GAAV 2007 - Lukas Synek - Ústav experimentální botaniky

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GAAV 2007 - Lukas Synek - Ústav experimentální botaniky
Grantová agentura Akademie věd ČR
Základní list A1
Návrh juniorského badatelského grantového projektu
Identifikační kód KJB600380802
01 Grantový projekt
Název
Transkriptom mutanta exo70A1 a buněčné funkce EXO70A1, předpokládané podjednotky komplexu
exocyst, u Arabidopsis thaliana
02 Navrženo k projednání v oborové radě
6
podobor
608
03 Doba řešení grantového projektu (v rocích) 3 tj. od začátku roku 2008 do konce roku 2010
04 Charakteristika grantového projektu
Komplex exocyst se v buňce účastní finálních kroků exocytózy. Vzhledem k odlišnostem živočišných
a kvasinkových buněk, kde byl doposud studován, od buňky rostlinné můžeme u rostlin
předpokládat i řadu rozdílných funkcí. O významu komplexu exocyst u rostlin svědčí například
dramaticky změněný fenotyp mutantů Arabidopsis v genu EXO70A1 kódujícím velmi
pravděpodobně jednu z osmi podjednotek tohoto komplexu. Na popis mutantů exo70A1, který
jsme provedli v rámci předchozího výzkumu, bychom chtěli navázat jejich detailnější analýzou.
Zejména analýza kompletního transkriptomu mutantů exo70A1 pomocí tzv. DNA čipu jistě odhalí
interagující geny a ukáže na další možné funkce EXO70A1. Tento projekt by měl přispět pomocí
molekulárně biologických a mikroskopických technik (např. imunolokalizace, GFP fúze,
farmakologie) k detailnějšímu poznání role EXO70A1, resp. exocystu, v rostlinné buňce.
05 Uchazeč (I)
Oficiální název instituce
Ústav experimentální botaniky AV ČR, v. v. i.
Poslední změna: 19.4.2007 14:20:01
1/23
Grant Agency of the Academy of Sciences of the Czech Republic
Cover Sheet A1/A
Grant Application
Identification code KJB600380802
01 Project
Title
Transcriptome of the exo70A1 mutant and cellular roles of EXO70A1, a putative subunit of the
exocyst complex, in Arabidopsis thaliana
02 Summary
The exocyst complex is involved in final steps of exocytosis. Cell functions of the exocyst have been
studied in yeast and animal cells, however due to sessile biology of the plant cell, plant specific
functions could be expected. As suggested by a discernible phenotype of mutants in EXO70A1
gene, coding for one of eight putative exocyst subunits, the importance of the exocyst in plants
(Arabidopsis) is evident. In this project, we would like to continue detailed analysis of exo70A1
mutants that were generally described in our previous research. We plan to begin with
transcriptomic approach using microarray DNA chips analysis that could reveal gene interactions of
EXO70A1 and could point to its functions. Employing techniques of molecular biology and
microscopy such as immunolocalization, GFP fusions and pharmacology, this project should
contribute to more detailed understanding of functions of EXO70A1 and exocyst, respectively, in
the plant cell.
Poslední změna: 19.4.2007 14:20:01
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Grantová agentura Akademie věd ČR
Základní list A2/I
06 Uchazeč (I) - Navrhovatel
Tituly
Jméno
Příjmení
Mgr.
Lukáš
Synek
Věd. hodnost
Státní příslušnost
Česká republika
Oficiální název instituce
Ústav experimentální botaniky AV ČR, v. v. i.
Doplňující údaje o pracovišti (např. u VŠ fakulta, katedra nebo ústav)
Oficiální zkratka názvu:
ÚEB
Typ organizace:
VVI
IČ:
61389030
Ulice
Místo
Rozvojová 263
Praha 6 - Lysolaje
PSČ
Tel.
Fax
165 00
225106111
225106456
E-mail
[email protected]
Bankovní spojení / příslušnost k resortu
Banka
Organizační jednotka: název
Kód
07 Kontaktní adresa navrhovatele
Název
Ústav experimentální botaniky AV ČR
Ulice
Místo
Rozvojová 263
Praha 6 - Lysolaje
PSČ
Tel.
165 00
225106458
Fax
E-mail:
[email protected]
08 Údaje o řešitelském týmu na pracovišti uchazeče (I)
Počet
tvůrčích pracovníků
1
, doktorandů
1
, ostatních
0.45
, doktorandů
0.25
, ostatních
, doktorandů
1
, ostatních
, doktorandů
0.25
, ostatních
Přepočtená pracovní kapacita
tvůrčích pracovníků
09 Souhrnné údaje o řešitelském týmu
Počet
tvůrčích pracovníků
1
Celková přepočtená pracovní kapacita
tvůrčích pracovníků
0.45
Poslední změna: 19.4.2007 14:20:01
3/23
Grantová agentura Akademie věd ČR
Základní list A3
10 Kódová označení oboru
UNESCO: 2415.00
CEP & RIV: EA
2417.19
EB
11 Adresa web-stránky projektu, pokud existuje
12 Klíčová slova
česky
anglicky
EXO70; buněčná morfogeneze; exocytóza; exocyst; Arabidopsis thaliana;
transkriptom
EXO70; cell morphogenesis; exocytosis; exocyst; Arabidopsis thaliana;
transcriptome
13 Upozornění na vhodné oponenty v daném oboru - Specialisté (zejména zahraniční), kteří
se mohou kvalifikovaně k projektu vyjádřit (úplná adresa, e-mail, fax)
14 Ochrana duševního vlastnictví - Pokud je třeba, uveďte pracovníky (nejvýše 3, případně tým
jednoho pracoviště), kteří nemají být s návrhem seznámeni nebo kteří by se neměli k projektu
vyjadřovat.
