Dynamics of Arabidopsis Dynamin-Related Protein 1C and aClathrin Light Chain at the Plasma Membrane W OA
Catherine A. Konopka,a,1 Steven K. Backues,b and Sebastian Y. Bednareka,b,2
a Program in Cellular and Molecular Biology, University of Wisconsin, Madison, Wisconsin 53706b Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706
Plant morphogenesis depends on polarized exocytic and endocytic membrane trafficking. Members of the Arabidopsis
thaliana dynamin-related protein 1 (DRP1) subfamily are required for polarized cell expansion and cytokinesis. Using a
combination of live-cell imaging techniques, we show that a functional DRP1C green fluorescent fusion protein (DRP1C-
GFP) was localized at the division plane in dividing cells and to the plasma membrane in expanding interphase cells. In both
tip growing root hairs and diffuse-polar expanding epidermal cells, DRP1C-GFP organized into dynamic foci at the cell
cortex, which colocalized with a clathrin light chain fluorescent fusion protein (CLC-FFP), suggesting that DRP1C may
participate in clathrin-mediated membrane dynamics. DRP1C-GFP and CLC-GFP foci dynamics are dependent on
cytoskeleton organization, cytoplasmic streaming, and functional clathrin-mediated endocytic traffic. Our studies provide
insight into DRP1 and clathrin dynamics in the plant cell cortex and indicate that the clathrin endocytic machinery in plants
has both similarities and striking differences to that in mammalian cells and yeast.
INTRODUCTION
Dynamin and dynamin-related proteins (DRPs) constitute a
structurally related, yet functionally diverse, superfamily of large
GTPases (reviewed in Praefcke and McMahon, 2004; Konopka
et al., 2006). Dynamins and DRPs are involved in various aspects
of endomembrane and intracellular organelle dynamics, includ-
ing endocytosis and post trans-Golgi network (TGN) trafficking
(Damke et al., 1994; Henley et al., 1998; Jones et al., 1998;
Nicoziani et al., 2000), mitochondrial fusion and fission (Mozdy
et al., 2000; Wong et al., 2000), peroxisome inheritance (Koch
et al., 2003), chloroplast division (Gao et al., 2003), and actin
dynamics (McNiven et al., 2000; Schafer et al., 2002). Mamma-
lian dynamin 1 plays critical roles in several types of endocytosis,
including clathrin-mediated endocytosis (CME; Damke et al.,
1994).
In mammalian cells, CME is mediated by a coordinated inter-
play of accessory and regulatory proteins. Invagination of the
clathrin-coated bud is thought to occur through the polymeriza-
tion of clathrin triskelia, which is composed of heavy chain (CHC)
and light chain (CLC) subunits, the membrane remodeling activ-
ities of accessory proteins, and the force generated by actin
polymerization (Conner and Schmid, 2003). Dynamin 1 subunits
are recruited to and form rings and spirals around the necks of
the invaginating clathrin-coated buds (Damke et al., 1994)
through their lipid-interacting pleckstrin homology (PH) domain
and protein-interacting Pro-rich (PR) domain (Vallis et al., 1999).
It has been proposed that there is a conformational change of the
dynamin oligomer upon GTP hydrolysis (Roux et al., 2006) that
along with actin polymerization (Merrifield et al., 2005) and the
activity of additional membrane modifying proteins promotes the
release of the clathrin-coated vesicle from the plasma mem-
brane. Similar to mammalian cells, CME in the yeast Saccharo-
myces cerevisiae depends on a highly ordered assembly of
membrane and cytoskeletal-associated proteins to initiate the
recruitment of cargo proteins, budding, and release of endocytic
vesicles (Kaksonen et al., 2003, 2005; Sun et al., 2006). However,
a few striking differences exist, including the lack of an identified
DRP in yeast CME, which is required for endocytosis in mammals
(Yu and Cai, 2004; Kaksonen et al., 2005).
Clathrin-coated structures at the plasma membrane are evi-
dent in electron micrographs of several plant species (van
der Valk and Fowke, 1981; Emons and Traas, 1986; Derksen
et al., 1995; Robinson, 1996; Fowke et al., 1999; Dhonukshe
et al., 2007). Apparent homologs for CHC, CLC, AP-2 subunits,
and mammalian accessory proteins are present in the Arabidop-
sis thaliana genome (Barth and Holstein, 2004; Holstein and
Oliviusson, 2005). Expression of a dominant-negative CHC in-
hibits uptake of the lipophilic tracer dye FM4-64 and of several
plasma membrane proteins (Dhonukshe et al., 2007; Tahara
et al., 2007). In addition, use of the mammalian AP-2 adaptor
complex inhibitor tyrphostin A23 causes a reduction in endocy-
tosis (Dhonukshe et al., 2007). Although the existence of CME in
plants is now widely accepted, the molecular machinery respon-
sible for the regulated uptake of membrane and endocytic cargo
(Russinova et al., 2004; Robatzek et al., 2006; Sutter et al., 2007)
is less well characterized.
1 Current address: Department of Pharmacology, University ofWashington, Seattle, WA 98195.2 Address correspondence to [email protected] author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Sebastian Y.Bednarek ([email protected]).W Online version contains Web-only data.OA Open Access articles can be viewed online without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.108.059428
The Plant Cell, Vol. 20: 1363–1380, May 2008, www.plantcell.org ª 2008 American Society of Plant Biologists
The Arabidopsis genome contains 16 genes that are predicted
to encode DRPs (Hong et al., 2003b; Gao et al., 2006), which
have been assigned to six protein families based on primary
sequence, predicted domain structure, and functional analysis.
Members of the DRP2 family share the greatest similarity in
domain structure to mammalian dynamin 1, including the PH
and PR domains, and are required for Golgi-to-vacuole protein
trafficking (Jin et al., 2001). DRP2A interacts with the putative
Arabidopsis AP-1 subunit, g-adaptin (Jin et al., 2001), which, in
turn, interacts with clathrin and the Arabidopsis homolog of epsin
(Song et al., 2006), an adaptor protein required for clathrin-
dependent trafficking at the plasma membrane (Chen et al.,
1998) and TGN (Duncan et al., 2003). However, the direct
involvement of DRP2 in endocytosis has not been shown.
In contrast with DRP2, members of the plant-specific DRP1
protein subfamily, which lack the PH and PR domains found in
dynamin 1, are required for plant cell expansion and cytokinesis
(Kang et al., 2001, 2003b). The DRP1 protein subfamily consists
of five members, designated A through E. In a related study, we
have described the dynamics of DRP1A in comparison to DRP1C
and shown that DRP1C is not fully functionally redundant with
DRP1A (Konopka and Bednarek, 2008b). Limited work on
DRP1C has demonstrated protein localization to the de novo
plasma membrane at the cell plate and a striking enrichment in
the tips of expanding root hairs. Loss-of-function drp1C mutants
are male gametophytic lethal with pollen characterized by large
invaginations of the plasma membrane (Kang et al., 2003a).
These early data suggest that DRP1C may be involved in plasma
membrane dynamics. Using a combination of confocal laser
scanning microscopy (CLSM) and variable angle epifluores-
cence microscopy (VAEM), we show that DRP1C–green fluores-
cent protein (GFP) was recruited from the cytoplasm to the
plasma membrane flanking the tip in rapidly growing root hairs. In
epidermal cells, DRP1C-GFP was organized into discreet, dy-
namic foci that colocalized with and displayed similar dynamics
to the plasma membrane–associated, fluorescent fusion protein
of clathrin light chain (CLC-FFP). These analyses support the hy-
pothesis that DRP1C is a component of the clathrin-associated
machinery in plants.
RESULTS
DRP1C-mGFP5 Is Localized to the Division Plane,
Plasma Membrane, and Cytoplasm
To determine the cellular distribution of DRP1C, a DRP1C-
mGFP5 translational fusion (now referred to as DRP1C-GFP)
was constructed and introduced into DRP1C/drp1C-1 plants
(Kang et al., 2003a). To ensure normal expression and localiza-
tion of GFP-tagged DRP1C, a genomic fragment of DRP1C in-
cluding 2.9 kb of the native promoter, which is known to
complement the drp1C pollen defect (Kang et al., 2003a), was
positioned at the N terminus of GFP (Figure 1A). DRP1C-GFP
rescued the development of drp1C-1 pollen, and drp1C-1/
drp1C-1 plants transformed with DRP1C-GFP had no visual
defects. Immunoblot analysis of total protein extracts from plants
expressing DRP1C-GFP confirmed that the fusion protein was
intact and expressed at levels similar to native DRP1C (Figure
1B). Together, these data indicated that the DRP1C-GFP fusion
protein was functional in vivo.
