Plant Cell, Development & Ultrastructureds9.botanik.uni-bonn.de/zellbio/AG-Baluska-Volkmann/pdf/PCDU/11... · izmb.uni--bonn.de bonn.deizmb.uni Plant Actin-Binding Proteins Number

PCDU – Lecture

izmb.uni-bonn.de izmb.uni-bonn.de

Plant Cell, Development & Ultrastructure

Plant Cell Biology Labs

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http://goo.gl/111Tha

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) 11 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


Microinjection of purified AtFim1-Protein into Tradescantia

staminal hairs stabilizes the cytoplasmic strands

Kovar et al. 2000

Tradescantia virginiana

Chwil 2011

Plant Actin-Binding Proteins


Microinjection of Oregon-green labeled fimbrin 1

into Tradescantia staminal hairs

fimbrin alone fimbrin alone

Alexa488-phalloidin alone Alexa488-phalloidin +fimbrin

Two examples (A, B) of

microinjected fimbrin

decorating actin filaments

in the cortical actin network

Injection of Alexa488-labelled

Phalloidin (D). Co-injection of

unlabelled fimbrin with Alexa488-

labelled phalloidin stabilizes the

finer actin structures.

Plant Actin-Binding Proteins

Calponin-Homology Domains of Fimbrin izmb.uni-bonn.de izmb.uni-bonn.de

ABD1 ABD2 Calponin is a smooth muscle

protein. it binds to F-actin and

blocks Actin-Myosin interaction.

Ca2+-dependent Phosphory-

ation releases Calponin from the

Actin filament.

Banuelos et al. 1998

CH1 CH2 CH3 CH4 Fimbrin

Klein et al. 2004

ABD1

ABD2

Ca-binding

EF-hand domain

helix F

helix E

loop

Calponin-Homology Domain Proteins izmb.uni-bonn.de izmb.uni-bonn.de

ABD1 ABD2

Calponin is a smooth muscle

protein. it binds to F-actin and

blocks Actin-Myosin interaction.

Ca2+-dependent Phosphory-

ation releases Calponin from the

Actin filament. Banuelos et al. 1998

Sr, spectrin repeat

PH, pleckstrin homology domain

EF, calmodulin-like EF-hand

Igr, immunoglobulin-like repeat

coiled-coil, a region with a propensity

to form a coiled-coils

PR, plectin repeat;

CR, calponin repeat;

DH, Dbl homology domain;

DAG, diacylglycerolbindingdomain

PR, Prolin-rich repeat

Calponin-Homology Domain izmb.uni-bonn.de izmb.uni-bonn.de

ABD1 ABD2

Footprint of ABD2 on the actin filament

Sjöblohm et al. 2008

ABD2-GFP izmb.uni-bonn.de izmb.uni-bonn.de

The ABD2-domain of fimbrin can be used as a molecular reporter of

actin filaments in live cells

pGFP-ABD2

35S GFP ABD2 pA

amp

ABD2-GFP as Reporter for F-Actin izmb.uni-bonn.de izmb.uni-bonn.de

Voigt et al. 2005

EUR J Cell Biol 84: 595-608

mouse monoclonal

Actin at the polar crosswalls in the root transition zone

A. thaliana

Zea mays

GFP-ABD2

Markus Schlicht, unpublished

10µm 20µm

50µm Xylem Primanen











Plant Actin Sequestering Proteins izmb.uni-bonn.de izmb.uni-bonn.de

Profilin („prolin-loving“, 12-16kDa) binds monomeric actin and enhances

the exchange of ADP against ATP. Hence G-actin is „recharged“ and

made competent for actin filament nucleation.

In Plants Profilin is the Major G-Actin Sequestering Protein.

In Vertebrate Animals it is both Thymosin-beta-4 (5kD) and Profilin.

Profilin Thymosin

CAP1 (Cyclase Associated Protein)


activated by

S/T-Kinase

CAP = (cyclase-associated protein) is required for nucleotide exchange in an adenylyl-cyclase dependent fashion (Chaudhry et al. 2007).

PKA = protein kinase A. S/T-Kinase = serin/threonin kinase PIP2 = phosphatidyl-inositol-4,5-bisphoshate

activated by

adenylyl cyclase

PIP2

ADF

Profilin-Actin Pool CAP1 Recycles G-Actin

from ADF-G-Actin to

Profilin-G-Actin


Regulation of Profilin-Actin Interactions

PIP2-binding

PIP2 (Phosphatidyl-inositol-4,5-bisphosphate) weakens the interaction between Profilin and G-Actin,

because it binds to the N-terminus of G-Actin, sterically interfering with profilin binding. The activity

of profilin is also regulated by a ser/thr protein kinase.

Plant Actin Sequestering Proteins

PIP2

PIP2

PIPI2 Sequesters Profilin in “Inactive" form.

Inactive Profilin can be Released by PLC.

Profilin izmb.uni-bonn.de izmb.uni-bonn.de

The dynamic of the actin cytoskeleton is severely changed after

microinjection of Profilin into Tradescantia staminal hair cells

control

Profilin izmb.uni-bonn.de izmb.uni-bonn.de

Completion of cytokinesis (fusion of the cell plate with the plasma membrane)

is inhibited after microinjection with profilin

control











Archer et al. 2005

Plant Gelsolin Family izmb.uni-bonn.de izmb.uni-bonn.de

plant gelsolin

plant villin

6 Gelsolin Repeat Domains

Gelsolin is the Most Potent Member of the

Actin-Severing Gelsolin/Villin Superfamily.

