Download Microtubules Contribute to Tubule Elongation and

Microtubules Contribute to Tubule Elongation and
Anchoring of Endoplasmic Reticulum, Resulting in
High Network Complexity in Arabidopsis1[W][OPEN]
Takahiro Hamada 2, Haruko Ueda, Takashi Kawase, and Ikuko Hara-Nishimura*
Graduate School of Science, Kyoto University, Kyoto 606–8502, Japan
The endoplasmic reticulum (ER) is a network of tubules and sheet-like structures in eukaryotic cells. Some ER tubules dynamically
change their morphology, and others form stable structures. In plants, it has been thought that the ER tubule extension is driven by
the actin-myosin machinery. Here, we show that microtubules also contribute to the ER tubule extension with an almost 20-fold
slower rate than the actin filament-based ER extension. Treatment with the actin-depolymerizing drug Latrunculin B made it possible
to visualize the slow extension of the ER tubules in transgenic Arabidopsis (Arabidopsis thaliana) plants expressing ER-targeted green
fluorescent protein. The ER tubules elongated along microtubules in both directions of microtubules, which have a distinct polarity.
This feature is similar to the kinesin- or dynein-driven ER tubule extension in animal cells. In contrast to the animal case, ER tubules
elongating with the growing microtubule ends were not observed in Arabidopsis. We also found the spots where microtubules are
stably colocalized with the ER subdomains during long observations of 1,040 s, suggesting that cortical microtubules contribute to
provide ER anchoring points. The anchoring points acted as the branching points of the ER tubules, resulting in the formation of
multiway junctions. The density of the ER tubule junction positively correlated with the microtubule density in both elongating
cells and mature cells of leaf epidermis, showing the requirement of microtubules for formation of the complex ER network. Taken
together, our findings show that plants use microtubules for ER anchoring and ER tubule extension, which establish fine network
structures of the ER within the cell.
The endoplasmic reticulum (ER) is a complex network composed of tubules and sheet structures. The ER
network’s morphology changes dynamically by elongation and shrinkage of tubules, sheet expansion, and
sliding junctions. For example, an ER tubule elongates
straight forward from a cisterna and subsequently, fuses
to another cisterna, producing a linkage between two
cisternae. If an elongating tubule fails to fuse to another
cisterna, the tubule contracts into the original cisterna.
However, the ER has stable anchoring points that associate with other cellular structures, such as the plasma
membrane or cytoskeleton. When an elongating ER
tubule reaches an association point, it forms a stable ER
anchor (i.e. establishment of the ER anchoring points
forms stable ER tubules). Hence, increasing the number
of ER anchoring points produces fine ER meshwork.
1
This work was supported by the Japan Society for the Promotion
of Science (research fellowship no. 11J01084 to T.H., Grants-in-Aid for
Scientific Research nos. 21200065 and 25440132 to H.U., and Grant-inAid for Specially Promoted Research no. 22000014 to I.H-.N.).
2
Present address: Graduate School of Arts and Sciences, University of Tokyo, Tokyo 153–8902, Japan.
* Address correspondence to [email protected].
The authors responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described in the Instruction to Authors (www.plantphysiolol.org) is:
Ikuko Hara-Nishimura ([email protected]).
[W]
The online version of this article contains Web-only data.
[OPEN]
Articles can be viewed online without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.114.252320
ER dynamics in eukaryotes depend on the cytoskeleton. In plants, major contributors for ER organization are
actin filaments (Quader et al., 1989; Knebel et al., 1990;
Lichtscheidl and Hepler, 1996; Sparkes et al., 2009a) and
the actin-associated motor proteins (myosins; Prokhnevsky
et al., 2008; Peremyslov et al., 2010; Ueda et al., 2010).
However, it had generally been thought that microtubules are not involved in ER organization in plants,
because microtubule-depolymerizing drugs do not induce obvious changes in the ER network (Quader et al.,
1989; Knebel et al., 1990; Lichtscheidl and Hepler, 1996;
Sparkes et al., 2009a). Nevertheless, involvement of microtubules in plant ER organization has been suspected
from several electron microscopy observations that
showed microtubules located close to the ER membrane
in Vicia faba guard cells, Nicotiana alata pollen tubes, and
Funaria hygrometrica caulonemata (Lancelle et al., 1987;
Hepler et al., 1990; McCauley and Hepler, 1992).
