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Single microfilaments mediate the early steps of microtubule bundling during
preprophase band formation in onion cotyledon epidermal cells
Authors
Miyuki Takeuchia, b, Ichirou Karaharac, Naoko Kajimurad*, Akio Takaokad, Kazuyoshi
Muratae, Kazuyo Misakif, Shigenobu Yonemuraf, L. Andrew Staeheling and Yoshinobu
Mineyukia†
Affiliations
aGraduate School of Life Science, University of Hyogo, 2167 Shosha, Himeji, Hyogo
671-2201, Japan; bGraduate School of Agricultural and Life Sciences, University of
Tokyo, 1-1-1 Yayoi, Bunkyou-ku, Tokyo 113-8657, Japan; cGraduate School of Science
and Engineering, University of Toyama, 3190 Gofuku, Toyama 930-8555, Japan;
dResearch Center for Ultra-High Voltage Electron Miceroscopy, Osaka University, 7-1
Mihogaoka, Ibaraki 567-0047, Japan; eNational Institute for Physiological Sciences, 38
Nishigonaka Myodaiji, Okazaki 444-8585, Japan; fRIKEN Center for Life Science
Technologies, 2-2-3 Minatojima-minamimachi, Chuou-ku, Kobe 650-0047, Japan;
gMCD Biology, University of Colorado, Boulder, CO 80309-0347, USA
Running Head. Actin-microtubule interaction in plants
Address Correspondence to: Yoshinobu Mineyuki (mineyuki@sci.u-hyogo.ac.jp).
* Present address: Graduate School of Frontier Biosciences, Osaka University, 1-3
Yamadaoka, Suita, Osaka, 565-0871 Japan
http://www.molbiolcell.org/content/suppl/2016/04/03/mbc.E15-12-0820v1.DC1.html
Supplemental Material can be found at:
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†This work was partly supported by JSPS grant 17207006 and MEXT grant 17049019
to YM.
Abbreviations used: ADZ, actin-depleted zone; CD, cytochalasin D; MF, microfilament;
MT, microtubule; PM, plasma membrane; PPB, preprophase band
ABSTRACT
The preprophase band (PPB) is a cytokinetic apparatus that determines the site of cell
division in plants. It originates as a broad band of microtubules (MT) in G2 and narrows
to demarcate the future division site during late prophase. Studies with fluorescent
probes have shown that PPBs contain F-actin during early stages of their development,
but become actin-depleted in late prophase. Although this suggests that actins contribute
to the early stages of PPB formation, how actins contribute to PPB-MT organization
remains unsolved. To address this question, we have used electron tomography to
investigate the spatial relationship between microfilaments (MFs) and MTs at different
stages of PPB assembly in onion cotyledon epidermal cells. We demonstrate that the
PPB actins observed by fluorescence microscopy correspond to short single MFs. A
majority of the MFs were bound to MTs, with a subset forming MT-MF-MT bridging
structures. During the later stages of PPB assembly the MF-mediated links between MTs
were displaced by MT-MT linkers as the PPB MT arrays matured into tightly packed MT
bundles. Based on these observations, we propose that the primary function of actins
during PPB formation is to mediate the initial bundling of the PPB MTs.
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INTRODUCTION
Precise control of the site where the newly formed cell plate attaches to the parental cell
walls is essential for plant morphogenesis (Sinnott, 1960). This site is thought to be
determined before karyokinesis during preprophase band (PPB) formation (Gunning,
1982). The initial PPB appears during G2 in the form of a broad band of microtubules
(MTs), which narrows during the progression of the cell cycle and disappears when the
nuclear envelope breaks down (Wick and Duniec, 1983, 1984; Mineyuki et al., 1988).
Because the position of the PPB predicts the future division site where the newly
formed cell plate is inserted into the parental cell wall at the end of cytokinesis, the PPB
is thought to play an essential role in the establishment of the division plane (Mineyuki,
1999). Since their discovery (Pickett-Heaps and Northcote, 1966a, b), PPBs have been
observed in many different types of land plants undergoing somatic cell divisions
(Mineyuki, 1999). A number of molecules have been localized to PPBs, but how these
molecules contribute to PPB MT organization remains unclear (McMichael and
Bednarek, 2013; Li et al., 2015).
Interactions between the MTs and actins are required for many fundamental
processes of animals and plants (Rodrigues et al., 2003; Collings, 2008). Such
interactions also appear to play a critical role in PPB formation. The presence of actins
in the PPB was first reported in 1987 by three different groups (Kakimoto and Shibaoka,
1987; Palevitz, 1987; Traas et al., 1987). Since then many other laboratories have
confirmed these findings using a variety of fluorescence microscopy techniques such as
immunolabeling, fluorescence-phalloidine labeling, microinjections of fluorescently
tagged phalloidine or recombinant actin/actin-interacting molecules in living cells, and
visualizing green fluorescent protein-tagged actin-binding-domain proteins in living
cells (Lloyd and Traas, 1988; Palevitz, 1988; McCurdy et al., 1988; McCurdy and
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Gunning, 1990; Mineyuki and Palevitz, 1990; Cho and Wick, 1991; Eleftheriou and
Palevitz, 1992; Katsuta et al., 1990, Katsuta and Shibaoka, 1992; Liu and Palevitz,
1992; Panteris et al., 1992; Cleary et al., 1992a, b; Cleary, 1995; Cleary and Mathesius,
1996; Baluska et al., 1997; Zachariadis et al., 2001; Hoshino et al., 2003; Sano et al.,
2005; Li et al., 2006; Higaki et al., 2007; Panteris et al., 2007). The presence of F-actin
like microfilaments (MFs) in PPBs has also been observed in electron micrographs
(Ding et al., 1991a).
Cells exhibiting an early broad PPB also possess a broad cortical F-actin array.
