Plant Cell, Development & Ultrastructureds9.botanik.uni-bonn.de/zellbio/AG-Baluska-Volkmann/pdf...izmb.uni--bonn.de bonn.de Roles of Plant Formins in Cell Polarity polar localization

PCDU – Lecture

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Plant Cell, Development & Ultrastructure

Plant Cell Biology Labs

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Myosins V are Related to Plant VIII & XI Myosins


Heptad-Repeat: a b c d e f g h - a b c d e f g h - a b c d e f g h

Hydrophobic amino acids at positions „a“ and „d“: I, L, V, M, F

DIL: dilute domain, cargo binding C-terminus. dilute was first discovered as a mutation in mice causing color dilution of the fur.

The affected gene codes for a myosin V which moves melanosomes from the cell center to the periphery in skin melanocytes.

Plant Myosins


Plant Myosins

Nakano et al. 2012


Plant Myosins of Class XI

Gallus gallina

Homo sapiens

Saccharomyces cerevisiae

Nicotiana tabacum

Chara corallina

Nakano et al. 2012


Regulation of Myosin XI

Nakano et al. 2012

no Cargo no Cargo

Calmodulin dissociation

Myosin Cross-Bridge Cycle izmb.uni-bonn.de izmb.uni-bonn.de

H5C2

N-Ethylmaleimide (NEM)

C6H7NO2 Mr 125.1

NEM BDM

2,3-Butanedione Monoxime (BDM)

Sulfhydryl alkylalting agent, blocks

SH-groups, prevents disulfide bridges

Synthetic inhibitor against

myosin ATPase activity in

the head domain of myosin

head swings forward

power stroke

Inhibition of Myosin Cross-Bridge Cycle izmb.uni-bonn.de izmb.uni-bonn.de

BDM

„Rigor“

NEM

BDM Inhibits ATP-Hydrolysis NEM Inhibits Release of Head

HMM/S1-Decoration of Actin Filaments izmb.uni-bonn.de izmb.uni-bonn.de

S1

HMM

*

*

In the Absence of ATP, Myosin is Locked on F-Actin Resulting in the Arrow Head Pattern

HMM - Heavy Meromyosin

Phylogenetic Tree of Myosins

17 classes

35 classes curently recognized

Anireddy et al. 2001

Plasma Membrane

Plasmodesmata

Cortical Cytoplasm

Organellar Motors

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


Arabidopsis Myosin XI family

Nakano et al. 2012

13 Members in the Myosin XI Family

(16)

(6 / MYA2)

(5 / MYA1)

(8)

(15)

(17)

Cargo of Plant Class XI Myosins izmb.uni-bonn.de izmb.uni-bonn.de

Reisen & Hanson 2007

*

MYA2: peroxisomes XI-B: unknown organelle

XI-K: peroxisomes +? XI-J: ? XI-I: ? + nucleus

MYA1: peroxisomes

Cargo of Plant Class XI Myosins izmb.uni-bonn.de izmb.uni-bonn.de

YFP-Myosin16-tail overlay

Myosin XI-J (16) co-localizes with mitochondria


Plant Myosin-Dependent Processes

BDM, 5 mM, 3h Control

Shape and motility of the ER depend on functionally active myosin and

structurally intact actin filament system

Helling & Menzel 2001

Lat B, 10 µM, 1.5h


Plant Myosin-Dependent Processes

BDM, 5 mM, 3h Control

Shape and motility of the ER depend on functionally active myosin and

structurally intact actin filament system

Helling & Menzel 2001

Lat B, 10 µM, 1.5h

Ueda et al., 2010

triple knock-out

XI-K, XI-1, XI-2



The DIL-domain binds mostly to peroxisomes, but also to Golgi, mitochondria,

nuclear envelope, plasma membrane, plastids and unidentified vesicles.

DIL: dilute domain, cargo binding C-terminus. dilute was first discovered as a mutation in mice causing color dilution of the fur.

The affected gene codes for a myosin V which moves melanosomes from the cell center to the periphery in skin melanocytes.



The PAL-domain binds to Golgi, mitochondria, nuclear envelope, peroxisomes

ER, plasma membrane and plastids


Arabidopsis Myosins XI – Cargo Recognition

Arabidopsis Mya1 yeast Myo2

Redundancy of cargo recognition

Peroxisome XI-F

Golgi XI-1, XI-K, XI-I, XI-H, XI-C, XI-E

Chloroplast (stromules) XI-F

Mitochondria XI-J, XI-K, XI-G, XI-C, XI-E

Nuclear envelope XI-1, XI-I, XI-G, XI-F, XI-D, XI-B

N

C

N

C

µm


Myosin XI-K is Involved in Tip Growth

Growth of root hairs is reduced in

myosin XI-K knock out mutant

Park et al., 2013

Over-expression of myosin XI-K GTD

inhibits root hair growth

µm


Correct localization of myosin XI-K

depends on the integrity of the

actin cytoskeleton

Park et al., 2013

Myosin XI-K is Involved in Tip Growth


Cargo of Plant Class XI Myosins

MyoB links myosin XI-K to the membrane of target organelles

metal-binding

domain transmembrane

domain

myosin-binding

domain


Plant Myosins of Class VIII

CAT-GFP

CAT-GFP

Head Tail

Neck

4/6 IQ Motifs

CC Whole Molecule

GFP : : Tail Domain

Head Domain : : GFP

putative

targeting site GESGAGKT

CC


Plant Myosins

Head-GFP GFP-Tail

bean leaf epidermis cell Helling & Menzel 2003

Transient Expression of ATM1-GFP Fusion Proteins



Transient expression of GFP-ATM1-tail

in onion bulb epidermis cell after gold particle bomardment

Helling, Menzel 2003


Plant Myosins VIII

Arabidopsis root Helling, Beck & Menzel 2003

ATM1-GFP accumulates at a lesion afflicted by a micro-

needle. Callose counterstained with aniline blue

ATM1 Callose


Plant Myosins VIII

Immunofluorescence co-localization of

GFP::ATM1-tail expression in roots of

transgenic A. thaliana



Plant Myosins VIII

Co-localization of Callose with GFP::ATM1-tail in

stably transformed tobacco BY2 cells

ATM1-tail Callose

Class VIII Myosins

Co-localization with

Callose in/at Plasmodesmata

of Aspen tree pith parenchyma

Plant Myosins VIII at Plasmodesmata izmb.uni-bonn.de izmb.uni-bonn.de


ATM1

Sadot et al. 2008

ATM1 involved in endocytosis ATM1 associated with pit fields/plasmodesmata

Plant Myosins VIII at Plasmodesmata


Plant Myosins VIII

GFP: ATM 2 tail: Transient expression

in onion bulb epidermis cells

DIC

GFP:ATM2

GFP:VIIIb

Diana Helling

D. Menzel 2003

BrdU-Cy3, control BrdU-Cy3,

Myosin siRNA knock down

Philimonenko et al. 2004

nucleoli in HeLa-cells ca. 30% reduction

of rDNA-transcription

BrdU= Brom desoxyuridine

thymidine/uridine analogon

indicator of DNA replication or transcription



Myosin VIII - Endocytic Motor in Plants


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