* Lehrstuhl für Entwicklungsgenetik, Universität Tübingen, D-72076 Tübingen, Federal Republic of Germany; Max-Planck-Institut für Entwicklungsbiologie, D-72076 Tübingen, Federal Republic of Germany; and § Plant Molecular Biology
and Biotechnology Research Center, Gyeongsang National University, Chinju, 660-701, Korea
In higher plant cytokinesis, plasma membrane and cell wall originate by vesicle fusion in the plane of cell division. The Arabidopsis KNOLLE gene, which is required for cytokinesis, encodes a protein related to vesicle-docking syntaxins. We have raised specific rabbit antiserum against purified recombinant KNOLLE protein to show biochemically and by immunoelectron microscopy that KNOLLE protein is membrane associated. Using immunofluorescence microscopy, KNOLLE protein was found to be specifically expressed during mitosis and, unlike the plasma membrane H+-ATPase, to localize to the plane of division during cytokinesis. Arabidopsis dynamin-like protein ADL1 accumulates at the plane of cell plate formation in knolle mutant cells as in wild-type cells, suggesting that cytokinetic vesicle traffic is not affected. Furthermore, electron microscopic analysis indicates that vesicle fusion is impaired. KNOLLE protein was detected in mitotically dividing cells of various parts of the developing plant, including seedling root, inflorescence meristem, floral meristems and ovules, and the cellularizing endosperm, but not during cytokinesis after the male second meiotic division. Thus, KNOLLE is the first syntaxin-like protein that appears to be involved specifically in cytokinetic vesicle fusion.
AFTER nuclear division, cytokinesis partitions the cytoplasm of the dividing cell. Whereas mitotic segregation of chromosomes is highly conserved among
eukaryotes, cytokinesis can be carried out in fundamentally different ways. In animal cells, the existing plasma membrane is pulled in by means of a contractile acto-myosin- based ring, and the daughter cells are eventually pinched
off (Fishkind and Wang, 1995 Little is known about the mechanism of cell plate formation, although the sequence of events at the plane of division has been well described at the electron microscope
level (Samuels et al., 1995 A small number of mutants that show cytokinesis defects
have been described in pea (cyd: Liu et al., 1995 Plant Material and Growth Conditions
The Arabidopsis thaliana ecotype Landsberg erecta used as wild-type was
the nonmutagenized parental strain in which the knolle alleles X37-2,
3-496, and AP6-16 were induced (Lukowitz et al., 1996 Generation of KNOLLE-specific Antiserum
A KNOLLE (KN)1 cDNA fragment lacking the sequence for the hydrophobic COOH terminus of the protein (Lukowitz et al., 1996 Preparation of Protein Extracts and Western
Blot Analysis
Plant material as specified in Results was ground in liquid nitrogen. After
boiling in sample buffer (Laemmli, 1970 Cell Fractionation
Inflorescences were cut into small pieces and ground with mortar and pestle in homogenization buffer (100 mM Hepes-Tris, pH 7.8, 300 mM sucrose, 5 mM EDTA, 2.5 mM DTT, 1 mM phenylmethyl sulfonyl fluoride,
0.5% [wt/vol] BSA). The homogenate was passed through one layer of
MiraclothTM (Calbiochem, La Jolla, CA) and centrifuged at 10,000 g at
4°C for 10 min. For density gradient centrifugation, the supernatant (S10)
was stirred briefly with a glass homogenizer (Potter-Elvejhem) and centrifuged through a linear 15- 45% sucrose gradient at 107,000 g at 4°C for 18 h. Fractions of 500 µl were checked by refractometry before Western blot
analysis of aliquots. To test for membrane association of KN protein, aliquots
of the supernatant (S10) were centrifuged at 100,000 g for 90 min to give
soluble fractions (S100) and pellets (P100). The pellets were resuspended
in the original volumes of homogenization buffer without additives (control), with 1% Triton X-100, with 1 M NaCl, or with 0.1 M Na2CO3 (pH
10.9), incubated on ice for 40 min, and again centrifuged at 100,000 g for
90 min, giving wash fractions S100 Immunofluorescence Localization
The procedure for whole-mount preparations was modified from Webb and
Gunning (1990) Immunogold Labeling and Electron Microscopy
For immunolabeling of membrane fractions, aliquots from a sucrose density gradient were adsorbed to glow-discharged pioloform/carbon-coated grids, washed with blocking buffer (PBS containing 0.5% BSA and 0.2%
gelatine), incubated with KNOLLE antiserum (diluted 1:200) for 15 min,
washed five times with blocking buffer, and labeled with protein A-15-nm
gold conjugates. After extensive washes with PBS and H2O, grids were
negatively stained with 1% uranyl acetate and viewed in a transmission
electron microscope (model CM10; Philips Electronic Instruments Co.,
Mahwah, NJ) at 60 kV. For the ultrastructural characterization, embryos
were fixed with 4% paraformaldehyde in MTSB at room temperature for
30 min and, after addition of 1% glutaraldehyde at 4°C for 17 h, postfixed with 1% OsO4 in PBS on ice for 40 min and, after rinsing with aqua bidest,
treated with 1% aqueous uranyl acetate at 4°C for 40 min. Samples were
dehydrated through a graded series of ethanol, infiltrated with ethanol/
resin mixtures, and finally embedded in Spurr's epoxy resin. Ultrathin 60-nm
serial sections were stained with uranyl acetate and lead citrate and
viewed as above.
KNOLLE Protein Is Associated with Membranes in
Dividing Cells
The nucleotide sequence of the KNOLLE cDNA predicted that the KN protein, like other syntaxins, consists of
an NH2-terminal variable region, a conserved region of 68 amino acids, and a COOH-terminal putative hydrophobic
membrane anchor (Lukowitz et al., 1996
KN mRNA was shown previously to accumulate preferentially in organs with actively dividing tissues, such as developing flowers and siliques (Lukowitz et al., 1996 The subcellular distribution of KN protein was determined by sucrose density gradient centrifugation of cell extracts (Fig. 1 c). KN protein was found in all gradient fractions, although the highest levels were observed in denser
fractions, some of which contained the PM ATPase (Villalba
et al., 1991 The predicted amino acid sequence of the KNOLLE gene
product includes a COOH-terminal hydrophobic stretch
that could serve as a membrane anchor (Lukowitz et al.,
1996 Intracellular Localization of KN Protein
To determine the intracellular localization of KN protein
throughout the cell cycle, whole-mount preparations of
single cells from squashed embryos were doubly labeled
with DAPI and with KN-specific antiserum (Fig. 2). No
KN signal was detected in interphase cells (Fig. 2 a). KN-positive material accumulated in large patches during early
M phase (Fig. 2 a) and predominantly in the plane of division in telophase cells (Fig. 2 b). Cells exiting from mitosis
showed more diffuse KN signals near the plane of division (Fig. 2 c). To determine the sequence of events more precisely, dividing embryonic cells were double-stained with
KN-specific antiserum and with anti-
To address whether the membrane of the cell plate is labeled by a marker for the plasma membrane, we examined
the distribution of PM ATPase in cytokinetic cells. The PM
ATPase did not accumulate in the forming cell plate as demonstrated by double labeling with KN (Fig. 4). Although
the surface of the cytokinetic cell was decorated with PM
ATPase, the developing cell plate only displayed the KN
signal. Even when the KN signal was confined to the periphery of the nearly complete cell plate, PM ATPase was
excluded (Fig. 4, c and f). Since the entire surface of surrounding interphase cells was labeled with PM ATPase, we
presume that PM ATPase entered the newly formed
plasma membrane only after the KN protein had disappeared from the cell plate. Thus, the developing cell plate appears to constitute a membrane compartment that is
topologically distinct from the plasma membrane and is
formed by a specialized set of cytokinetic transport vesicles (see Discussion).