Foissner et al. (2009) have suggested that microtubules are involved in motility and orientation of cortical
ER in Characean algae (Nitella translucens, Nitella flexilis,
Nitella hyalina, and Nitella pseudoflabellata) internodal cells.
Characean cortical ER is spatially separated from inner
cytoplasmic streaming by the middle layer of fixed
chloroplasts. The cortical ER forms a tight meshwork of
predominantly transverse ER tubules that frequently
coalign with microtubules, and microtubule depolymerization reduces the transverse ER tubules and increases mesh size (Foissner et al., 2009). Consistently,
Hamada et al. (2012) have shown in Arabidopsis (Arabidopsis thaliana) that microtubule depolymerization increases
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Hamada et al.
mesh size in young elongating cells. In addition, stable
ER tubule junctions are often colocalized with cortical
microtubules (Hamada et al., 2012), suggesting that
microtubules stabilize ER tubule junctions to form fine
ER meshes. Oryzalin-induced ER nodulation (Langhans
et al., 2009) was not observed in our experimental
conditions.
Here, we showed that ER tubules elongate along microtubules in plant cells. In addition, we revealed that the
ER is stably anchored to defined points on cortical microtubules. The stable anchoring points are the basis of
various ER shapes, such as three-way, two-way, or deadend ER tubules. These microtubule-ER interactions, together with the actin-myosin system, contribute to ER
network organization.
RESULTS
Actin Filament-Independent ER Tubule Extension
We generated Arabidopsis plants that expressed both
lifeact-venus and tag Red Fluorescent Protein (RFP) with
a signal peptide (SP) at the N-terminus and an ER retention signal at the C-terminus (SP-tagRFP-HDEL) to
visualize actin filaments and the ER, respectively. We
took time-lapse images to analyze dynamic ER organization based on actin filaments. Orientation of actin
filaments and ER morphology dramatically changed
every 2 s (Fig. 1A; Supplemental Movie S1). From 4 to
6 s, an ER tubule elongated along an actin filament bundle
(Fig. 1A, arrowheads). Next, we treated the transgenic
plants with Latrunculin B (Lat B) for 30 min to depolymerize actin filaments. After Lat B treatment, all of the
fine actin filaments disappeared, but immobile actin
filament bundles still remained (Fig. 1B). It seems that
these bundles were stabilized by the excess stabilization
effects of lifeact-venus (van der Honing et al., 2011). We
found that ER tubule extension still occurred in the
presence of Lat B, although most ER movements were
abolished (Fig. 1B). Interestingly, the ER tubule extension was observed in the area that lacked bundled actin
filaments (Fig. 1B, arrowheads; Supplemental Movie S2).
The elongating ER tubule is shown by red lines in Figure 1B.
In addition, the velocity of the tubule extension in the
presence of Lat B was obviously slower than that in the
absence of Lat B (Fig. 1B; Supplemental Movie S2). We
named the actin filament-independent ER tubule extension as slow ER tubule extension.
Slow ER Tubule Extension Occurs along Microtubules
The straightforward movement of ER tubule extension
implied involvement of other cytoskeletal components.
Microtubules, which are abundant in the cortical region
of plant cells, are a top candidate for the base of the slow
ER tubule extension. To examine the relationship between microtubules and the slow ER tubule extension,
we used Arabidopsis seedlings expressing both GFPtubulin and SP-tagRFP-HDEL to visualize microtubules
Figure 1. Actin filament-independent ER tubule extension in the presence
of Lat B. ER and actin filaments were observed in hypocotyl epidermal
cells of 5-d-old Arabidopsis seedlings expressing SP-tagRFP-HDEL and
lifeact-venus. A, ER tubule extension along actin filaments. B, ER tubule
extension without actin filaments. Seedlings were treated with 0.1%
dimethyl sulfoxide (DMSO; A) or 2 mM Lat B (B) for 30 min. Inactive
bundled actin filaments that did not have normal actin dynamics were
left in Lat B treatment. Photos were taken every 2 (A) or 5 s (B; corresponding to Supplemental Movies S1 and S2, respectively). Arrowheads
show elongating ER tubules. The DSLT and drawing columns show illustrations of ER and actin filament dynamics corresponding to the AF
and ER columns. Actin filaments and ER are shown in magenta and
green, respectively, in the DSLT column and black and gray in the
drawing column. Red lines in the drawing column indicate growing ER
tubules. AF, Actin filament.