DRP1C-GFP was expressed throughout pollen germination.
GFP fluorescence was first observed in hydrated pollen and
localized to the plasma membrane (Figure 1D) but was not de-
tected in pollen from untransformed plants (Figure 1C). During
pollen grain germination, plasma membrane–localized DRP1C-
GFP converged to the region near the aperture through which the
pollen tube would emerge (Figure 1D). As the pollen tube
continued to grow, DRP1C-GFP remained in the distal end of
the pollen tube in the cytoplasm and along the plasma mem-
brane (Figure 1E; see Supplemental Video 1 online).
As previously observed in roots using immunofluorescence
(Kang et al., 2003a), DPR1C-GFP localized to the division plane
in the transition zone of roots (Figure 1F) and to the distal region
of elongating root hairs (Figure 1G). In addition, live-cell imaging
showed that DRP1C-GFP associated with the plasma mem-
brane in these cell types. In aboveground tissue, DRP1C-GFP
was expressed in hypocotyls (see Supplemental Figure 1A
online), leaf pavement, and socket cells (Figure 1H) and in de-
veloping leaf trichomes (Figure 1H), where it localized to both the
cell cortex and the cytoplasm.
DRP1C-GFP was not observed to be associated with any
cytoskeletal structures or mitochondria in dividing or nondividing
cells as was previously reported (Hong et al., 2003a; Jin et al.,
2003). DRP1C-GFP did not colocalize with the mitochondrial
tracer dye MitoTracker Orange CM-H2XRos in root epidermal
cells (see Supplemental Figure 1B online) or cortical cells. To
confirm the lack of mitochondrial localization, roots expressing a
GFP-tagged mitochondria signal sequence fusion protein (ss-
b-ATPase-GFP; Logan and Leaver, 2000) were fixed and probed
with an antibody specific for DRP1C (Kang et al., 2003a). Again,
a-DRP1C did not associate with the ss-b-ATPase-GFP mito-
chondrial marker (see Supplemental Figure 1C online).
DRP1C-GFP Is Enriched at the Lateral Tip Plasma
Membrane of Growing Root Hairs
Previous studies have shown that Arabidopsis DRP1s are in-
volved in polarized cell expansion (Kang et al., 2003a, 2003b).
Root hairs are a model cell for the study of polar cell growth as
they expand through membrane addition exclusively at the tip.
Many signaling and membrane trafficking pathways that are
required to establish and maintain this polar cell growth have
been characterized (reviewed in Carol and Dolan, 2002; Samaj
et al., 2006). Thus, we analyzed the in vivo dynamics of DRP1C-
GFP in these tip-growing cells. DRP1C-GFP fluorescence was
not evenly distributed throughout the root hair but was primarily
localized at the distal end (toward the tip) during all stages of root
hair development, beginning with the appearance of the first
bulge and continuing through to termination of root hair growth
(Figure 2A). DRP1C-GFP fluorescence levels at the distal end of
the root hair decreased as root hairs stopped growing and
eventually reached levels similar to the rest of the root hair
plasma membrane.
To investigate the dynamics of DRP1C-GFP during root hair
growth with greater time resolution, seedlings were grown at a
308 horizontal angle through half-strength Murashige and Skoog
1364 The Plant Cell
(MS) þ 0.5% agar medium (Murashige and Skoog, 1962) on a
glass cover slip (Konopka and Bednarek, 2008a) to allow root hairs
to elongate in one plane without disturbance. Root hairs that
grew along the surface of the glass cover slip were imaged using
CLSM. Root hairs appeared morphologically normal throughout
the 3 h of imaging. Their growth rate oscillated over time, with
rates varying between 0.4 and 1.7 mm/min, which was consistent
with root hair growth dynamics observed previously (Monshausen
et al., 2007). DRP1C-GFP localized to the plasma membrane
primarily at the tip apex, which is the site of membrane addition
(Shaw et al., 2000), and the lateral plasma membrane within ;15
mm of the apex (referred to as tip lateral flanks) as well as
throughout the circulating cytoplasm (Figure 2B; see Supple-
mental Video 2 online). As the root hair elongated, the plasma
membrane–associated DRP1C-GFP fluorescence intensity fluc-
tuated both at the lateral flanks and at the tip apex throughout the
2 h of imaging (Figure 2C). The highest DRP1C-GFP fluorescence
intensities at the tip apex were observed when the growth rate of
the root hair was lowest (Figure 2C). At the fastest growth rates
(;1.7 mm/min), only 6% of total membrane-associated DRP1C-
GFP fluorescence was located at the tip apex. By contrast, at the
slowest rate, 60% of the total plasma membrane–associated
DRP1C fluorescence was present at the root hair tip apex. This
analysis suggested an inverse relationship between the locali-
zation of DRP1C at the tip apex, where exocytosis occurs, and
the rate of root hair growth during expansion (Figure 2C).
DRP1C-GFP Is Recruited from a Cytoplasmic Pool to the
Plasma Membrane
We performed a FRAP analysis to determine how DRP1C is
recruited to the plasma membrane at the root hair tips. DRP1C
could be recruited directly from the cytoplasm and/or delivered
to the tip apex via an exocytic pathway and then diffuse into the
root hair tip flanks during periods of rapid growth. To test these
models, the diffusion of plasma membrane–associated DRP1C-
GFP at the tip lateral flanks of expanding root hairs was analyzed.
If DRP1C-GFP were recruited directly from the cytoplasm, the
fluorescence in the entire photobleached area would recover at a
uniform rate. Alternatively, if DRP1C-GFP localization depended
on initial delivery to the root hair apex and subsequent diffusion,
the initial fluorescence recovery would occur at the edge of the
photobleached area. As a control, the fluorescence recovery rate
of the cytoplasmic pool was determined, which recovered to its
original intensity (corrected for photobleaching) in 17.1 6 5.1 s
(n ¼ 9; Figures 3A, 3C, and 3D, black circles). The plasma
membrane–associated fluorescence recovered in 47.6 6 15.8 s
(n ¼ 18; Figures 3B to 3D), indicating that DRP1C-GFP did not
freely diffuse at the plasma membrane. The photobleached area
of the root hair tip flank was divided into two regions, one
comprising the peripheral two-thirds of the photobleached area
(Figures 3C and 3D, white boxes) and the other comprising the
inner one-third of the bleached area (Figures 3C and 3D, black
Figure 1. Localization of the Functional DRP1C-GFP Fusion Protein.
(A) Schematic of the DRP1C-GFP construct. T, Nos terminator. The four–amino acid linker between DRP1C and GFP is shown.
(B) Immunoblot analysis of total protein extracts from seedlings of wild-type (lanes 1 and 5), wild-type transformed with DRP1C-GFP (lanes 2 and 6),
DRP1C/drp1C-1 (lanes 3 and 7), and drp1C-1/drp1C-1 transformed with DRP1C-GFP (lanes 4 and 8) probed with a-DRP1C antibody (lanes 1 to 4),
a-GFP antibody (lanes 5 to 8), or a-PUX1 (lanes 1 to 8, loading control). Relative mobilities of DRP1C, DRP1C-GFP, and PUX1 are indicated. *,
breakdown product of DRP1C-GFP; **, cross-reactant with either the a-GFP antibody or an endogenous biotin conjugated protein recognized by
streptavidin–horseradish peroxidase (HRP).
(C) to (E) Pollen from an untransformed wild-type plant (C) or a drp1C-1/drp1C-1 plant transformed with DRP1C-GFP ([D] and [E]) imaged with LCSM.
DRP1C-GFP (green) and autofluorescence of pollen coat (red) are evident.
(D) DRP1C-GFP is concentrated around the periphery of the vegetative cell and at the germination exit site (yellow arrow).
(E) Four frames from a CLSM time series (see Supplemental Video 1 online) of a growing pollen tube taken from a drp1C-1/drp1C-1 plant transformed
with DRP1C-GFP. Times indicate time from start of series.
(F) to (H) Confocal slices of drp1C-1/drp1C-1 plants transformed with DRP1C-GFP, showing the root cortex and epidermal cells (F), growing root hair
(G), and an average projection Z-stack reconstruction of a first leaf, which includes pavement cells, socket cells, and a stage 4 bibranched trichome
(H). Autofluorescence from chloroplasts (red) is evident in (H). DRP1C-GFP–labeled cell plates are marked with yellow arrowheads ([F] and [H]). Bars¼10 mm.
Dynamics of Arabidopsis DRP1C and CLC 1365
boxes). In 83% of the root hairs (n¼ 18), the peripheral and inner
regions recovered with equal kinetics, suggesting that DRP1C-
GFP was primarily recruited from the cytoplasm (Figures 3B to
3D; see DPR1C-GFP forms discreet foci at the plasma mem-
brane below).