Analysis of knolle Mutant Cells
We examined the expression of KN protein in whole-mount
preparations of single cells from embryos mutant for each
of the three knolle alleles described so far: 3-496 is a stop-codon mutation that truncates the KN protein at amino
acid 70, AP6-16 is a frame-shift mutation, resulting in a
KN protein without the conserved region and the COOH-terminal hydrophobic anchor, and X37-2 is a large deletion that removes about half the coding region and 3
To address whether the lack of KN protein interfered
with the transport of membrane vesicles to the plane of cell
division, we analyzed the intracellular distribution of the
membrane-associated Arabidopsis dynamin-like (ADL1)
protein in cytokinetic cells, using an antiserum raised
against recombinant ADL1 (Park et al., 1997 To visualize the cytokinesis defect in more detail, we
prepared serial sections of knX37-2 mutant embryos for EM
analysis. Fig. 6 a shows a diagram of an enlarged interphase cell displaying the characteristic features of incomplete cytokinesis, such as several nuclei and fragments of
internal cell walls. In addition, bands of vesicles extend some of the wall fragments. At higher magnification, a
nearly 1-µm-wide band of vesicles appears to extend from
the cell surface to the interior, interrupted by islands of vesicle aggregates that enclose stretches of material that resembles the middle lamella of the cell wall (Fig. 6 b). The
vesicles are fairly uniform in size, measuring 60-80 nm in diameter, and appear to be coated by electron-dense material.
The spaces between adjacent vesicles are free of ribosomes and paved with homogeneous material. We followed the extension of the vesicle band in adjacent sections and determined that the vesicles were actually arranged in
a disc-shaped structure that eventually joined a mature cell
wall (data not shown). These data imply that the lack of KN
protein does not block vesicle transport but impairs vesicle
fusion during the formation of the cell plate.
KN Protein Is Expressed in Dividing Somatic Cells and
in the Cellularizing Endosperm but Not during
Cytokinesis of Male Meiotic Cells
Most cell divisions during plant development involve the
phragmoplast-assisted formation of a cell plate by vesicle
fusion in the plane of division. However, there are exceptions to this type of cytokinesis. For example, the endosperm, which surrounds the developing embryo, passes
through a syncytial stage of nuclear multiplication ("free
nuclear endosperm") before cellularization by the simultaneous formation of cell walls between adjacent nuclei (Mansfield and Briarty, 1990a, b; for review see Olsen et al., 1995 To determine whether the KN protein is generally involved in cytokinesis or only in divisions that deploy the
phragmoplast-assisted formation of a cell plate, we studied
the accumulation of KN protein in various tissues undergoing cell division (Fig. 7). In addition to embryonic cells
(see Fig. 3), KN-positive material was found in dividing cells
in the seedling root (Fig. 7, a-d), in the inflorescence apex
(Fig. 7, e-h), and in developing flowers (Fig. 7 i-l). Thus, these somatic tissues appear to use KN protein in cytokinesis. Of the exceptional cases we analyzed, the endosperm
expressed KN protein. During cellularization, hexagonal
arrays of KN-positive material separated adjacent nuclei
(Fig. 7 m), and later on, the asynchronously dividing endosperm cells accumulated KN-positive material in the plane
of cell division in much the same way as dividing embryo
cells (Fig. 7 n). The only truly exceptional case was the cytokinesis of the male meiotic cell. The sporogenous tissue
of anthers, which generates the male meiotic cells, expressed KN protein essentially like somatic cells (Fig. 7 o).
As expected, surrounding tapetum cells that were undergoing endomitosis, but not cytokinesis, did not give a KN
signal (Fig. 7 p). Also, no KN-positive material accumulated during the cytokinesis of the male meiotic cell (Fig. 7,
q-s) although phragmoplast-like microtubule arrays formed
between the four nuclei of the tetrad after the second nuclear division (Fig. 7, t-v). Thus, the cytokinesis of the
male meiotic cell appears to occur via a KN-independent
mechanism.
Current models of higher plant cytokinesis suggest that
Golgi-derived vesicles are transported along the phragmoplast to the plane of cell division, where they fuse with one
another to form an immature cross-wall, the cell plate,
covered with a new plasma membrane (Samuels et al.,
1995 Mechanistic studies of plant cytokinesis require knowledge
of the molecules involved. One way to identify relevant
molecules is to isolate mutants with specific phenotypes,
such as incomplete cell walls, and to clone the affected
genes. The KN gene thus identified encodes a syntaxin-
related protein and is expressed in a cell cycle-dependent
manner (Lukowitz et al., 1996 Role of KNOLLE in Membrane Fusion during Cell
Plate Formation
The cytokinesis defect of kn mutant cells suggested that
KN protein may be involved in vesicle trafficking to, or
vesicle fusion at, the plane of cell division (Lukowitz et al.,
1996 In kn mutant cells, daughter nuclei are not or are incompletely separated by a cell wall (Lukowitz et al., 1996 Is the Cell Plate a Distinct Transient
Membrane Compartment?
The expression of KN protein is tightly regulated in time
and space. Not only is the KN mRNA made during a brief
period of the cell cycle and turned over rapidly (Lukowitz
et al., 1996 What is the biological significance of the tight regulation
of KN protein expression? The Golgi apparatus is the site
of departure for vesicle traffic to a variety of membrane
compartments. During cytokinesis, much of the post-Golgi
vesicle traffic has to be routed to the developing cell plate.