and the ER, respectively. In the presence of Lat B, an
ER tubule emerged from an ER cisterna (0 s in Fig. 2;
Supplemental Movie S3), elongated straightforward along
microtubules (15 s in Fig. 2), and finally reached another
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ER Tubule Extension and Anchoring on Microtubules
Arabidopsis expressing both Endoplasmic Reticulum
Retention Defective2 (ERD2)-GFP (a cis-Golgi/ER marker)
and SP-tagRFP-HDEL (an ER marker). No Golgi body
was found at the tip of elongating ER tubules in the
presence of Lat B, indicating that the slow ER tubule
extension occurred independent of Golgi body movement (Supplemental Fig. S3; Supplemental Movie S6).
These results show that the driving force for slow ER
Figure 2. ER tubule extension along cortical microtubules in the presence
of Lat B. ER and microtubules were observed in hypocotyl epidermal cells
of 5-d-old Arabidopsis seedlings expressing SP-tagRFP-HDEL and GFPtub6. Seedlings were treated with 2 mM Lat B for 30 min. Most of the ER
was immobile, but some was elongated along microtubules (arrowheads). Note that the tip of an elongating ER tubule was associated with
a microtubule from 10 to 25 s. The speed of elongation was dramatically
slower compared with the actin-driven ER tubule elongation. Still images corresponding to Supplemental Movie S3 are shown. The DSLT and
drawing columns of ER dynamics correspond to the MT and ER columns. Microtubules and ER are shown in magenta and green, respectively, in the DSLT column and black and gray in the drawing column.
Red lines in the drawing column indicate growing ER tubules. MT,
Microtubule.
ER cisterna (30 s in Fig. 2). Other ER tubules and sheets
were mostly immobile during 50 s (Supplemental Movie S3).
Importantly, all of the slow ER tubule extensions (as
many as 67) in our 90-min observation period were along
microtubules. For example, 11 ER tubule extensions occurred during a 10-min observation, and all of them were
along microtubules (Supplemental Movie S4). The slow
ER tubule extension along microtubules was occasionally observed, even in the absence of Lat B (Supplemental
Fig. S1), suggesting that the slow ER tubule extension
occurs in intact cells.
We tested the possibility of involvement of factors
other than microtubules in the slow ER tubule extension. Seedlings were treated with Lat B and oryzalin (a
microtubule-depolymerizing drug) to depolymerize both
actin filaments and microtubules. ER tubules and sheets
vibrated and changed their shape slowly; however, directional ER tubule elongation did not occur anymore
(Supplemental Fig. S2; Supplemental Movie S5). Next,
we checked the involvement of Golgi bodies in the
slow ER tubule extension, because movements of Golgi
bodies associated with ER can induce ER tubule extension (Sparkes et al., 2009b). We used transgenic
Figure 3. The tip of the ER tubule associates with microtubules during
tubule extension. The tip of an ER tubule slid along a microtubule, but
the following part of the ER tubule was apart from the microtubule.
From 70 to 80 s, the tip of an ER tubule changed direction of elongation with a different microtubule. Seedlings were treated with 2 mM
Lat B for 30 min before observation. Still images corresponding to
Supplemental Movie S7 are shown. The DSLT and drawing columns of
ER dynamics correspond to the MT and ER columns. Microtubules and
ER are shown in magenta and green, respectively, in the DSLT column
and black and gray in the drawing. Red lines in the drawing column
indicate growing ER tubules. MT, Microtubule.
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Hamada et al.
tubule extension is generated in a microtubuledependent manner.
Characterization of the Slow ER Tubule Extension
To characterize the microtubule-dependent ER tubule
extension, we carefully analyzed the time-lapse images.
