems may exist in promoting/controlling the
translocation of organelles along the tube. As information
on this particular topic is lacking, we can only make a
72
Cai et al.-Pollen Tube Cytoskeleton
hypothesis based on information from other cell systems.
Cells use the two main cytoskeleton motor machineries in
order that MT- and AF-based motors frequently accomplish a concerted action rather than overlapping functions
(Schliwa, 1999). In this context, 'concerted action' means
that a particular organelle could have both AF- and MTmotors on the surface and that the transport of membranebounded organelles to specific cell regions may possibly
require switching from one system to the other. Research
during recent years has revealed that many membranebounded organelles move along both AFs and MTs and
that the coordinate regulation of MT motors, myosins and
linker proteins is strictly associated with the processes of
membrane trafficking (Allan and Schroer, 1999). In some
cases, it seems likely that the two classes of motors can
transiently interact, thus allowing the fine-tuning of
organelle transport along both cytoskeleton systems
(Huang et al., 1999).
AFs and MTs in plant cells usually co-localize (mostly in
the cortical region of cells). Although several papers
indicate interactions between AFs and MTs, their nature
is unknown [e.g. see Collings et al. (1998) and references
therein]. One hypothesis is that the cortical actin arrays may
be dependent on the arrangement of MTs, but it is also
consistent with the fact that MTs may be simply co-aligned
with AFs in response to the hydrodynamic forces created by
cytoplasmic streaming (Foissner and Wasteneys, 1999). Coalignment of AFs and MTs has also been observed in the
pollen tube (Pierson et al., 1986; Lancelle and Hepler,
1991). Nevertheless, it seems that disarrangement of AFs or
MTs does not alter the organization of the rest of the
cytoskeleton network, and, additionally, has specific effects
on both cytoplasmic organization and tube growth
(Heslop-Harrison et al., 1988; Lancelle and Hepler, 1988;
Joos et al., 1994; Astrm et al., 1995). Although AFs and
MTs in the pollen tube are arranged in parallel and
spatially related, observations reported in literature (see
previous citations) suggest that the two main cytoskeleton
filaments are independently regulated and have different
functions. As a working hypothesis, AFs are involved in the
translocation of organelles along the tube, while MTs may
participate in the cytoplasmic organization of the cell and
(see below) in the control of the GC and VN translocation.
Nuclear positioning in many cells is a MT-dependent
process but, in some cases, AFs are also involved (Lloyd
et al., 1987; Grolig, 1998). Two basic mechanisms of MTdependent nuclear positioning are known to occur in
eukaryotic cells, the 'MT organizing centre-dependent
nuclear positioning' and the 'nuclear tracking along MTs'
(Reinsch and Gonczy, 1998). In tip-growing cells, like
fungal hyphae, the positioning of nuclei depends on
cytoskeleton components and mechanochemical motor
proteins (Fischer, 1999), the majority of which are MTdependent motors (Inoue et al., 1998). In the pollen tubes,
movement of the GC and VN almost certainly occurs along
AFs, while the MT cytoskeleton is likely to control the
translocation rate of the GC. This working model derives
from both inhibitory experiments (Heslop-Harrison et al.,
1988; Astr6m et al., 1995) and localization of different
myosins on the surface of the GC and VN (Tang et al.,
1989; Miller et al., 1995; Tirlapur et al., 1995). These
findings suggest that AFs and myosin motors are the
primary support for the translocation of the GC and VN;
however, recent experiments have weakened this hypothesis
by showing that SCs from Plumbago do not possess myosin
molecules on their surface, although they move along actin
cables (Zhang and Russell, 1999). The finding suggests that
the movement of SCs along AFs is much more complex
than previously thought. The role of MTs is uncertain, but
experiments with MT inhibitors have implied a regulatory
function (Astr6m et al., 1995). Whether MTs could simply
anchor the GC/VN surface or actively move these
structures using motor proteins is not known. Furthermore,
MT motors have not been detected in association with the
outer surface of both GC and VN. As already speculated
(Cai et al., 1997), MTs may perhaps organize the actin
cytoskeleton around the GC in order to promote the finely
regulated interaction between AFs and myosin molecules.
