First published online November 7, 2006
doi: 10.1242/10.1242/dev.02654
Development 133, 4679-4689 (2006)
Published by The Company of Biologists 2006
The Arabidopsis elch mutant reveals functions of an ESCRT component in cytokinesis
Christoph Spitzer1,
Swen Schellmann1,*,
Aneta Sabovljevic1,
Mojgan Shahriari1,
Channa Keshavaiah1,
Nicole Bechtold2,
Michel Herzog3,
Stefan Müller4,
Franz-Georg Hanisch5 and
Martin Hülskamp1,*
1 University of Köln, Botanical Institute III, Gyrhofstr. 15, 50931
Köln, Germany.
2 MEDICAGO Inc., 1020, route de l'Église, bureau 600, Sainte-Foy,
Québec G1V 3V9, Canada.
3 Laboratoire de Génétique Moléculaire des Plantes,
CNRS/Universite J. Fourier BP 53, 38041 Grenoble Cedex 09, France.
4 Centre for Molecular Medicine Cologne, University of Cologne,
Joseph-Stelzmann-Str. 52, 50931 Köln, Germany.
5 Institute of Biochemistry II, Medical Faculty, University of Cologne,
Joseph-Stelzmann-Str. 52, 50931 Köln, Germany.
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Fig. 1. elc mutant trichome phenotype. (A) Wild-type
trichome. (B) elc mutant trichome mimicking a trichome
cluster. (C) DAPI-stained sti trichome. Arrowhead marks the
polyploid nucleus. (D) DAPI-stained sti elc double mutant
trichome with two stems, each containing a nucleus (arrowheads). (E-H)
elc mutant trichome in which a peroxisome marker is expressed. The
boxed area in E is shown in F-H at higher magnification at different time
points (s, seconds). The path of a single peroxisome from one trichome to the
next is indicated by the arrowhead.
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Fig. 2. Cytokinesis defects in elc mutants. The nuclear phenotypes
of wild type and elc were shown by DAPI staining. (A)
Wild-type trichome with one nucleus located at the branch point. (B-F)
elc mutant trichomes with two nuclei in one stem (B), one nucleus in
each stem (C), with three nuclei (D) and with four nuclei (E). (F) Higher
magnification of E. (G) Occasionally, trichomes with a wild-type
appearance have two nuclei. (H) Multiple nuclei in epidermal pavement
cells. (I) Multiple nuclei in hypocotyl cells. (J) Incomplete
cell wall in an epidermal pavement cell (red arrow). (K) DAPI staining
of J to indicate the position of the two nuclei in the cell (black arrows);
the white arrow marks the incomplete cell wall.
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Fig. 3. DNA content of nuclei in elc mutants. Comparison of the DNA
content of trichome nuclei of wild-type (top) and elc mutant
trichomes containing one (middle) or two nuclei (bottom). The relative
distribution and the average nuclear DNA content of individual nuclei are very
similar in all three situations.
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Fig. 4. Molecular analysis of the ELC gene. (A) Schematic of
the chromosomal region of the ELC gene. The fragment used for the
rescue experiments is indicated. (B) The chromosomal region of the
ELC gene shows two insertions in the mutant. (C) The insertion
in the ELC gene results in a truncated protein. A sequence alignment
of the wild-type and mutant proteins and a schematic highlighting their domain
structure are shown. (D) Sequence alignment of ELC (At3g12400) and
ELC-like (At5g13860) from Arabidopsis thaliana (At), ELC from
Oriza sativum (Os) (BAD28453), yeast Vps23 (Sc) (Af004731), human
TSG101 (Hs) (U82130) and Drosophila TSG101 (Dm) (NM_079396). Letters
in a black background indicate identity, dark gray backgrounds indicate strong
similarity and a light gray background indicates weak similarity. (E)
Schematic of the protein domain arrangement of ELC, TSG101 and Vps23. The
characteristic absence of a cysteine conserved in UBC domains (star) is
depicted below.
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Fig. 5. Expression analysis of ELC. (A) Expression of
ELC and ELC-like in different tissues. As control for
genomic contamination, the different RNAs were used as templates without
reverse transcriptase. (B) Semi-quantitative RT-PCR to compare the RNA
levels of wild type (Ws2), elc mutant and a 35S:ELC
overexpression line.
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Fig. 6. Ubiquitin binding of ELC. Pull-down assays were performed using
ubiquitin coupled to agarose and the ELC-HA protein was detected on the
western blot using an anti-HA antibody. Protein G coupled to agarose was used
as a control.
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Fig. 7. ELC is a component of a large protein complex. Protein extract was
loaded on a Superose 6 10/300 GL gel filtration column, separated on an
SDS-PAGE gel and ELC-HA detected by western blotting. The following size
markers were used: thyroglobulin (660 kDa), -amylase (200 kDa), alcohol
dehydrogenase (140 kDa) and ovalbumin (43 kDa).
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Fig. 8. Co-immunoprecipitation of ESCRT-I proteins with ELC-HA. Using
anti-HA beads, ELC-HA was immunoprecipitated from protein extracts and
separated by PAGE. MALDI-TOF analysis revealed indicative fragments for the
three bands marked by a star: ELC-HA, VPS37-1 and VPS28-1.
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Fig. 9. ELC localizes to endosomes. (A-D) Protoplasts of
Arabidopsis cultured cells expressing YFP-ELC (A) were labeled with
FM4-64 (B). The small dots with YFP-ELC (white arrows) were also stained by
FM4-64. Note that few dots labeled by FM4-64 were not marked by YFP-ELC (arrow
heads). (E-L) CFP-ELC (E,I) was co-transfected with Ara6-GFP (F) and
with GFP-Ara7 (J) into protoplasts of Arabidopsis cultured cells. The
dots with CFP-ELC co-localized with Ara6-GFP (yellow arrows) and also with
GFP-Ara7 (pink arrows). Scale bar: 20 m.
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Fig. 10. Analysis of the elc kis-T1 double mutant. (A-C)
Comparison of kis-T1 (A), elc (B) and kis-T1 elc
(C) mutant rosette leaves. (D-F) Comparison of kis-T1 (D),
elc (E) and kis-T1 elc (F) mutant trichomes. Note that the
cluster frequency is strongly enhanced in the double mutant. (G-I)
Comparison of kis-T1 (G), elc (H) and kis-T1 elc
(I) mutant epidermal pavement cells. Cell size is reduced in the double
mutant. (J-L) Comparison of kis-T1 (J), elc (K) and
kis-T1 elc (L) sections of rosette leaves. The integrity of the leaf
is strongly disturbed in the double mutant. (M-O) Higher magnification
of propidium iodide-stained leaf sections from kis-T1 elc double
mutants. Multinucleated cells are frequently observed in the kis-T1
elc double mutants. Nuclear anomalies like nuclear bridges (arrowhead, M)
and high numbers of nuclei per cell (N) were found frequently. In very rare
cases, multinucleated stomata with incomplete cell walls were observed
(arrowhead, O).
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© The Company of Biologists Ltd 2006