Poslední změna: 19.4.2007 14:20:01
4/23
Grantová agentura Akademie věd ČR
List A4/I
10 Seznam členů řešitelského týmu a jejich pracovní kapacita
Uchazeč
Ústav experimentální botaniky AV ČR, v. v. i.
Pracovní kapacita
Tituly, jméno, příjmení a vědecká hodnost
(%)
Navrhovatel: Mgr. Lukáš Synek
Rodné číslo
45
790822/1123
25
840425/8082
Tvůrčí pracovníci
Doktorandi
Ivan Kulich
Ostatní pracovníci
Celková pracovní kapacita
70
Navrhovatel souhlasí s tím, že údaje uvedené v návrhu projektu budou uloženy v interním
databázovém systému GA AV a že s návrhem (s výjimkou rodných čísel) budou seznámeny osoby,
které se budou podílet na jeho hodnocení. V případě udělení grantu souhlasí s předáním údajů do
centrální evidence projektů výzkumu a vývoje.
Datum:
Navrhovatel (podpis):
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_________________________
5/23
Grant Agency of the Academy of Sciences of the Czech Republic
Sheet B
Justification of the proposal
Transcriptome of the exo70A1 mutant and cellular roles of EXO70A1, a putative subunit
of the exocyst complex, in Arabidopsis thaliana
1) Introduction
Precise regulation of localized cell expansion and cell division is essential for plant cell
morphogenesis. The major morphogenetic process in plant cell, exocytosis, consists in interaction
of vesicle trafficking and cytoskeleton. Many regulatory proteins such as RAB GTPases and ARF
GTPases participate in delivery of secretory vesicles to the plasma membrane. Before the fusion of
vesicles to the plasma membrane mediated by SNARE proteins, a multimeric complex called
exocyst acts as a tethering complex. The exocyst specifies the vesicle docking site on the plasma
membrane which is demonstrated by its localization to specific domains of the plasma membrane
characterized by local maxima of secretion. Thus, the exocyst is crucial for spatially localized
secretion.
In yeast, the exocyst localizes at sites of extensive polarized vesicle exocytosis (Hsu et al., 1999):
e.g. the tip and neck of budding yeast (TerBush and Novick, 1995); at the furrow of dividing fission
yeast (Fielding et al., 2005). In polarized animal epithelial cells, the exocyst is responsible for
targeting proteins to the basolateral membrane and localizes to the region of tight junctions
(Yeaman et al., 2004); in neuronal cells, the exocyst is present at the tip of growing neurite (Vega
and Hsu, 2001).
The exocyst was originally described in yeast as the so called sec6/8 complex (TerBush et al.,
1996). Based on sequence homology the mammalian exocyst was subsequently characterized (Kee
et al., 1997). In both yeast and mammals, the exocyst complex consists of eight subunits called
Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 (Guo et al., 1999; Matern et al., 2001).
Evidence for the exocyst complex in plants
Homologs to all eight exocyst subunits have been identified in silico in plant genomes, including
Arabidopsis thaliana (Cvrčková et al., 2001; Eliáš et al., 2003; Jurgens and Geldner, 2002). It
remains to be demonstrated whether exocyst subunits assemble in a complex and share the same
functions in plants as in yeast and animals. However, recent studies performed in our laboratory
suggest that the homologous proteins do form a complex similar to the yeast and animal exocyst
(Hála and Žárský, unpublished). In addition, an electron tomographic analysis of cell plate
formation during cytokinesis of somatic cells and pollen development in Arabidopsis (Otegui and
Staehelin, 2004; Segui-Simarro et al., 2004) uncovered the existence of 24 nm long structures
that tethers membrane vesicles, resembling the mammalian exocyst as observed in the electron
microscope (Hsu et al., 1998). Furthermore, a maize roothairless1 mutation, which is manifested
by the failure of root hair primordia to elongate properly, was shown to result from a disruption of
SEC3 gene (Wen et al., 2005). Phenotypic analysis of a series of T-DNA insertion mutants in the
Arabidopsis SEC8 gene revealed that the putative SEC8 exocyst subunit is necessary for pollen
tube germination (Cole et al., 2005). These findings suggest that the exocyst is conserved in plants
and may be involved in cytokinesis and polarized exocytosis.
Plant homologs of exocyst subunits
Exocyst subunits are typically encoded by single genes. However, bioinformatic analysis of plant
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genomes performed in our laboratory revealed that plant homologs of exocyst subunits are often
encoded by multiple genes in contrast to yeast and animals (Eliáš et al., 2003; Synek et al., 2006).
For example, Arabidopsis has only SEC6 and SEC8 subunits represented as single-copy genes. On
the other hand, it has two SEC3, SEC5, SEC10 and SEC15 paralogs and three EXO84 paralogs. The
total number of EXO70 paralogs, however, reaches to 23 genes (one of them unfunctional) that can
be classified into eight clusters. The same number of EXO70 paralogs was also identified
independently by Cannon et al. (2004). Even larger family of EXO70 genes was found in rice,
where the total number of likely functional EXO70 genes is 39. Also in the poplar genome (Populus
trichocarpa), multiple EXO70 genes count 26 paralogs and one probable pseudogene. The
multiplication of the EXO70 gene seems to be common in angiosperm plants. The ongoing research
aims to elucidate whether multiplied EXO70 genes possess redundant, overlapping or different
functions.
Structure and functions of EXO70
Plant EXO70 proteins typically comprise 600-700 amino acid residues, with predicted molecular
weight mostly in the range between 70 and 80 kDa, which is very similar to the size of the human
(684 aa / 78.1 kDa) and S. cerevisiae (623 aa / 71.3 kDa) Exo70. Recently Dong et al. (2005) and
Hamburger et al. (2006) determined the structure of the yeast Exo70 (except the very N-terminus)
and revealed that the protein forms a rod composed of contiguous a-helical bundles comprising 19
helices separated by loops.