Tip-Localized DynamicsofDRP1C-GFPAre Associatedwith
Growth Rate in Inhibitor-Treated Root Hairs
Root hair growth has been shown to require calcium gradients
and reactive oxygen species as signaling modules, phosphatidyl-
inositol metabolism, actin dynamics, and a functional secretory
pathway (Carol and Dolan, 2002; Samaj et al., 2006). To deter-
mine if these processes are also required for DRP1C-GFP
polarity, DRP1C-GFP dynamics in root hairs were analyzed
before, during, and after the addition of inhibitors of the above
processes, including, 0.1% DMSO (control treatment; Figure
4A), 20 mM brefeldin A (secretory trafficking inhibitor; Figure 4B;
Geldner et al., 2003), 30 mM cytochalasin D (F-actin inhibitor;
Figure 4C; Ketelaar et al., 2003), or 30 mM tyrphostin A23 (CME
inhibitor; Figure 4D; Banbury et al., 2003). Unlike the control
treatment, which did not affect growth (Figure 4A), each inhibitor
caused a cessation of root hair growth 10 to 30 min after inhibitor
addition (Figures 4B to 4D). With the exception of tyrphostin A23
Figure 2. DRP1C-GFP Localization to the Tips of Root Hairs Is Dependent on Growth.
(A) Z-stack reconstructions of one cell file of root hairs in a drp1C-1/drp1C-1 plant transformed with DRP1C-GFP. Numbers indicate the length of the
root hair from the cell base.
(B) Heat map of DRP1C-GFP fluorescence in growing root hair with white indicating the highest intensity and purple indicating the lowest intensity.
Bar ¼ 5 mm.
(C) Graph plotting threshold area (i.e., the number of pixels at or above a set intensity level; green line) at the tip of the root hair and growth rate of the
root hair (blue line) over time.
1366 The Plant Cell
Figure 3. DRP1C-GFP Is Recruited from the Cytoplasm to Sites on the Plasma Membrane.
(A) and (B) Time course of photobleaching and recovery of DRP1C-GFP pools in the cytoplasm (A) and lateral tip plasma membrane (B) in growing root
hairs. Numbers represent time in seconds from the bleach. White shapes indicate bleached areas. Bars ¼ 10 mm.
(C) Enlarged confocal images of root hairs at time 0 in (A) and (B). The photobleached region indicated in (B) is subdivided into peripheral and middle
areas (indicated by dashed and solid squares, respectively) used for the graph in (D).
(D) GFP fluorescence intensity was measured for regions shown in (C) and plotted versus time. The images and graphs shown are representative of 18
separate FRAP experiments.
Dynamics of Arabidopsis DRP1C and CLC 1367
(tyrA23), polarized localization of DRP1C-GFP at the apical and
lateral plasma membrane was abolished within 15 min of growth
cessation, resulting in a nonpolar distribution of DRP1C-GFP
throughout the root hair (Figures 4B and 4C). These results sug-
gested that the known requirements for active root hair tip
growth are most likely not directly required for DRP1C-GFP
recruitment or dynamics at the plasma membrane but that
DRP1C-GFP localization at the tip of root hairs is intimately tied
with active growth. This is supported by the result that DRP1C-
GFP was not mislocalized in several root hair mutants, including
the tip growth expansion mutants rhd2, rhd4, cow1, tip1, cob-1,
cob-2, and erh1 (see Supplemental Figure 2 online).
tyrA23 inhibits mammalian AP-2 binding of endocytic cargo
and has been shown to block CME in mammalian cells (Banbury
et al., 2003) and endocytic traffic in plants (Dhonukshe et al.,
2007). Although CME has not yet been shown to be required for
tip growth in Arabidopsis, clathrin-coated structures have been
observed at the flanks of root hair tips (Emons and Traas, 1986).
We sought to determine if disrupting CME would alter the
dynamics of DRP1C-GFP in root hairs. Within ;15 min of
tyrA23 application, root hair growth slowed and stopped. (Figure
4D). In contrast with the other inhibitor treatments, DRP1C-GFP
fluorescence at the apical or lateral plasma membrane de-
creased but never completely disappeared upon growth cessa-
tion. Instead, DRP1C-GFP remained localized at the tip up to 30
min after growth of the root hair had ceased. A further analysis of
the effects of tyrA23 on DRP1C-GFP dynamics is presented
below.
DPR1C-GFP Forms Discreet Foci at the Plasma Membrane
To examine DRP1C-GFP dynamics at the plasma membrane of
transformed plants, VAEM was used, which allows for imaging of
the plant cell cortex with a high signal-to-noise ratio (Konopka
and Bednarek, 2008a). The distribution of DRP1C-GFP was visu-
alized using VAEM in root hair tips and flanks (Figures 5A and 5B),
expanding epidermal cells of the differentiation and elongation
zone of the root (Figures 5E and 5F; see Supplemental Video 3
online), mature atrichoblasts in the root, leaf pavement cells,
hypocotyl epidermal cells, and guard cells (see Supplemental
Figure 4. DRP1C-GFP Apical Localization and Growth Was Uncoupled Using the Clathrin Inhibitor tyrA23.
Root hairs expressing DRP1C-GFP were treated with 0.1% DMSO (A), 20 mM brefeldin A (B), 30 mM cytochalasin D (C), or 30 mM tyrA23 (D). Red arrow
indicates time of drug application. DRP1C-GFP fluorescence threshold area at the tip was plotted against the distance grown by the root hair tip. Bars¼10 mm.
1368 The Plant Cell
Figure 3 online). In all cell types, DRP1C-GFP was prominently
organized into discreet cortical-associated foci (Figures 5A to
5C; see Supplemental Video 3 online). To eliminate any differ-
ences that may occur in different cell types, DRP1C-GFP foci
were initially analyzed in expanding root epidermal cells. In
addition, only foci that had lifetimes (defined as the length of time
that a focus was visible in the imaging plane) of >2 s were
included in the analysis. Each of the DRP1C-GFP foci that
appeared also disappeared during the 5 min of imaging. The
average lifetime of the DRP1C-GFP foci was 17.7 6 8.8 s (n¼ 175;
Figure 5. DRP1C-GFP Forms Discreet, Dynamic Foci at the Plasma Membrane.
DRP1C-GFP root hairs ([A] and [B]) and expanding root epidermal cells ([E] to [G]) imaged with VAEM.
(B) Time series of boxed area in (A). Numbers indicate elapsed time from start of imaging in seconds.
(C) Fluorescence intensity profile of two foci indicated in (B) with arrow (red) and arrowhead (green).
(D) Average normalized fluorescence (black line) and SD (gray lines) of 22 foci from one cell over time. The peak fluorescence was centered at time 0.
(E) and (F) Images from a time series, acquired at 0.5-s intervals. Blue arrow indicates the same focus in (E) and (F). A focus that split off from the original
is indicated by the yellow caret in (E).
(G) Images from a time series, acquired at 0.5-s invervals. The focus indicated with an orange caret merges with the focus indicated by the green arrow.
(H) The average density (6SD) of foci for four expanding (gray bars) and four nonexpanding (white bars) cells averaged from five different time points in
two locations for each cell. Expanding cells were root epidermal cells just distal (toward the root tip) to the first root hair bulge. Nonexpanding cells were
atrichoblasts (non-hair cells) in regions of the root with mature root hairs.
(I) and (J) Roots expressing DRP1C-GFP were incubated in 50 mM tyrA23 for the time indicated and imaged in the presence of the inhibitor. The same
root was imaged for the 1- and 3-min time points. Numbers indicate time elapsed from addition of inhibitor.
Bars ¼ 1 mm in (A), (B), (E) to (G), and (I) and 10 mm in (J).
Dynamics of Arabidopsis DRP1C and CLC 1369
Figure 5C). When first appearing, the foci had low but detectable
fluorescence (Figures 5B to 5D) that increased steadily. Upon
reaching peak fluorescence intensity, the fluorescence rapidly
decreased to background levels and the foci disappeared from
the cell cortex (Figures 5B to 5D). This behavior was character-
istic of DRP1C-GFP foci as suggested by the average, normal-
ized fluorescence intensity of 25 foci, which were centered at
peak intensity (Figure 5D). In addition, the foci were largely
immobile (Figure 5B), with 55% of the foci exhibiting no lateral
movement in the imaging plane. The remaining foci were immo-
bile for >90% of their lifetime at the cell cortex but were mobile
within the image plane and exhibited additional behaviors, in-
cluding foci splitting (Figure 5E), foci fusion (Figure 5G), and
dimming without disappearing (Figure 5F). In addition, short-
lived (<5 s) foci that did not fluctuate in fluorescence intensity and
long-lived (>45 s) foci that underwent several rounds of fluores-
cence intensity fluctuations were also present (Figures 5E and
5F). Besides having limited mobility in the image plane, the foci
exhibited movement just out of the plane of focus as they
disappeared from the cell cortex. Seventy percent (n ¼ 220) of
foci were observed moving out of focus, presumably deeper into
the cytoplasm, within 1 s of completely disappearing, most likely
due to cytoplasmic streaming.