Our results suggest that this is achieved by creating a specialized set of cytokinetic vesicles marked by KN protein:
KN-positive vesicles are formed only before the onset of
cytokinesis and are transported only along the phragmoplast to the plane of cell division. Vesicles normally destined to the plasma membrane are excluded from this transport route, as evidenced by the sharp boundary between the KN-positive cell plate and the PM ATPase-positive cell surface at the end of cytokinesis. Previous studies
established that the newly formed cell plate differs in composition from the interphase cell wall, suggesting that cytokinetic vesicles also contain a specific cargo (Samuels et al.,
1995 Different Modes of Plant Cell Division
The prevalent center-out mode of higher plant cell division involves a phragmoplast for directed vesicle transport
to the plane of division, where the vesicles fuse to form a
cell plate that expands laterally to reach the parental cell
surface (Wick, 1991 In reality, the situation may be more complex because
the fusion of the cell plate with the parental cell wall,
which completes center-out cytokinesis, requires an active
contribution of the cortical site that was marked earlier by
the preprophase band (Mineyuki and Gunning, 1990). During this process, the
surface of the plasma membrane is extended by the fusion
of large membranous vesicles at the flanks of the cleavage
furrow (Byers and Armstrong, 1986
). In higher plants, cytokinesis is initiated at the center of the division plane, and
a new plasma membrane is formed by vesicle fusion (Staehelin and Hepler, 1996
). Plant cell division is assisted by specific arrays of cytoskeletal elements. Between the separating anaphase chromosomes, two groups of microtubules interdigitate with their plus ends at the plane of division,
forming a cylindrical structure. This so-called phragmoplast also contains two opposing sets of actin microfilaments
that, however, do not overlap or directly abut (Zhang et al.,
1993
). The phragmoplast mediates the accumulation of
Golgi-derived vesicles that fuse to form the cell plate, consisting of an immature cross-wall bounded by an incipient
plasma membrane. As the phragmoplast moves out to the
periphery, the disc-shaped cell plate expands laterally, eventually reaching the parental cell surface (Staehelin and Hepler, 1996
). Cytokinesis is completed by the fusion of the
cell plate with the parental wall at a site that was transiently marked by a cortical preprophase band of microtubules at the onset of mitosis (Wick, 1991
).
). Biochemical assays have been
used to identify relevant molecules, such as polypeptides
with microtubule-translocating activity from phragmoplasts of tobacco cells, that may be involved in transporting membrane vesicles to the site of cell plate formation (Asada and Shibaoka, 1994
), and one candidate motor molecule, the kinesin-related TKRP125 protein, has been isolated (Asada et al., 1997
). Another approach involved the
study of plant homologues of proteins with defined roles
in other systems. For example, an Arabidopsis kinesin-like
protein, KatAp, has been localized to the phragmoplast in
dividing cells (Liu et al., 1996
). Two other plant homologues, AtCdc48 (Feiler et al., 1995
) and the dynamin-like
GTPase phragmoplastin (Gu and Verma, 1996
, 1997
), show cell cycle-dependent intracellular redistributions, accumulating at the plane of cell division during cytokinesis.
AtCdc48 is related to the yeast N-ethylmaleimide-sensitive ATPase Cdc48p involved in homotypic ER-ER fusion events (Latterich et al., 1995
). Phragmoplastin and its
Arabidopsis homologue, ADL1 (Park et al., 1997
), bear resemblance to animal dynamins that are required for the
pinching off of vesicles from membranes (Baba et al., 1995
).
However, the functions that these plant homologues have
during cytokinesis are not known.
) and Arabidopsis (knolle: Lukowitz et al., 1996
; keule: Assaad et al.,
1996
). The KNOLLE gene was isolated by map-based cloning, and its mRNA appears to accumulate in a cell cycle-
dependent manner (Lukowitz et al., 1996
). The predicted
34-kD KNOLLE protein is related to a family of membrane-anchored proteins, the syntaxins, which are implicated in
directing intracellular vesicle trafficking, with specific syntaxins mediating fusion events in different pathways
(Bennett and Scheller, 1993
). Although syntaxins form a
target-membrane component of the vesicle-docking complex, t-SNARE, in heterotypic membrane fusion (Ferro-Novick and Jahn, 1994
; Pfeffer, 1996
), a new syntaxin has
been recently reported to mediate homotypic vacuolar fusion in yeast (Nichols et al., 1997
). Plant cytokinesis resembles homotypic fusion because there is no target membrane, and vesicle fusion itself forms the de novo plasma
membranes separating the daughter cells. To determine its
role in cytokinesis, we raised specific antiserum against
KNOLLE protein and analyzed its membrane association,
tissue distribution, and intracellular localization. We also
examined, by immunofluorescence and electron microscopy, the cytokinetic defect of knolle mutant cells. Our results suggest that KNOLLE, which is specifically expressed
in mitotically dividing cells and in the cellularizing endosperm, mediates vesicle fusion in the plane of cell division.
Materials and Methods
). Plants were grown
as previously described (Mayer et al., 1991
).
) was expressed in Escherichia coli from the His-tag expression vector pQE 60 (Qiagen, Chatsworth, CA). Recombinant protein was purified by Ni2+ affinity chromatography (Qiagen) according to the manufacturer's instructions. After preparative SDS-PAGE and electroelution (Schleicher & Schuell, Inc., Keene, NH), 300 µg of purified protein in complete Freund's
adjuvant emulsion was injected into a rabbit. After boosting with 500 µg
recombinant protein in incomplete Freund's adjuvant on day 28, serum
was collected once or twice a week, starting on day 8 after boosting. The
antibody was purified by affinity chromatography with purified KN protein coupled to cyanbromide-activated Sepharose and elution of bound
antibodies with 2.5 M and 4 M MgCl2 (Harlow and Lane, 1988
). Purified
antibody gave the same signals as the whole antiserum in immunolocalization experiments.
) at 95°C for 10 min, the homogenate was centrifuged at 13,000 g for 15 min to remove insoluble debris. For
Western blot analysis, proteins were separated by 12% SDS-PAGE
(Laemmli, 1970
) and electroblotted onto a polyvinylidene difluoride membrane (Pharmacia Biotech, Piscataway, NJ). The filter was blocked with
5% (wt/vol) nonfat dry milk in Tris-buffered saline with 0.2% Tween 20 (TBST: 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.2% Tween 20), probed
with primary antibody, washed with TBST plus 400 mM NaCl and 0.8%
Tween 20, incubated with horseradish peroxidase-conjugated secondary antibody diluted 1:2,000 in blocking buffer, and washed again before detection with the BM chemiluminescence system (Boehringer Mannheim
Corp., Indianapolis, IN). Primary antibodies were used at the following
concentrations: anti-KNOLLE, 1:1,000 to 1:8,000; anti-ADL1, 1:1,000; anti-
plasma membrane H+-ATPase (PM ATPase), 1:1,000.
and pellets. This procedure was repeated to give wash fractions S100
and washed pellets P100
. All fractions were subjected to Western blot analysis.