This activity implies the presence of AF/MT-interacting
proteins, as yet unidentified.
ROLE OF THE CYTOSKELETON IN THE
DIFFERENTIAL DISTRIBUTION OF
ORGANELLES BETWEEN THE APICAL
AND SUBAPICAL REGION
A distinct zonation of organelles is frequently observed in
the pollen tube (Cresti et al., 1977; Heslop-Harrison and
Heslop-Harrison, 1990; Pierson et al., 1990), but how this
differentiation is maintained during tube growth is not
known. For example, larger organelles are nearly absent in
the apical region, which is instead characterized by the
accumulation of secretory vesicles. The role of the cytoskeleton in determining this polar zonation is not clear,
although AFs are likely to play a major role in maintaining
this differential distribution of membrane material (Derksen et al., 1995; Cai et al., 1997). Possible control mechanisms of cytoskeleton organization between the apical and
subapical domains will be discussed in the next section.
The relatively different distribution of membranebounded organelles in the subapical and apical regions
could be related to the dissimilar organization of the
cytoskeleton between the two cellular domains. Observations on physically fixed or on living cells microinjected
with FITC-phalloidin have underlined the scarcity of AFs
in the apical region of the tube (Miller e al., 1996).
Furthermore, observations of pollen tubes from plants that
express GFP-talin have emphasized the absence of wellstructured AFs in the tube apex (Kost et al., 1998, 1999).
The tip region is expected to contain either a pool of
monomeric actin (likely to be incorporated in new or
preexisting filaments) or short AFs that are synthesized at
the level of the plasma membrane and then incorporated
into preexisting filaments. The identification of Rho-family
proteins in the apical area (Lin et al., 1996), the specific
inhibition of which arrests tube growth (Lin and Yang,
1997), suggests that assembly of new AFs occurs in the
apical region and that Rho GTPases could control
cytoplasmic streaming. In addition, Rac homologues have
also been found in the pollen tube apex and shown to be
Cai et al.-Pollen Tube Cytoskeleton
necessary for two activities: organization of AFs and
control of polar growth (Kost et al., 1999). The finding
that AFs are randomly arranged at the margin between the
apical and subapical regions (Kost et al., 1999) suggests
that AFs could also act as a molecular filter, preventing
access of larger organelles to the apex (Heslop-Harrison
and Heslop-Harrison, 1990). AFs may have more than one
role in the apical region. In addition to their role in
transporting secretory vesicles to their final destination,
AFs may also maintain the structural integrity of the tube
apex. In fact, the consequences of cytochalasin treatment
on pollen tube growth have long been known (Mascarenhas
and Lafountain, 1972; Tiwari and Polito, 1990), but it is
unlikely that the rapid cessation of tube growth after
cytochalasin treatment exclusively results from the suspension of vesicle delivery. AFs may also be used for
organizing the molecular architecture that underlies the
apical plasma membrane and stabilizes the tip area. Since
AFs and spectrin molecules are part of the membrane
cytoskeleton in other cells, the evidence that spectrin
antigens are found at the cytoplasmic side of the apical
plasma membrane is in agreement with this hypothesis
(Derksen et al., 1995; Derksen, 1996).
The role of AFs in the delivery of secretory vesicles to the
tip membrane is unclear. The movement of vesicles in the
apical domain appears to be random (de Win et al., 1998)
and is hardly convertible into a linear-directed movement
(as expected if occurring along a cytoskeleton track). It is
also consistent with the fact that myosin molecules have
been identified in association with vesicular structures
(Tirlapur et al., 1995) and in the very tip domain (Yokota
et al., 1995). This association could suggest two alternatives: (1) myosins are important in the delivery of vesicles to
the tip membrane; or (2) they represent motors that have
been inactivated in the tube apex.
The role of MTs in preventing the movement of
organelles to the apical domain is not clearly understood.