Endocytic membrane trafficking is more active in expanding
than in nonexpanding cells (Emons and Traas, 1986). To deter-
mine if the distribution or dynamics of DRP1C-GFP foci were also
different, we compared the density and behavior of foci in the
expansion zone of root epidermal cells with those in fully ex-
panded root epidermal cells. Although the focus lifetimes were
quite variable within a single cell, ranging from 2 to 60 s in both
expanding and nonexpanding epidermal root cells, the average
lifetime of the DRP1C-GFP foci did not vary widely between cell
types. The average lifetime was 17.7 6 8.8 s in expanding root
epidermal cells and 24.2 6 12.4 s in nonexpanding root epider-
mal cells. By contrast, the density of DRP1C-GFP foci varied
between cell types, with the highest density being measured in
actively expanding cells (Figure 5H). In addition, more foci
exhibited splitting and fusion in expanding cells (45%, n ¼ 182)
than in nonexpanding cells (28%, n ¼ 182).
Inhibiting CME Disrupts DRP1C-GFP Foci Dynamics
To test the hypothesis that DRP1C plays a role in endocytosis,
drp1C-1/drp1C-1 seedlings transformed with DRP1C-GFP were
treated with 50 mM tyrA23, which was previously shown to inhibit
endocytic traffic in cultured plant cells (Dhonukshe et al., 2007).
After 3 min of treatment with tyrA23, ;15% of the cortical-
associated foci imaged with VAEM began to increase in size and
fluorescence intensity (Figure 5I) and the cytoplasmic pools of
DRP1C-GFP began to amass into immobile structures with high
GFP fluorescence as viewed by time-lapse CLSM (Figure 5J; see
Supplemental Video 4 online). Thirty min after the addition of
tyrA23, the cortical and cytoplasmic-associated DRP1C-GFP
fluorescent signal was observed in immobile cortical-associated
foci and cytoplasmic structures, respectively (Figures 5I to 5J).
After 30 min of tyrA23 treatment, <1% (n ¼ 600) of the cortical
foci entered or disappeared from the cell cortex during 2 min of
imaging. By contrast, the phosphotyrosine analog tyrphostin
A51, which does not inhibit mammalian clathrin/AP2–cargo
interaction (Crump et al., 1998; Banbury et al., 2003), had no
effect on localization or behavior of DRP1C-GFP (see Supple-
mental Figure 4A online). The effect of tyrA23 was rapidly
reversible, with a complete disappearance of immobile cyto-
plasmic DRP1C-GFP structures within 5 min of washout (see
Supplemental Figure 4B online). tyrA23 most likely does not
inhibit the GTPase activity of DRP1C, as tyrA23 had no inhibitory
effect on the GTPase activity of Escherichia coli expressed
DRP1A (Supplemental Figure 4C online), which is 88% identical
to DRP1C in its GTPase domain.
Dynamics of Cortical-Associated CLC
Cortical-associated DRP1C-GFP displayed characteristics that
were reminiscent of the dynamics of dynamin 1 during CME
(Merrifield et al., 2002). Based on this and our finding that tyrA23
inhibits DRP1C-GFP dynamics, CLC fluorescent fusion proteins
were created and expressed in planta for real-time imaging of
clathrin dynamics at the plasma membrane. Fluorescent fusions of
CLC have been successfully used for illuminating clathrin dynam-
ics in yeast (Sun et al., 2007) and mammalian cells (Merrifield et al.,
2002). C-terminal fusions to mGFP5 (Konopka and Bednarek,
2008a), mOrange, or enhanced cyan fluorescent protein (ECFP)
were used. All three CLC fusion proteins exhibited similar local-
izations (Figures 6A to 6F): to intracellular organelles that colocal-
ized with the TGN marker, N-sialyl transferase-YFP (Figures 6D to
6F; Batoko et al., 2000), within the cytoplasm, and at the plasma
membrane of root epidermal cells (Figures 6A to 6C) and root hairs
(Figure 6G). Plasma membrane–associated CLC-GFP exhibited a
similar distribution as DRP1C-GFP in growing root hairs. In time-
lapse images (see Supplemental Video 5 online), CLC-GFP was
localized to the tip flanks in growing root hairs and redistributed to
the tip-most region of the plasma membrane as growth ceased.
CLC-GFP localization at the plasma membrane was examined
using VAEM. Similar to DRP1C-GFP, plasma membrane–
associated CLC-FFPs were organized into dynamic, discreet
foci (Figures 6H to 6J; see Supplemental Video 6 online). CLC-
GFP foci had a similar lifetime distribution (Figure 6J) as DRP1C-
GFP in expanding root epidermal cells, with the average lifetime
of 19.7 6 6.8 s. The density of CLC-GFP foci was also similar in
expanding root epidermal cells (3.48 6 0.55 foci/mm2) to that of
DRP1C-GFP (3.54 6 0.62 foci/mm2).
DRP1C and CLC-FFP Localization in Expanding Root
Epidermal Cells Were Perturbed upon Cytoskeleton
and Sterol Disruption
Actin dynamics and membrane sterols are required for efficient
CME in mammals and yeast (Lamaze et al., 1997; Rodal et al.,
1999; Engqvist-Goldstein and Drubin, 2003; Toshima et al.,
2006). To determine cellular components that are required for
DRP1C-GFP and CLC-FFP dynamics, seedlings were treated
with molecular inhibitors of cytoskeletal dynamics, sterol syn-
thesis, and membrane and protein trafficking and then imaged
with VAEM. Specifically, 1 and 50 mM latrunculin B (latB; F-actin
inhibitor), 10 mM oryzalin (microtubule inhibitor; Kandasamy and
Meagher, 1999), 50 mM 2,3-butanedione monoxime (BDM;
1370 The Plant Cell
myosin inhibitor; Paves and Truve, 2007), 10 mg/mL fenpropi-
morph (sterol synthesis inhibitor; He et al., 2003), 50 mM
wortmannin (Jaillais et al., 2006), and 50 mM brefeldin A (Jaillais
et al., 2006) were assessed and their effects were compared with
mock treatment with 0.1% DMSO (except for fenpropimorph,
which was compared with half-strength MS). Inhibitors were
used at concentrations previously shown to effectively inhibit
their corresponding cellular processes, and where possible,
inhibition was visually confirmed as described below. All inhib-
itors, except wortmannin and brefeldin A, had significant effects
on foci dynamics (Figures 7A to 7C).
Seedling roots were treated with low (1 mM) and high (50 mM)
concentrations of latB for 20 min to inhibit cytoplasmic streaming
independent and dependent processes, respectively. F-actin
depolymerization was verified using the actin probe GFP-ABD2
(Sheahan et al., 2004). At 1mM latB, there was no effect on
DRP1C-GFP foci dynamics (data not shown) and a small, but
significant increase in CLC-GFP average focus lifetime (from
19.7 6 6.8 s in control cells to 31.1 6 23.8 s; Figure 7A, striped).
However, at latB concentrations that inhibited cytoplasmic
streaming as viewed in bright-field images (i.e., 50 mM), >95%
of the cortical-associated immobile DRP1C-GFP and CLC-GFP
foci that were present at the beginning of imaging were present
after 5 min. Besides this immobile population, ;30% of CLC-
GFP foci were in constant motion in and out of the plane of focus,
which was not observed in mock-treated CLC-GFP seedlings.
To determine if the effects of actin inhibition on foci dynamics
were due to cytoplasmic streaming, seedlings were incubated
with BDM, a myosin inhibitor, for 20 min. Both DRP1C-GFP and
CLC-GFP foci became immobile. Foci that were present at the
beginning of imaging rarely (<1%) disappeared from the cell
cortex. CLC-GFP also formed constantly mobile foci, similar to
those observed after treatment with 50 mM latB. In addition,
DRP1C-GFP foci increased in size and fluorescence intensity,
similar to those observed after tyrA23 treatment (Figure 7C).
Microtubules are the major cytoskeletal component of the
cortex of plant cells (Ehrhardt and Shaw, 2006). Thus, microtu-
bule organization may be important for regulating processes at
Figure 6. CLC Localization and Foci Dynamics at the Plasma Membrane.