An ER tubule started to elongate along a microtubule (0 s
in Fig. 3; Supplemental Movie S7). The tip of the ER tubule moved along the microtubule, but the lateral side of
the following ER tubule did not align with the microtubule in this case (from 10 to 40 s in Fig. 3; Supplemental
Movie S7). The elongating ER tubule shrank one time and
reelongated (Supplemental Movie S7). Interestingly,
the tip of the elongating ER tubule on one microtubule track transferred to another microtubule track (80 s
in Fig. 3; Supplemental Movie S7). In contrast, we did
not find an ER tubule extension that synchronized with
the growing ends of the microtubules (Supplemental
Movie S4).
Next, we examined the elongation direction of ER
tubules against microtubule polarity. Microtubule polarity is distinguishable by observing microtubule dynamics; plus ends of microtubules show faster growing
and shortening than minus ends. Figure 4A is an example image that shows the elongation direction of ER
tubules (Fig. 4A, red arrows) and the plus-end direction
of microtubules (Fig. 4A, blue arrows; Supplemental
Movie S4). During a 10-min observation, ER tubule extension occurred 11 times in total, consisting of 4 times
bound to the plus end, 5 times bound to the minus
end, and 2 times that were uncharacterized extensions
(Fig. 4A; Supplemental Movie S4). We analyzed the
elongation direction of 64 ER tubules in Supplemental
Movies S1 to S8, and each is composed of time-lapse
images over 10 min (Fig. 4D). The frequencies of ER
tubule extensions bound to plus and minus ends of
microtubules were 0.25 6 0.05 and 0.46 6 0.07 times
per minute, respectively, indicating that minus enddirected ER tubule extensions are nearly 2 times
higher than plus end-directed ER tubule extension.
Figure 4. Characterization of microtubule-dependent ER tubule extension.
A, Bidirectional ER tubule extension
along microtubules. The ER and microtubule dynamics in Supplemental
Movie S4 were analyzed and are
shown in a still image from time 0.
Microtubules are indicated by blue arrows. The direction of the blue arrows
indicates the plus ends of the microtubules. The direction of ER extensions is
represented by red arrows. Slow ER
tubule extension occurred in both directions of microtubule polarities. This
figure does not have time information.
Seedlings were treated with 2 mM Lat B
for 30 min. B, Distributions of run distances of ER tubule extension. Numbers of samples are 23 and 41 for plus
end- and minus end-directed ER tubule
extensions, respectively. C, Distributions of velocities of ER tubule extension. Numbers of samples are 23 and
41 for plus end- and minus enddirected ER tubule extensions, respectively. D, Statistical analyses for average
rate, frequency of ER extension, average of run distance, and proportions of
switch-track movement. Numbers of
samples are 23, 41, and 64 for plus-end
directed, minus-end directed, and sum,
respectively.
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ER Tubule Extension and Anchoring on Microtubules
We estimated the velocities of ER tubule elongation
(Fig. 4D). The average velocity of the slow ER tubule
elongation in the presence of Lat B was 0.39 6 0.07 mm s21,
although the average velocity of ER tubule elongation
in the control was 7.8 6 1.7 mm s21. The velocity of the
slow ER tubule elongation is similar to velocities reported
for microtubule-driven elongation of ER tubules in animal cells (Waterman-Storer and Salmon, 1998). We also
investigated the plus end- and minus end-directed slow
ER tubule elongations. There are no significant differences of average velocities of tubule elongation between
the plus-end direction (0.40 6 0.05 mm s21) and the
minus-end direction (0.39 6 0.08 mm s21) and no differences of average lengths between plus-end direction
(4.38 6 1.91 mm) and minus-end direction (5.40 6 2.41
mm; Fig. 4, B–D).
ER Anchoring Sites on Cortical Microtubules Are
Maintained during ER Reorganization
During observations in the presence of Lat B, we found
that dead end-shaped ER often associated with microtubules (Fig. 5; Supplemental Movie S4). In most cases, the
tips of dead end-shaped ER were paused and stably
anchored to microtubules. It has been reported that ER
tubule junctions often colocalize with microtubules in
young elongating cells under normal conditions (Hamada
et al., 2012). However, ER anchoring on microtubules
has not been observed previously.
