Plant Cell, Development &...

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

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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

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Plant Myosins

Nakano et al. 2012

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Plant Myosins of Class XI

Gallus gallina

Homo sapiens

Saccharomyces cerevisiae

Nicotiana tabacum

Chara corallina

Nakano et al. 2012

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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

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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

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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

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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

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Arabidopsis Myosin XI family

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.

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Arabidopsis Myosin XI family

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

ER, plasma membrane and plastids

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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

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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

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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

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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

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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

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Plant Myosins

Head-GFP GFP-Tail

bean leaf epidermis cell Helling & Menzel 2003

Transient Expression of ATM1-GFP Fusion Proteins

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Plant Myosins of Class VIII

Transient expression of GFP-ATM1-tail

in onion bulb epidermis cell after gold particle bomardment

Helling, Menzel 2003

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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

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Plant Myosins VIII

Immunofluorescence co-localization of

GFP::ATM1-tail expression in roots of

transgenic A. thaliana

Helling, Menzel 2003

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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

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ATM1

Sadot et al. 2008

ATM1 involved in endocytosis ATM1 associated with pit fields/plasmodesmata

Plant Myosins VIII at Plasmodesmata

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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

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Plant Myosins of Class VIII

Myosin VIII - Endocytic Motor in Plants

Helling, Menzel 2003

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