(A) to (G) Confocal sections of seedling roots ([A] to [F]) or root hair (G) expressing CLC-GFP ([A] and [G]), CLC-mOrange (B), CLC-ECFP ([C] to [F]),
and/or sialyltransferase-YFP ([D] to [F]), with single color channels ([A] to [E]) and a merged image of the images in (D) and (E) in (F). Cell plates are
indicated with yellow arrowheads ([A] and [B]).
(H) Three expanding root epidermal cells expressing CLC-GFP, imaged with VAEM. Cortical Golgi are indicated by yellow arrows, which can be seen
circulating in Supplemental Movie 6 online. Individual foci used for analysis are smaller and more stable than the Golgi.
(I) Time series of boxed area in (H). Numbers indicate elapsed time from start of imaging in seconds.
(J) Fluorescence intensity profile of two foci indicated in (I) with arrow (blue) and arrowhead (red).
Bars ¼ 10 mm in (A) to (G) and 1 mm in (H) and (I).
Dynamics of Arabidopsis DRP1C and CLC 1371
the plasma membrane. To examine the role of the microtubule
cytoskeleton on cortical-associated DRP1C and CLC dynamics,
expanding epidermal root cells expressing DRP1C-GFP or CLC-
GFP were imaged after 20 min incubation with 10 mM oryzalin,
which caused complete depolymerization of microtubules, as
assessed by a microtubule binding domain–GFP reporter
(Granger and Cyr, 2001). Both DRP1C-GFP and CLC-GFP foci
had a wider lifetime distribution than with 0.1% DMSO treatment
(Figures 7A and 7B, white bars), and the average focus lifetime of
DRP1C-GFP and CLC-GFP with oryzalin treatment was 2.6 and
1.6 times, respectively, that of mock-treated roots. In addition,
DRP1C-GFP and CLC-GFP had a greater mobility within the
plane of the cell cortex after treatment with oryzalin. Twice as
many cortical-associated DRP1C-GFP foci were observed to
split and/or merge, and more than twice as many CLC-GFP foci
moved laterally in the cell cortex compared with mock-treated
roots. Interestingly, foci dynamics were greatly inhibited in the
absence of both the actin and microtubule cytoskeletons (Fig-
ures 7A and 7B, gray bars). In seedlings incubated with 10 mM
oryzalin and 1 mM latB for 20 min, DRP1C and CLC foci displayed
an average focus lifetime of 135.2 6 71.3 s and 46.2 6 35.5 s (n¼106), respectively (as opposed to 17.7 6 8.8 s and 19.7 6 6.8 s
in mock-treated cells, respectively). In addition, the mobile
DRP1C-GFP and CLC-GFP foci that were present in mock-
treated seedlings or with either drug alone were not apparent in
seedlings treated with both oryzalin and latB, with <2% of
DRP1C and CLC foci being mobile within the cell cortex.
Plant cell expansion and division are greatly affected by sterols
present in the plasma membrane (He et al., 2003; Schrick et al.,
2004). CME in mammalian cells also requires the presence of
sterols in the plasma membrane (Rodal et al., 1999). Fenpropi-
morph has been used as a sterol synthesis inhibitor to probe the
Figure 7. DRP1C-GFP and CLC-GFP Foci Dynamics Are Inhibited by Cytoskeleton Disruption.
Roots expressing CLC-GFP (A) or DRP1C-GFP ([B] and [C]) were incubated with inhibitors for 20 min ([A] and [B]) or time indicated ([C], white
numbers), transferred to a slide, and imaged in the presence of the inhibitor with VAEM. Roots were incubated with 0.1% DMSO ([A] and [B], black), 10
mM oryzalin ([A] and [B], white), 1 mM latB ([A], striped), 10 mM oryzalin þ 1 mM latB ([A] and [B], gray), or 50 mM BDM (C). The foci dynamics were
analyzed for 20 to 30 foci in each of 6 to 10 cells from four different roots and the lifetime distribution was plotted ([A] and [B]). Seedlings expressing
CLC-GFP (D) or DRP1C-GFP (E) were germinated and grown on either half-strength MS (black bars) or half-strength MS þ 10 mg/mL fenpropimorph
(white bars), then transferred to a slide and imaged by VAEM in half-strength MS media. The foci dynamics were analyzed for 10 to 20 foci in each of 7 to
10 cells from two to seven different roots, and the lifetime distribution was plotted ([D] and [E]). Bars ¼ 1 mm.
1372 The Plant Cell
effects of altered membrane sterol profiles on plant morphology
(He et al., 2003; Schrick et al., 2004). Seedlings germinated and
grown on half-strength MS þ 10 mg/mL fenpropimorph had
altered DRP1C-GFP and CLC-GFP foci dynamics (Figures 7D
and 7E). The average DRP1C-GFP foci lifetime from seedlings
grown on fenpropimorph was 39.3 6 20.9 s (n¼ 200), which was
1.9 times that of seedlings grown on 0.53 MS media (Figure 7D).
Likewise, the average lifetime of CLC-GFP foci when seedlings
were grown on fenpropimorph was 34.0 6 23.2 s (n¼ 90), which
was 1.7 times that of seedlings grown on half-strength MS
(Figure 7E). Together, these inhibitor studies indicated that the
cortical environment was critical for efficient DRP1C-GFP and
CLC-GFP foci dynamics.
DRP1C-GFP and CLC-GFP Foci Colocalize at
the Plasma Membrane
As cortical-associated DRP1C-GFP and CLC-GFP foci dis-
played very similar dynamics, we decided to test whether these
foci colocalize in the cell cortex. Plants expressing both DRP1C-
GFP and CLC-mOrange were imaged using dual fluorescence
color VAEM imaging (Figure 8A; see Supplemental Video 7
online). As demonstrated by single fluorophore imaging, both
DRP1C and CLC fusion proteins formed discreet foci in the cell
cortex (Figure 8A, yellow arrowheads). CLC-mOrange was also
found in larger structures that represent TGNs (Figure 8A, blue
arrowhead, and Figures 6D to 6F). We found that 48.8% (n ¼ 3
cells) of pixels that had green fluorescence above an intensity
threshold that included all fluorescence within the cell bound-
aries also contained mOrange fluorescence above the intensity
threshold. Conversely, only 15.3% (n ¼ 3 cells) of pixels that
contained orange fluorescence above threshold intensity also
contained green fluorescence above the threshold intensity. This
was most likely due to the TGN-associated CLC-mOrange
fluorescence signal. On the other hand, when only foci were
analyzed for the presence of green or orange fluorescence,
95.1% of all CLC-mOrange foci analyzed (n ¼ 1044) overlapped
with DRP1C-GFP foci, while 94.6% of all DRP1C-GFP foci
analyzed (n ¼ 1049) overlapped with CLC-mOrange foci. The
total percentage of overlapping foci was 90.4% (n ¼ 1100). To
eliminate the possibility that the high percentage of colocaliza-
tion was due to random overlap of the highly dense foci in the cell
cortex of elongating epidermal cells, the red channel image from
eight different cells was rotated 1808 with respect to the green
channel, an analysis technique that has been used previously to
show nonrandom colocalization (Delcroix et al., 2003; Dedek
et al., 2006). The average peak distance for the original image
(3.29 pixels; n ¼ 256) was significantly different from that of the
rotated images (6.43 pixels; n ¼ 253; P < 0.000001), indicating
that the colocalization was statistically significant.
The dynamics of DRP1C-GFP and CLC-mOrange were
analyzed for 88 foci from expanding epidermal root cells in 10 in-
dividual roots that displayed overlapping DRP1C-GFP and CLC-
mOrange localization. In 85% of these foci, the DRP1C-GFP and
CLC-mOrange fluorescence decreased within 1 s of each other
(Figures 8B and 8C), suggesting that DRP1C-GFP and CLC-
mOrange resided on the same structure in the cell cortex. Of
those that displayed simultaneous disappearance, 65% of foci
(n ¼ 68) had simultaneous rise in fluorescence (Figure 8B),
indicating that the fluorophores were recruited concurrently,
while 28% of foci were characterized by a peak in CLC-mOrange
fluorescence before DRP1C-GFP (Figure 8C), suggesting a
stepwise recruitment. Furthermore, 9% of all foci had concurrent
recruitment of the DRP1C-GFP and CLC-mOrange but did not
have simultaneous disappearance (Figure 8D). The time differ-
ence between the disappearances of the two foci ranged from
2.5 to 7 s. Finally, 6% of foci did not display coordinated
dynamics of DRP1C-GFP and CLC-mOrange (Figure 8E).
DISCUSSION
Members of the DRP1 family are required for cell plate and
plasma membrane dynamics during cytokinesis and cell expan-
sion, respectively (Otegui et al., 2001; Kang et al., 2003a, 2003b).