In both yeast and animals the Exo70 subunit is known, together with Sec3, as a spatial marker at
the plasma membrane (Matern et al., 2001; Boyd et al., 2004; Roumanie et al., 2005), where the
exocyst complex is eventually being assembled.
Apart from interactions with other exocyst subunits, Exo70 is known to interact with Rho3 and
Rho4 GTPases, modulating cytoskeleton dynamics in S. cerevisiae (Adamo et al., 1999; Robinson
et al., 1999). In the fission yeast Sch. pombe, interaction of Rho3 with Exo70 is instrumental in the
last step of cytokinesis called abscission (Wang et al., 2003). There are preliminary reports about
Exo70-interacting proteins in yeast and mammals that point to the possibility that Exo70 may have
a role in the nucleus as a chromatin component (e.g. BIND database at http://bind.ca - complex
11988). Exo70 was also shown to co-localize with microtubules and mitotic spindles in rat kidney
cells (Wang et al., 2004). Similar to Sec5, Sec6 and Sec15, Exo70 was capable of inhibiting tubulin
polymerization, modulating, thus, microtubule dynamics.
2) Preliminary data
After finding multiple EXO70 genes within the Arabidopsis genome, we wanted to gain insight into
the developmental and organ-specific regulation of the expression of individual EXO70 genes. We
analyzed the publicly available Affymetrix ATH1 Arabidopsis genome array data in the
Genevestigator database (Zimmermann et al., 2004). Except for EXO70A3 (probably an inactive
pseudogene), EXO70H4, and EXO70H6, all other 20 EXO70 genes are detectable at least in one
microarray experiment.
We identified EXO70A1 as the most strongly expressed EXO70 gene in Arabidopsis (except for
pollen). In addition, EXO70A1 is the most likely candidate for the genuine exocyst subunit as it has
the highest sequence similarity to other eukaryotes. Therefore, we characterized two independent
T-DNA insertional mutants (SALK Institute) of the EXO70A1 gene (exo70A1-1 and exo70A1-2).
Heterozygous EXO70A1/exo70A1 plants appear normal and segregate in 1:2:1 ratio, suggesting
that neither male nor female gametophytes are affected by the EXO70A1 disruption. However, both
exo70A1-1 and exo70A1-2 homozygotes exhibit a suite of phenotypic defects (Synek et al., 2006).
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Homozygotes are dwarfish, organs are generally smaller, and plants show loss of apical dominance.
Inflorescences are recurrently branched due to ectopic initiation of lateral inflorescences instead of
flowers. The life span of exo70A1-1 and exo70A1-2 is about five months, i.e. more than twice as
long as wild-type Arabidopsis plants.
Mutant roots grow slower, exhibit skewing direction opposite to the wild type and lack waving
phenotype. Last two features are tempting to speculate that tubulin cytoskeleton could be affected
by EXO70A1 disruption. Polar growth of root hairs is blocked or reduced depending on the content
of sucrose in growing medium. Involvement of EXO70A1 in polar growth would be consistent with
the known function of the exocyst in polarized exocytosis.
Etiolated 7-day-old seedlings of both exo70A1 mutants develop 30% shorter hypocotyls than wild
type. Detailed analysis revealed no difference in average lengths of epidermal cells in hypocotyls,
but number of epidermal cells forming one file was significantly decreased. Nevertheless, a class of
longest cells (> 800 mm) present normally in wild type was missing in mutant hypocotyls. These
results show that EXO70A1 is involved in both cell division and cell elongation in etiolated
hypocotyls.
Both exo70A1 mutants have dramatically reduced fertility because homozygous exo70A1 mutants,
in contrast to heterozygotes, are impaired in production of mature pollen. Thus, seeds from
homozygotes can be harvested only very rarely. Additionally, elongation of stigmatic papillae is
disturbed similar to polar growth of root hairs.
These observations suggest that the putative exocyst subunit, EXO70A1, is involved in cell and
organ morphogenesis. Many phenotypic defects of exo70A1 mutants could be explained by
dysfunction of the exocyst complex.
We raised polyclonal antibodies against several putative subunits of the plant exocyst, EXO70A1,
EXO70G1, SEC3, SEC5, SEC6 and SEC8. Using anti-SEC3, anti-SEC6 and anti-SEC8 antibodies,
respective proteins were localized by indirect immunofluorescence in tips of tobacco pollen tubes.
Furthermore, we have studied intracellular localization of EXO70A1, EXO70G1, SEC6, SEC8 and
SEC10 using transient expression assays of GFP-fusions overexpressed under the 35S promoter in
tobacco leaves. Proteins were localized along plasma membrane, accumulated in invaginations and
elongations, and in the cytoplasm of epidermal cells. EXO70G1-GFP was also partly localized in the
nucleus (Eliáš et al., 2003; Drdová, unpublished).
The yeast two-hybrid screening identified three possible interactors of EXO70A1: tRNA ligase and
two unknown proteins.
We also started to characterize the putative exocyst subunits biochemically, employing different
types of chromatography followed by immunodetection and/or mass spectroscopy. Preliminary
results showed that seven subunits of the predicted exocyst, including EXO70A1, co-fractionate as
a high-molecular weight complex in Arabidopsis thaliana suspension culture.
3) Aims of the project
This project is connected to the finishing project IAA6038410 "The characterization of Exo70
subunit family of tethering complex exocyst in Arabidopsis" that was focused on the multiplicity of
EXO70 gene family in Arabidopsis and on the interaction of EXO70 with other subunits of the
putative plant exocyst complex. The project IAA6038410 revealed EXO70A1 as the main EXO70 in
Arabidopsis. Therefore, we would like to continue detailed study of EXO70A1 plant and cellular
function.
