Plant Cell, Development &...
Transcript of Plant Cell, Development &...
PCDU – Lecture
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Plant Cell, Development & Ultrastructure
Plant Cell Biology Labs
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Construction of the Actin Filament
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Fine Structure of the Actin Filament izmb.uni-bonn.de izmb.uni-bonn.de
Polymerization Kinetics of the Actin Filament izmb.uni-bonn.de izmb.uni-bonn.de
Parthasathy et al. 1985
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Re-Annealing of Actin Filament Fragments izmb.uni-bonn.de izmb.uni-bonn.de
Merged micrographs of red and green fluorescence. Equal
concentrations (0.25 uM) of red (rhodaminephalloidin)-and
green (Alexa® green-phalloidin)-labeled actin filaments
were mixed, sheared through a 26-gauge needle, and
allowed to anneal for up to 60 min before dilution and
absorption to a coverslip coated with poly-L-lysine. Bar, 10
um. (A) Actin filaments alone allowed to anneal for 5 min.
(B) Actin filaments alone allowed to anneal for 60 min.
Actin Isoform Diversity izmb.uni-bonn.de izmb.uni-bonn.de
Petunia has more than 100 Actin genes
Arabidopsis has 8 Actin genes and 2 pseudogenes
Actin isoformes were formed from a single ancestral gene
some 350-500 Mio years ago
Actin classes in Arabidopsis
vegetative
reproductive
pseudogenes
McDowell et al. 1996
Phylogenetic Tree of Actin Genes izmb.uni-bonn.de izmb.uni-bonn.de
An et al. 1999
green algae
Higher and
Lower Plants
Actin isoformes evolved from a
single ancestral gene
some 350-500 Mio years ago
Actin Inhibitors
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Latrunculin A Latrunculin B
From the red marine sponge Latrunculia magnifica, inhibits polymerization of G-actin
by binding to the nucleotide pocket actin
G-Actin
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L
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Phalloidin MW 790Da
Bicyclic heptapeptide, Thioester between Tryptophan and Cystein
Binds to filamentous actin from most sources one phallotoxin molecule per actin subunit
from plants and animals.
Shifts the equilibrium from G-actin to F-actin, stabilizes F-actin
Does not readily cross membranes
Knollenblätterpilz
Amanitas phalloides
Actin Inhibitors
Rh-Phall
Actin Inhibitors
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Cytochalasin D
Fungal metabolite, shifts the equilibrium to smaller fragments of F-actin,
eventually leading to large scale depolymerization of actin filaments and
consequently a breakdown of the actin cytoskeleton
CD
Actin Inhibitors izmb.uni-bonn.de izmb.uni-bonn.de
Cytochalasin D Effect on Trichome Morphogenesis
Actin Inhibitors
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Disruption of the Actin Cytoskeleton
Inhibits the following processes in plants
- Rapid cell elongation
- Gravitropic bending
- Organelle motility
- Endocytosis/Exocytosis
- Establishment of polarity
- Tip growth (pollen tubes, root hairs)
- Trichome cytomorphogenesis
- stomata closure
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Cortex and exodermis of A. thaliana root
Voigt et al. 2002
Actin Cytoskeleton, Interphase
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Voigt et al. 2002
Actin Cytoskeleton in Special Cell Types
leaf epidermis
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Voigt & Menzel 2002
GFP-ABD2 Arabidopsis root
Actin Cytoskeleton in Special Cell Types
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Tobacco BY2 cells Preprophase band
Mitosis
Cytokinesis
Actin Cytoskeleton Through the Cell Cycle
Actin Filament disruption coincides with K+ Channel activation
Blue light or
Fusicoccin induce