and Goodbody and Lloyd (1994)
. Ovules or anthers were
fixed in 4% paraformaldehyde in microtubule-stabilizing buffer (MTSB:
50 mM Pipes, 5 mM EGTA, 5 mM MgSO4, pH 6.9-7.0) at room temperature under vacuum for 1 h. Embryos and endosperm were dissected from
washed ovules and squashed onto gelatine-coated microscopic slides, and
anthers were squashed directly to release sporogenous cells, meiocytes,
and developing pollen. Coverslips were removed after dipping the slides
into liquid nitrogen. After drying for at least 15 min, specimens were rehydrated in MTSB for 10 min. Cell walls were partially digested with 2%
(wt/vol) driselase (Sigma Chemical Co., St. Louis, MO) for 30 min. The plasma membrane was permeabilized with 0.5 or 3% Nonident P40 in
10% DMSO-MTSB for ~1 h. Unspecific interactions were blocked with
1% (wt/vol) BSA in MTSB overnight at 4°C, and antibodies were diluted
in 3% (wt/vol) BSA in MTSB and incubated at 37°C for ~3 h. In double-labeling experiments, the antibodies were concomitantly incubated after
performing appropriate controls. For cryosections, material was fixed in
4% paraformaldehyde in MTSB overnight at 4°C, washed in buffer, embedded in 30% (wt/vol) sucrose in MTSB (inflorescences and flowers) or
only in MTSB (root tips), and frozen in tissue-tek on dry ice or liquid nitrogen. Sections 5-7 µm thick were cut at
15°C to
20°C, using a cryomicrotome (model 2700-Frigocut; Reichert-Jung; Leica, Wetzlar, FRG),
washed, and incubated in 5% (wt/vol) nonfat dry milk in PBT (PBS with 0.2% Triton X-100) to block nonspecific antibody binding. Primary antibodies were used at the following concentrations: anti-KNOLLE, 1:2,000;
anti-ADL1 (Park et al., 1997
), 1:500; anti-
-tubulin YOL 1/34 (Harlan
Sera-Lab), 1:25 or 1:50; anti-
-tubulin N356 (Amersham Corp., Arlington
Heights, IL), 1:600; anti-PM ATPase (a kind gift from W. Michalke, Biologie III, University of Freiburg, FRG; Villalba et al., 1991
), 1:500 or 1:1,000.
Fluorochrome-conjugated secondary antibodies (Dianova, Hamburg,
FRG) were used at the following concentrations: anti rabbit-FITC, 1:300;
anti-rabbit-Cy3TM, 1:500; anti-rat-TRITC, 1:300; anti-mouse-TRITC, 1:200;
anti-mouse-Cy3TM, 1:500. DNA was stained with 1 µg/ml 4
-6-diamidino-2-phenylindole (DAPI; Sigma Chemical Co.). Mounting was done in
Citifluor (Amersham). Photographs were taken on Fujichrome Provia 400 color films (Tokyo, Japan), using appropriate filters for FITC, TRITC,
Cy3TM, and DAPI fitted to a microscope (model Axiophot; Carl Zeiss, Inc.,
Thornwood, NY). Confocal laser-scanning was performed with an inverse
fluorescence microscope (Carl Zeiss, Inc.), using the Comos program (Bio-Rad Labs, Hercules, CA). Standard scanning conditions: 100× objective, twofold zoomed, 10× Kalman filter. Images were processed with PhotoshopTM 3.0 (Adobe, Mountain View, CA) and Aldus Freehand 7.0 (Macromedia, San Francisco, CA) software.
Results
). Recombinant
KN protein without the hydrophobic COOH terminus was purified from E. coli for generating polyclonal antiserum
(for details see Materials and Methods). The specificity of
the antiserum was determined by Western blot analysis
of recombinant protein (Fig. 1 a) and by immunostaining
of knolle mutant embryos (described below). In Western
blots of plant cell extracts, the KN-specific antiserum detected a protein of ~40 kD and, if used at higher concentration, an additional ~55-kD protein (Fig. 1 a).
Fig. 1.
KNOLLE protein from plant cell extracts. (a-d) Western blots. (a) Specificity of anti-KN antiserum. Equal amounts of
protein from inflorescences were loaded in lanes 1-7. Lanes: 1,
primary antibody only; 2, secondary antibody only; 3, preimmune
serum diluted 1:500; 4-7, antiserum diluted 1:8,000, 1:4,000, 1:2,000, 1:1,000, respectively; 8, 2.5 ng recombinant protein, antiserum 1:1,000. (b) Organ distribution of KN protein. Equal amounts of protein were loaded. (c) Subcellular distribution of KN protein as
determined by 15 to 45% linear sucrose density gradient centrifugation. Gradient fractions (1, top; 24, bottom) were probed with
anti-KN, -ADL1, and -plasma membrane H+-ATPase antibodies;
S, supernatant of 10,000 g centrifugation; C, total cell extract. (d)
Membrane association of KN protein (differential centrifugation, anti-KN, and anti-ADL1 antiserum at 1:1,000). Cell extract
was centrifuged at 10,000 g, and aliquots of supernatant (S10,
lanes 1) were centrifuged at 100,000 g to give supernatants S100
(lanes 2). Pellets (P100) were resuspended in homogenization
buffer (control), supplemented with 1% Triton X-100, 1 M NaCl,
or Na2C03 (pH 10.9) and washed twice by centrifugation at
100,000 g (supernatants S100, lanes 3; S100
, lanes 4) to give the
washed pellets (P100
, lanes 5). For details, see Materials and
Methods. (e and f) Electron micrographs of KN protein immunogold localization on membranous material from (e) gradient
fraction 18 (see Fig. 1 c) and (f) S100 supernatant (Fig. 1 d, control, lane 2). Note abundance of vesicles in f. Bar, 0.1 µm.
[View Larger Version of this Image (50K GIF file)]
). Essentially the same organ distribution was observed for KN
protein (Fig. 1 b). Developing flowers and immature siliques,
which contain developing embryos, showed the highest
levels of KN protein. Roots and callus cells contained less
KN protein while almost none was detectable in young
seedlings and leaves. This distribution suggested that KN protein, like its mRNA, is expressed in dividing cells.
). The KN protein showed essentially the same
distribution as the Arabidopsis dynamin-like (ADL1) protein, which was recently shown to be localized to membranes (Park et al., 1997
). Samples of several fractions were processed for electron microscopy and stained with
KN antiserum and protein A-gold. KN-positive material was
observed on the surface of membranous material (Fig. 1 e).