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Our working hypothesis is that EXO70A1 represents a subunit of the plant exocyst complex.
Although the presence of the exocyst in the plant cell has not been published yet, data from our
laboratory and recent articles suggest that plants do very likely have the exocyst complex (Cole et
al., 2005; Wen et al., 2005). More detailed knowledge in EXO70A1 localization, interaction and
functions is crucial to prove our hypothesis.
We intend to compare the complete transcriptome of the exo70A1 mutants to the wild type using
microarray approach. This efficient high-throughput screening should uncover molecular
mechanisms related to EXO70A1 functions.
Employing methods of both GFP fusions and indirect immunofluorescence, we would like to
visualize EXO70A1 and describe its localization within the cell cycle.
We are going to determine the relationship of EXO70A1 and cytoskeleton, because our observation
on mutant roots has suggested a possible interaction. Using split GFP system we would like to
verify possible EXO70A1 interactors.
Last but not least, we are planning to continue characterization of exo70A1 mutants. Our
preliminary data suggest that at least some features of the mutant phenotype result from
deficiency in exocytosis as expected for compromised function of a mutant exocyst subunit.
4) Experimental plan
A) Transcriptome analysis
As the first attempt for transcriptome analysis we will cultivate a large amount of exo70A1 and
wild-type seedlings in darkness. Etiolated mutant seedlings exhibit 30% shorter hypocotyls. Thus,
deregulation of plant morphogenesis caused by EXO70A1 disruption will be pronounced. RNA from
mutant and control 7-day-old seedlings will be extracted and send for commercial microarray
analysis (NASC's International Affymetrix Service) using Arabidopsis DNA chip. Acquired data will
be analyzed. Results gained from this survey might consequently help to specify or justify further
experiments.
B) Localization of EXO70A1
In addition to previously constructed EXO70A1-GFP fusion expressed under the strong 35S
promoter, we intend to prepare an expression construct of EXO70A1-GFP under the EXO70A1
native promoter. We would also like to construct EXO70A1 with GFP inserted in an internal loop of
the protein. These constructs will be used for transient Agrobacterium-mediated expression of
EXO70A1-GFP in tobacco leaves, and stable expression in Arabidopsis. After the previous basic
localization of EXO70A1, we would like to describe its localization within the cell cycle. We will also
transform exo70A1 mutants with these constructs to complement EXO70A1 disruption.
We are also going to visualize EXO70A1 within the cell cycle using indirect immunofluorescence.
The material for immunolocalization will vary from whole-mount Arabidopsis seedlings and
Arabidopsis suspension to pollen tubes. Although we raised a mouse polyclonal anti-EXO70A1
antibody, it will be necessary (due to limited amount of this stock) to produce a new recombinant
EXO70A1, and immunize more mice. In case of high specificity of new anti-EXO70A1 antibody, we
will perform immuno-gold electron microscopy (in collaboration with Prof. Derksen, University of
Nijmegen).
C) Relationship of EXO70A1 to the cytoskeleton
First, using purified recombinant EXO70A1 protein, we plan to perform the Tubulin polymerization
in vitro assay as described in Wang et al. (2004) to reveal possible effect of EXO70A1 on tubulin
polymerization as it has in animal cells.
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Second, will cross exo70A1 mutants to lines expressing MAP4-GFP (a marker of tubulin
cytoskeleton) and to talin-GFP (a marker of actin cytoskeleton) which will provide an insight to
possible changes of the cytoskeleton in exo70A1 mutants.
Third, we intend to use whole-mount immunolocalization to visualize both tubulin and actin
cytoskeleton in mutant seedlings, namely in roots, and compare the pattern to the wild type. We
will also try to perform double labeling of EXO70A1 and cytoskeleton to consider possible
co-localization.
Fourth, we would like to find out causes for the opposite skewing (spirality) of mutant roots.
Therefore, we plan to cultivate plants on vertical agar plates containing microtubule-stabilizing and
microtubule-destabilizing drugs such as taxol, oryzalin, propyzamid, nocodazole (and
actin-destabilizing drug cytochalasin). Phenotype of treated roots could help to recognize the
relationship of EXO70A1 to cytoskeleton. We also intend to observe (by immunolabeling of
cytoskeleton) the recovery of cytoskeleton after a short-time treatment with drugs mentioned
above.
D) EXO70A1 interactors
The yeast two-hybrid screening done previously identified three possible interactors of EXO70A1:
tRNA ligase and two unknown proteins. We would like to obtain Arabidopsis mutants in these
genes, perform a basic analysis and cross them to exo70A1 mutants.
We are planning to take advantage of the split GFP system and involve it in testing for interactions
of EXO70A1 with candidate proteins (incl. putative exocyst subunits). In contrast to the yeast
two-hybrid system, this latest system allows to identify protein interactions within the cytoplasm
and with membrane proteins.
E) Phenotypic analysis
We will continue especially detailed microscopic analysis of exo70A1 mutants, for example stomata
function, stigmatic papillae elongation, root and floral meristem anatomy, length of root cells,
hypocotyl cells and cells of stamen filaments.
Crossing to mutants in other putative plant exocyst subunits (SEC3, SEC6, SEC8, SEC15 and
EXO84) and analysis of hybrid plants is also planned.
5) Time schedule
1st year:
We will start this project with the transcriptome analysis. Constructs for EXO70A1-GFP expression
and for the split GFP system will be prepared and sequenced. We will continuously perform the
detailed phenotypic analysis of the exo70A1 mutants. The exo70A1 mutants will be crossed to
cytoskeleton marker lines and to chosen mutants in other putative exocyst subunits. We will order
and select mutants in the three EXO70A1 interacting genes. All experiments concerning
cytoskeleton drugs will be also performed this year.
