Download Functions of the exocyst complex in secretion and cell wall biogenesis

Functions of the exocyst complex in secretion and cell
wall biogenesis
Nemanja Vukašinović
Charles University in Prague
Faculty of Science
Department of Experimental Plant Biology
Summary
Ph.D. Thesis
2016
ABSTRACT
The mechanical strength of plant tissues and organs can be attributed to
specific properties of the cell wall. In many cases, in order to establish their final shape,
cells deposit various cell wall materials in a localized manner. This is achieved by highly
organized action of the endomembrane system which is essential for biosynthesis and
secretion of cell wall proteins and polysaccharides. The exocyst complex is a conserved
tethering complex in eukaryotes and it is involved in tethering of secretory vesicles to the
sites of secretion at the plasma membrane. In this study, we address several aspects of the
plant exocyst complex architecture and cell wall development using molecular biology
techniques and advanced confocal microscopy. We demonstrated that two SEC10 exocyst
subunits are present in Arabidopsis thaliana and share redundant functions. We also
showed that the architecture of the plant exocyst complex shares several structural
features with the yeast one. We demonstrated the importance of the functional EXO84b
exocyst subunit for normal tracheary element development and showed that the main
constituents of the secondary cell walls are deposited normally in exocyst mutants. We
described a clear difference in the exocyst microtubule-independent dynamics in
epidermal cells vs. cell type specific microtubule-driven exocyst recruitment in
developing tracheary elements. In Arabidopsis pollen tubes, we showed that the depletion
of EXO70C1 and EXO70C2 isoforms leads to a complete male-specific transmission
defect due to inefficient pollen tube growth. Both proteins localize in the cytoplasm of
pollen grains, pollen tubes and root hair cells. We suggest that EXO70C1/C2 may not act
as subunits of the exocyst complex but rather acquired a different function as regulators
of polar growth. Taken together, our findings extend the current knowledge about the
exocyst complex in plant cells and further support its role in targeted secretion.
TABLE OF CONTENTS
1. INTRODUCTION .......................................................................................................... 1
1.1. The plant cell wall .............................................................................. 1
1.2. Role of the endomembrane system in the plant cell wall deposition . 1
1.3. The exocyst complex .......................................................................... 2
2. AIMS OF THE THESIS ................................................................................................ 3
3. RESULTS ........................................................................................................................ 4
3.1. PAPER No. 2 ...................................................................................... 4
3.2. PAPER No. 3 ...................................................................................... 5
3.3. PAPER No. 4 ...................................................................................... 5
4. CONCLUSIONS ............................................................................................................ 6
5. REFERENCES ............................................................................................................... 7
6. APPENDIX ..................................................................................................................... 9
1. INTRODUCTION
1.1. The plant cell wall
Plants would be unable to achieve their large variety of shapes and forms
without their exoskeleton – the cell wall. The presence of cell wall also underlies the
most basic physiological principles in plant bodies. The plant cell wall is a highly
complex and surprisingly dynamic structure that is constantly modulated in response to
biotic and abiotic signals from environment. Thanks to its stiffness and elasticity, it
allows the cell to grow without bursting, by resisting turgor pressure generated by
expanding central vacuole, on one side, and allows the cell to expand, on the other. The
three main groups of carbohydrates within the cell walls (cellulose, hemicelluloses and
pectins) are able to interact in covalent and non-covalent manner, in order to form strong
and extensible networks. Cell wall structural proteins and remodeling enzymes represent
another layer of complexity within the cell walls, because the former can affect wall
strength, assembly, hydration and permeability while the latter can reshape carbohydrates
by creating and breaking bonds between molecules or by adding and removing different
chemical modifications (Cosgrove, 2005). Inspite of intensive research, the molecular
mechanisms of cellulose and matrix polysaccharides deposition to the apoplast remain
mostly elusive.
1.2. Role of the endomembrane system in the plant cell wall deposition
Plant cells are encircled by cell walls which prevent their migration, and once
the cell expansion is finished and the cell wall maturated, the final cell shape is locked. It
means that morphogenesis is highly dependent on precise regulation of cell divisions and
growth that both rely on correct deposition of cell wall building blocks and their
modulators. Some cell types, in order to fulfill their functions, build special types of cell
walls with complicated morphology and the cell wall deposition must be precisely
localized. Best examples are phloem sieve elements and xylem vessels with sieve plates
and spiral thickenings, respectively, or Casparian strips of endodermal cells. Highly
polarized delivery of the cell wall material is also essential in cells exhibiting tip growth,
such as pollen tubes or root hairs, to meet their extreme demands for fast cell wall
synthesis at one specific site.