However, the specific processes in which DRP1 isoforms func-
tion and their interaction with the molecular machinery required
for membrane trafficking are largely uncharacterized (Backues
et al., 2007). In this study, we used live-cell imaging of a
functional DRP1C-GFP to examine its localization and dynamics
at the plasma membrane of various plant cell types. DRP1C-GFP
was found to be associated with dynamic foci containing CLC.
The coordinated dynamics of DRP1C and CLC in the cell cortex
suggest that they may function together in CME in plant cells.
Similarly, recent studies have suggested a role for DRP1A in CME
in Arabidopsis (Collings et al., 2008; Konopka and Bednarek,
2008b). Interestingly, DRP1A, CLC, and DRP2B, a DRP that
contains PH and PR domains, were recently shown to form
cortical foci when heterologously expressed in tobacco Bright
Yellow 2 cells (Fujimoto et al., 2007). However, only short-term
(<6 s), single color imaging was performed, so it is not clear
whether the dynamics of DRP1A and DRP2B are similar.
DRP1C and CLC Colocalize into Foci at Regions of Active
Membrane Trafficking
Recent studies have begun to elucidate the endocytic molecular
machinery in plants. The auxin efflux carrier, PIN1, was shown to
be constitutively endocytosed in a clathrin-dependent manner
(Dhonukshe et al., 2007), while the endocytic pathway for the
hormone receptor BRI1 (Russinova et al., 2004) and plant de-
fense receptor FLS2 (Robatzek et al., 2006) has not been
identified. The central importance of clathrin-dependent traffick-
ing in plant cells was demonstrated by the expression of a
dominant-negative CHC, which disrupted internalization of sev-
eral cargos (Dhonukshe et al., 2007). Here, we demonstrated that
CLC-GFP was associated with the distal plasma membrane in
expanding root hairs (Figure 6G), and at the cell plate in dividing
root cells (Figures 6A to 6C), in agreement with previous mor-
phological studies (Emons and Traas, 1986; Fowke et al., 1999;
Otegui et al., 2001; Segui-Simarro et al., 2004; Segui-Simarro
and Staehelin, 2006).
Components of the endocytic machinery in yeast and mam-
mals have been shown to colocalize as punctate structures at the
plasma membrane (Merrifield et al., 2002, 2005; Kaksonen et al.,
2003, 2005; Elde et al., 2005; Le Clainche et al., 2007) by total
internal reflection fluorescence microscopy. Using VAEM, which
Dynamics of Arabidopsis DRP1C and CLC 1373
Figure 8. DRP1C-GFP and CLC-mOrange Foci Colocalize at the Plasma Membrane.
(A) Expanding root epidermal cells expressing CLC-mOrange and DRP1C-GFP imaged with VAEM. Three foci that had both mOrange and
GFP fluorescence are indicated (yellow arrowheads). A larger internal structure that only had mOrange fluorescence is indicated (blue arrowhead).
Bars ¼ 1 mm.
(B) to (E) Intensity profiles of GFP (green) and mOrange (red) fluorescence in overlapping foci. Corresponding mOrange (top), GFP (middle), and merged
(bottom) images for which the intensities were measured are below each time point indicated in the graph. White circles in the first frames indicate
measured regions for fluorescence intensity.
1374 The Plant Cell
gives a comparable signal to noise ratio in plant cells as to-
tal internal reflection fluorescence microscopy (Konopka and
Bednarek, 2008a), we similarly observed that DRP1C-GFP and
CLC-FFPs organized into discreet dynamic foci at the cell cortex.
Both DRP1C-GFP and CLC-FFP dynamics in these foci were
altered by compounds that affect cytoskeletal dynamics (Figures
7A and 7B), membrane sterol content (Figures 7C and 7D), and
tyrA23, further supporting the model that DRP1C and CLC are
part of the same machinery. DRP1C-GFP also formed foci at the
root hair tip flanks, where clathrin-coated structures are abundant
(Emons and Traas, 1986). Although the density of DRP1C-GFP
foci at the root hair tip prevented kinetic imaging of individual foci,
FRAP studies suggested that DRP1C-GFP was recruited to the
flanks of growing root hairs in the same manner as in expanding
root epidermal cells.
Arabidopsis DRP1C and CLC Display Distinct Dynamics
from Dynamin 1 and Clathrin in Mammals
DRP1C-GFP and CLC-GFP dynamics were suggestive of grad-
ual accumulation of DRP1C and clathrin network formation at
plasma membrane sites (Figures 5B to 5D, 6I, and 6J). Unlike the
recruitment of dynamin 1, which is thought to require an
established clathrin network (Merrifield et al., 2002, 2005; Conner
and Schmid, 2003), DRP1C-GFP and CLC-mOrange were re-
cruited concurrently in more than half of the foci where the FFPs
colocalize (Figure 8B). Only 30% of foci exhibited the step-
wise recruitment (Figure 8C) previously described for dynamin
1 (Merrifield et al., 2002).
The rapid disappearance of CLC-GFP foci most likely corre-
sponds to vesicle release from the plasma membrane (Merrifield
et al., 2002, 2005; Kaksonen et al., 2005). In mammalian cells,
dynamin 1 foci disappear concomitantly with vesicle fission
(Merrifield et al., 2002) as a result of dynamin 1 release from the
vesicle membrane. DRP1C-GFP disappearance could likewise
be due to its release from the vesicle. Alternatively, DRP1C-GFP
disappearance may represent movement of the oligomer away
from the plasma membrane, possibly on the clathrin-coated
structure. In support of the latter model, both CLC-GFP and
DRP1C-GFP foci moved laterally out of the plane of focus upon
disappearance from the cell cortex. Cytoplasmic streaming did
not permit long-term imaging of the internalized structures and
so their subsequent fate is unknown. DRP1C-GFP also displayed
additional behaviors, including foci splitting and fusion, not
reported for dynamin 1 (Merrifield et al., 2002), but which have
been reported for CLC foci in mammalian cultured cells (Yarar
et al., 2005).
Besides differences in the dynamics of CLC in plants and
animals, the regulation of CME in plants also differs, especially
regarding the role of the cytoskeleton. ARP2/3-dependent actin
polymerization is thought to provide the force needed to drive the
clathrin-coated vesicle away from the plasma membrane in both
yeast (Kaksonen et al., 2005; Sun et al., 2006) and mammalian
cells (Merrifield et al., 2005; Yarar et al., 2005). However, directly
coupled actin polymerization, if required, is most likely not ARP2/
3 dependent in plants. Arabidopsis mutants in the ARP2/3
complex (crk, dis1 arp3, wrm arp2, and dis2) or its nucleators
(dis3 scar2, grl, pir, sra1, itb1, and brk1) do not display dramatic
morphological defects (Deeks and Hussey, 2003), which would
be expected if ARP2/3-dependent actin polymerization were
required for endocytosis. Interestingly, high concentrations of
latB (50 mM) were needed to completely disrupt DRP1C-GFP
dynamics in epidermal cells. It is possible that vesicle fission and
movement away from the plasma membrane does not need to be
directly coupled to actin polymerization via an activator of
polymerization like cortactin but instead uses the force gener-
ated by cytoplasmic streaming.
Unlike in mammals and yeast cells, in which there is little
evidence of microtubule involvement in CME, microtubules were
required for efficient DRP1C and CLC dynamics. This was
surprising because neither DRP1C-GFP nor CLC-GFP foci or-
ganized in filamentous-like arrays or moved in a linear fashion,
similar to other microtubule-associated proteins (Paredez et al.,
2006; Konopka and Bednarek, 2008a). However, clathrin-coated
structures clustered around cortical microtubules have been
observed in micrographs (Fowke et al., 1999). It is plausible that
cortical microtubules act as a trellis for stabilization of the
endocytic protein network or as a diffusion barrier similar to the
cortical actin network in mammalian cells (Giner et al., 2007).
The local environment, including protein, membranes, and
physical forces is likely very different in an epidermal root cell
than in mammalian cultured cells; most striking is the presence of
turgor pressure and the force of cytoplasmic streaming. There-
fore, it should not be surprising that the proteins facilitating or
regulating CME in plants are also different. In fact, two important
interactors of mammalian dynamin, amphiphysin, which can
remodel membranes, and cortactin, an activator of actin nucle-
ation, are not evident in plants. This is consistent with our finding
that CME in plants involves members of the DRP1 subfamily,
which do not contain the same identifiable lipid or protein binding
domains as mammalian dynamin. Together, these observations
point to significant differences in CME between plants and
animals. DRP1A also forms foci that colocalize with CLC and
DRP1C, albeit with lower frequency and different dynamics
(Konopka and Bednarek, 2008b). Other recent studies have
shown that DRP2B also forms foci at the plasma membrane,
although its dynamics have not been studied (Fujimoto et al.,
2007). This suggests the possibility that multiple CME pathways
may exist in plants that use different dynamin isoforms, although
further research is needed in this area. It may be that the need for
multiple dynamin isoforms in plant CME is due to different
cortical environments that a cell encounters as it differentiates
or the wide variety of intracellular and extracellular stimuli in-
volved in plant signaling.