As the PPB MTs narrows, the actin band narrows, and when the PPB MTs reach their
narrowest configuration, the fluorescent signals of actins start to disappear from the cell
cortex region occupied by the MT band, giving rise to the actin-depleted zone (ADZ)
(Cleary et al., 1992b; Liu and Palevitz, 1992). The ADZ forms prior to the breakdown
of the nuclear envelope and persists throughout mitosis (Cleary et al., 1992b; Liu and
Palevitz, 1992). At that point, it becomes a negative marker of the future division site.
In addition to the microscopic evidence for cortical actins in the PPB,
pharmacological studies have provided indirect evidence for the idea that actin plays a
critical role in organizing the PPB MTs. The narrowing of the PPB MTs is prevented by
F-actin disrupters such as cytochalasin D (CD) (Mineyuki and Palevitz, 1990;
Eleftheriou and Palevitz, 1992) and latrunculin A (Granger and Cyr, 2001, Li et al.,
2006). These actin disrupters also cause a re-broadening of previously narrowed MT
bands (Mineyuki and Palevitz, 1990; Granger and Cyr, 2001; Eleftheriou and Palevitz,
1992). These studies imply that actin is necessary for PPB formation, but how actins
interact with MTs during PPB formation remains unknown.
The effect of CD on PPB actin is unique. It is known that cytochalasins block
actin polymerization and promotes depolymerization of cytoplasmic F-actins. However,
the F-actins are not completely depolymerized in CD-treated onion cells (Palevitz,
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1988). Indeed, F-actins have been observed in onion root tips and in cotyledon
epidermial cells treated with CD (Palevitz, 1988; Mineyuki and Palevitz, 1990). Using
improved immunofluorescent techniques we have recently demonstrated that in onion
root tip cells treated with 20µM CD for 30 min, the PPB MT bands of late prophase
cells increase in width, and that actin co-localizes with these broadened PPBs (Takeuchi
and Mineyuki, 2014).
To address the question as to how F-actins interact with other PPB
components requires structural studies with electron microscope level resolution. MFs
bound to interphase cortical MTs have been observed in several ultrastructural studies
of chemically fixed plant cells (Seagull and Heath, 1979; Hardham et al., 1980; Heath
and Seagull, 1982; Tiwari et al., 1984) as well as in cells preserved by
cryofixation/freeze-substituted techniques (Tiwari et al., 1984; Lancelle et al., 1987;
Tiwari et al., 1988, Ding et al., 1991b; Murata et al., 2002). Single MFs associated with
PPBs have been reported in electron micrographs of high-pressure frozen tobacco root
tip cells (Ding et al., 1991a). However, the functional significance of this binding has
never been explored in a systematic manner. In the present study, we have characterized
MFs of the PPB by means of electron tomography with high pressure freezing
/freeze-substitution techniques. This approach has enabled us to obtain quantitative
information on the number and length of PPB MFs, as well as on the spatial relationship
between MFs and MTs in PPBs. Our results show that ~170 nm-length MFs bind to and
crosslink the MTs and that they are involved in the process of MT bundling during the
early stages of PPB assembly.
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RESULTS
We have investigated the role of MT-associated MFs in PPB formation in 3-day-old
onion cotyledon epidermal cells preserved by high pressure freezing and
freeze-substitution techniques, because PPB assembly and the role of endocytosis in the
creation of PPB memory structures in such cells is well characterized (Mineyuki et al.,
1989; Mineyuki and Palevitz, 1990; Karahara et al., 2009).
As shown in Figure 1, electron tomography can be used to map the 3-D
organization of MT bundles and associated MFs in the cell cortex of PPB forming cells.
Figure 1A illustrates a tomographic slice image of a tangentially sectioned cortical
region of an epidermal cell containing a PPB, and Figure 1B an electron
tomogram-based model of the same PPB. In this model, the thick magenta lines
correspond to MTs, and the thin yellow lines to MFs. The reconstructed cell cortex
region covers an area of ~4 µm2, and the boxes mark the free MF depicted in Figure 1C,
and the MT-associated MF shown in Figure 1D, respectively. The diagrams in Figure 1E
explain the planes of the tomographic sections investigated in this study, and Figure 1A
corresponds to the (a) plane shown in the Figure 1E.
PPB formation stages can be determined both by nuclear morphology and by the
width of the PPB in mid-longitudinal sections of the cells
To determine the sequential changes in PPB architecture during PPB formation, we
have staged the cells via nuclear morphology and PPB width in mid-longitudinal
sections of the cells (Figure 2). The changes in nuclear staining due to differences in
chromosome condensation during PPB formation are illustrated in Figure 2, A-F.
Typical of interphase nuclei, the nucleus in Figure 2A displays dispersed chromosomes
that give rise to a fine network of darkly staining dots. During prophase the
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chromosomes condense into an increasingly coarser dark network that eventually can be
resolved into individual chromosomes during the prophase/prometaphase transition
stage (Figure 2F). In this paper the “late prophase” cells have nuclei with highly
condensed chromatin (Figure 2, D and E), whereas the “prophase” cells have more
dispersed chromatin (Figure 2, B and C).
The changes in PPB assembly depicted in Figure 2, G-I, illustrate
cross-sectional views of the PPB regions in Figure 2, A, E and F, respectively. The PPB
widths were determined using these images. These micrographs demonstrate that the
PPB MTs form increasingly larger and tighter bundles as the width of the PPB decreases
- compare Figure 2G (PPB width = 10 µm) with Figure 2 H (PPB width = 3.6 µm) - and
that the number of MTs in a PPB decreases when the cells enter the prophase to
prometaphase transition stage (Figure 2I; PPB width = 3.5 µm). In this study, the
process of PPB formation is divided into three stages based on the extent of nuclear
condensation and the width of the MT band. Cells with a “prophase” nucleus whose
PPB MT width is >7µm are classified as “prophase 1” stage cells, cells with a
“prophase” nucleus and a narrow PPB (<7µm) as “prophase 2” stage cells, and cells
with a nucleus of the “late prophase” type are termed “late prophase“ stage cells. All of
the late prophase cells examined during this study had a narrow PPB.