A common feature of all these processes is that the cell wall material needs to
be secreted in the right place at the right time, which is orchestrated by the vesicle
trafficking machinery. Secretory vesicles originate from the endomembrane system
where they move in two main directions: 1) Anterograde pathway, starts at the
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endoplasmic reticulum (ER), goes through the Golgi apparatus and ends at the plasma
membrane (PM) where vesicles deliver their cargo or PM proteins. 2) Retrograde
pathway starts with endocytotic vesicles that fuse with trans-Golgi network/early
endosome (TGN/EE) compartment and ends with the delivery of the endocytosed cargo
to the vacuole (Bassham et al., 2008). There are multiple crosstalk events between these
two pathways that enable the eukaryotic cell to continuously maintain its homeostasis
(Wu et al., 2013).
At the molecular level, the vesicle trafficking includes a vesicle budding from a
donor compartment that involves different coat proteins (e.g. COPI, COPII, clathrin).
Once the vesicle reaches its final destination, it is tethered through activated RAB
GTPases and their effectors (e.g. tethering complexes). Finally, SNAREs (Soluble Nethylmaleimide-sensitive Factor Attachment Protein Receptors) mediate the fusion of the
vesicle with the target membrane, and its cargo is released into the target compartment.
1.3. The exocyst complex
The exocyst is an octameric complex mediating tethering of secretory vesicles
to the PM, involved intimately also in the regulation of cell polarity (He and Guo, 2009;
Heider and Munson 2012). Its eight subunits (Sec3, Sec5, Sec6, Sec8, Sec10, Exo70 and
Exo84) form a stable complex in yeast (Heider at al., 2016). Yeast and mammalian Sec3
and Exo70 subunits interact directly with the activated RHO GTPases and membrane
lipids and are proposed to serve as landmarks for delivery of secretory vesicles to a
specific PM domain (Boyd et al., 2004; Wu et al., 2008; Pleskot et al., 2015) Rab
GTPases Sec4 (in yeast) or RAB11 (in animals) and v-SNARE Snc2 interact with Sec15
and Sec6 respectively, to link the complex to the secretory vesicle (Guo et al., 1999; Shen
et al., 2013).
Due to its engagement in the final stages of exocytosis, i.e. also in the cell wall
biogenesis, plant cell cytokinesis and defence, the plant exocyst complex has attracted
more attention as compared to other tethering complexes in plants. The first Arabidopsis
genome analysis (Eliáš et al., 2003) immediately indicated a peculiar feature of the land
plant exocyst complex - a multiplicity of EXO70 paralogs with more than twenty-three
genes in Arabidopsis and forty genes encoded by the rice genome (Cvrčková et al., 2012).
The first phenotypic analyses of loss of function exocyst subunit mutants in Arabidopsis
and maize clearly supported the expected involvement of the exocyst in the targeting of
secretory vesicles that regulate plant cell polarity (Cole et al., 2005; Wen et al., 2005;
Synek et al., 2006). The plant exocyst was partially purified and shown to exist as a
biochemical entity with all eight basic subunits present (Hála et al., 2008; Fendrych et al.,
2010). Over the last ten years its participation has been documented in many exocytosisrelated processes in plants (Žárský et al., 2009, Žárský et al., 2013). Reviewed also in the
Paper No1. Of this thesis (Vukašinović and Žárský, 2016).
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2. AIMS OF THE THESIS
The exocyst complex is an important regulator of cell polarity and secretion. In
this thesis, following questions about the plant exocyst architecture and aspects of its
involvement in xylem development and pollen tube growth are addressed:
- How many SEC10 exocyst subunit isoforms are present in Arabidopsis
thaliana and if more than one, do they have redundant functions? What is the position of
the SEC10 within the plant exocyst complex?
- What is the role of the exocyst complex during the secondary cell wall
deposition in xylem? What molecular mechanisms underlie the targeting of the exocyst
complex to the plasma membrane in xylem cells?
- What EXO70 isoforms are involved in pollen tube growth in Arabidopsis?
What is the subcellular localization of the dominant EXO70 isoforms in growing pollen
tubes and how do they interact with other exocyst subunits?
