Current Biology 17, 2047–2053, December 4, 2007 ª2007 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2007.10.

040

Report
Plant Cytokinesis Requires
De Novo Secretory Trafficking
but Not Endocytosis
Ilka Reichardt,1 York-Dieter Stierhof,2 Ulrike Mayer,1 Results and Discussion
Sandra Richter,1 Heinz Schwarz,3 Karin Schumacher,4
and Gerd Jürgens1,* Trafficking of Cytokinesis-Specific Syntaxin
1Entwicklungsgenetik KNOLLE Highlights the Secretory Pathway
Center for Plant Molecular Biology in Dividing Cells
University of Tübingen Results from ultrastructural studies suggest that the
Auf der Morgenstelle 3 partitioning membrane formed in plant cytokinesis—
D-72076 Tübingen the cell plate—is made from Golgi-derived vesicles
Germany that fuse with one another in the plane of cell division
2Mikroskopie [2, 6, 7]. The developing cell plate also accumulates
Center for Plant Molecular Biology plasma-membrane proteins, a process that has been
University of Tübingen attributed to endocytic trafficking [3]. To assess the
Auf der Morgenstelle 5 relative contributions of secretory and endocytic traf-
D-72076 Tübingen ficking to the formation of the cell plate, one must map
Germany out the trafficking routes used during cytokinesis and
3Max-Planck-Institut für Entwicklungsbiologie then specifically interfere with one or the other pathway.
Spemannstr. 35 Visualization of the secretory pathway in dividing cells
D-72076 Tübingen requires a newly synthesized protein marker, such as
Germany the cytokinesis-specific syntaxin KNOLLE which plays
4Pflanzenphysiologie an essential role in cell-plate formation [5], whereas
Center for Plant Molecular Biology the endocytic pathway can be easily visualized with
University of Tübingen the fluorescent dye FM4-64, which is actively internal-
Auf der Morgenstelle 1 ized in a clathrin-dependent manner [8–10]. KNOLLE is
D-72076 Tübingen only present in dividing cells, where it initially accumu-
Germany lates in punctate structures that might correspond to
Golgi stacks [5] or endosomes [3], then translocates to
the plane of division and accumulates in the laterally
expanding cell plate, and finally disappears from the
Summary completed cell plate [5]. However, the exact trafficking
pathway of KNOLLE has not yet been elucidated.
During plant cytokinesis membrane vesicles are effi- To identify the endomembrane compartments
ciently delivered to the cell-division plane, where they through which KNOLLE moves in dividing cells, we
fuse with one another to form a laterally expanding cell performed double labeling with specific subcellular
plate [1, 2]. These membrane vesicles were generally be- markers (Figure 1) and localized KNOLLE by immuno-
lieved to originate from Golgi stacks. Recently, however, gold labeling (Figure 2) in dividing seedling root cells of
it was proposed that endocytosis contributes substan- Arabidopsis during the early and late stages of mitosis.