The morphological characterization of clathrin-coated struc-
tures suggests a structural role for clathrin in plant endocytosis
(van der Valk and Fowke, 1981; Emons and Traas, 1986; Derksen
et al., 1995; Fowke et al., 1999; Dhonukshe et al., 2007). If DRP1C
functions similarly to dynamin 1 in CME in mammals, questions
about the nature of the regulatory network arise. DPR1C lacks
the PH and PR domains required for the recruitment and function
of dynamin 1 during endocytosis. Also, direct homologs of many
of the dynamin 1–associated proteins (cortactin and amphiphy-
sin) have not been identified in the Arabidopsis genome, although
proteins with similar domains, such as ANTH, BAR, and SH3,
have been identified (Holstein and Oliviusson, 2005; Koizumi
Dynamics of Arabidopsis DRP1C and CLC 1375
et al., 2005; Jaillais et al., 2006). If and how these proteins interact
with DRP1C or clathrin is unknown. Identification and charac-
terization of the putative CME core machinery and endocytic
membrane cargo, together with CLC and DRP1C dynamics, will
help further advance the understanding of CME and its regulation
in plants.
METHODS
Construction and Transformation of Fluorescent-Tagged DRP1C
and CLC
The DPR1C-GFP plant expression vector was constructed as follows: A
SalI/HpaI DRP1C genomic construct from pBK03K (Kang et al., 2003a),
which included 1.8 kb of 59 untranslated (UTR) and promoter elements,
and 39UTR of DRP1C were subcloned into pPZP211 (Hajdukiewicz et al.,
1994) containing mGFP5 and the nopaline synthase (NOS) terminator
(Kang et al., 2003b) using SmaI and SalI sites (pSB29). The native 39UTR
was removed from pSB29 by restriction digestion using KpnI and SacI
sites and a DRP1C coding DNA fragment amplified with primers
59-AGAGAGTCGATATGATT GCTGCACGTAGA-39 and 59-CTAGAGCTC-
CTTCCAAGCCACTGCATCGATGTC-39 was inserted after digestion with
KpnI and SacI (pSB31).
The CLC-mOrange plant expression vector was constructed as fol-
lows: The coding sequence for mOrange (Shaner et al., 2004) was PCR
amplified from pRSET-B mOrange (a gift from R. Tsien) using primers
59-TTGAGCTCATGGTGAGCAAGGGCGAGGAGAATAACATGG-39 and
59-TTGAGCTCTTACTTGTACAGCTCGTCCATGCCGCCGGTG-39, sub-
cloned as a SalI fragment into a pPZP221-B vector (Kang et al., 2001), re-
sulting in pPZP221B-mO-NOS. A genomic fragment of At2g40060 (CLC)
was PCR amplified from BAC T28M21 (ABRC) with primers 59-CTGCAG-
GAGTCGGAGATGATGATTATGATG-39 (CLC for) and 59-GAGCTCAG-
CAGCAGTAACTGCCTCAGTGGGC-39 (CLC rev) and subcloned with PstI
and SacI into pPZP221B-mO-NOS.
CLC-ECFP was constructed as follows: The coding sequence for ECFP
was PCR amplified from pECFP-C1 (Clontech) using primers 59-GAGCT-
CATGGTGAGCAAGGGCGAGGAG-39 and 59-GAGCTCTTAGTACAGCT-
CGTCCATGCCGAGAGTGA-39, subcloned as a SacI fragment into a
modified pPZP221-B vector (Kang et al., 2001) resulting in pPZP221B-
ECFP-NOS. A genomic fragment of At2g40060 (CLC) was PCR amplified
from BAC T28M21 (ABRC) with primers CLC for and CLC rev and
subcloned as a PstI-SacI fragment into pZP221B-ECFP-NOS, resulting in
pPZP211B-CLC-ECFP. The DNA sequence of all constructs was verified.
Arabidopsis thaliana ecotype Wassilewskija wild-type or DRP1C/
drp1C-1 (Kang et al., 2003a) plants were transformed with the constructs
for CLC-FFP or DRP1C-GFP, respectively, using the Agrobacterium
tumefaciens–mediated floral dip method (Clough and Bent, 1998). Trans-
genic plants were selected either on solid medium (0.6% phytagar), half-
strength MS medium (Caisson Labs) containing 40 mg/mL kanamycin
(DRP1C-GFP) or on soil, sprayed once with 20 mg/mL ammonium
glusofinate (CLC-ECFP; Liberty).
Immunoblot Analysis
To determine expression level of the transgenic DRP1C-GFP construct,
total protein extracts were prepared from wild-type, wild-type trans-
formed with DRP1C-GFP, DRP1C/drp1C-1, and drp1C-1/drp1C-1 trans-
formed with DRP1C-GFP seedlings grown vertically on half-strength
MSþ 1% phytagar. Seven-day-old seedlings were homogenized in SDS-
PAGE sample buffer (Laemmli, 1970), incubated at 658C for 15 min, and
insoluble debris was cleared by centrifugation at 16,000g for 10 min at
room temperature. The supernatant was separated on a 12.5% (w/v)
SDS-polyacrylamide gel and analyzed by immunoblotting as described
(Kang et al., 2001) using anti-DRP1C (Kang et al., 2003a) and biotin-
conjugated anti-GFP antibodies (Rockland Immunochemicals). HRP-
conjugated anti-rabbit secondary antibodies (GE Healthcare) and
HRP-conjugated streptavidin (Rockland Immunochemicals) were used
to detect the primary antibodies, and anti-DRP1C and anti-GFP, respec-
tively.
Immunofluorescence and Live-Cell Confocal Microscopy
For indirect immunolocalization of DPR1C in plants expressing ss-b-
ATPase-GFP (Logan and Leaver, 2000), 5-d-old seedlings that were
grown vertically on half-strength MS þ 0.6% phytagar were fixed in 4%
(w/v) formaldehyde (Ted Pella) in PME (55 mM PIPES-KOH, 2.5 mM
MgSO4, and 1 mM EGTA, pH 6.9) for 1 h under vacuum. All subsequent
incubations were conducted at room temperature in a humid chamber
unless otherwise noted. Fixed seedlings were washed in PME, placed on
Probe-On-Plus slides (Fisher Scientific), dried on a slide warmer at 558C,
incubated in permeabilization buffer (0.1% Nonidet P-40 in PME) for 10
min, and treated with 0.1% (w/v) pectolyase and 0.5% (w/v) macro-
enzyme in PME for 20 min. The roots were then washed in 0.1% Triton
X-100 in PME and then in PME. For immunolabeling, the roots were
blocked with 3% BSA (w/v) in PME for 1 h and probed with anti-DRP1C
(Kang et al., 2003a) in PME þ 3% BSA overnight at 48C. Roots were then
washed in PME þ 3% BSA and incubated with Cy3-conjugated anti-
rabbit antibodies (Jackson ImmunoResearch) in PME þ 3% BSA for 1 h.
Afterward, roots were washed with PME þ 3% BSA, covered with
Vectashield (Vector Laboratories), topped with a cover slip, and sealed
with fingernail polish.
To examine the association of DRP1C with mitochondria, 5-d-old
drp1C-1/drp1C-1 seedlings transformed with DRP1C-GFP were incu-
bated with 5 mM MitoTracker Orange CM-H2TMRos (Molecular Probes) in
half-strength MS for 30 min at room temperature and rinsed for 2 min in
half-strength MS before imaging.
All CLSM images, except for those of the FRAP experiments, were
captured using a Nikon TE2000-U inverted confocal laser scanning
microscope fitted with a 360/1.4 numerical aperture (NA) PlanApo VC
objective lens and EZ-C1 acquisition software (Nikon). For colocalization
studies in epidermal and cortical cells, DRP1C-GFP and b-ATPase-
ss-GFP were excited at 488 nm (Melles Griot) while MitoTracker Orange
and Cy3 (anti-DRP1C) were excited at 543 nm (Melles Griot). CLC-FPs
were detected using 488-nm light (GFP), 543-nm light (mOrange), or 408-
nm light (ECFP; Melles Griot). All dual-color imaging was performed using
sequential scans to prevent bleed through fluorescence.
For imaging of trichomes expressing DRP1C-GFP, seedlings with one
to two pairs of true leaves were selected and the roots were removed.