). To determine whether KN is indeed an integral membrane protein, we investigated its association with membranes under various experimental conditions, such as
alkaline pH, high salt concentration, or presence of detergent (Fig. 1 d). Cell extract was centrifuged at 10,000 g. Aliquots of the supernatant fraction (S10, Fig. 1 d, lanes 1)
were centrifuged at 100,000 g to give supernatant (S100,
lanes 2) and pellet fractions. The pellets were resuspended
in different buffers and recentrifuged at 100,000 g, yielding
wash fractions (S100
, lanes 3) and pellets. The pellets
were washed as before, resulting in wash fractions (S100
,
lanes 4) and washed pellets (P100
, lanes 5). Western blotting detected a KN double band in the S10 supernatant fraction (lanes 1) but only the upper band in the S100
supernatant fractions (lanes 2). By contrast, the lower
band was detected in the washed pellets (lanes 5) except
after treatment of the pellet with 1% Triton X-100, which
released KN protein into the wash fraction S100
(lane 3).
The other two treatments, high salt concentration and alkaline pH, released little or no KN protein from the pellet, respectively, suggesting that KN is an integral membrane
protein. For comparison, ADL1 protein was recovered
from the washed pellets (lane 5) after treatment with Triton X-100 but released into the wash fraction (lane 3)
when the pellet had been resuspended at pH 10.9, suggesting that ADL1 is a peripheral membrane protein. In the
presence of Triton X-100, ADL1 appears to be released
from the membrane but forms a pelletable complex of 400 -
600 kD (Park et al., 1997
). To determine whether the nonpelletable KN protein was soluble or associated with a specific membrane fraction, we processed an aliquot of the
S100 supernatant for electron microscopy and immunogold labeling. As shown in Fig. 1 f, KN protein was attached to the surface of small vesicles as well as to lumps of membranous material. These data suggest that KN protein is tightly associated with membranes.
-tubulin antibody to
visualize the microtubule cytoskeleton (Fig. 3). At the onset of mitosis, a preprophase band of cortical microtubules
marks the future plane of cell division (Lambert, 1993
).
Some cells displaying the preprophase band contained large
patches of KN-positive material while others did not, suggesting that the expression of KN protein started at or
soon after the onset of mitosis (Fig. 3, a and b). From prophase to anaphase, KN staining was detected only in large
patches within the cytoplasm; during these stages, mitotic
spindles formed and separated the sets of daughter chromosomes (Fig. 3, c-e). The phragmoplast forms during
anaphase between the two sets of daughter chromosomes
and narrows down in the center of the division plane during early telophase (Staehelin and Hepler, 1996
). In early
telophase cells, KN-positive material started to accumulate
in the center of the phragmoplast (Fig. 3 f). As the phragmoplast was displaced toward the periphery of the cell, the
KN-positive material extended from the center to the advancing edge of the phragmoplast (Fig. 3 g). When the
phragmoplast reached the lateral cortex of the cell, the KN-positive disc extended across the entire plane of division, although the KN signal appeared weaker in the center than
at the periphery (Fig. 3, h and i). In the early interphase,
KN staining was rarely found, and if still present, it appeared fragmented (see Fig. 2 c). Thus, the time course of
KN protein accumulation at the plane of cell division reflected the formation and lateral expansion of the cell plate.
Fig. 2.
Cell cycle-dependent distribution of KN protein.
Whole-mount preparations of single cells from squashed wild-type
embryos were stained with DAPI (blue) to visualize the stage of
the cell cycle and with KN-specific antiserum (Cy3, orange). (a)
Mitotic cells show KN-positive patches; interphase cells are KN
negative. (b) Two telophase cells with KN-positive material in
plane of division. (c) Early interphase cell with diffuse KN staining
near division plane (arrow). No KN signal in other interphase cells.
[View Larger Version of this Image (42K GIF file)]
Fig. 3.
Confocal laser scanning microscopy of temporal sequence of KN protein redistribution in dividing embryonic cells.
Whole-mount preparations of single cells from squashed wild-type embryos were stained with anti-KN antiserum (FITC, green)
and anti--tubulin antibody (TRITC, red). (a) Onset of mitosis:
early preprophase with preprophase band (arrow). KN signals
are barely detectable. (b) Preprophase: KN-positive material in
large patches away from preprophase band (arrow). (c) Late preprophase: only remnants of preprophase band are detectable (arrow), while mitotic spindles (asterisk) are forming. (d) Metaphase: mitotic spindles (asterisk) flank the metaphase plate. (e)
Anaphase: mitotic spindles (asterisk) extend toward the poles;
KN-positive material still scattered in large patches. (f) Early telophase: formation of phragmoplast (red; arrowhead) signals onset of cytokinesis; KN-positive material accumulates in the center
(yellow) of the phragmoplast. (g) Mid-telophase: phragmoplast
(arrowhead) and associated KN-positive material (yellow) coextend laterally toward the periphery of the cell. (h and i) Late telophase and end of cytokinesis: phragmoplast (arrowhead) is at lateral cell surface; KN-positive fuzzy disc extends from the center
(green) to the edge of the phragmoplast (yellow); (h) side view;
(i) face view. Arrows, preprophase bands; asterisks, mitotic spindles; arrowheads, phragmoplasts. Bar, 10 µm.
[View Larger Version of this Image (33K GIF file)]
Fig. 4.
Confocal laser scanning microscopy (three Z layers each
0.5-µm thick) of KN protein and plasma membrane H+-ATPase
distribution during cytokinesis. Double-immunofluorescence staining of cells from squashed wild-type embryos with anti-KN (FITC) and anti-PM ATPase (Cy3) antibodies. Cytokinetic cells are marked with arrows. (a and d) Early, (b and e) late, and (c and f) final stages of cytokinesis stained for PM ATPase (a-c) and KN protein (d-f). Note presence of PM ATPase in surrounding plasma membrane and its absence from the forming cell plate
until the stage when only remnants of KN signal are present at
the edges (arrows in c and f). Bar, 10 µm.
[View Larger Version of this Image (116K GIF file)]
-
adjacent sequences (Lukowitz et al., 1996
). Mutant cells
were analyzed during telophase as visualized by DAPI
staining. No KN-positive material was observed in knX37-2
mutant cells (Fig. 5, a and b) and in mutant cells for the
two other kn alleles (data not shown). Thus, all three mutations appear to interfere with the accumulation of stable
mutant KN protein.
Fig. 5.
Cytokinetic knolle
mutant cells. Cells from
squashed embryos were
stained with DAPI (blue)
and with anti-KN antibody
(b) or anti-ADL1 antibody (c
and d) followed by Cy3-conjugated secondary antibody
(orange). (a and b) Telophase cell from knX37-2 mutant embryo: no KN-positive
material. Inset in b shows KN
signal from wild-type telophase cell photographed under identical conditions. (c and d) Cytokinetic cell from wild-type (c) and knX37-2 mutant (d) embryo:
ADL1 accumulates in the plane of cell division. Note enlarged
mutant cell in d. Bar, 10 µm.