Despite the identification of PKH in the pollen tube apex
(see below), the presence and organization of MTs in the
apical domain is not established. Although some studies
have shown the presence of short MTs randomly arranged
in the apical region [see Del Casino et al. (1993) and
references therein], these observations have not been further
supported (Lancelle et al., 1987; Lancelle and Hepler,
1992). One hypothesis suggests that MTs are organized as
bundles along the pollen tube, while the apical region may
possibly contain individual MTs available to be incorporated into the bundles (Del Casino et al., 1993). According
to this model, the apical region of the tube might represent
a site of MT organization or growth. This hypothesis would
be confirmed by the localization of a pericentriolar related
antigen in the apical plasma membrane (Cai et al., 1996),
but not by the diffused localization of y-tubulin (Palevitz
et al., 1994). The different organization of MTs between the
apical and subapical regions requires either the presence of
distinctive MT-associated proteins (MAPs) in the two areas
or a differential regulation of similar MAPs. Although
MAPs have been characterized in plant cells as well
(Chang-Jie and Sonobe, 1993; Schellenbaum et al., 1993;
Vantard et al., 1994; Bokros et al., 1995; Marc et al., 1996;
73
Rutten et al., 1997), no corresponding evidence exists in
the pollen tube, with the exception of polypeptides that cosediment with taxol-stabilized MTs (Tiezzi et al., 1987).
REGULATION OF CYTOSKELETON
ORGANIZATION AND ORGANELLE
TRANSPORT: EFFECTS OF Ca 2 +
A higher concentration of Ca2 + in the tube apex,
particularly in the very tip region, characterizes the pollen
tube, as well as other cells with apical growth [for a review
of the role of Ca2 + in promoting and guiding pollen tube
growth, see Franklin-Tong (1999)]. The dissipation of the
Ca 2+ gradient can remove the typical organelle zonation
present in the pollen tube (Pierson et al., 1994), suggesting
that some control mechanisms could be based on Ca 2+ and
Ca 2 +-dependent proteins. A current model predicts that
Ca 2+ , through a series of intermediates, promotes the
fusion of secretory vesicles in specific areas of the tip
membrane (Malh6 and Trewavas, 1996). Although the role
of Ca 2+ during tube growth is becoming increasingly clear,
little information is available about the links between Ca 2+ ,
cytoskeleton and organelle transport in pollen tubes (see
below).
Ca 2+ has been shown to influence the structure and
organization of AFs (Kohno and Shimmen, 1987), but the
molecular mechanism at the basis of this finding is unclear.
There is a major gap in understanding due to the poor
information on the presence of actin-binding proteins
(ABPs) in pollen tubes, which could participate in the
Ca 2+-dependent control of AFs. Profilin is a well-known
ABP, whose function in the pollen tube is not completely
clear (Mittermann et al., 1995). Profilin is likely to be part
of a mechanism that controls the organization of AFs in the
pollen tube, but whether this function is also carried out in
the apical region is not yet known. In addition, immunocytochemical techniques have shown that profilin is not
associated with specific regions of the pollen tube (Vidali
and Hepler, 1997), despite the fact that other authors have
described the association of profilin isoforms with the
plasma membrane of pollen (von Witsch et al., 1998).
However, profilin has been suggested to play a role in the
signalling pathways of pollen tubes by altering the
phosphorylation state of cytosolic proteins (Clarke et al.,
1998). Other ABPs identified in pollen are represented by
115 and 135 kD-proteins, which could induce AFs to form
bundles (Nakayasu et al., 1998; Yokota et al., 1998). The
135-kD ABP is unlikely to be involved in the apical and
subapical organization of AFs, but it is expected to play a
role in directing the cytoplasmic streaming in pollen tubes
as it assembles AFs into bundles with identical polarity
(Yokota and Shimmen, 1999). Actin-depolymerizing
factors (ADFs) have been identified in pollen and in other
plant tissues (Lopez et al., 1996), but have not been
characterized in the pollen tube. Nevertheless, information
on ADFs in other plant cells is promising. For example,
ZmADF3, which is located in the apex of root hairs, is
interesting (Jiang et al., 1997) as it is activated through
phosphorylation by a Ca2+-stimulated protein kinase
(Smertenko et al., 1998). Pollen homologues of ZmADF3
74
Cai et al.-Pollen Tube Cytoskeleton
could elucidate the relationship between Ca 2+ and AFs in
the apical region, although Ca2+-dependent protein kinases
could affect the cytoskeleton through other ABPs, not only
by having an effect upon ADFs. To conclude, Ca2+ is
probably able to modulate the organization of AFs in the
apical/subapical regions of the pollen tube and, in doing so,
could participate in establishing the polar organization of
organelles.