We investigated whether ER anchoring occurs under
normal conditions (i.e. in the absence of Lat B). During
long observations of 1,040 s, we found more than 10 ER
anchoring points (Fig. 6; Supplemental Movie S8). An
ER anchoring site at 0 s functioned as a three-way junction of ER tubules (0 s in Fig. 6, arrowheads). The ER
tubule junction kept its shape for 240 s (160 s in Fig. 6;
from 0 to 240 s in Supplemental Movie S8). The ER
morphology surrounding the anchoring site dramatically
Figure 6. ER anchoring to cortical microtubule is retained during ER
reorganization. Three-way ER tubule junctions (arrowheads at 0 s)
change to one-way (dead end; at 480–640 s) and subsequently, twoway (linear; 720–880 s) junctions, while keeping microtubule-ER interaction. Arrowheads point to an ER anchoring site on a cortical
microtubule. Still images corresponding to Supplemental Movie S8 are
shown. The DSLT images of ER dynamics corresponding to the MT and
ER columns. Microtubules and ER are shown in magenta and green,
respectively, in the DSLT column. MT, Microtubule.
changed by bulk ER flow; however, the ER anchoring site
still held the ER cisterna and formed dead end-shaped ER
(480 s in Fig. 6; from 320 to 640 s in Supplemental Movie S8).
Other ER flow reached the anchoring site and formed
the two-way linear ER tubule (from 720 to 880 s in Fig. 6
and Supplemental Movie S8). Interestingly, the ER tubule
was thicker, especially at the ER anchoring site. Finally,
new ER tubules elongated from the ER anchoring site
and formed three-way ER tubule junctions again (from
960 to 1,040 s in Supplemental Movie S8). These ER
anchoring sites on cortical microtubules were relatively
stable during dynamic ER reorganization but were not
permanent structures. These results suggest that cortical
microtubules contribute to providing ER anchoring sites
and forming stable junctions or dead-end ER tubules.
Density of ER Tubule Junctions Positively Correlates with
Microtubule Density in Both Elongating and
Expanded Cells
Figure 5. Dead end-shaped ER often associates with microtubules in the
presence of Lat B. Four examples were taken from Supplemental Movie S4.
Dead end-shaped (one-way) ER tubules were often observed in the presence of Lat B. Arrowheads indicate ER anchoring sites on the microtubules.
Seedlings were treated with 2 mM Lat B for 30 min. MT, Microtubule.
We examined the contribution of microtubules to the
architecture of the ER network under different developmental conditions of cells. Both tubule-tubule and sheettubule junctions were associated with microtubules in
expanded cells (Fig. 7A, right) as well as elongating cells
(Hamada et al., 2012; Fig. 7A, left). We analyzed the
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Hamada et al.
correlation between microtubule density and ER junction
number in 80 independent regions of interest of 10 3 10 mm
from both elongating and expanded cells. The number of ER junctions positively correlated with microtubule
density (Fig. 7B). The correlation was observed even in a
single cell (Fig. 7B). Taken together with previous reports that microtubule depolymerization induced coarse
ER meshwork (Foissner et al., 2009; Hamada et al.,
2012), these results clearly show that microtubules contribute to the increasing density of the ER meshwork
throughout cell development.
DISCUSSION
Mechanism of Microtubule-Dependent ER-Tubule
Extension and ER Anchoring
Figure 7. Association of ER tubule junctions with cortical microtubules in expanded cells. A, Left, Elongating epidermal leaf cells of 5-dold Arabidopsis seedlings were used. Small ER tubule patches and
crowded microtubules are shown. ER tubule junctions were often associated with microtubules. A, Right, Expanded epidermal leaf cells of
10-d-old Arabidopsis seedlings were used. The density of microtubules
and the size of ER tubule patches decreased. ER tubule junctions still
associated with microtubules. ER and microtubules are shown in green
and magenta, respectively. Yellow and red dots represent microtubuleassociated ER junctions and free ER junctions, respectively. B, Correlation between the number of ER tubule junctions and microtubule
density. The microtubule density and the number of ER tubule junctions were measured in 80 regions of interest of 10 3 10 mm from both
elongating and expanded cells. Blue dots and red squares correspond
to data from elongating and expanded cells, respectively. Microtubule
densities were calculated by area of threshold images converting from
microtubule images. MT, Microtubule.