[View Larger Version of this Image (78K GIF file)]
). It had been
shown previously that the dynamin-like phragmoplastin (PDL) from soybean was expressed in dividing as well as
in nondividing cells and that a substantial fraction of PDL
redistributed to the cell plate during cytokinesis (Gu and
Verma, 1996
). We made the same observation for ADL1,
and Fig. 5 c shows a wild-type cell that accumulated ADL1
at the newly forming cell plate during cytokinesis (Fig. 5 c).
We also detected ADL1 at the plane of cell division in
knX37-2 mutant cells, suggesting that membrane vesicles accumulated there in the absence of KN protein (Fig. 5 d).
Fig. 6.
Ultrastructure of a
knolle mutant cell. (a) Diagram of a single large interphase cell from a knX37-2 mutant torpedo-stage embryo.
Note multiple nuclei (light
shading), nucleoli (dark
shading), incomplete internal
cell walls (solid lines), and
bands of vesicles (dotted
lines). (b) Higher-magnification electron micrograph of
region boxed in a. A band of
vesicles (asterisks) is interrupted by local aggregates of
vesicles that enclose electron-dense material resembling the middle lamella of
cell wall (arrowhead). Note
nearly uniform size of vesicles. cw, outer cell wall of
multinucleate cell. Bars: (a)
10 µm; (b) 0.5 µm.
[View Larger Version of this Image (55K GIF file)]
).
The cell walls grow in from the surface, and masses of vesicles ~70 nm in diameter and microtubules have been observed near the growing tips of cell walls (Mansfield and
Briarty, 1990b). A special case of cytokinesis also occurs in
male meiotic cells. After the two nuclear divisions, the new
cell walls grow in from the cell surface, although microtubule arrays form a complex network of phragmoplast-like
structures (Owen and Makaroff, 1995
; Peirson et al., 1997
).
Fig. 7.
Accumulation of KN-positive material in dividing tissues. Cryosections (a-l) or squashed tissues (m-v) were stained
with DAPI (blue) and with anti-KN antiserum (b, d, f, h, j, l-p,
and r) or anti-tubulin antibody (u) followed by Cy3-conjugated
secondary antibody (yellow or orange). (a-d) Seedling root tip;
basal end to the right. Slightly oblique section. KN-positive mitotic cell (arrowheads in c and d) visible at higher magnification
of region boxed in a and b. (e-h) Inflorescence apex with flower
primordia. Longitudinal section. I, Inflorescence meristem; F,
flower primordia. KN-positive mitotic cell (arrowheads in g and
h) visible at higher magnification of region boxed in e and f. (i-l)
Pistil of developing flower with ovules (O). Cross section. KN-positive mitotic cell (arrowheads in k and l) visible at higher magnification of region boxed in i and j. (m) Cellularizing endosperm.
Note cages of KN-positive material (orange) surrounding nuclei
(blue). (n) Cellular endosperm with KN-positive material in planes
of cell division. (o) Male sporogenous tissue. Asterisk, metaphase;
arrowhead, telophase. (p) Tapetum cell in early telophase (arrowhead) of endomitosis. No KN signal. (q-v) Cytokinesis after
male second meiotic division. (q and t) DAPI staining. (r) No KN
signal. (u) Phragmoplast-like arrangement of microtubules. (s
and v) Corresponding Nomarski images. Note ingrowing cell
walls (arrowheads).
[View Larger Version of this Image (117K GIF file)]
Discussion
; Staehelin and Hepler, 1996
). This mode of cytokinesis appears totally different from the pulling-in of the existing plasma membrane during animal cytokinesis and may
thus involve a unique mechanism.
). Those findings raised the
possibility that KN protein may be specifically involved in
cytokinetic vesicle trafficking. In this study, we have made
the following observations to determine the role of KN
protein in cytokinesis: (a) KN protein is tightly associated with membranes. (b) KN protein is expressed only in dividing cells from the onset of mitosis, it accumulates at the
plane of division in a centrifugal progression and disappears upon the completion of the cell plate. (c) In kn mutant cells, membrane vesicles accumulate at the plane of
cell division, but their fusion appears to be impaired. (d)
KN protein was not detected in male meiotic cells, which
undergo a distinct mode of cytokinesis.
). Our observation that ADL1 protein, the Arabidopsis homologue of the dynamin-like phragmoplastin, accumulates at the division plane of kn mutant cells makes it
unlikely that KN protein is essential for the transport of
Golgi-derived vesicles along the phragmoplast. This conclusion is supported by our electron microscopic study of
kn mutant cells, which revealed bands of free vesicles interrupted by scattered patches of membrane-bounded material. What role ADL1/phragmoplastin plays in cell plate
formation has yet to be established. However, arguments
have been put forward to support the idea that these dynamin-like proteins act at an early step in cytokinesis.
Their animal counterpart, dynamin, appears to attach to
clathrin-coated vesicle buds of membranes, where it oligomerizes to form a collar-like structure or, under certain
conditions, a tubular structure (Baba et al., 1995
; Hinshaw
and Schmid, 1995
; Takei et al., 1995
). These structures resemble the tubular extensions of vesicles observed in the
early cell plate of dividing plant cells (Samuels et al., 1995
;
Gu and Verma, 1996
). In addition, a GFP fusion of phragmoplastin, the soy bean homologue of ADL1, still accumulates in the division plane of transgenic tobacco cells
treated with caffeine, a drug that inhibits maturation of the
cell plate but has a less pronounced effect on the early
steps in cell plate formation (Gu and Verma, 1997
; see also
Samuels and Staehelin, 1996
).
). To
explain this phenotype as the consequence of a primary
defect in cytokinetic vesicle fusion, two interpretations are
conceivable. Considering its similarity to syntaxins that act
as t-SNAREs, docking specific vesicles to target membranes (heterotypic fusion), KN protein might mediate heterotypic fusion of later-arriving vesicles with a short stretch
of cell plate membrane that may have formed initially via
a KN-independent mechanism. In this scenario, the same
KN-independent mechanism may be used to form the incomplete cell walls found in kn mutant cells. However,
there is no evidence for such a two-step formation of the
cell plate during cytokinesis. Furthermore, KN protein accumulates in the plane of cell division essentially at the
same time when the phragmoplast forms in the center of the early telophase cell. We thus favor the alternative interpretation that the entire cell plate arises by KN-mediated
homotypic fusion of Golgi-derived vesicles. A recent study
of the yeast vacuolar compartment has established that
homotypic fusion can indeed be mediated by specific v-
and t-SNAREs (Nichols et al., 1997
). In that case, vesicle
fusion was impaired but still occurred with low efficiency
when the v-SNARE was absent and was almost abolished in the absence of the syntaxin-related t-SNARE (Nichols
et al., 1997
). A likely candidate for a v-SNARE complementary to the t-SNARE KNOLLE would be the product
of the KEULE gene, which has not been isolated. Mutant
keule cells display very similar defects in cytokinesis, although slightly weaker and more variable (Assaad et al.,
1996
).