Proton efflux and K+ influx
Modulation of the actin cytoskeleton by natural factors
hv
closed open
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Actin Cytoskeleton Through the Cell Cycle
Proton ATPase
Fusicoccin
Fusicoccum amygdali
causes wilting disease
Filamin
Spectrin, Vinculin (membrane binding)
Talin (focal adhesion)
Dystrophin, Utropin (muscle-specific)
Tropomyosin
Formin (+end binding) Severin/Gelsolin
Actin-Associated Proteins izmb.uni-bonn.de izmb.uni-bonn.de
AiP (+end binding) Myosin
Fimbrin, Villin
Polymer binding
ADF/ cofilin
Profilin (monomer sequestering)
Fascin
alpha-actinin
Barbed end capping
Green indicates occurrence in plants
Filamin
Spectrin, Vinculin (membrane binding)
Talin (focal adhesion)
Dystrophin, Utropin (muscle-specific)
Tropomyosin
Formin (+end binding) Severin/Gelsolin
Actin-Associated Proteins izmb.uni-bonn.de izmb.uni-bonn.de
AiP (+end binding) Myosin
Fimbrin, Villin
Polymer binding
ADF/ cofilin
Profilin (monomer sequestering)
Fascin
alpha-actinin
Barbed end capping
Green indicates occurrence in plants
Plant Actin-Binding Proteins izmb.uni-bonn.de izmb.uni-bonn.de
Number of gene copies in A. thaliana
• Fimbrin (F-actin crosslinking) 3 genes
• Profilin (G-actin sequestering) 5 genes
• ADF/cofilin (F-actin destabilizing) 10 genes
• AiP1 (barbed end binder, co-factor of ADF) 2 genes
• Villin (F-actin crosslinking) 5 genes
• Gelsolin (F-actin destabilizing) single copy gene
• Arp2/3 (F-actin branching) single copy genes
• Formin (F-actin binding, polymerization) 21 genes
interacting
partners
Gymnosperm
Maciver et al. 2002
Plant ADF genes
show a higher diversity than
animal ADFs
Plant Actin-Binding Proteins izmb.uni-bonn.de izmb.uni-bonn.de
Cofilin/ADF destabilizes actin filaments by increasing the torque in the filament
Bamburg et al. (1999) Trends
Cell Biol 9(9): 364-370
DosRemidos et al. 2003
F-actin
F-actin/cofilin 1:1
Plant Actin-Binding Proteins izmb.uni-bonn.de izmb.uni-bonn.de
Ono 2003
+ AiP
Plant Actin-Binding Proteins izmb.uni-bonn.de izmb.uni-bonn.de
Blanchoin et al. 2010
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Addition of actin subunits
Dynamic behavior of actin filaments
in a reconstituted in vitro system
components: •ATP-G-Actin (fluorochrome labelled) •Formin-coated polystyrene beads •ADF1 •CAP
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+G-actin
+ Formin-beads
+G-actin
+ Formin-beads
+G-actin
+ Formin-beads
+ADF
+ Cap
Plant Actin-Binding Proteins izmb.uni-bonn.de izmb.uni-bonn.de
Chen et al. 2002
GFP-NtADF1
Distribution of ADF in
Nicotiana pollen tubes
ADF is regulated by intracellular pH
Lovy-Wheeler et al. 2006
ADF fragments actin filaments.
The fragmentation exposes new plus
ends that in turn enhance new
polymerization of actin
Plant Actin-Binding Proteins izmb.uni-bonn.de izmb.uni-bonn.de
Expression Levels of ADF-Isoforms in Arabidopsis Hypocotyls
* *
* pollen-specific bundling activity
Plant Actin-Binding Proteins izmb.uni-bonn.de izmb.uni-bonn.de
Daher et al. 2011
male gametophyte-specific ADF-Isoforms in Arabidopsis
Comparison Between ADF1 and ADF9 izmb.uni-bonn.de izmb.uni-bonn.de
Superimposition of the crystal structure of ADF1 (red)
and predicted structure of ADF9 (yellow).
53% identity
78% similarity
loop
loop
Tholl et al. 2011
F-actin binding site
Some divergence in the actin-
binding site (shaded area),
otherwise very similar tertiary
structure despite substantial
differences in sequence.
Regulation of ADF Activity izmb.uni-bonn.de izmb.uni-bonn.de
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Chen et al. 2003
The activity of ADF is inactivated
by LIM-Kinase, which in turn is
regulated by signal cascades
LIM-kinases are LIM-domain proteins
also binding to actin.