PPB assembly starts with the alignment of cortical MTs and the formation of
closely associated MT pairs that coalesce into increasingly large MT clusters
The 3-D changes in MT organization and bundling associated with PPB formation are
shown in the tomographic models Figure 3, A-C, which highlight the changes in MT
organization during this process. As reported previously (Mineyuki and Palevitz, 1990),
most cortical MTs are not organized transversely but distributed randomly in interphase
cells of the basal region of the onion cotyledon epidermis (Figure 3A). At the onset of
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PPB formation, the MTs become organized into loose, irregular arrays oriented at right
angles to the longitudinal axis of the cells (Figure 3B). Upon completion of this MT
reorganization stage, groups of two or three MTs initiate the formation of MT bundles
by moving closer together and becoming more aligned (Figure 3C). At this stage, the
nascent PPBs exhibit their greatest width (~10 µm in the PPB shown in Figure 3C).
During the subsequent PPB maturation process, pairs of closely aligned MTs serve as
templates for the assembly of increasingly large MT clusters (Figure 1B).
Electron tomography provides the 3-D resolution needed to map the spatial
relationship between individual MFs and MTs
As documented in Figure 1, electron tomography can provide precise 3-D information
on the distribution of both MTs and MFs in the PPB region of onion cotyledon
epidermal cells. One of the most striking features of Figure 1B is that while the PPB
region contains some randomly distributed MFs, most of the MFs appear to be closely
associated with MTs, and in many instances the MFs appear to be laterally connected to
a MT. This relationship can be seen both in longitudinal (Figure 1D and Figure 3, E and
F) as well as in cross-sectional views (Supplemental Figure S1A) of the MTs. In such
images MTs have a diameter of 25.1 ± 2.6 nm (mean ± SD, n = 137), and the associated
cross-sectioned MFs, which appear as electron dense dots, a diameter of 5.9 ± 1.2 nm (n
= 104), consistent with the expected diameter of F-actins (Supplemental Figure S1E).
To demonstrate that the dots marked by arrowheads in Figure S1 A are indeed filaments
and not globular protein complexes, we rotated the tomographic images by 90o
(Supplemental Figure S1, B-D).
Closer analysis of the MF-MT images shown in Figure 3, E and F
demonstrates that the association of MFs with the MTs is maintained even during MT
depolymerisation. This is most clearly seen in Figure 3F, where the end of the upper MT
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displays a horned end (typical of depolymerizing MTs), while its associated MF is seen
to extend beyond the flaring MT end to the adjacent MT.
Bundled-MFs have been observed previously in electron micrographs of the
plant cell cortex in some interphase cells preserved by rapid freezing (e.g. plate 31 in
Gunning and Steer, 1996). However, in none of our tomograms of high pressure frozen
cells have we detected any MF bundles in PPBs. All of the PPB MFs we have observed
were single, relatively short (168 ± 14 nm; mean of the means of 9 tomograms ±
standard error) filaments with a diameter of ~6 nm (Supplemental Figure S1E). This
contrasts with the significant number of longer (>500 nm) MFs seen in interphase cell
cortex cells, most of which are not bound to MTs (Figure 3A and Supplemental Figure
S1F).
In addition to the linear and straight MFs documented in Figure 1, C and D
and Figure 3, D-F, the MTs in the forming PPBs are also associated with short, curved
and branched filamentous structures (Figure 3, H and I, arrowheads). These branched
filaments are seen to form connections to both cortical MTs and to the adjacent plasma
membrane (PM) (Figure 3, J-M).
The cortical PPB MFs exhibit the same distribution as actin in
immunofluorescence studies and are sensitive to CD
If the MFs observed in our tomograms are F-actins, the density of such MFs should
decrease significantly in PPB regions of late prophase cells as the ADZ is formed.
Figure 4A illustrates a tomographic model of a late prophase PPB in which the dense
packing of the bundled MTs makes it difficult to discern the associated MFs. In Figure
4B we have left out the MTs and only show the MFs. As predicted by the ADZ
hypothesis, the density of the MFs is reduced within the PPB region (bracketed area)
compared to the area outside of the PPB where the cortical MFs are randomly oriented.
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To verify that the MFs in our tomograms are F-actins, we examined the
distribution of MFs after subjecting the cells to the F-actin depolymerizing drug CD. In
previous fluorescence microscopy studies (Mineyuki and Palevitz, 1990; Takeuchi and
Mineyuki, 2014) we observed that CD did not completely eliminate F-actins from PPBs,
and that the treatment led to PPB broadening in prophase cells. Our electron
tomography data demonstrate the presence of short MFs in the PPBs of CD-treated cells
(Figs. 4C and D), and that the CD-induced broadening of the band of PPB MTs (11.6 ±
5.0 µm wide; mean ± SD from 3 PPBs) is associated with the binding of the shortened
MFs to the MTs. In the presence of CD, the average length of the MFs decreased by
~36% when compared to control prophase 2-type PPBs (90 nm versus 140 nm, mode
from histograms). We also observed that the shortened MFs were unable to form bridges
between MTs (Supplemental Figure S2A), and the extent of their binding to MTs
appeared to be reduced (Figure 4, C and D).
The length of the MT-associated MFs increases during prophase 1 and reaches a
maximum during prophase 2, whereas the MF to MT length ratio is greatest in
prophase 1 and smallest in late prophase
As evidenced in the tomographic models shown in Figure 1B, Figure 3 and Figure 4,
A-D, the spatial organization of MFs and MTs in PPBs undergoes major changes during
PPB formation. To further characterize these changes we have determined both the
length of the MFs at different stages of PPB formation as well as the ratio of the length
of the MFs to the length of the MTs.
