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 sybednar@wisc.edu.The 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 (sybednar@wisc.edu).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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