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3. RESULTS
3.1. PAPER No. 2
Dissecting a Hidden Gene Duplication: The Arabidopsis thaliana SEC10 Locus
Vukašinović, N., Cvrčková, F., Eliáš, M., Cole, R., Fowler, J. E., Žárský, V., & Synek, L.
(2014). PloS One, 9(4), e94077.
Summary: Repetitive sequences present a challenge for genome sequence
assembly, and highly similar segmental duplications may disappear from assembled
genome sequences. Having found a surprising lack of observable phenotypic deviations
and non-Mendelian segregation in Arabidopsis thaliana mutants in SEC10, a gene
encoding a core subunit of the exocyst tethering complex, we examined whether this
could be explained by a hidden gene duplication. Re-sequencing and manual assembly of
the Arabidopsis thaliana SEC10 (At5g12370) locus revealed that this locus, comprising a
single gene in the reference genome assembly, indeed contains two paralogous genes in
tandem, SEC10a and SEC10b, and that a sequence segment of 7 kb in length is missing
from the reference genome sequence. Differences between the two paralogs are
concentrated in non-coding regions, while the predicted protein sequences exhibit 99%
identity, differing only by substitution of five amino acid residues and an indel of four
residues. Both SEC10 genes are expressed, although varying transcript levels suggest
differential regulation. Homozygous T-DNA insertion mutants in either paralog exhibit a
wild-type phenotype, consistent with proposed extensive functional redundancy of the
two genes. By these observations we demonstrate that recently duplicated genes may
remain hidden even in well-characterized genomes, such as that of A. thaliana. Moreover,
we show that the use of the existing A. thaliana reference genome sequence as a guide for
sequence assembly of new Arabidopsis accessions or related species has at least in some
cases led to error propagation.
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3.2. PAPER No. 3
Cell Type Specific Microtubule-Dependent Targeting of the Exocyst Complex in
Xylem
Nemanja Vukašinović, Yoshihisa Oda, Přemysl Pejchar, Lukáš Synek, Tamara
Pečenková, Anamika Rawat, Juraj Sekereš and Viktor Žárský
Summary: Cortical microtubules (MTs) play the major role in patterning of
secondary cell wall (SCW) thickenings in tracheary elements (TE) by determining the
sites of SCW deposition. EXO70A1 subunit of exocyst secretory-vesicles tethering
complex was implicated to be important for TE development via VETH1/2 and COG2
MT interaction. We investigated subcellular localization of several core exocyst subunits
in xylem and proposed a mechanism for exocyst recruitment to the sites of SCW
deposition. Live cell imaging of fluorescently tagged exocyst subunits in TE using
spinning disk and laser-scanning confocal microscopy was performed and yeast twohybrid system and FRET analyses were used to confirm interaction between COG2 and
core exocyst subunits. In TEs all exocyst subunits localized to the sites of SCW
deposition in a microtubule-dependent manner. We propose that the mechanism of
exocyst targeting to microtubules involves a direct interaction of several exocyst subunits
with COG2-VETH1/2 complex. Additionally, we demonstrate the importance of a
functional EXO84b for normal TE development and show that the three main
constituents of the SCWs are normally deposited in exocyst mutants. We conclude that
the exocyst complex is an important factor bridging the pattern defined by cortical
microtubules with localized secretion of the SCW in TEs.
3.3. PAPER No. 4
EXO70C2, a putative exocyst subunit, is responsible for efficient pollen tube growth
in Arabidopsis
Lukáš Synek, Nemanja Vukašinović, Michal Hála, Matyáš Fendrych, Viktor Žárský
Summary: The exocyst tethering complex as an effector of small GTPases is
involved in regulation of targeted exocytosis which underlies polarized growth in
eukaryotic cells. In plants, several exocyst subunits are encoded by double or even
multiple genes (e.g. the EXO70 family). Here, we inspected Arabidopsis EXO70
isoforms that are expressed in the male gametophyte. Genetic and microscopic analyses
of mutants in putative pollen EXO70 genes (EXO70A2, C1, C2, F1, H3, H5, H6) revealed
that an exo70C2 mutant exhibits a significant male-specific transmission defect due to
aberrant pollen tube growth. A pollen-specific knock-down of EXO70C1 (the closest
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paralog to EXO70C2) in the exo70C2 mutant background resulted in a complete
transmission defect. Both EXO70C1 and EXO70C2, tagged with GFP and expressed
under their native promoters, showed comparable localization in the cytoplasm of pollen
grains, pollen tubes, and root trichoblast cells. We conclude that EXO70C2 is a key factor
for the efficient pollen tube growth, while EXO70C1 plays a partially redundant function
to EXO70C2.