In interphase cells, the MFs are distributed randomly like most of the MTs
and few are associated with MTs (Figure 3A). However, as the MTs assume a transverse
orientation and MT bundling starts, a majority of the MFs become aligned with and
many become bound to the MTs (Figure 3, B and C). This spatial relationship is
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maintained through prophase 2 (Figure 1B) until late prophase, where the PPB MFs
break down giving rise to the ADZ (Figure 4). Some of the PPB MFs are connected
only at one end to the surface of a MT (arrowheads in Figure 1B, Figure 3, B and C).
This type of MF attachment to a MT is also seen in CD treated PPBs (Figure 4D).
The changes in MF length during these different stages of PPB formation are
illustrated in the histograms shown in Figure 5. During the earliest stages of PPB
formation (prophase 1) the MFs are relatively short (mode of histogram 70 nm, average
length 126±80 nm; Figure 5A). As the PPB matures the length of MFs increases to 140
nm (mode) during prophase 2 (Figure 5B) and maintained at ~140 nm (mode) during
late prophase (Figure 5C). The average length of MFs in prophase 2 and late prophase
were 187±126 nm and 178±89 nm, respectively.
Figure 5E highlights a different feature of the relationship between the
MT-associated MFs and the MTs, namely the ratio of the total length of the
MT-associated MFs to the length of the MTs in PPBs, which provides information on
the extent of MF binding to the MTs. The ratio is very low in interphase cells without a
PPB. In prophase 1 the ratio is the highest (~0.3), in prophase 2 the ratio drops to ~0.2,
and in late prophase the ratio falls to ~0.07.
MF-mediated bridging of MTs is observed mostly during early, and the
appearance of MT-MT linkers during later stages of PPB development
To elucidate the role of the MT cross-linking structures in PPB formation, we have
characterized the length, the distribution and the timing of the appearance of the
different types of MF-MT and the MT-MT bridging structures (Figure 6, A-E and Figure
7). Based on length measurements from 8 tomograms, most of the MF-MT linkers in
prophase 1 were ~14 nm long (13.7 ± 4.4 nm, the mean ± SD of 187 MF-MT linkers)
and those in late prophase were ~17 nm long (16.7 ± 7.2 nm, the mean ± SD of 144
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MF-MT linkers). CD had no effects on the length of the MF-MT linkers (Supplemental
Figure S3). During early stages of PPB formation, the number of MFs forming bridges
between MTs is typically quite low (Figure 3, B and C). However, as the development
of the PPBs progresses, the percentage of MFs forming bridges between MTs increases
significantly (Figure 1). MFs running between two adjacent MTs can form connections
to both MTs, but the linkers to the two MTs are not in the same plane (Figure 6, B-F).
In contrast to the MT-MF linkers, the length of the MT-MT linkers is more
variable (Figure 7, A-E). The mean length of all of the MT-MT linkers was 25.3 ± 9.9
nm (mean ± SD from 1109 linkers from 10 tomograms), but the shortest linkers were
only ~30% (Figure 7C) of the length of the longest ones (Figure 7D). Since we were not
able to discern individual peaks in the MT-MT linker length histogram of all linkers, we
determined the length distribution for each stage of PPB development (Figure 7, F-H).
Although few in number, distinct >50 nm bridge structures (Figure 7D) were seen in all
PPB developmental stages. The histogram of prophase 1 cells (Figure 7F) exhibits a
typical Gaussian distribution with a peak of ~27 nm and the average length of 29.0 ± 9.3
nm. This is the stage during which groups of two to three MTs begin to form bundles
(Figure 8A). As the small MT bundles start to coalesce into MT clusters during
prophase 2 (Figure 8B), the average length of the linkers decreases to ~25 nm (Figure
7G). The linker length histogram of late prophase cells (Figure 7H) exhibits a typical
Gaussian distribution with a peak of ~23 nm. During this phase the small MT clusters
consolidate into large MT clusters. At the end of late prophase a PPB typically consists
of 2-3 large, 3-D MT clusters with each composed of ~30 MTs held together by MT-MT
linkers. These bundled PPB MTs are also tethered to the PM via cross-bridges between
PM and MTs (Figure 6, G-I and Supplemental Figure S4).
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DISCUSSION
In this electron tomography study of high-pressure frozen/freeze substituted PPBs in
onion cotyledon epidermal cells, we demonstrate that PPBs contain two types of
filamentous structures, single, linear 6 nm filaments (MFs) that are often bound to MTs
and run parallel to the plane of the PM (Figure 1, C and D, Figure 3 D-F, Figure 6, A-D
and Supplemental Figure S1, A-D and F), and short, branched and angled MF-like
structures that form links between the MTs and the PM (Figure 3, H and I). The single,
linear MFs appear to correspond to F-actins that have been predicted to exist based on
various types of fluorescence microscopy studies (see Introduction), whereas the
identity of the branched structures is unknown.
The single MFs start to associate with MTs during the onset of PPB assembly
(Figure 3, A-C), and both the number and the length of the MT-associated MFs
increases during prophase 2 (Figure 1 and Figure 5). A significant drop in the number of
MFs was observed in late prophase PPBs (Figure 4, A and B). These changes in MF
distribution and number during PPB formation correlate with the appearance of PPB
actins during early stages of PPB development and the loss of actin documented by
immunofluorescence microscopy during late prophase (Liu and Palevitz, 1992;
Takeuchi and Mineyuki, 2014). The CD-induced re-distribution of MFs in late-prophase
PPBs (Figure 4, C and D) matches the changes in F-actin distribution in the CD-treated
and re-broadened PPBs shown by fluorescence microscopy (Mineyuki and Palevitz,
1990; Takeuchi and Mineyuki, 2014). Together, these results support the idea that the
PPB-associated single MFs are F-actins, although we can not formally claim that the
microfilaments in the tomograms are F-actins.