Archer et al. 2005

The Gelsolin Superfamily izmb.uni-bonn.de izmb.uni-bonn.de

also present

in plants

present in

plants


I

II IV

V

VI

III

3D-Structure of Gelsolin

In vitro Assay of Plant Gelsolin Functions izmb.uni-bonn.de izmb.uni-bonn.de

Actin Actin plus gelsolin

Actin plus gelsolin plus 15nM Ca2+ Actin plus gelsolin plus 160nM Ca2+

Huang et al. 2004

F-actin is labelled with Rh-Phalloidin plant gelsolin = 80kDa protein

Gelsolin – Cleavage Mechanism izmb.uni-bonn.de izmb.uni-bonn.de

mem

bra

ne

mem

bra

ne

Ca2+

Gelsolin Activity is Stimulated by

Calcium and Inhibited by PIP2











not found in plants

Actin-related proteins

Actin Filament Branching izmb.uni-bonn.de izmb.uni-bonn.de

P16 = crooked, Arabidopsis mutant

Activator of Arp2/3


Svitkina & Borisy 1999

Actin Filament Branching

Summary of Actin Dynamics

In Animal and Plant Cells


Pollard & Borisy

AiP1

Formin stabilizes plus ends at membranes

CAP1

ser/thr kinase

PAK = P21-activated

protein kinase

P21 = Rho-GTPase

Boundary Extension in Cells

via Actin Polymerization


Mathur 2005

bipolar

expansion growth

Diffuse growth

polar tip growth


Mutations in proteins

of the ARP-complex

cause defects in cellular

morphogenesis

Boundary Extension in Cells

via Actin Polymerization


Arp2/3 Mutants in Arabidopsis

wildtype

wildtype wildtype

wildtype wildtype

dis/wrm

dis/wrm dis/wrm

dis/wrm dis/wrm

Arp2

Arp3

Arp2/3 Mutants in Arabidopsis izmb.uni-bonn.de izmb.uni-bonn.de

wrm

dis1-1

wt

wt

Actin cytoskeleton

in wild type leaf

trichomes

Arp2 mutant

Arp3 mutant

The brick Mutant in Zea mays izmb.uni-bonn.de izmb.uni-bonn.de

brick mutant wild type

brick mutant wild type Frank et al 2003

cell walls not interlocking

actin cytoskeleton

brick is an activator of the Arp2/3 complex

Expanding Cell Boundaries in Pavement Cells

MT-rich zone restricts

expansion of Cell A

MT-depleted, actin-rich zone

promotes expansion of Cell B











Domain Structure of Plant Formins izmb.uni-bonn.de izmb.uni-bonn.de

GBD/RhoGAP: G-protein bindig domains

DAD: diaphanous autoinhibitory domain

DID: diapanous inhibitory domain

DD: dimerization domain

Yeast formin Bni1p

PR: prolin-rich domain

PTEN: Phosopholipid-binding domain

SP: membrane insertion signal

TM: transmembrane domain

Many Formins are RHO-GTPases Effector Proteins

Yeast

3 Formins

Humans

15 Formins

Arabidopsis

21 Formins

Formin izmb.uni-bonn.de izmb.uni-bonn.de

Formin promotes the addition of G-actin subunits to the plus end of actin filaments.

During that process formin remains bound to the plus end, while subunits are added


Tertiary Structure of the FH2-Domain

N

Association with the actin filament plus end

Visualization of formin

adding subunits at the

plus end of an actin

filament in-vitro


Formin

Kovar 2005

members in the formin

family differ by their

efficiency as plus-end

polymerizers


Some formins hold on to

membranes while

polymerizing actin filaments.

This is one way, how polar

organization is controlled, as

for instance in the yeast

cytoskeleton


This feature may be

combined with Arp2/3 based

filament branching

3 Plant Classes in the Formin Family Tree izmb.uni-bonn.de izmb.uni-bonn.de

Prasinophytes

Lycophytes

all lower and

higher plants

all lower and

higher plants

Functions of Arabidopsis Formins izmb.uni-bonn.de izmb.uni-bonn.de








Intracellular Localization of Plant Formins izmb.uni-bonn.de izmb.uni-bonn.de

In plant cells, some formins

such as AtFH5

are found in the developing

cell plate and in endosomal

vesicles

Ingouff et al. 2005

Time series of cortical root tissue, AtFH5–GFP shown in green, cell wall in red,

scale bar is 10µm.

5min 0min

10min 20min AtFH5-T-DNA-mutant

shows defects in cell division

as does the diaphanous

mutant in Drosophila

Roles of Plant Formins in Cell Polarity izmb.uni-bonn.de izmb.uni-bonn.de

polar localization of plant formins

Baluska & Hlavacka 2005

mouse monoclonal

A. thaliana,

root transition zone

GFP-ABD2 10µm 20µm

Zea mays,

root transition zone

Documents

Plant Cell, Development & Ultrastructureds9.botanik.uni-bonn.de/zellbio/AG-Baluska-Volkmann/pdf/PCDU/11... · izmb.uni--bonn.de bonn.deizmb.uni Plant Actin-Binding Proteins Number