tially to cell-plate formation [3]. To determine the relative Before cell-plate formation, KNOLLE-positive puncta
contributions of secretory and endocytic traffic to cyto- were distinct from adjacent medial Golgi labeled by
kinesis, we specifically inhibited either or both traffick- GFP-tagged N-acetylglucosaminyltransferase I (NAG-
ing pathways in Arabidopsis. Blocking traffic to the divi- GFP [11, 12]), and partially overlapped with trans-Golgi
sion plane after the two pathways had converged at the labeled by YFP-tagged rat sialyltransferase (N-ST-YFP
trans-Golgi network disrupted cytokinesis and resulted [12–15]). However, KNOLLE was clearly distinct from
in binucleate cells, whereas impairment of endocytosis both NAG and N-ST during cell-plate formation (Figures
alone did not interfere with cytokinesis. By contrast, 1A, 1B, 1D, and 1E; Figures S1A, S2A, and S2B in the
inhibiting ER-Golgi traffic by eliminating the relevant Supplemental Data available online). By contrast, the
BFA-resistant ARF-GEF [4] caused retention of newly vacuolar H+-ATPase (VHA) subunit a1, which localized
synthesized proteins, such as the cytokinesis-specific to the trans-Golgi network (TGN) (Figure S2C [13, 16,
syntaxin KNOLLE in the ER [5], and prevented the forma- 17]), labeled KNOLLE-positive punctate structures be-
tion of the partitioning membrane. Our results suggest fore and during cytokinesis (Figures 1C and 1F; Figures
that during plant cytokinesis, unlike animal cytokinesis, S1B and S1C), a finding supported by quantitative mea-
protein secretion is absolutely essential, whereas endo- surement of colocalization (Figure 1M). Consistent with
cytosis is not. this finding, treatment of the seedlings with the mem-
brane-trafficking inhibitor brefeldin A (BFA) trapped
KNOLLE and VHA-a1 in large BFA compartments [12,
13, 18, 19] surrounded by N-ST-YFP-labeled Golgi
stacks (Figures 1J and 1K; Figure S1D). Furthermore,
EM analysis performed with cryosections of cryofixed
*Correspondence: gerd.juergens@zmbp.uni-tuebingen.de root tips localized immunogold-labeled KNOLLE to the
Current Biology Vol 17 No 23
2048
TGN and the expanding cell plate (Figures 2A and 2B;
Figure S2D). GFP-KNOLLE colocalized with the endo-
cytic tracer FM4-64, which labels the TGN immediately
after internalization [13, 17] and is rapidly targeted to de-
veloping cell plates [3, 13] (Figure 3D). Thus, a major sta-
tion on the KNOLLE-trafficking pathway to the cell plate
is the TGN, where secretory and early endocytic traffick-
ing pathways converge [13, 17].
After cell-plate formation, KNOLLE remained detect-
able in punctate structures that were also positive for
the TGN marker VHA-a1 (Figure 1M) and, unlike during
Figure 1. KNOLLE Localization in Mitotic
Cells
(A–L) Root cells stained for KNOLLE (green),
markers (red), and DAPI (blue).
(A and D) KNOLLE and the Golgi marker NAG-
GFP do not colocalize.
(B and E) KNOLLE and the trans-Golgi marker
N-ST-YFP colocalize in early- (B) but not in
late- (E) mitotic stages.
(C and F) KNOLLE and the TGN marker VHA-
a1-RFP colocalize in early mitosis (C) and
during cytokinesis (F).
(G–I) Increasing partial colocalization of
KNOLLE with MVB marker ARA7-GFP from
metaphase (G) to telophase (H) to the end of
cytokinesis (I).
(J–L) Colocalization of KNOLLE with VHA-a1-
RFP (K), but not with N-ST-YFP (J) or ARA7-
GFP (L) in BFA compartments (50 mM BFA,
1 hr).
(M) Quantitative measurements of colocaliza-
tion between KNOLLE and markers in early-
and late-mitotic cells.
(N) Schematic of subcellular marker locali-
zation. ER, endoplasmic reticulum; MVB,
multivesicular body; PM, plasma membrane;
TGN, trans-Golgi network; and VSR, vacuolar
sorting receptor. The scale bar represents
5 mm.
early telophase, was observed to largely colocalize
with the vacuolar sorting receptor (VSR) BP80. This ob-
servation suggests that KNOLLE takes the VSR traffick-
ing pathway from the TGN to the multivesicular body
(MVB) [20] after its return from the cell plate (Figure 1M;
Figures S1E–S1G). This idea is supported by the obser-
vation that, during late mitosis, BFA treatment trapped
KNOLLE and the TGN marker VHA-a1 in large BFA com-
partments [12, 13, 18, 19] surrounded by N-ST-YFP-
labeled Golgi stacks (Figure 1J and 1K; Figure S1D).
This is important because the MVBs, which correspond
Membrane Trafficking in Cytokinesis
2049

Figure 2. KNOLLE-Trafficking Pathway in Dividing Cells

(A and B) KNOLLE on cell-plate ([A], ‘‘CP’’) and neighboring vesicles, (B) TGN (early telophase; overview: Figure S2D).