In the present study we have characterized the single MFs in terms of their
3-D organization and spatial relationship to the PPB MTs. We also present quantitative
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information on their length. Our measurements show that most MT-associated MFs are
relatively short, ~70 nm (mode) in prophase 1, and ~140 nm (mode) in prophase 2 and
late prophase (Figure 5). These results suggest that the initial step of PPB formation
involves the binding of short MF fragments to the MTs (arrowheads in Figure 1B,
Figure 3, B and C, Figure 4D and Figure 9A) followed by MF elongation along the MTs,
possibly mediated or enhanced by the linkers, during prophase (Figure 9B). Live cell
imaging has demonstrated that F-actins start to elongate along cortical MTs following
washout of lantrunculin B in interphase cells (Sampathkumar et al., 2011). This type of
growth may also occur during early stages of PPB formation (Figure 9, A-C).
In contrast to the short MFs in the PPB, longer MFs were also frequently
observed in the interphase cells of our samples (Figure 3A and Supplemental Figure
S1F), as reported in pollen tubes (Lancelle et al., 1987) and tobacco interphase cells
(Ding et al., 1991b). This implies that the MFs in the PPB are maintained in a short
configuration for their function. Possible candidate proteins for keeping MFs short
include a heterodimeric MF capping protein that has been shown to bind tightly to the
barbed ends of F-actins in Arabidopsis (Huang et al., 2003, 2006; Li et al., 2012), and
F-actin severing proteins such as ADF/cofilin (Staiger and Cande, 1991; Staiger et al.,
2010).
Analysis of the changes in MT clustering during PPB formation (Figure 8)
shows that the MT band narrowing process can be divided into three stages: (1)
Formation of MT pairs to initiate MT bundle formation, (2) organization of the MT
bundles into loose MT clusters, and (3) reduction of the MT-MT spacing to form tight
MT clusters. As demonstrated in Figures. 3, 4 and 6, the early events of PPB assembly
appear to be mediated by MF bridging structures, whereas the latter ones involve
different types of MT-MT linkers (Figure 7). Based on these data we have developed the
PPB assembly model shown in Figure 9.
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The actin-mediated events of PPB assembly start with the binding of short
(~70 nm) MFs to the MTs and the subsequent alignment of the MFs with the MTs as
more linkers connect the two structures (Figure 1B, Figure 3, B and C, and Figure 9A
and B). Whether these events involve one or two types of MF-MT linkers has yet to be
determined. During prophase 2, lengthening of the bound MFs (Figure 5, A and B)
enables them to from bridges between MTs that are separated by spaces that are too big
to be bridged by MT-MT linker proteins (Figure 1B, Figure 3, E-G, and Figure 9, C and
D). As the zippering up of the MF-linked MTs continues, the MTs come closer together
with the MFs becoming sandwiched between the two parallel MTs to which they are
connected via alternating crosslinks (Figure 6, B-F and Figure 9E). As this occurs, the
MF-mediated crosslinks are replaced by direct MT-MT bridging structures (Figure 7,
Figure 9, F and G, and Supplemental Figure S4).
Our observation shows that single MFs bound to MTs play an essential role in
MT bundling during early stages of PPB formation. The linkers that connect the MFs to
the PPB MTs are ~15 nm long. 10-15 nm long crossbridges have been observed
connecting single MFs and MTs in tobacco pollen tubes and tissues preserved by
cryofixation/freeze-substitution techniques (Lancelle et al., 1987; Ding et al., 1991b).
The recent demonstration that F-actin fragments are translocated along MTs
(Sampathkumar et al., 2011) in jasplakinolide-treated interphase cells suggests an
interesting mechanism for bringing F-actins attached to one MT into contact with a
second MT, so that the MTs can become crosslinked by an F-actin bridge. Molecules
capable of forming crosslinks between F-actins and MTs have an actin-binding domain
at one end and a MT-binding motif at the other, or they can be composed of a protein
complex with one subunit having an actin-binding motif and the second MT-binding
motif (Petrasek and Schwarzerova, 2009). Candidate motor proteins for this activity
include the kinesin-14 protein family with actin-binding, calponin-homology domains
16
(KCHs) (reviewed in Schneider and Persson 2015). Members of the KCH protein
family capable of forming MF-MT cross-bridges have been identified in a variety of
plant species and tissues: in cotton fibers (GhKCH1, Preuss et al., 2004; GhKCH2, Xu
et al., 2009), rice coleoptiles OsKCH1 (Frey et al., 2009), and tobacco cells (Frey et al.,
2009). In addition, two kinesin-14 family proteins (NtKCH, Klotz and Nick, 2012) and
KingG (Bushmann et al., 2011) have been shown to localize to PPBs. The formin
isoform AFH14 of Arabidopsis has been detected in PPBs and has been suggested to
interact with both actin and MTs as demonstrated by in vitro experiments and the
knockdown of AFH14 in mitotic cells (Li et al., 2010).
Based on their length, PPB MT-MT linkers can be divided into two groups:
MAP-M, the dominant type in Prophase 1, and MAP-S, the major one in late prophase
(Figures 7 and 9). MAP-M is replaced by MAP-S linkers during MT band narrowing.
Several MT associated proteins have been localized to PPBs (McMichael and Bednarek
2013, Hamada 2014). Further studies will be necessary to identify the MAPs that
correspond to the MAP-M and MAP-S structures.