The ER tubule extension mediated by microtubules
is relatively slow (0.14 mm s21) compared with the velocity of ER tubule extension mediated by actin filaments
(7.8 mm s21). In animals, ER tubule extension occurs along
microtubules with a velocity of approximately 0.1 mm s21
(Waterman-Storer and Salmon, 1998), which is similar to
our observations. In animals, two different mechanisms of
ER tubule extension along microtubules have been identified: a sliding mechanism driven by kinesin/dynein
motor proteins and a Tip Attachment Complex mechanism
through microtubule plus end-accumulating protein
END BINDING1 (EB1; Friedman and Voeltz, 2011). In
plants, EB1-like protein is reported to link microtubule
dynamics to endomembrane organization (Mathur et al.,
2003). However, our observations revealed that slow ER
tubule extension did not synchronize with the microtubule growing plus ends (Supplemental Movie S4). In
addition, ER tubule tips are associated with microtubules during the slow ER tubule extensions. These results suggest that the slow ER tubule extension in plants
is generated by the motor protein-dependent sliding
mechanism but not the EB1-dependent Tips Attachment
Complex mechanism. It should be noted that the tip
association of ER tubules with microtubules is necessary
and sufficient for slow ER tubule extension, although the
lateral association of the ER tubules that is occasionally
observed is not required for ER tubule extension. A
similar manner was reported for the actin-mediated ER
tubule extension (Yokota et al., 2011).
In addition, we found that ER tubules elongated in both
directions of microtubule polarity (Fig. 4; Supplemental
Movie S4). In animal cells, bidirectional ER tubule extension has also been observed. Two different proteins
(kinesins and cytoplasmic dynein) that direct to the plus
and minus ends of microtubules, respectively, mediate
ER tubule extension (Woźniak et al., 2009). Many kinesin homologs occur widely in eukaryotes, including
plants (Richardson et al., 2006), although plants lack
cytoplasmic dynein. It has been suggested that plants
have special minus end-directed kinesins, such as some
of the kinesin-14 family (Chen et al., 2002; Marcus et al.,
2002, 2003; Ambrose et al., 2005). These members
are candidate drivers for ER tubule extension to the
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ER Tubule Extension and Anchoring on Microtubules
minus-end direction. In animals, kinesins require adapter
proteins, such as kinectin, to induce ER tubule elongation (Toyoshima et al., 1992). Plants lack obvious
kinectin homologs. Identification of ER-related kinesins and adapter proteins is required.
Animal cells have ER binding microtubules-associated
proteins, such as STROMAL INTERACTION MOLECULE1 and CYTOSKELETON-LINKING MEMBRANE
PROTEIN63 (Friedman and Voeltz, 2011), for ER anchoring
to microtubules. However, plants have no obvious homologs of these animal proteins. In plants, overexpression
of Arabidopsis formin14 induced the alignment of ER
tubules along cortical microtubules (Deeks et al., 2010).
This finding seems to be a secondary effect of the
microtubule-actin filaments interaction, because formin
could bind to both actin filaments and microtubules.
Recently, the ER membrane-associated protein VAMP/
SYNAPTOBREVIN-ASSOCIATED27 (VAP27) directly
bound to microtubules in vitro (Wang et al., 2014). VAP27
may be involved in ER anchoring to microtubules.
Contribution of Microtubules to ER Network Organization
Based on the slow ER tubule extension and ER anchoring, microtubules provide fine ER tubule meshwork
at the cortex in plant cells. Although the exact functions
of the fine ER meshwork remain unclear, the fine ER tubule meshwork has at least two advantages compared
with the coarse ER tubule meshwork and ER sheet. First,
the fine ER tubule meshwork has a large surface area per
volume. Increasing the ER surface area may strengthen
the protein synthesis ability and signal transduction between the cytoplasm and ER lumen. Second, the fine ER
tubule meshwork provides lots of high-curvature faces on
the ER. The ER exit site, which mediates protein transport
from the ER to Golgi bodies, accumulates, especially at
high-curvature faces of the ER (Okamoto et al., 2012).
Plants may enhance ER functions by forming fine ER
tubule meshwork with regulation of cortical microtubules.