2nd year:
We will transform Agrobacteria and consequently Arabidopsis plants (incl. exo70A1 mutants) with
prepared constructs, select transformants and observe EXO70A1-GFP localization and
complementation of EXO70A1 disruption. We will start phenotypic analysis of mutants in EXO70A1
interacting genes, crosses with cytoskeleton marker lines and crosses with mutants in putative
exocyst genes. The split GFP system will be established. New anti-EXO70A1 antibody will be
prepared and EXO70A1 will be localized using indirect immunofluorescence.
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3rd year:
We are going to continue localization of EXO70A1 by both immuno and GFP approach. Phenotypic
analysis will be completed. Work in the last year will be probably adjusted based on the previous
results. Finally, we will finish all experiments and prepare a manuscript.
6) Conditions for the implementation of the project
Mgr. Lukáš Synek will continue research started during his PhD. study in this project. In the
previous study on putative plant exocyst complex, he has obtained experience in techniques of
plant molecular biology, phenotypic analysis, microscopy and transcriptome analysis in silico. He is
going to finish his PhD. study in October 2007.
Ivan Kulich will continue research started during his diploma thesis as a part of his PhD. study. He
is experienced especially in the yeast two-hybrid system, indirect immunofluorescence and
microscopy. He is going to defend his diploma thesis in June 2007.
Implementation of no new experimental methods will be required except for the split GFP system.
All basic procedures of molecular biology (conventional cloning, Gateway cloning, transformation of
plants, DNA-biolistics, immunodetection, indirect immunofluorescence) and Arabidopsis research
have been routinely used in our laboratory. The laboratory is also completely technically equipped
for plant molecular biology research including a fluorescence microscopes and access to laser
scanning confocal microscopes.
7) Collaboration
Our team has collaborated with the laboratory of Prof. Marie-Theres Hauser (University of Natural
Resources and Applied Life Sciences, Vienna) specialized in phenotypic analysis of Arabidopsis
mutants (Synek et al., 2006). Their experience could contribute to the further detailed analysis of
exo70A1 mutants.
Another cooperation has been established with laboratory of Prof. John E. Fowler (Oregon State
University, USA), where other putative exocyst subunits in Arabidopsis has been studied using
genetic approach. Comparison and crossing of exo70A1 to other exocyst mutants could help to
reveal other EXO70A1 functions.
8) Overlap with other projects
There is a relationship but not overlap to the research center LC06034 "Regulation of plant cell and
organ morphogenesis (Remorost)" that is focused on biochemical characterization of the putative
plant exocyst complex and on functional characterization of other putative subunits (SEC3, SEC5,
SEC6, SEC15 and EXO84).
9) Expected outcome
Preliminary results will be presented at an international conference every year. Final results of this
project will be published in at least one scientific article in an internationally recognized
peer-reviewed journal, and will contribute to the basic plant cell science.
10) References
Adamo, J.E., Rossi, G. and Brennwald, P. (1999) The Rho GTPase Rho3 has a direct role in
exocytosis that is distinct from its role in actin polarity. Mol. Biol. Cell, 10, 4121-4133.
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Boyd, C., Hughes, T., Pypaert, M. and Novick, P. (2004) Vesicles carry most exocyst subunits to
exocytic sites marked by the remaining two subunits, Sec3p and Exo70p. J. Cell. Biol., 167,
889-901.
Cannon, S.B., Mitra, A., Baumgarten, A., Young, N.D. and May, G. (2004) The roles of segmental
and tandem gene duplication in the evolution of large gene families in Arabidopsis thaliana. BMC
Plant Biol., 4, 10.
Cole, R.A., Synek, L., Žárský, V. and Fowler, J.E. (2005) SEC8, a subunit of the putative
Arabidopsis exocyst complex, facilitates pollen germination and competitive pollen tube growth.
Plant Physiol., 138, 2005-2018.
Cvrčková, F., Eliáš, M., Hála, M., Obermeyer, G. and Žárský, V. (2001) Small GTPases and
conserved signalling pathways in plant cell morphogenesis: From exocytosis to Exocyst. In Cell
Biology of Plant and Fungal Tip Growth (Geitmann, A. and Cresti, M., eds). Amsterdam: IOS Press,
pp. 105-122.
Dong, G., Hutagalung, A.H., Fu, C., Novick, P. and Reinisch, K.M. (2005) The structures of exocyst
subunit Exo70p and the Exo84p C-terminal domains reveal a common motif. Nat. Struct. Mol. Biol.,
12, 1094-1100.
Eliáš, M., Drdová, E., Žiak, D., Bavlnka, B., Hála, M., Cvrčková, F., Soukupová, H. and Žárský, V.
(2003) The exocyst complex in plants. Cell Biol. Int., 27, 199-201.
Fielding, A.B., Schonteich, E., Matheson, J., Wilson, G., Yu, X., Hickson, G.R., Srivastava, S.,
Baldwin, S.A., Prekeris, R. and Gould, G.W. (2005) Rab11-FIP3 and FIP4 interact with Arf6 and the
Exocyst to control membrane traffic in cytokinesis. EMBO J., 24, 3389-3399.
Guo, W., Grant, A. and Novick, P. (1999) Exo84p is an exocyst protein essential for secretion. J.
Biol. Chem., 274, 23558-23564.
Hamburger, Z.A., Hamburger, A.E., West, A.P. and Weis, W.I. (2006) Crystal structure of the S.
cerevisiae exocyst component Exo70p. J. Mol. Biol., 356, 9-21.