ADF is the only substrate.
LIM is a double zinc-finger domain found
in 14 classes of regulatory proteins
LIM-kinase
Zn
Zn
Plant Actin-Binding Proteins izmb.uni-bonn.de izmb.uni-bonn.de
15-19 kDa
A. thaliana ADF1 yeast COF1
Maciver & Hussey 2002
10 members in the ADF gene family in Arabidopsis
ADF-activity
is pH regulated,
inhibited by Ptins-4,5-bisphosphate (PIP2)
Phosphorylation by LIM-Kinase inhibits F-actin binding
Plant Actin-Binding Proteins izmb.uni-bonn.de izmb.uni-bonn.de
Number of gene copies in A. thaliana
• Fimbrin (F-actin crosslinking) 3 genes
• Profilin (G-actin sequestering) 5 genes
• ADF/cofilin (F-actin destabilizing) 9 genes
• AiP1 (barbed end binder, co-factor of ADF) 2 genes
• Villin (F-actin crosslinking) 5 genes
• Gelsolin (F-actin destabilizing) single copy gene
• Arp2/3 (F-actin branching) single copy genes
• Formin (F-actin binding, polymerization) 21 genes
• LIM-domain actin-binding proteins 6 genes
Plant Actin-Binding Proteins izmb.uni-bonn.de izmb.uni-bonn.de
LIM is an acronym derived from the
three gene products in animals:
lin-11, isl-1 and mec-3)
WLIM - widely expressed LIM-proteins
Eliasson et al. 2000
actin actin
+WLIM1
Thomas et al. 2007
Effect of LIM-protein overexpression in tobacco epidermis
A: actin-YFP reporter. B: tobacco WLIM1-GFP reporter
expression driven by cauli flower mosaic virus promotor 35S
A B
Effect of LIM-protein on F-actin in vitro
A: F-actin alone B: F- actin plus tobacco WLIM1
Even a single LIM-domain can
crosslink actin filaments
A: LIM1-domain
B: full LIM-protein
A
B
Thomas et al. 2009
Plant Actin-Binding Proteins izmb.uni-bonn.de izmb.uni-bonn.de
Eliasson et al. 2000
Full LIM-Protein with two double Zinc-finger domains
Linker
Plant Actin-Binding Proteins izmb.uni-bonn.de izmb.uni-bonn.de
Number of gene copies in A. thaliana
• Fimbrin (F-actin crosslinking) 3 genes
• Profilin (G-actin sequestering) 5 genes
• ADF/cofilin (F-actin destabilizing) 9 genes
• AiP1 (barbed end binder, co-factor of ADF) 2 genes
• Villin (F-actin crosslinking) 5 genes
• Gelsolin (F-actin destabilizing) single copy gene
• Arp2/3 (F-actin branching) single copy genes
• Formin (F-actin binding, polymerization) 21 genes
Plant Actin-Binding Proteins
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Ono 2003
AIP1 caps F-actin plus
ends and enhances the
activity of ADF.
ADF/AIP-Interaction
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1: buffer
2: 500nM AIP
3: 50nM ADF
4: 50nM ADF + 100nM AiP
5: 50nM ADF + 250nM AiP
6: 50nM ADF + 500nM AiP
1 2 3 4 5 6
1
3
5
2
Shi et al. 2012
Plant Actin-Binding Proteins izmb.uni-bonn.de izmb.uni-bonn.de
AIP is a WD40 repeat protein, characterized by two
mirror-imaged beta propellers consisting of 7 blades
each. A single blade, representing one WD40 repeat
motif, is build of a four-stranded antiparallel beta
sheet
Two Aip isoforms in the Arabidopsis genome
Ono 2003
Plant Actin Binding Proteins izmb.uni-bonn.de izmb.uni-bonn.de
Blocked expression of AIP1 by RNAi causes overstabilization of actin filament bundles, i.e. a
decrease of F-actin turnover. The effect is that expansion during cell growth is compromized.
RNAi-treated plants develop a dwarf phenotype.
Overexpression of AIP1
untreated RNAi-knock down overexpression
Manipulation of AIP1 levels