17
MATERIALS AND METHODS
Plant Material
Onion (Allium cepa L. cv. Highgold Nigou, Sakata Seed Co., Yokohama) seeds were
sown on pieces of filter paper soaked with 0.05M sucrose in water. They were grown
in the dark at 25ºC for 2 days. Seedlings were transferred to 0.1M sucrose in water and
cultured for 1 day. For CD treatment, the 3d-old seedlings were incubated on 20 µM CD
solution containing 0.1 % (v/v) dimethylsulfoxide for 30 min in the dark. CD was
obtained from Sigma Chemicals Co. (St. Louis, MO, USA) and 20 mM stock dissolved
in dimethylsulfoxide were kept at -20ºC until use.
Sample Preparation for High-pressure Freezing and Freeze Substitution
Three-day-old seedlings were used for sample preparation by high-pressure freezing
and freeze substitution following the method described in Murata et al. (2002). Briefly,
3 mm -long basal parts of the cotyledons were cut from the onion seedlings and
immediately frozen using a high-pressure freezer (BAL-TEC HPM 010, Bal-Tec AG,
Liechtenstein now, Leica, Wetzlar, Germany). The 0.1M sucrose dissolved in water was
used as cryoprotectant. The high-pressure frozen tissues were freeze-substituted in 2%
OsO4 in acetone at -80ºC for 3 days, at -20ºC for 1 day, at 4ºC overnight, and at room
temperature for 2 h. Following the incubation of samples with 2% (w/v) OsO4 solution
at 40ºC for 2 h, the samples were treated with 5% uranyl aceteate in methanol at 4ºC for
2 h. After washing with methanol and acetone at room temperature, they were infiltrated
with a graded series of Spurr's resin (Polysciences, Inc., Warrington, PA, USA).
Polymerization was performed at 70ºC for 16 h.
18
Specimen Preparation for Electron Tomography
We prepared serial tangential sections and serial cross sections of epidermal cells from
the embedded tissues for quantitative analysis. Tangential sections of PPBs (Figure
1E-a) were used to analyze MF-MT relationships, and cross sectional views (Figure
1E-b) to determine nuclear stages and PPB width. Cross sections were also used to
analyze MT-MT and MT-PM linkers. Serial tangential sections (250 -nm thick) of
epidermal cells were prepared from the embedded tissues. The sections were mounted
on formvar-coated slot grids. Each series of sections contained from 50 to 100 serial
sections and included mid-longitudinal plane and outer surface wall sections of the cells.
Sections containing longitudinal plane views of the nuclei were examined in a TEM
(JEOL 1010, JEOL Ltd., Akishima) to identify prophase cells. Prophase cells were
distinguished by condensation level of chromosomes (Figure 2). Once a prophase cells
was identified, the other sections were examined to find a section with a
mid-longitudinal view of the nucleus and the corresponding cross-sectional view of the
PPB, as well as a tangential section through the cell cortex for a tangential view of the
PPB). PPB width was determined on the section containing the mid-longitudinal view
of the nucleus. Serial cross sections (500-nm thick) were cut at the level of the nuclei
and sections containing MTs in an outer cortical region of the target prophase cell were
processed for electron tomography.
Electron Tomography
The sections were double-stained with 2% (w/v) uranyl acetate in 60% (v/v) methanol
and Reynold's lead citrate and then colloidal gold particles were added to both sides of
the grids. The gold particles were used as fiducial markers to align series of tilted
images. 10-nm colloidal gold particles (BBI solutions, Cardiff, UK) were used for
250-nm thick sections and 25-nm colloidal gold particles conjugated with BSA
19
(AURION, Wageningen, the Netherlands) were used for 500-nm thick sections. For
dual-axis electron tomography, the 250-nm-thick tangential sections were observed
either using a Hitachi H-9500 (Hitachi) operating at 300 kV, a TF30 intermediate
-voltage Tecnai EM (FEI) operating at 300 kV or a JEM-1000 high-voltage microscope
(JEOL) operating at 750 kV. The images were taken from 60º to -60º tilt with 1º
increments about two orthogonal axes. When a Hitachi H-9500 was used, the images
were taken at 15,000 × with a resolution of 2048 × 2048 pixels at a pixel size of 1.23
nm using a CCD camera F224HD (TVIPS, Gauting, Germany). The detailed operating
conditions for TF30 and JEM-1000 were described elsewhere (Karahara et al. 2009).
For single-axis tomography of 500-nm thick sections, images were obtained using a
H-1250M (Hitachi High-Technologies Co., Tokyo) operating at 1,000 kV and the
images were taken at 10,000 ×. The images on films were scanned at 4000 dpi and used
for image processing. Tomograms were generated from the tilted image series and the
tomograms were analysed with IMOD, which is a group of programs for 3D
reconstruction and modeling (Kremer et al. 1996). 20 tomograms of intact cells and 4
tomograms of CD treated cells were used for this work.
ACKNOWLEDGEMENTS
A part of this work was supported by “Advanced Characterization Nanotechnology
Platform, Nanotechnology Platform Program of the Ministry of Education, Culture,
Sports, Science and Technology (MEXT), Japan” at the Research Center for Ultra-High
Voltage Electron Microscopy (Nanotechnology Open Facilities) in Osaka University
and by the Cooperative Study Program of National Institute for Physiological Sciences.
This work was also partly supported by JSPS grant 17207006 and MEXT grant
17049019 to YM.
20
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Figure 1. Tomographic images and model of MTs and MFs of a tangentially-sectioned
PPB in an onion cotyledon epidermal cell. (A) A tomographic slice image from a
tangentially sectioned cell cortex of a prophase cell containing a PPB. CW, cell wall.
Bar = 500 nm. (B) Tomography-based reconstruction of the PPB region illustrated in
(A). The thick magenta lines correspond to MTs and the thin yellow lines to MFs.