4. CONCLUSIONS
The Arabidopsis thaliana genome was the first plant genome to be fully
sequenced and assembled. However, even such a well characterized genome contains
errors caused by imperfect computational assembling at regions of tandem duplications.
Gene duplication events are common in plant evolution and that also applies for the genes
coding for subunits of the exocyst. Initiated by puzzling outputs from genetic analysis of
sec10 exocyst subunit mutants, we re-sequenced and manually assembled the SEC10
locus of A. thaliana and discovered that it contains two genes in fact - SEC10a and
SEC10b, instead of a sole gene in the current genome annotation. We showed that the two
proteins, which exhibit a very high degree of sequence identity, share the same set of
interactors within the exocyst complex. Since also the expression patterns and subcellular
localization of the two proteins are identical, we conclude that SEC10a and SEC10b act
redundantly as interchangeable parts of the exocyst complex.
Based on studies of physical interactions among exocyst subunits, together
with the localization analyses of exocyst subunits in different exocyst mutant
backgrounds, we suggest that the plant exocyst complex is organized in two relatively
stable modules similarly as proposed in yeast cells (Heider et al., 2016): SEC3-SEC5SEC6-SEC8 and SEC10-SEC15-EXO70-EXO84 binding one another.
The EXO70A1 exocyst subunit was shown to play a role in xylem
development in several studies. Therefore, we tested the involvement of other exocyst
subunits in this process, and we propose that whole exocyst complex localizes to the sites
of secondary cell wall deposition at the plasma membrane. Exocyst subunits are observed
there in static foci co-aligned with cortical microtubules. Unlike microtubule-independent
exocyst targeting in rhizodermal cells, we uncovered a strong xylem-specific dependence
on microtubule cytoskeleton in the exocyst recruitment. This association coincides with
the start of differentiation of the tracheary elements as documented using an advanced
inducible system for tracheary-element differentiation. In comparison to exocyst foci in
root epidermal cells, the foci in developing xylem cells have much longer dwelling time,
probably reflecting a higher demand for cell wall material secretion in cells generating
secondary cell walls. We also described a novel phenotype in exo84b-1 mutants:
interrupted protoxylem cells with considerably thinner cell walls in comparison to the
wild-type morphology. All three major constituents of the secondary cell wall (cellulose,
lignin and xylans) were deposited normally in xylem cell walls of exocyst mutants,
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therefore further research will be needed for the molecular identification of exocystmediated cargo that is delivered to secondary cell walls. Finally, we propose a model of
the exocyst recruitment to the sites of secondary cell wall deposition through a direct
interaction of several exocyst subunits with the COG2/VETH1/2 complex. Together,
these data indicate the the exocyst complex is essential for normal xylem development.
The importance of the exocyst complex for pollen tube growth has been well
documented. However, previous studies were focused the “core” exocyst subunits. Here,
we inspected putative pollen EXO70 isoforms for their possible role in pollen tube
growth. EXO70C1 and EXO70C2 paralogs emerged as dominant regulators of the polar
growth, since their simultaneous knock-down resulted in a complete transmission defect
of mutant alleles via pollen. Surprisingly, these EXO70s do not localize to the plasma
membrane at the apex a growing pollen tube, unlike SEC8 and SEC10a exocyst subunits.
These findings, together with the fact that EXO70C2 does not physically interact with
other exocyst subunits, suggest that they may function outside of the exocyst complex,
perhaps as regulators rather than direct mediators of vesicle tethering. Despite the
molecular mechanism of their action remains to be elucidated, we conclude that
EXO70C1 and EXO70C2 are key factors for the efficient pollen tube tip growth.
5. REFERENCES
Bassham, D. C., Brandizzi, F., Otegui, M. S., & Sanderfoot, A. A. (2008). The secretory system of
Arabidopsis. Arabidopsis Book, e0116.
Boyd, C., Hughes, T., Pypaert, M., & 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.
Cole, R. A., Synek, L., Zarsky, V., & Fowler, J. E. (2005). SEC8, a subunit of the putative Arabidopsis
exocyst complex, facilitates pollen germination and competitive pollen tube growth. Plant Physiol, 138.
Cosgrove, D. J. (2005). Growth of the plant cell wall. Nat Rev Mol Cell Biol, 6, 850-861.