Myosins have been shown to be responsible for organelle
movement along the tube, but their role in the accumulation of secretory vesicles in the apex is not yet clear. The
question of why secretory vesicles gather in the apex, while
larger organelles invert their course after approaching this
region, is not easy to answer. Regulatory mechanisms
underlying this process are likely to be composed of many
different factors that interact with each other. An interesting
working model predicts that a Ca2+-calmodulin (CaM)
complex can modulate the activity of myosins in the region
of tube growth (Moutinho et al., 1998). CaM is a
ubiquitous protein that is involved in many functions
within cells, and therefore in the pollen tube as well. The
distribution of CaM, as depicted by microinjection of
FITC-CaM (Moutinho et al., 1998), seems to be uniform
along the tube axis, except for the subapical region of the
pollen tube where a V-shaped area of high concentration is
found. Because of its location at the margin of the growth
region, this CaM-enriched area is likely to be involved in
the regulation of tube growth. Based on the information
currently available, one oversimplified model may perhaps
be drawn to explain the diverse distribution of organelles
and vesicles in the apex. The key point of this model
predicts that secretory vesicles and organelles exhibit
different myosins on their surface and that the Ca 2+-CaM
complex is able to selectively inhibit the vesicle-associated
myosin, but not the organelle-associated motor. Consequently, vesicles would be moved to the apex, whereas
organelles make use of the inversely-oriented AFs to move
back. Evidence backing up this hypothesis includes the
occurrence of different forms of myosin in association with
organelles and vesicles (Miller et al., 1995), and the fact that
Ca 2+ is able to inhibit the activity of the 170-kD pollen
myosin (Yokota et al., 1999), probably by releasing a
myosin-associated CaM.
Although evidence exists that Ca 2+ affects the arrangement of AFs, we know almost nothing about the relation
between Ca 2+, MTs and organelle movement in the pollen
tube. Evidence that associates Ca2 + with MTs and MAPs
(mobile and structural) in plants is scarce (Durso and Cyr,
1994; Moore et al., 1998); therefore, we ignore whether (and
how) Ca 2 + controls the architecture of MTs in the pollen
tube apex and whether this activity influences the MTdependent delivery of specific components. We speculate
that MTs could control the directionality of tip growth, as
in root hairs (Bibikova et al., 1999), and that this activity
may be determined by connections with the molecular
apparatus that preserves the Ca2 + gradient in the tip.
However, supporting evidence is missing. Although the
role of MTs in the process of organelle/vesicle movement
is still questionable, it should be noted that the PKH has
been localized in the pollen tube apex by conventional
immunofluorescence microscopy (Tiezzi et al., 1992). The
finding suggests that this putative kinesin may have a role in
the process of tube growth, for example in the accumulation
of specific vesicles or during endocytosis. Whether this
motor protein may be controlled by Ca 2+ is not known. In
recent years, a subfamily of kinesin, which is characterized
by a CaM-binding domain, has been identified in plants
(Reddy et al., 1996a,b; Wang et atl., 1996). These kinesins,
the activity of which is controlled by the Ca2+-CaM
complex (Narasimhulu et al., 1997; Song et al., 1997;
Deavours et al., 1998), seem to be involved in the process of
cell division (Bowser and Reddy, 1997). The identification
of these particular kinesins suggests that members of this
family could also be directly controlled through the activity
of Ca2+ and Ca 2+-binding proteins and raises the question
of whether the pollen tube may contain analogous motor
proteins regulated by the high concentration of Ca 2 + in the
apical domain.
Based on the information currently available in literature,
at least one model may be made describing the control of
the cytoskeleton-dependent movement of organelles during
tube growth (Fig. 1). It is evident that most of the steps in
this model are only speculative, as few of the reported
activities have been clearly identified.