Hsu, S.C., Hazuka, C.D., Foletti, D.L., Heuser, J. and Scheller, R.H. (1998) Subunit composition,
protein interactions and structures of the mammalian brain sec6/8 complex and septin filaments.
Neuron, 20, 1111-1122.
Hsu, S.C., Hazuka, C.D., Foletti, D.L. and Scheller, R.H. (1999) Targeting vesicles to specific sites
on the plasma membrane: The role of the sec6/8 complex. Trends Cell Biol., 9, 150-153.
Jurgens, G. and Geldner, N. (2002) Protein secretion in plants: from the trans-Golgi network to the
outer space. Traffic, 3, 605-613.
Kee, Y., Yoo, J.S., Hazuka, C.D., Peterson, K.E., Hsu, S.C. and Scheller, R.H. (1997) Subunit
structure of the mammalian exocyst complex. Proc. Natl Acad. Sci. USA, 94, 14438-14443.
Matern, H.T., Yeaman, C., Nelson, W.J. and Scheller, R.H. (2001) The Sec6/8 complex in
mammalian cells: characterization of mammalian Sec3, subunit interactions, and expression of
subunits in polarized cells. Proc. Natl Acad. Sci. USA, 98, 9648-9653.
Otegui, M.S. and Staehelin, L.A. (2004) Electron tomographic analysis of post-meiotic cytokinesis
during pollen development in Arabidopsis thaliana. Planta, 218, 501-515.
Robinson, N.G., Guo, L., Imai, J., Toh-E, A., Matsui, Y., Tamanoi, F. (1999) Rho3 of Saccharomyces
cerevisiae, which regulates the actin cytoskeleton and exocytosis, is a GTPase which interacts with
Myo2 and Exo70. Mol. Cell. Biol. 19: 3580-3587.
Roumanie, O., Wu, H., Molk, J.N., Rossi, G., Bloom, K. and Brennwald, P. (2005) Rho GTPase
regulation of exocytosis in yeast is independent of GTP hydrolysis and polarization of the exocyst
complex. J. Cell Biol., 170, 583-594.
Segui-Simarro, J.M., Austin J.R 2nd, White, E.A. and Staehelin, L.A. (2004) Electron tomographic
analysis of somatic cell plate formation in meristematic cells of Arabidopsis preserved by
high-pressure freezing. Plant Cell, 16, 836-856.
Synek, L., Schlager, N., Eliáš, M., Quentin, M., Hauser, M.T., Žárský, V. (2006) AtEXO70A1, a
member of a family of putative exocyst subunits specifically expanded in land plants, is important
for polar growth and plant development. Plant Journal, 48, 54-72.
TerBush, D.R., Maurice, T., Roth, D. and Novick, P. (1996) The Exocyst is a multiprotein complex
Poslední změna: 19.4.2007 14:20:01
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required for exocytosis in Saccharomyces cerevisiae. EMBO J., 15, 6483-6494.
TerBush, D.R. and Novick, P. (1995) Sec6, Sec8, and Sec15 are components of a multisubunit
complex which localizes to small bud tips in Saccharomyces cerevisiae. J. Cell Biol., 130, 299-312.
Vega, I.E. and Hsu, S.C. (2001) The exocyst complex associates with microtubules to mediate
vesicle targeting and neurite outgrowth. J. Neurosci., 21, 3839-3848.
Wang, H., Tang, X. and Balasubramanian, M.K. (2003) Rho3p regulates cell separation by
modulating exocyst function in Schizosaccharomyces pombe. Genetics, 164, 1323-1331.
Wang, S., Liu, Y., Adamson, C.L., Valdez, G., Guo, W., Hsu, S.C. (2004) The mammalian exocyst, a
complex required for exocytosis, inhibits tubulin polymerization. J. Biol. Chem. 279: 35958-66.
Wen, T.J., Hochholdinger, F., Sauer, M., Bruce, W. and Schnable, P.S. (2005) The roothairless1
gene of maize encodes a homolog of sec3, which is involved in polar exocytosis. Plant Physiol.,
138, 1637-1643.
Yeaman, C., Grindstaff, K.K. and Nelson, W.J. (2004) Mechanism of recruiting Sec6/8 (exocyst)
complex to the apical junctional complex during polarization of epithelial cells. J. Cell Sci., 117,
559-570.
Zimmermann, P., Hirsch-Hoffmann, M., Hennig, L. and Gruissem, W. (2004) GENEVESTIGATOR.
Arabidopsis microarray database and analysis toolbox. Plant Physiol., 136, 2621-2632.
Poslední změna: 19.4.2007 14:20:01
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Grant Agency of the Academy of Sciences of the Czech Republic
Sheet C1/I
Curriculum vitae
Mgr. Lukáš Synek
Ústav experimentální botaniky AV ČR, v. v. i.
Mgr. Lukáš Synek
Curriculum Vitae
born on 22. 8. 1979 in Příbram
Education:
1997 – 2002
Charles University in Prague, Faculty of Sciences, specialization in molecular biology and virology,
Diploma thesis theme: "Interaction of the major capsid protein, VP1, of mouse polyoma virus with
cell structures in Saccharomyces cerevisiae."