Arrowheads, contact sites of an end of MF and a MT; Arrows, MFs where an end
contact to a MT and another end to another MT; Asterisks, MFs that run parallel
between two MTs and linked with them by cross bridging structures (see images in
Figure 7B-F). (C) and (D) Tomographic slices of images of a single free MF (arrowhead
in (C)) and of a single MF running along a MT (arrowhead in (D) highlighted in the
28
boxed areas of (B). Bars = 100 nm. (E) Schematic illustrations depicting the plane of the
sections used in this study. (a) Tangential section of the cotyledon showing the surface
view of the cell. Surface view of the PPB is seen in the x-z plane. (b) Tangential section
of the cotyledon showing the mid-longitudinal plane of the cell. Cross sectional view of
the PPB is seen in the x-z plane. (c) Cross section of the cotyledon showing the
transverse plane of the cell. View of the PPB from the top face is seen in the x-y plane.
Figure 2. Changes in nuclear structure and cortical MT organization in the PPB region
of onion cotyledon epidermal cells from interphase to the end of prophase. (A) to (I)
Electron microscopic images of nuclei ((A) to (F)) and PPBs ((G) to (I)) in
mid-longitudinal sections of epidermal cells of onion cotyledons as used for cell cycle
staging. (A) to (F) Nuclei of cells in interphase (A), prophase ((B) and (C)), late
prophase ((D) and (E)) and prophase-prometaphase transition stage (F). The nuclear
staging was based on the extent of chromosome condensation. Bar = 5 µm. (G) to (I)
29
Cross sectional views of PPB MTs at different stages of PPB formation. Higher
magnification views of the cortex regions of cells shown in (A), (E) and (F). (G)
Initial stage of MT gathering in an early, broad PPB. (H) Late prophase PPB. (I)
Prophase-prometaphase transition stage PPB. PPB width determined for the cells
depicted in (A) to (F) were 10.0, 9.3, 6.8, 4.5, 3.6 and 3.5 µm, respectively. Bar = 200
nm.
Figure 3. Changes in MT-MF association during PPB formation and a gallery of images
of cortical MFs and branched MF-like filaments in the PPB region of onion cotyledon
epidermal cells. (A) to (C) Electron tomography models depicting the distribution of
MFs and MTs in the cortical cytoplasm of tangentially sectioned cells. Yellow lines and
magenta lines show MFs and MTs, respectively. Arrowheads, contact sites between a
MF end and a MT. Bar = 500 nm. (A) Interphase cell with randomly oriented cortical
30
MTs and MFs. Very few MT-MF interactions are seen. Note the great variability in MF
length (There are 92 MFs in this tomogram and their length distributed from 57 nm to
1086 nm, and 14 MFs are longer than 500 nm). (B) Interphase cell with some
transversely oriented MTs. Note that more MFs are bound to MTs compared to (A).
(C) Prophase cell with a broad band of PPB MTs (prophase 1). A high proportion of the
MFs are bound to MTs. (D) to (F) Images of MFs (arrowheads) from tomograms of
tangentially sectioned cell cortex regions. Bars = 100 nm. (D) A single MF located
between the PM and two MTs and which is not aligned with the MTs. (E) and (F) Two
tomographic slice images showing a MF (arrowheads) associated with a depolymerizing
MT just beneath the PM. The image shown in (F) is ~10 nm below (E). In (E) the
tomographic slice passes close to the top surface of the MT and the bound MF runs
parallel to the MT, whereas the image in (F) shows a mid-longitudinal view of the MT,
which has a horned end (*). The MF is seen in a grazing view. (G) A tomographic
model showing the MF and MTs illustrated in (E) and (F). MT, microtubule; MF,
microfilament. (H) and (I) MT- and PM-bound, branched and angled MF-like structures
in PPBs. The tomograms were reconstructed from tangentially sectioned prophase cells.
The arrowheads point to filaments that are connected to other filaments, to MTs and to
the PM. PM, plasma membrane. Bar = 100 nm. (J) and (L), and (K) and (M) Models
of MF-like structures shown in (H) and (I), respectively. The light-blue lines correspond
to PM-bound MF-like filaments and the green lines to the other branched and angled
MF-like filaments.
31
Figure 4. Distribution of MFs and MTs in the PPB region of a late prophase, onion
cotyledon epidermal cell, and effects of CD on PPB structure. (A) and (B) A model of
MFs and MTs (A) and of MFs (B) in which the expected reduction of MFs in the ADZ
is clearly visible. (A) Model of a 3.5 µm wide, late prophase PPB reconstructed from
two tomograms illustrating the organization of MFs (yellow lines) and MTs (magenta
lines). Tomographic images of a tomogram used for this model are shown in Figure 3 of
Karahara et al. (2009). Bar = 500 nm. (B) Same model as shown in (A) but without the
MT images to illustrate the difference of MF distribution between the inside and the
outside of the PPB. The MF-depleted zone is clearly visible. The white bracket indicates
the width of the PPB. (C) and (D) MF fragmentation in a CD-treated late-prophase PPB.
(C) A tomographic slice from a tangentially sectioned cell cortex of a CD-treated
late-prophase cell containing a broadened-PPB MTs. The onion seedling was incubated
with 20 µM CD for 30 min before high pressure freezing. The PPB width in this cell
was 8.7 µm. Bar = 500 nm. (D) A MF-MT distribution model reconstructed from the
tomogram shown in (C). Thick purple lines show MTs and the thin yellow lines MFs.
The arrowheads, mark the contact sites between the end of MFs and MTs.