IS THE CYTOSKELETON-DEPENDENT
MOVEMENT OF ORGANELLES
CONTROLLED BY EXTRACELLULAR
MOLECULES?
The pollen tube is a cell that grows by penetrating the intercellular spaces of other cells. A wide range of mechanisms is
likely to control the growth direction of the pollen tube from
the stigma surface down to the ovary [for example, proteins
of the extracellular matrix (Sanders and Lord, 1992), the
activity of specific stylar glycoproteins (Cheung et al., 1995;
Lind et al., 1996), the action induced by lipid molecules
(Wolters-Arts et al., 1998), the control exerted by the female
gametophyte on pollen tube guidance (Ray et al., 1997)]. A
problem, still unresolved, is whether the mechanisms of
signal transduction can affect the cytoskeleton-dependent
movement of organelles. The amount of information available that associates cytoskeleton proteins to signals coming
from the surrounding environment is rather small. In other
cells, for example, small Rho and Rac GTPases are part of
complexes that recruit proteins to form focal contacts as a
reaction to external signals (Tapon and Hall, 1997). In the
pollen tube, inhibition of Rho GTPases can suppress pollen
tube growth and cytoplasmic streaming, suggesting that the
activity of these proteins is also linked to the process of
organelle movement (Lin and Yang, 1997). Profilin has also
been suggested to have a role in the signalling pathways of
pollen tubes (Clarke et al., 1998), but how this activity is
connected to the organization of AFs is not known. An
additional handicap is the technical difficulty involved in
analysing the cytoskeleton and organelle movement in
in vivo-grown pollen tubes. In vitro growing conditions are
most appropriate to study several aspects of the pollen tube,
but they could mask many features of the cytoskeleton,
specifically those concerning the relationship between the
Cai et al.-Pollen Tube Cytoskeleton
Extracellular
signal?
Extracellular
Ca 2 '
Extracellular
signal?
75
signal transduction pathway and organelle movement.
Isolating native stylar components could allow both cytoskeleton dynamic movement and organelle movement to be
observed in response to these molecules (Du et al., 1994;
Cheung et al., 1995, 1996; Wu et al., 1995; Lind et al., 1996).
ACKNOWLEDGEMENTS
We sincerely thank Dr E. S. Pierson (Laboratory of Plant
Cell Biology, Department of Experimental Botany, Catholic University of Nijmegen, Nijmegen, The Netherlands) for
critically reading the manuscript and for suggestions.
LITERATURE CITED
Mfi
Myosin
~
Actin filament
Microtubule
m:
Rho/Rac proteins
FIG. . Schematic representation of a hypothetical model for the
control of the cytoskeleton activity and structure during tube growth. It
is suggested that the localized increase of Ca 2+ concentration,
produced by both extracellular influx and/or Ca2+-release from
intracellular stores (such as endoplasmic reticulum), might influence
coordinately the process of tube elongation and the structure and
function of the cytoskeleton. The flow of Ca 2+ from intracellular stores
may possibly be the result of hypothetic stimulation by IP 3, which
could, in turn, be triggered by extracellular signals. Ca2+ might
possibly control both the organization of AFs and MTs as well as the
activity of motor proteins. The actin cytoskeleton could be regulated at
different levels through the consecutive activation of Ca2+-dependent
CaM-independent protein kinases (CDPK) and ABPs/ADFs, thus
controlling the architecture, extension, bundling activity, and stability
of AFs. The identification and characterization of ABPs are required
to understand these mechanisms of control. Profilin is an ABP which
is thought to have different effects on actin in living cells and also
plays important roles in the regulation of the actin cytoskeleton in the
pollen tube. Profilin also affects protein phosphorylation, probably by
interacting with protein kinases (PKs). The interactions between AFs
and MTs have not been shown in the figure because they are only
hypothetical and nothing is known about their characteristics and
control. A hypothetical cascade of events, based on the information
available from literature, is shown. IP 3, Inositol 1,4,5-triphosphate;
ADF, actin-depolymerizing factor; ABP, actin-binding proteins; CaM,
calmodulin; AF, actin filament; MT, microtubule. The aspect ratio
and spatial arrangement of different objects has been substantially
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