), but also the KN protein accumulates during
M phase and disappears at the completion of cytokinesis.
Topologically, the KN protein has only been localized to
large cytoplasmic patches that resemble JIM 84-positive
Golgi membranes in maize (Satiat-Jeunemaitre and Hawes,
1992
) and to the developing cell plate. Our attempts to analyze these membrane compartments in more detail by
immunogold labeling were not successful, presumably because the KN protein was no longer accessible after embedding of the tissue for electron microscopy; an alternative
procedure, preembedding immunogold labeling of cytokinetic cells, gave distinct signals, but the necessary detergent
treatment destroyed the ultrastructure (our unpublished
observation). We attempted to identify the KN-positive cytoplasmic patches by double-labeling with the JIM 84 mAb. However, we did not observe cross-reaction signals
in Arabidopsis cells, although we were successful in JIM 84 labeling of maize root cells (our unpublished observation).
Nevertheless, the most likely interpretation seems that KN
specifically localizes to post-Golgi vesicles destined to form
the cell plate.
, and references therein). The existence of a specific
route for vesicle transport to the cell plate is also supported by the observation that kn mutant cells do not
seem to be impaired in other aspects of membrane traffic,
including increase in cell surface area as well as tip growth
of root hairs and pollen tubes (Lukowitz et al., 1996
). Although apparently not required for the transport of cytokinetic vesicles, KN protein may ensure that only vesicles
with the correct membrane composition and cargo fuse
with one another to form the cell plate and may thus help
to establish and maintain this transient membrane compartment.
). Although cellularization of the endosperm involves ingrowth of cell wall from the surface,
both microtubules and free vesicles have been observed
near the growing cell wall tips (Mansfield and Briarty,
1990b). Subsequently, the center-out mode of division also
operates in endosperm cells (Olsen et al., 1995
). In all these cases, KN protein accumulates in the plane of division. The only exception we have found so far is the male
meiotic cell, which undergoes cytokinesis by cell wall ingrowth from the periphery after meiosis II (Owen and
Makaroff, 1995
; Peirson et al., 1997
) and does not express
KN protein. Our observation is consistent with the recent
report that cytokinesis of the male meiotic cell is specifically affected by mutations in a different Arabidopsis gene,
STUD (Hülskamp et al., 1997
). Thus, at least two distinct mechanisms of cell division appear to operate in higher
plants.
, and
references therein). Furthermore, when cell plate formation is disrupted by caffeine, cell wall stubs are formed,
which may reflect a default mechanism for cytokinesis by
ingrowth from the cell surface (Röper and Röper, 1977
).
From an evolutionary perspective, this may not be surprising since two separate mechanisms, actin-based cleavage
initiated at the parental wall and microtubule-dependent
formation of a cell plate in the center, combine to achieve
cytokinesis in the alga Spirogyra (McIntosh et al., 1995
;
Sawitzky and Grolig, 1995
). Considering the important role
of the KN protein in cell plate formation, the isolation of
KN homologues from lower plants may shed light on the
evolution of the center-out mode of cytokinesis in higher plants.
Received for publication 28 July 1997 and in revised form 29 September 1997.
This work was funded by the European Communities' BIOTECH Programme, as part of the Project of Technological Priority 1993-1996, and by the Leibniz Programme of the Deutsche Forschungsgemeinschaft.We thank W. Michalke (Biologie III, Universität Freiburg, Germany) for generously providing the anti-PM ATPase monoclonal antibody, M. Heese for establishing a protocol for recombinant KN protein expression in E. coli, I. Zimmermann for help with the EM analysis, and J. Berger for advice on confocal laser scanning microscopy. We thank M. Grebe, M. Heese, M. Hülskamp, T. Laux, and K. Schrick for critically reading the manuscript.
ADL1, Arabidopsis dynamin-like protein;
DAPI, 4,6-diamidino-2-phenylindole;
KN, KNOLLE;
MTSB, microtubule-stabilizing buffer;
PM ATPase, plasma membrane H+-ATPase.
1. |
Asada, T., and
H. Shibaoka.
1994.
Isolation of polypeptides with microtubule-translocating activity from phragmoplasts of tobacco BY-2 cells.
J. Cell Sci.
107:
2249-2257
|
2. |
Asada, T.,
R. Kuriyama, and
H. Shibaoka.
1997.
TKRP125, a kinesin-related
protein involved in the centrosome-independent organization of the cytokinetic apparatus in tobacco BY-2 cells.
J. Cell Sci.
110:
179-189
|
3. | Assaad, F., U. Mayer, G. Wanner, and G. Jürgens. 1996. The KEULE gene is involved in cytokinesis in Arabidopsis. Mol. Gen. Genet. 253: 267-277 |
4. | Baba, T., H. Damke, J.E. Hinshaw, K. Ikeda, S.L. Schmid, and D.E. Warnock. 1995. Role of dynamin in clathrin-coated vesicle formation. Cold Spring Harbor Symp. Quant. Biol. 60: 235-242 |
5. | Bennett, M.K., and R.H. Scheller. 1993. The molecular machinery for secretion is conserved from yeast to neurons. Proc. Natl. Acad. Sci. USA. 90: 2559-2563 [Abstract]. |
6. | Byers, T.J., and P.B. Armstrong. 1986. Membrane protein redistribution during Xenopus first cleavage. J. Cell Biol. 102: 2176-2184 [Abstract]. |
7. | Feiler, H.S., T. Desprez, V. Santoni, J. Kronenberger, M. Caboche, and J. Traas. 1995. The higher plant Arabidopsis thaliana encodes a functional CDC48 homologue which is highly expressed in dividing and expanding cells. EMBO (Eur. Mol. Biol. Organ.) J. 14: 5626-5637 [Abstract]. |
8. | Fishkind, D.J., and Y.-L. Wang. 1995. New horizons for cytokinesis. Curr. Opin. Cell Biol. 7: 23-31 |
9. | Ferro-Novick, S., and R. Jahn. 1994. Vesicle fusion from yeast to man. Nature. 370: 191-193 |
10. | Goodbody, K.C., and C.W. Lloyd. 1994. Immunofluorescence techniques for analysis of the cytoskeleton. In Plant Cell Biology. A Practical Approach. N. Harris, and K.J. Oparka, editors. IRL Press, Oxford. 221-243. |
11. | Gu, X., and D.P.S. Verma. 1996. Phragmoplastin, a dynamin-like protein associated with cell plate formation in plants. EMBO (Eur. Mol. Biol. Organ.) J. 15: 695-704 [Abstract]. |
12. |
Gu, X., and
D.P.S. Verma.