(C) KNOLLE label reduced on TGN, increased on internal vesicles of MVB (arrowhead) (early interphase; overview: Figure S2E).
(A–F and H) Immunogold labeling of cryosections from cryofixed root tips.
(D and E) GFP-KNOLLE labeling of vesicles inside MVB ([D], arrowhead) and intra-vacuolar vesicles ([E], arrows) and residual labeling of the
plasma membrane ([E], arrows).
(F–H) GFP-ARA7 on the membrane of MVBs ([F]; [H], arrowhead); (G) MVB. CP, cell plate; CW, cell wall; G, Golgi stack; MVB, multivesicular body;
T, trans-Golgi network (TGN); V, vacuole. The scale bar represents 250 nm.
(G) Resin section of cryofixed root-tip cells.

to prevacuolar compartments (PVCs) [20], are not sensi-
tive to BFA. Rather, MVBs are sensitive to wortmannin
and are labeled by the GTPase ARA7, which is related
to endosomal Rab5 [21–23] (Figure 1L; Figures S1H
and S1I; Figures 2F–2H). KNOLLE and ARA7-GFP sig-
nals increasingly overlapped from cytokinesis on, sug-
gesting that KNOLLE passes through ARA7-GFP-posi-
tive MVBs on its way to the vacuole (Figures 1G–1I).
Indeed, immunogold labeling detected GFP-KNOLLE
in internal vesicles of MVBs in late-mitotic cells and in
vacuoles after cytokinesis (Figures 2C–2E; Figures S1J
and S2E). In summary, KNOLLE is retrieved from the
cell plate, possibly via the TGN, and targeted via MVBs
to the lytic vacuole for degradation (Figure 4F).
Inhibiting Traffic at the Trans-Golgi Network Disrupts
Cytokinesis, but Impairment of Endocytosis
Does Not Affect Cell-Plate Formation
The V-ATPase inhibitor concanamycin A (ConcA) blocks
all trafficking at the TGN but does not interfere with
the formation of endocytic vesicles at the plasma mem-
brane [13, 24, 25]. ConcA-treated root tips showed
binucleate cells, and internalized FM4-64 accumulated
at the TGN (Figures 3A and 3B). Neither FM4-64 nor
Current Biology Vol 17 No 23
2050
Figure 3. Concanamycin A but not Wortmannin Prevents Cell-Plate
Formation
(A–N): Fluorescence imaging of root cells.
(A–C) Nuclear GFP (green) and FM4-64: (A) control; (B) ConcA-
treated (2 hr); (C) wortmannin-treated (2 hr). Note the absence of la-
beled intracellular vesicles in ([C], circle) compared to (A).
(D–F) GFP-KNOLLE (green) and FM4-64: (D) control; (E) ConcA-
treated (2 hr); (F) wortmannin-treated (2 hr). Note the absence of
FM4-64 label in vacuolar membrane (arrowheads) in (F) compared
to (E).
(G–N) Time-course of FM4-64 accumulation in cell plate.
(G–J) H2B-YFP (green) and FM4-64.
(K–N) H2B-CFP (green) and FM4-64; wortmannin-treated (2 hr). Ar-
rowheads: (E and F) vacuolar membrane. Asterisks: (D, F, I, and M)
cell plate, (B) binucleate cell, (E) cell-wall stubs. Circles: (A and C)
FM4-64 internalization. The scale bar represents 5 mm.
GFP-KNOLLE reached the division plane, and cell-plate
formation was severely impaired, which resulted in cell-
wall stubs (Figure 3E). We also found that ConcA-treated
mitotic cells accumulated unfused membrane vesicles
that were similar to unfused vesicles in dividing knolle
mutant cells [5] (Figure 3O). Binucleate early-interphase
cells exhibited a vesiculated, fragmented cell-plate-like
structure (Figure 3P). Therefore, traffic from the TGN to
the plane of cell division is essential for cytokinesis.