32
Figure 5. Changes in the length and frequency of MT-associated MFs during PPB
formation and in response to CD treatment in onion cotyledon epidermal cells. (A) to
(C) Histograms showing the distribution of MF length in PPBs of stages “prophase 1”
(A), “prophase 2” (B) and “late prophase” (C). (D) MF length distribution in the
broadened PPBs of CD-treated cells. Onion seedlings were incubated with 20 µM CD
for 30 min before high pressure freezing. Cells used for the tomograms were late
prophase and possessed broad MT bands. The mean ± SD of the length of MFs, and the
mode of the length of MFs are shown in each column. The mode of each histogram is
marked by an arrow. The number of MFs used for the histogram (A), (B), (C) and (D)
were 93, 172, 79 and 140, respectively. (E) Changes in the ratio of the total length of the
MT-associated MFs to that of the MTs in PPBs during PPB formation. Cells were
33
divided into 4 groups according to nuclear stages and PPB width: interphase (interphase
cell without a PPB), prophase1 (prophase cell with a broad PPB whose width is equal to
or more than 7µm), prophase2 (prophase cell with a narrow PPB whose width is less
than 7µm) and late prophase (a late-prophase cell with a PPB). The extent of MFs
associated with MTs was determined by measuring the total length of the MT-associated
MFs to the total length of the MTs in each stage. The mean and ± SD is shown in each
group. The data points were calculated from 3 (interphase), 6 (prophase 1), 4
(prophase 2) and 6 (late prophase) tomograms, respectively.
Figure 6. Gallery of images of MF-MT and MT-PM bridges during PPB formation of
onion cotyledon epidermal cells. (A) A tomographic slice view of a PPB showing a MF
(arrowhead) associated with a MT. The MF is connected to the MT by cross-bridging
structures (arrows). (B) to (D) Three tomographic slices from a small volume of a PPB.
34
The volume contains two MTs and a MF. (A) to (F) are the same magnifications. Bars =
100 nm. (B) and (C) The single MF (arrowhead) is seen to be bound to two adjacent
MTs, MTa (B) and MTb (C), via cross-bridges (arrows). The image (C) was a
tomographic slice tilted about 60 degrees around the axis of the MF depicted in (B) (D)
Another view of the volume illustrated in (B) and (C). The MF cannot be seen in this
plane; instead the image was chosen to highlight the parallel arrangement of MTa and
MTb. (E) and (F) Tomographic models of the arrangement of the two MTs (colored
green and red) and one MF illustrated in (B) to (D). Magenta, MTs; yellow, MF; light
blue, MT-MF bridges. The two MTs are bundled via linkers to the single MF. Note that
centers of the two MTs and MF are not on the same plane. (G) to (I) Anchoring of
bundled MTs to PM in a late-prophase PPB. (G) and (H) Tomographic slices of a
500-nm thick mid-cross section showing MTs running along PM. The MT- PM (arrows)
and MT-MT (arrowhead) linkers have different length. PM, plasma membrane, CW, cell
wall. Bar = 100 nm. (I) A tomographic model showing the MF, MTs, PM and cross
bridges in (H). The length of bridging structures between PM and MTs varied from 20
nm to 60 nm.
35
Figure 7. Gallery of images of MT-MT bridges and length-distribution histograms of
MT-MT bridges during PPB formation of onion cotyledon epidermial cells. (A) to (E)
Gallery of images of MT-MT bridges in PPBs. Bars = 100 nm. (A) to (D) Tomographic
slices from tangential sections through PPBs. The arrows point to bridging structures
between adjacent MTs. The length of the bridges in (A), (B), (C), and (D) are 30 nm, 20
nm, 12 nm, and 57 nm, respectively. (E) A tomographic slice from tangential sections
showing a cross sectional view of a PPB. A cluster of MTs is seen beneath the PM, and
bridging structures between the MTs are evident (arrows). CW, cell wall; PM, plasma
membrane. (F) to (I) Comparison of the length of MT-MT bridges in PPBs of prophase
1 (F), prophase 2 (G) and late prophase (H), and that in CD-treated late-prophase PPBs
(I) from 2, 4, 3, and 4 tomograms, respectively. The mean ± SD is shown in each
column. Each developmental stage is explained in Figure 7. For CD treatment, onion
seedlings were incubated with 20 µM CD for 30 min before high pressure freezing and
36
the cells used for tomograms were late prophase and they all possessed wide MT bands.
Analysis of the length distribution was performed by using PeakFit software with a
Gaussian distribution function with R2= 0.99 in the case of cells in the early prophase 1
or the late prophase stage, and 0.91 and 0.96 in early prophase 2 and CD, respectively.
The number in a parenthesis of each column shows the value of each peak. The fitted
curves suggest that dominant size of the bridges was 26.9 nm in prophase 1 and 22.5 nm
in late prophase. A significant difference was found between the distributions of (F) and
(H) (Kolmogorov-Smirnov test, two-tailed, P<0.01).
Figure 8. Changes in MT-bundle cluster sizes during PPB formation in onion cotyledon
epidermal cells. (A) to (C) MT- distribution models from tomograms of PPBs in
tangentially sectioned cell cortex in prophase 1 (A), in prophase 2 (B) and in late
prophase (C). The colored lines correspond to individual MTs. MTs that were connected
each other via MT-MT bridges are shown in the same MT color. Bar = 500 nm.
37
Figure 9. A model showing how single F-actins contribute to MT bundling during the
early stages of PPB and are replaced during later stages by MT-MT linkers. (A) PPB
formation starts when one end of a short F-actin fragment attaches to a MT via a linker
protein. (B) The F-actin fragment aligns along the MT by linkers. (C) The short F-actin
fragment bound to the MT elongates. (D) The free end of the MT-associated F-actin
extends to a neighboring MT to which it becomes attached. (E) Addition of more
MF-MT crosslinks pulls the MTs together until they form zipper-like structures where
the MF is connected to the two MTs through alternating linkers. This tight coupling
enables the two MTs to become crosslinked through short MT-MT bridges. (F) and (G)
The MT-MT bridges are formed by MAPs with different lengths. In earlier stages the
longer MAPs are more numerous, whereas in prophase 2 and in late prophase the
shorter MAPs become the dominant form.