2002
Master Degree, cum laudum
since 2002
Charles University in Prague, Faculty of Sciences, specialization in plant molecular biology, PhD
thesis theme: "Characterization of chosen subunits of the putative plant exocyst komplex")
2007
PhD thesis submitted
Courses:
2003 - Training course in methods of indirect immunofluorescence (Dr. František Baluška,
Universität Bonn, Germany)
2003 - Training course in electron microscopy (Prof. Jan Derksen, University of Nijmegen, The
Netherlands)
International conferences:
XI. International Conference on Plant Embryology, 1. - 3. 9. 2003, Brno, Czech Republic (poster
presentation)
13th International Workshop on Plant Membrane Biology, 6. - 10. 7. 2004, Monpellier, France
(poster presentation)
17th International Botanical Congress, 17. – 23. 7. 2005, Wien (poster presentation)
Activities:
1997 - 8th place in the National Olympiad in Biology
Poslední změna: 19.4.2007 14:20:01
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since 2002 - employed in the Institute of Experimental Botany ASCR
2004 - principal investigator of the FRVŠ project "Localization of proteins regulating secretion in
plants by the indirect immunofluorescence"
Publications:
Synek, L., Schlager, N., Eliáš, M., Quentin, M., Hauser, M.T., Žárský, V. (2006). AtEXO70A1, a
member of a family of putative exocyst subunits specifically expanded in land plants, is important
for polar growth and plant development. Plant Journal, 48: 54 - 72.
Cole, R.A., Synek, L., Žárský, V., Fowler, J.E. (2005). SEC8, a subunit of the putative Arabidopsis
exocyst complex, facilitates pollen germination and competitive pollen tube growth. Plant
Physiology 138: 2005 - 2018.
Žárský, V., Eliáš, M., Drdová, E., Synek, L., Quentin, M., Kakešová, Z., Žiak, D., Hála, M.,
Soukupová, H. (2004): Do exocyst subunits in plants form a complex? Acta Physiol. Plant., 26:
146.
Synek, L. (2003): Struktura a funkce mitotického aparátu Saccharomyces cerevisiae. Biologické
listy 68 (1).
Poslední změna: 19.4.2007 14:20:01
15/23
Grant Agency of the Academy of Sciences of the Czech Republic
Sheet C2/I
Results of previous funding by GA AV
Name of the scientist:
Lukáš Synek
Duration
Registration No.
Investigator
IAA6038410
Viktor Žárský
(years)
4
Last year of the project
2007
Title of the project
The characterization of Exo70 subunit family of tethering complex exocyst in Arabidopsis
Summary of results, including references to publications
Among six mutants in EXO70 genes only exo70A1 mutants showed a discernible phenotype. They
are small, show a loss of apical dominance, indeterminate growth and dramatically reduced
fertility. Polar growth of root hairs and stigmatic papillae is disturbed. These results, together with
expression data, suggest that EXO70A1 is involved in plant morphogenesis, and is probably the
main EXO70 in Arabidopsis. EXO70A1 was preliminary localized (antibody, GFP fusion) along the
plasma membrane and in the cytoplasm of tobacco epidermal cells and in Arabidopsis roots. In the
yeast 2-hybrid screening, three possible interactors of EXO70A1 were identified (tRNA ligase and
two unknown proteins). Preliminary results showed that seven subunits of the predicted exocyst,
including EXO70A1, co-fractionate as a high-molecular weight complex in Arabidopsis thaliana
suspension culture. Synek, L., Schlager, N., Eliáš, M., Quentin, M., Hauser, M.T., Žárský, V.
(2006). AtEXO70A1, …. Plant J., 48: 54-72.
Poslední změna: 19.4.2007 14:20:01
16/23
Grantová agentura Akademie věd ČR
List D
Identifikační kód KJB600380802
Návrhy projektů a projekty související s předloženým
návrhem
1. Současně s předloženým návrhem projektu je nebo bude v letošním roce podána u jiného
poskytovatele žádost o podporu projektu, který je buď shodný, nebo se s předkládaným
návrhem výrazně tematicky překrývá, takže v případě podpory obou projektů by došlo
k duplicitnímu financování:
Název projektu
Transkriptom mutanta exo70A1 a buněčná funkce EXO70A1, podjednotky komplexu exocyst, u
Arabidopsis thaliana
Jméno pracovníka, který zpracoval
Doba řešení
Poskytovatel, u kterého je (bude)
návrh překrývajícího se projektu
Uchazeč
projektu
podána žádost o podporu
Mgr. Lukáš Synek
Ústav experimentální
od 2008
Grantová agentura České
botaniky, v.v.i. AVČR
do 2010
republiky (postdoktorské
projekty)
Název projektu
Jméno pracovníka, který zpracoval
návrh překrývajícího se projektu
Uchazeč
Doba řešení
Poskytovatel, u kterého je (bude)
projektu
podána žádost o podporu
od
do
2. Probíhající projekty na jejichž řešení se předkladatelé návrhu podílejí a které řeší obdobnou
problematiku (předpokládá se, že část výsledků bude shodných).
Identifikační kód a název projektu
LC06034 Regulation of plant cell and organ morphogenesis (Remorost)
Doba řešení
Jméno řešitele
Uchazeč
projektu
Poskytovatel
Eva Zažímalová
Ústav experimentální
od 2006
MŠMT ČR
botaniky, v.v.i., AVČR
do 2010
Název projektu
Jméno pracovníka, který zpracoval
návrh překrývajícího se projektu
Uchazeč
Doba řešení
Poskytovatel, u kterého je (bude)
projektu
podána žádost o podporu
od
do
Poslední změna: 19.4.2007 14:20:01
17/23
Grant Agency of the Academy of Sciences of the Czech Republic
Sheet F1/I
Financial Proposal
Principal investigator (I): Mgr. Lukáš Synek
Institution name:
Ústav experimentální botaniky AV ČR, v. v. i.
COST OF INVESTMENTS
We do not ask any investments.
MATERIAL COST
Operating cost
Materials
Materials comprise of expenses for bacteria and Arabidopsis cultivation media, selection antibiotics,
polymerases for cloning and plant genotyping, restriction endonucleases for conventional cloning,
BP and LR clonases for Gateway cloning, new destination vectors for the Gateway system, isolation
kits for DNA and RNA, DNA purification kits, oligonucleotide synthesis, microtubule and actin
destabilizing drugs. Further operating cost will cover laboratory plastics and consumption material
for microscopy. We also intend to order several insertional Arabidopsis mutants (SALK Institute).