Specific inhibition of endocytic trafficking in cytokine-
sis is difficult to achieve. Previously used genetic or
drug interference, such as that caused by a dominant-
negative clathrin variant or the adaptor-complex in-
hibitor tyrphostin A23 [10], affects not only endocytic
trafficking but also other trafficking processes from
the TGN to the cell plate, and it might have additional
deleterious effects [26]. The inhibitor wortmannin im-
pairs endocytosis and also induces swelling of MVBs
(Figure S1I) [3, 9]. However, wortmannin can be used
to explore the role of endocytosis in cytokinesis be-
cause trafficking from the plasma membrane to the
cell plate does not pass through the late-endosomal
MVBs, as described above. Wortmannin treatment im-
paired FM4-64 internalization (Figures 3A and 3C, cir-
cles), which resulted in weaker and delayed accumula-
tion of the endocytic tracer at early cell plates than
in the control. However, cell-plate formation was not
affected (Figures 3K–3N; compare to Figures 3G–3J).
Consequently, cytokinesis was completed and there
were no binucleate cells, which contrasts with ConcA-
induced defects (Figure 3C; compare to Figure 3B). Fur-
thermore, GFP-KNOLLE accumulated in the vacuoles of
neighboring wortmannin-treated cells, indicating that
these cells were newly formed daughter cells. Remark-
ably, FM4-64 labeled only the plasma membranes of
these cells and did not label their vacuolar membranes
(Figure 3F; compare to Figure 3E; arrowhead). These
results suggest that a strong reduction or inhibition of
endocytosis does not prevent cytokinesis, which is in
striking contrast to inhibition of membrane trafficking
from the TGN.
Inhibition of ER-Golgi Trafficking Blocks Cytokinesis
To examine the role of secretory traffic in cytokinesis, we
used the inhibitor BFA to target BFA-sensitive large
ARF-GEFs, such as GNOM, that act in endosomal recy-
cling [19]. In BY-2 cells, BFA treatment inhibits cell-plate
formation [27]. However, the specific BFA target in this
biological context is not known. Therefore, it is not clear
whether secretory and/or endocytic trafficking is af-
fected by BFA treatment. In contrast, there is no com-
parable BFA effect in dividing cells of Arabidopsis [3].
(O–Q) EM analysis of cell-plate formation in ConcA-treated cryofixed
and resin-embedded root-tip cells.
(O) Abnormal Golgi stack morphology and dilated Golgi/TGN-
derived unfused vesicles (arrowheads) in cell-division plane. Inset:
Fusion-incompetent vesicles (arrowheads) in a knolle mutant cell,
shown at the same magnification.
(P and Q) Early-interphase binucleate cell ([Q], overview; [P], higher
magnification of boxed area) with vacuolated, fragmented cell plate
(arrowheads); Inset: control cell plate (arrowheads). ER, endoplas-
mic reticulum; G, Golgi stack; M, mitochondrion; N, nucleus. The
scale bar represents 500 nm.
Membrane Trafficking in Cytokinesis
2051

Figure 4. Inhibition of ER-Golgi Trafficking Strongly Affects Cell-Plate Formation

(A–C) KNOLLE (green) localizes to ER (arrows) in BFA-treated (25 mM, 2 hr) BFA-sensitive GNL1 seedling root cells: (A) metaphase, (B) telophase,
(C) post-cytokinesis. Counterstained with DAPI (blue). The scale bar represents 5 mm.
(D and E) Electron micrographs of BFA-treated gnl1 homozygous seedling root-tip cells after cryofixation and resin embedding: (D) Binucleate
cell with cell-wall stubs (boxed area); n, nucleus. The scale bar represents 5 mm. (E) Higher magnification of cell-wall stub (cws) from ([D], boxed
area). The scale bars represent 5 mm (D) and 500 nm (E).
(F) Vesicle trafficking during cell-plate formation (model). Secretory (green arrow) and endocytic (red arrow) trafficking pathways converge at the
TGN to reach the cell plate (brown arrow). Traffic from the cell plate to the vacuole passes through the TGN (left) or only through the MVB (right).