Seedlings were inverted onto a cover slip within water and covered by a
glass slide.
Pollen was allowed to germinate in vitro as described previously (Kang
et al., 2003a) on a glass slide for 0 to 12 h, covered with a cover slip, and
imaged with CLSM as described above, except a 3100/1.4 NA PlanApo
lens was used.
For time-lapse imaging of growing root hairs, seedlings were grown on
half-strength MS þ 0.5% phytagar coated cover slips as described
(Konopka and Bednarek, 2008a) and imaged by CLSM with either the
360 or 3100 objective lens. One hundred and fifty microliters of half-
strength MS þ 1% (w/v) sucrose was pipetted on top of the agar and
covered with parafilm to prevent desiccation during imaging.
FRAP experiments were performed on a Zeiss 510 Meta confocal
microscope (Carl Zeiss) equipped with a 363x/1.4 NA PlanApo Chromat
objective. Circular or rectangular areas were photobleached using 30
iterations of the 488-nm line from a 200-mW argon laser operating at
100% laser power. Fluorescence recovery was monitored at 1-s intervals.
1376 The Plant Cell
VAEM
Cortical-associated DRP1C-GFP and CLC-FFP dynamics were imaged
using variable angle fluorescence microscopy as described (Konopka
and Bednarek, 2008a). Briefly, seedlings were transferred from vertically
growing plates to a glass slide with 150 mL of half-strength MS and
covered with a cover slip. Plants were imaged with a Nikon Eclipse
TE2000-U fitted with the Nikon T-FL-TIRF attachment and a Nikon 3100/
NA 1.45 CFI Plan Apo TIRF objective. For double fluorophore imaging,
GFP and mOrange were excited with a 488- and 543-nm laser, respec-
tively, and the fluorescence emission spectra were separated with a
540LP dichroic mirror and filtered through either a 515/30 (GFP) or 585/65
(mOrange) filter in a Dual View filter system (Photometrics).
Image Analysis
For trichomes and root hairs, z-series were recombined using the average
projection command on Image J 1.36b (National Institutes of Health;
http://rsb.info.nih.gov/ij/). Root hair length was calculated from XYZ
coordinates of the tip and base of the root hair. For growing root hairs,
time series images were captured every 8 to 10 s and compiled using
Image J. Growth rate was determined using MetaMorph’s (Molecular
Devices) track points application. An intensity threshold value was
assigned to each root hair so that the maximum number of pixels in the
plasma membrane was included, and the number of pixels in the
cytoplasm was limited. Threshold area was calculated using the region
measurements application in Metamorph (Molecular Devices). Analysis of
FRAP, DRP1C-GFP, CLC-GFP, and CLC-mOrange foci dynamics was
performed using Image J.
Fluorescence recovery in the FRAP experiments was corrected for
photobleaching as follows. The percentage of inherent photobleaching
(PIP) for each frame during recovery was determined by measuring the
change in fluorescence intensity of a region that was not initially photo-
bleached. This fluorescence intensity in the initial bleached regions was
multiplied by (1 þ PIP) for each frame.
A focus was identified by a local increase in intensity above a desig-
nated threshold assigned to each time-lapse image for >2 s. Foci
dynamics were analyzed for plants that were grown on half-strength
MS solid medium and imaged in half-strength MS liquid medium. Lifetime
was calculated from the first frame the focus appeared to the frame that it
disappeared from the imaging planes. The intensity profiles in Figures 5,
6, and 8 were generated using Image J’s Region of Interest (ROI) Multi
Measure Plugin. Circular ROIs included all pixels of the focus, and a mean
intensity for the ROI was recorded. To determine if colocalization was
random, the green channel of dual-color images was rotated 1808 relative
to the red image using Adobe Photoshop CS2 (Adobe Systems). The
distance from each focus in the green channel to the nearest focus in the
red channel was measured using MetaMorph. All images for figures were
processed in Adobe Photoshop CS2.
Inhibitor Studies
tyrA23, tyrphostin A51, cytochalasin D, wortmannin, brefeldin A, and latB
were purchased from EMD Biosciences; oryzalin was purchased from
Restek; lanthanum chloride and BDM were purchased from Sigma-
Aldrich. Stock solutions of lanthanum chloride and BDM were prepared in
deionized water. All other inhibitors were dissolved initially in 100%
DMSO for a stock solution. Inhibitors were diluted in half-strength MS (for
VAEM imaging of expanding root epidermal cells and confocal imaging of
the root tip) or half-strength MS þ 1% sucrose (for confocal imaging of
root hairs). The final DMSO concentration was 0.1% (v/v) or less in all
working solutions. For VAEM analysis, inhibitor treatment was for 20 min
unless otherwise stated. Foci lifetime analysis for inhibitors was com-
pared with a mock treatment in 0.1% DMSO, except for fenpropimorph.
For treatment of growing root hairs, inhibitors were pipetted on top of
agar and allowed to diffuse into the agar to reach the root hairs. Time
series imaging was initiated prior to the addition of the inhibitor to confirm
the growth status of the root hair. For treatment of root tips with inhibitors,
5- to 7-d-old vertically grown seedlings were transferred from 1% agar
plates to 3 mL of final working concentration in half-strength MS in a
12-well culture plate. After the indicated time, seedlings were transferred
to a glass slide with 150 mL of inhibitor solution, covered with a glass
cover slip, the excess liquid was wicked away, and they were imaged as
described above. For tyrA23 washout experiments, seedlings were
incubated with 50 mM tyrA23 in half-strength MS for 20 min, transferred
to media without the drug for 5 min, and subsequently imaged in half-
strength MS.
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome
Initiative database under accession numbers At2g40060 (CLC) and
At1g14830 (DRP1C).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. DRP1C Localizes to the Plasma Membrane
in Hypocotyls and Does Not Associate with Mitochondria.
Supplemental Figure 2. DRP1C-GFP Retains Its Tip Localization in
Several Root Hair Expansion Mutants.
Supplemental Figure 3. DRP1C-GFP Forms Cortical Foci in Various
Epidermal Cells.
Supplemental Figure 4. tyrA23, but Not tyrA51, Reversibly Inhibits
DRP1C Dynamics in Vivo but Not DRP1 GTPase Activity in Vitro.
Supplemental Video 1. Time-Lapse Images of a Growing Pollen
Tube Expressing DRP1C-GFP.
Supplemental Video 2. Time-Lapse Images of Growing Root Hair
Expressing DRP1C-GFP.
Supplemental Video 3. Time-Lapse Images of Expanding Root
Epidermal Cell Expressing DRP1C-GFP Imaged with VAEM.
Supplemental Video 4. Time-Lapse Images of Seedling Root Tip
Expressing DRP1C-GFP Treated with 50 mM tyrA23.
Supplemental Video 5. Growing Root Hair from Root Expressing
CLC-GFP.
Supplemental Video 6. Time-Lapse Images of Expanding Root
Epidermal Cell Expressing CLC-GFP Imaged with VAEM.
Supplemental Video 7. Time-lapse VAEM Images of Expanding Root
Epidermal Cell Expressing DRP1C-GFP (Middle Panel) and CLC-
mOrange (Left Panel), and Merged (Right Panel).
ACKNOWLEDGMENTS
We thank T. Martin and members of his lab for extensive use of their
epifluorescence-TIRF microscope. We thank R. Tsien for his generous
gift of the mOrange fluorescent protein construct. The University of
Wisconsin-Madison Plant Imaging Facility was partially supported by
funding from the National Science Foundation Grant DBI-0421266. We
thank members of our lab, especially D. Rancour, S. Park, and C.
McMichael, for critical reading of the manuscript and helpful discus-
sions. This research was supported by funding to S.Y.B. from the USDA
National Research Initiative Competitive Grants Program (Project
Dynamics of Arabidopsis DRP1C and CLC 1377
2004-03411). C.A.K. was supported by a Howard Hughes Medical
Institute Predoctoral Fellowship. C.A.K. and S.K.B. were supported by
National Institutes of Health, National Research Service Award T32
GM07215 from the National Institute of General Medical Sciences.
Received March 12, 2008; revised April 20, 2008; accepted May 6, 2008;
published May 23, 2008.
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1380 The Plant Cell
DOI 10.1105/tpc.108.059428; originally published online May 23, 2008; 2008;20;1363-1380Plant Cell
Catherine A. Konopka, Steven K. Backues and Sebastian Y. BednarekMembrane
Dynamin-Related Protein 1C and a Clathrin Light Chain at the PlasmaArabidopsisDynamics of
This information is current as of May 15, 2020
Supplemental Data /content/suppl/2008/05/09/tpc.108.059428.DC1.html
References /content/20/5/1363.full.html#ref-list-1
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