Plant Cell, Development & Ultrastructureds9.botanik.uni-bonn.de/zellbio/AG-Baluska-Volkmann/pdf/PCDU/10... · Plant Cell, Development & Ultrastructure ... by binding to the nucleotide

PCDU – Lecture

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Plant Cell, Development & Ultrastructure

Plant Cell Biology Labs

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plus end

minus end

Construction of the Actin Filament

1

2 4

3

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

minus end

plus end

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


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

plus end

minus end

L


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


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


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


Cortex and exodermis of A. thaliana root

Voigt et al. 2002

Actin Cytoskeleton, Interphase


Voigt et al. 2002

Actin Cytoskeleton in Special Cell Types

leaf epidermis


Voigt & Menzel 2002

GFP-ABD2 Arabidopsis root

Actin Cytoskeleton in Special Cell Types


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


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


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


Ono 2003

+ AiP


Blanchoin et al. 2010

+ - (ADF)

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

+ end

+G-actin

+ Formin-beads

+G-actin

+ Formin-beads

+G-actin

+ Formin-beads

+ADF

+ Cap


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


Expression Levels of ADF-Isoforms in Arabidopsis Hypocotyls

* *

* pollen-specific bundling activity


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

+ -

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

//upload.wikimedia.org/wikipedia/commons/9/9e/Lims.png


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











• LIM-domain actin-binding proteins 6 genes


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


Eliasson et al. 2000

Full LIM-Protein with two double Zinc-finger domains

Linker











Plant Actin-Binding Proteins


Ono 2003

AIP1 caps F-actin plus

ends and enhances the

activity of ADF.

ADF/AIP-Interaction


1: buffer

2: 500nM AIP

3: 50nM ADF

4: 50nM ADF + 100nM AiP



1 2 3 4 5 6

1

3

5

2

Shi et al. 2012


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

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Plant Cell, Development & Ultrastructureds9.botanik.uni-bonn.de/zellbio/AG-Baluska-Volkmann/pdf/PCDU/10... · Plant Cell, Development & Ultrastructure ... by binding to the nucleotide