1997.
Dynamics of phragmoplastin in living cells
during cell plate formation and uncoupling of cell elongation from the plane
of cell division.
Plant Cell.
9:
157-169
|
13. | Harlow, E., and D. Lane. 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 313-315. |
14. | Hinshaw, J.E., and S.L. Schmid. 1995. Dynamin self-assembles into rings suggesting a mechanism for coated vesicle budding. Nature. 374: 190-192 |
15. | Hülskamp, M., N.S. Parekh, P. Grini, K. Schneitz, I. Zimmermann, S.J. Lolle, and R.E. Pruitt. 1997. The STUD gene is required for male-specific cytokinesis after telophase II of meiosis in Arabidopsis thaliana. Dev. Biol. 187: 114 -124. |
16. | Lambert, A.-M. 1993. Microtubule-organizing centers in higher plants. Curr. Opin. Cell Biol. 5:116 -122. |
17. | Laemmli, U.K.. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227: 680-685 |
18. | Latterich, M., K.-U. Fröhlich, and R. Schekman. 1995. Membrane fusion and the cell cycle: Cdc48p participates in the fusion of ER membranes. Cell. 82: 885-893 |
19. | Liu, C.-M., S. Johnson, and T.L. Wang. 1995. cyd, a mutant of pea that alters embryo morphology is defective in cytokinesis. Dev. Genet. 16: 321-331 . |
20. |
Liu, B.,
R.J. Cyr, and
B.A. Palevitz.
1996.
A kinesin-like protein, KatAp, in the
cells of Arabidopsis and other plants.
Plant Cell.
8:
119-132
|
21. | Lukowitz, W., U. Mayer, and G. Jürgens. 1996. Cytokinesis in the Arabidopsis embryo involves the syntaxin-related KNOLLE gene product. Cell. 84: 61-71 |
22. | Mansfield, S.G., and L.G. Briarty. 1990a. Development of the free-nuclear endosperm in Arabidopsis thaliana. Arabidopsis Inf. Serv. 27: 53-64 . |
23. | Mansfield, S.G., and L.G. Briarty. 1990b. Endosperm cellularization in Arabidopsis thaliana. Arabidopsis Inf. Serv. 27: 65-72 . |
24. | Mayer, U., R.A. Torres, Ruiz, T. Berleth, S. Miséra, and G. Jürgens. 1991. Mutations affecting body organization in the Arabidopsis embryo. Nature. 353: 402-407 . |
25. | McIntosh, K., J.D. Pickett-Heaps, and B.E.S. Gunning. 1995. Cytokinesis in Spirogyra: integration of cleavage and cell-plate formation. Int. J. Plant Sci. 156: 1-8 . |
26. | Mineyuki, Y., and B.E.S. Gunning. 1990. A role for preprophase bands of microtubules in maturation of new cell walls, and a general proposal on the function of preprophase band sites in cell division in higher plants. J. Cell Sci. 97: 527-537 . |
27. | Nichols, B.J., C. Ungermann, H.R.B. Pelham, W.T. Wickner, and A. Haas. 1997. Homotypic vacuolar fusion mediated by t- and v-SNAREs. Nature. 387: 199-202 |
28. | Olsen, O.A., R.C. Brown, and B.E. Lemmon. 1995. Pattern and process of wall formation in developing endosperm. Bioessays. 17: 803-812 . |
29. | Owen, H.A., and C.A. Makaroff. 1995. Ultrastructure of microsporogenesis and microgametogenesis in Arabidopsis thaliana (L.) Heynh. ecotype Wassilewskija (Brassicaceae). Protoplasma. 185: 7-21 . |
30. |
Park, J.M.,
S.G. Kang,
K.T. Pih,
H.J. Jang,
H.L. Piao,
H.W. Yoon,
M.J. Cho, and
I. Hwang.
1997.
A dynamin-like protein, ADL1, is present in membranes as a high molecular weight complex in Arabidopsis thaliana.
Plant
Physiol.
115:
763-771
|
31. | Peirson, B.N., S.E. Bowling, and C.A. Makaroff. 1997. A defect in synapsis causes male sterility in a T-DNA-tagged Arabidopsis thaliana mutant. Plant J. 11: 659-669 |
32. | Pfeffer, S.R.. 1996. Transport vesicle docking: SNARE and associates. Annu. Rev. Cell Dev. Biol. 12: 441-461 . |
33. | Röper, W., and S. Röper. 1977. Centripetal wall formation in roots of Vicia faba after caffeine treatment. Protoplasma. 93: 89-100 . |
34. | Samuels, A.L., and L.A. Staehelin. 1996. Caffeine inhibits cell plate formation by disrupting membrane reorganization just after the vesicle fusion step. Protoplasma. 195: 144-155 . |
35. | Samuels, A.L., T.H. Giddings Jr., and L.A. Staehelin. 1995. Cytokinesis in tobacco BY-2 and root tip cells: a new model of cell plate formation in higher plants. J. Cell Biol. 130: 1345-1357 [Abstract]. |
36. |
Satiat-Jeunemaitre, B., and
C. Hawes.
1992.
Redistribution of a Golgi glycoprotein in plant cells treated with Brefeldin A.
J. Cell Sci.
103:
1153-1166
|
37. | Sawitzky, H., and F. Grolig. 1995. Phragmoplast of the green alga Spirogyra is functionally distinct from the higher plant phragmoplast. J. Cell Biol. 130: 1359-1371 [Abstract]. |
38. | Staehelin, L.A., and P.K. Hepler. 1996. Cytokinesis in higher plants. Cell. 84: 821-824 |
39. |
Takei, K.,
P.S. McPherson,
S.L. Schmid, and
P. De Camilli.
1995.
Tubular membrane invaginations coated by dynamin rings and induced by GTP-![]() |
40. | Villalba, J.M., M. Lützelschwab, and R. Serrano. 1991. Immunocytolocalization of plasma-membrane H+-ATPase in maize coleoptiles and enclosed leaves. Planta (Heidelb.). 185:458 - 461. |
41. | Webb, M.C., and B.E.S. Gunning. 1990. Embryo sac development in Arabidopsis thaliana I. Megasporogenesis, including the microtubular cytoskeleton. Sex Plant Reprod. 3: 244-256 . |
42. | Wick, S.M.. 1991. Spatial aspects of cytokinesis in plant cells. Curr. Opin. Cell Biol. 3: 253-260 |
43. | Zhang, D., P. Wadsworth, and P. Hepler. 1993. Dynamics of microfilaments are similar, but distinct from microtubules during cytokinesis in living, diving plant cells. Cell Motil. Cytoskel. 24: 151-155 . |