Black arrow, retrograde Golgi-ER traffic; CP, cell plate; ER, endoplasmic reticulum; MVB, multivesicular body; PM, plasma membrane, TGN,
trans-Golgi network.

Instead, in BFA-treated dividing cells KNOLLE accumu-
lates in a dumbbell-shaped structure that consists of
BFA compartments and the cell plate. This accumula-
tion is similar to that of FM4-64 [3] (Figure S3A). One
explanation for this finding might be that cell-plate
trafficking in Arabidopsis is mediated by one or more
BFA-resistant ARF-GEFs.
The ARF-GEF GNL1 was recently shown to mediate
ER-Golgi traffic and to be resistant to BFA [4, 28]. Endo-
cytosis was quantitatively similar between wild-type and
gnl1 mutant seedlings (Figures S3I–S3K). Therefore, we
examined whether BFA treatment of gnl1 seedlings
lacking BFA-resistant GNL1 compromised trafficking
during cytokinesis (Figure 4). KNOLLE localization was
not altered in untreated mutant seedlings (Figures S3B
and S3C). After BFA treatment, however, KNOLLE accu-
mulated in the ER and colocalized with the ER-relocated
Golgi marker N-ST-YFP [4], which indicated that traffick-
ing of newly synthesized KNOLLE from the ER to the cell
plate via the TGN was inhibited (Figures 4A–4C; Fig-
ure S3D). Consequently, KNOLLE was not readily de-
graded and failed to overlap with the phragmoplast
Current Biology Vol 17 No 23
2052
microtubules at the division plane (Figures S3E–S3H).
Most importantly, the BFA-induced inhibition of ER-
Golgi traffic resulted in binucleate cells that had cell-
wall stubs and resembled knolle mutant cells [29], which
indicates that secretion of newly synthesized proteins is
essential for plant cytokinesis (Figures 4D and 4E).
Essential Membrane-Trafficking Pathway
during Cytokinesis: A Model
Plant cytokinesis is a very rapid process, requiring the
timely delivery of much membrane material to the plane
of cell division. Although endocytosis might be a means
of achieving the rapid formation of the partitioning mem-
brane [3], there are no functional data that reveal the
necessity of endocytic trafficking in cytokinesis. Our
results show that only secretory trafficking of newly syn-
thesized proteins is essential, although endocytosed
plasma-membrane proteins might also be targeted to
the cell plate [3]. Like secretory trafficking in interphase
cells, trafficking to the division plane has been viewed as
a default pathway in dividing cells [1]. In this scenario,
secretory and endocytic trafficking pathways, which
converge at the TGN, are both deflected to the plane
of cell division, and constitutively cycling plasma-
membrane proteins accumulate at the cell plate rather
than being recycled to the plasma membrane (Figure 4F).
This targeting is essential for KNOLLE and other pro-
teins that are necessary for cytokinesis and are tightly
regulated during the cell cycle, and it might also enable
them to be recycled to the growing margin of the ex-
panding cell plate via the TGN and ensure that cytokine-
sis is completed on time.
Supplemental Data
Additional Results and Experimental Procedures as well as three fig-
ures are available at http://www.current-biology.com/cgi/content/
full/17/23/2047/DC1/.
Acknowledgments
We thank Frédéric Berger, Natasha V. Raikhel, David G. Robinson,
Markus Grebe, Axel Völker, Hanno Wolters, and Alexandra Schlereth
for kindly providing antisera or transgenic lines; Ulrike Hiller and
Dagmar Ripper for technical assistance; and Nadine Anders and
Niko Geldner for critical reading of the manuscript and helpful sug-
gestions. This work was supported by the Deutsche Forschungsge-
meinschaft through Arabidopsis Functional Genomics Network to
G.J.
Received: August 2, 2007
Revised: October 15, 2007
Accepted: October 16, 2007
Published online: November 8, 2007
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