At the cortex of plant cells, the slow ER tubule extension along cortical microtubules may be insignificant, because most of the ER tubule extensions are
actin-myosin-dependent. Considering the stability of
microtubule-dependent ER tubules, microtubules may
contribute to the stabilization of the ER network. In the
case of mitotic cells, the ER tubule extension along microtubules may significantly contribute to the organization of ER structures. ER tubules are associated with
the spindle and early phragmoplast from metaphase to
early telophase (Hepler and Wolniak, 1984; Gupton et al.,
2006). Future analyses based on identification of microtubule-ER-connecting proteins and their mutants’ analyses will shed light on the importance of microtubules to
ER organization in plant cells.
two decades ago (Hepler et al., 1990). However, the
functions of microtubules in ER network organization
are unclear compared with those of actin filaments. In
this study, we found that microtubules engage slow
ER tubule extension and ER anchoring in plant cells.
Our findings confirm the contribution of microtubules
to ER network organization in plant cells.
MATERIALS AND METHODS
Plant Materials and Growth Conditions
All material was Arabidopsis (Arabidopsis thaliana) ‘Heynh’ on Columbia-0
background. For labeling microtubules and ER, plants expressing both GFPb-TUBULIN6 (TUB6) and SP-TagRFP-HDEL were used as described by Hamada
et al. (2012). For labeling actin filaments, plants expressing lifeact-venus were
used (Era et al., 2009). For labeling Golgi bodies, plants expressing ERD2-GFP
were used (Takeuchi et al., 2000). Seeds were sterilized in 5% (w/v) sodium
hypochlorite and 1% (w/v) Triton X-100 for 5 min. After sterilization, seeds were
rinsed five times with sterile water and plated on agar containing 1% (w/v) Suc,
1% (w/v) agar, and one-half-strength nutrient solution as described by Haughn
and Somerville (1986). Plates were set in a near-vertical position at 22°C with
constant light. Five-day-old plants were used for analyses of elongating cells and
drug treatments. Ten-day-old plants were used for analyses of expanded cells.
Imaging Analyses and Drug Treatments
Observations were performed on a fluorescence microscope (Axio Observer;
Zeiss) equipped with a confocal spinning disc unit (CSU-X1; Yokogawa) with a
dual band-pass filter (Semrock) and a twin electron-multiplying CCD cameras
(Andor) system. Images were taken with IQ software (Andor) and analyzed with
Image J (National Institutes of Health; http://rsbweb.nih.gov/ij/). For drug
treatments, plants were soaked in small tubes containing 2 mM Lat B (Sigma) or
50 mM oryzalin (Sigma) and incubated for 30 min at 22°C in light. Direction-selective
local threshold (DSLT) is a modified local threshold method (T. Kawase,
S.S. Sugano, T. Shimada, and I. Hara-Nishimura, unpublished data). In brief,
DSLT measures the neighboring regions connected to the central point by line
segment kernels in various orientations rather than weighing the cubic or spherical
neighboring regions. The threshold at each pixel is the minimal weighed sum
calculated by convolving the original images with a line segment kernel.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Slow ER tubule extension in the absence of Lat B.
Supplemental Figure S2. Removal of both microtubules and actin filaments repressed ER tubule extension.
Supplemental Figure S3. ER tubule extension in the presence of Lat B is
independent of Golgi bodies.
Supplemental Movie S1. Dynamics of ER and actin filaments.
Supplemental Movie S2. Dynamics of ER and actin filaments in the presence of 2 mM Lat B.
Supplemental Movie S3. ER tubule extension along cortical microtubules
in the presence of Lat B.
Supplemental Movie S4. Dynamics of ER and microtubules for 10 min in
the presence of 2 mM Lat B.
Supplemental Movie S5. Dynamics of ER and microtubules in the presence of 50 mM oryzalin and 2 mM Lat B.
Supplemental Movie S6. Dynamics of ER and Golgi bodies in the presence
of 2 mM Lat B.
CONCLUSION
ER-microtubule interactions in plant cells were suggested by electron microscopy observations more than
Supplemental Movie S7. Dynamics of an ER tip and microtubules in the
presence of 2 mM Lat B.
Supplemental Movie S8. Dynamics of ER and microtubules.
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Hamada et al.
ACKNOWLEDGMENTS
We thank Atsuko Era, Akihiko Nakano, and Takashi Ueda (University of
Tokyo) for donation of the seeds of the Arabidopsis line expressing 35S:lifeactvenus or 35S:ERD2-GFP.
Received October 22, 2014; accepted October 30, 2014; published November 3,
2014.
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