Overhead
Overhead represents 15% of total material cost.
Social and medical insurances
Social and medical insurances represent 37% of total labor cost.
Services
In the first year, larger amount of total material cost will be devoted to services because we intend
to analyze transcriptome of the exo70A1 mutant. This service is commercially available in NASC's
International Affymetrix Service. Further, services comprise of payment for sequencing of prepared
constructs, printing of a poster for a conference.
Traveling cost
Traveling cost covers registration fee and (partially) traveling expenses for a scientific conference
where our preliminary results will be presented. It is not possible to specify a particular conference
yet, however, we consider the International Workshop on Plant Membrane Biology for the first
year.
LABOR COST
Poslední změna: 19.4.2007 14:20:01
18/23
The principle investigator does not ask salary, due to a participation in the LC 06034 project. Labor
cost covers bonus for the principle investigator, salary (0.25 work load) for the co-investigator and
other personal expenses for technical stuff (approx. 80 hours per year) for occasional cultivation of
a large amount of experimental plants.
Poslední změna: 19.4.2007 14:20:01
19/23
Grantová agentura Akademie věd ČR
List F2/I
Identifikační kód KJB600380802
Požadované finanční zabezpečení
na 1. rok řešení grantového projektu
(náklady se uvádějí v tis. Kč)
Uchazeč (I) - ÚEB
1. investiční náklady (GIN)
jednotlivé investiční položky
pořizovací cena
doba provozně
technické funkce
požadováno
celkem investiční náklady na 1. rok
2. věcné náklady (GVN)
provozní náklady
požadováno
- drobný dlouhodobý hmotný majetek (předměty, přístroje a zařízení do 40 tis. Kč)
- drobný dlouhodobý nehmotný majetek (např. software do 60 tis. Kč)
- materiál
35
- doplňkové (režijní) náklady
25
- povinné zákonné odvody
30
- jiné: (specifikovat, např. speciální literatura)
služby (na faktury)
60
cestovní náklady (včetně konferenčních poplatků a úhrady za pobyt pozvaných
pracovníků)
celkem věcné náklady na 1. rok
Poslední změna: 19.4.2007 14:20:01
17
167
20/23
List F2/I
3. mzdové náklady
3.1. platy (mzdy) požadované od GA AV (TMZ)
příjmení
tarifní roční plat
Kulich
požadováno
180
celkem mzdové náklady na platy na 1. rok
45
45
3.2. pohyblivá část mzdy (PMZ)
celkem pohyblivá část mzdy na 1. rok
30
3.3. ostatní osobní náklady (OON)
specifikace OON
požadováno
kultivace rostlin
6
celkem ostatní osobní náklady na 1. rok
6
Poslední změna: 19.4.2007 14:20:01
21/23
Grantová agentura Akademie věd ČR
List F3
Identifikační kód KJB600380802
Celkové předpokládané náklady na řešení
grantového projektu
(náklady se uvádějí v tis. Kč)
1. Účelová podpora požadovaná od GA AV
Účelová podpora požadovaná od GA AV
Investiční
Neinvestiční náklady
náklady GIN
GVN
TMZ
Celkem GA
PMZ
OON
AV
1. rok
0
167
45
30
6
248
2. rok
----
167
45
30
6
248
3. rok
----
167
45
30
0
242
4. rok
----
5. rok
----
501
135
90
12
738
celkem
0
2. Celkové předpokládané uznané náklady
Zdroje finančních prostředků
Účelová podpora požadovaná od GA AV celkem
1. rok
2. rok
248
3. rok
248
4. rok
5. rok
Celkem
242
738
Veřejné prostředky z ostatních zdrojů, nepatřící do státního rozpočtu
0
0
0
0
Neveřejné prostředky z ostatních zdrojů (např. vlastní prostředky u soukromých subjektů)
0
0
0
0
Celkové předpokládané uznané náklady na řešení projektu
248
248
Poslední změna: 19.4.2007 14:20:01
242
22/23
0
0
738
Grantová agentura Akademie věd ČR
List F3/I
Identifikační kód KJB600380802
Rozpis celkových předpokládaných nákladů na
řešení grantového projektu
(náklady se uvádějí v tis. Kč)
Uchazeč (I) - ÚEB
1. Účelová podpora požadovaná od GA AV na řešení projektu rozepsaná na jednotlivé roky
řešení (shrnutí finančních požadavků zdůvodněných na Sheet F1)
Účelová podpora požadovaná od GA AV
Investiční
Neinvestiční náklady
náklady GIN
GVN
TMZ
Celkem GA
PMZ
OON
AV
1. rok
0
167
45
30
6
248
2. rok
----
167
45
30
6
248
3. rok
----
167
45
30
0
242
4. rok
----
5. rok
----
501
135
90
12
738
celkem
0
2. Celkové předpokládané uznané náklady na celou dobu řešení projektu ze všech zdrojů
financování
Zdroje finančních prostředků
Účelová podpora požadovaná od GA AV celkem
1. rok
2. rok
248
3. rok
248
4. rok
5. rok
Celkem
242
738
Veřejné prostředky z ostatních zdrojů, nepatřící do státního rozpočtu
0
0
0
0
Neveřejné prostředky z ostatních zdrojů (např. vlastní prostředky u soukromých subjektů)
0
0
0
0
Celkové předpokládané uznané náklady na řešení projektu
248
248
Poslední změna: 19.4.2007 14:20:01
242
23/23
0
0
738

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