Cvrčková, F., Grunt, M., Bezvoda, R., Hala, M., Kulich, I., Rawat, A., & Zarsky, V. (2012). Evolution of
the Land Plant Exocyst Complexes. Front Plant Sci, 3, 159.
Elias, M., Drdova, E., Ziak, D., Bavlnka, B., Hala, M., Cvrckova, F., Soukupova, H., & Zarsky, V. (2003).
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Fendrych, M., Synek, L., Pečenková, T., Toupalová, H., Cole, R., Drdová, E., Nebesářová, J., Šedinová,
M., Hála, M., Fowler, J. E., & Žárský, V. (2010). The Arabidopsis exocyst complex is involved in
cytokinesis and cell plate maturation. Plant Cell, 22, 3053-3065.
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targeting secretory vesicles to sites of exocytosis. EMBO J, 18, 1071-1080.
Hála, M., Cole, R., Synek, L., Drdová, E., Pečenková, T., Nordheim, A., Lamkemeyer, T., Madlung, J.,
Hochholdinger, F., Fowler, J. E., & Žárský, V. (2008). An exocyst complex functions in plant cell growth
in Arabidopsis and tobacco. Plant Cell, 20, 1330-1345.
He, B. & Guo, W. (2009). The exocyst complex in polarized exocytosis. Curr Opin Cell Biol, 21, 537-542.
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Heider, M. R., & Munson, M. (2012). Exorcising the exocyst complex. Traffic, 13, 898-907.
Heider, M. R., Gu, M., Duffy, C. M., Mirza, A. M., Marcotte, L. L., Walls, A. C., Farrall, N., Hakhverdyan,
Z., Field, M. C., Rout, M. P., Frost, A. & Munson, M.(2016). Subunit connectivity, assembly determinants
and architecture of the yeast exocyst complex. Nat Struct Mol Biol, 23, 59-66.
Pleskot, R., Cwiklik, L., Jungwirth, P., Žárský, V., & Potocký, M. (2015). Membrane targeting of the yeast
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Shen, D., Yuan, H., Hutagalung, A., Verma, A., Kümmel, D., Wu, X., Reinisch, K., McNew, J. A., &
Novick, P. (2013). The synaptobrevin homologue Snc2p recruits the exocyst to secretory vesicles by
binding to Sec6p. J Cell Biol, 202, 509-526.
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
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Wen, T. J., Hochholdinger, F., Sauer, M., Bruce, W., & 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.
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6. APPENDIX
Curriculum vitae
Nemanja Vukašinović
Born in Užice, Serbia, September 17, 1985
2011-present: Ph.D. student in the lab of Viktor Žárský at the Faculty of Science at
Charles University in Prague and at the Institute of Experimental Botany AS CR.
2010: Diploma in Biology, Major: Plant Physiology, Faculty of Biology, University
of Belgrade, Serbia; Diploma thesis – “Effect of salicylic acid on the development of
induced heat tolerance in potato (Solanum tuberosum L.)”, done at the Institute for
Biological Research “Siniša Stanković” in Belgrade, Serbia.
2004: Grammar school, Užice, Serbia.
Publications:
Vukašinović, N., Cvrčková, F., Eliáš, M., Cole, R., Fowler, J. E., Žárský, V., & Synek, L. (2014).
Dissecting a hidden gene duplication: the Arabidopsis thaliana SEC10 locus. PloS One, 9, e94077.
Vukašinović, N., & Žárský, V. (2016). Tethering Complexes in the Arabidopsis Endomembrane
System. Front Cell Dev Biol, 4.
Bloch, D., Pleskot, R., Pejchar, P., Potocký, M., Trpkošová, P., Cwiklik, L., Vukašinović, N., Sternberg,
H., Yalovsky, S., & Žárský, V. (2016). SEC3a exocyst subunit is required for pollen tube development, is
differentially regulated by phosphoinositides and predicts growing site. Plant Physiol, submitted.
Vukašinović, N., Oda, Y., Pejchar, P., Synek, L., Pečenková, T., Rawat, A., Sekereš, J. & Žárský, V..
(2016). Cell Type Specific Microtubule-Dependent Targeting of the Exocyst Complex in Xylem. In
preparation.
Synek, L., Vukašinović, N., Hála, M., Fendrych, M., & Žárský, V. (2016). EXO70C2, a putative exocyst
subunit, is responsible for efficient pollen tube growth in Arabidopsis. In preparation.
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