PCDU – Lecture

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Plant Cell, Development & Ultrastructure

Plant Cell Biology Labs

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Plant MT-Based Motor Proteins izmb.uni-bonn.de izmb.uni-bonn.de

• Kinesin

Plus and Minus end Directed MT-Based Molecular Motors

(homodimer, 110-130kDa, about 150 members in the kinesin

gene family, more than 60 kinesin genes in plants of these

20 without homologues in other organisms)

• Dynein

Minus end Directed MT-Based Molecular Motors

(heteromultimer, almost completely lost from higher .

plants, some vestigial forms recently discovered)

Molecular Motors Convert the Chemical Energy Contained in

ATP into the Mechanical Energy of Movement. They Transport

Various Cellular Cargo by “Walking" Along Cytoskeleton.

Kinesin Domain Structure izmb.uni-bonn.de izmb.uni-bonn.de

MT-binding surface

neck-linker

ATP-binding

site

from Wilson et al. 2009

motor domain

Cargo

coiled-coil

neck

linker

tail head

CC-tail

http://www.bioquest.org

Function of Kinesin Neck Linker izmb.uni-bonn.de izmb.uni-bonn.de

replacement of

ADP for ATP

hydrolysis of ATP

& release of head

neck-linker

Czövek et al. 2011

T

T

neck-linker of

enganged head

http://www.rcsb.org

Examples of Kinesin Motor Proteins izmb.uni-bonn.de izmb.uni-bonn.de

N-terminal motor C-terminal motor

tetrameric, bipolar

N-terminal motor

N-terminal motor

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Position of Motor Domain

N-Terminal Position

Middle Position

C-Terminal Position

1, 2, 3, 4, 5, (6), 7, 8, 9, 10, 11, 12

in the 14 classes of kinesin

Miki et al. 2005

13

8

10

11

6

7

14A

14B

3

12

2

1

9

5

The Kinesin Superfamily

4

Miki et al. 2005

13

8

10

11

6

7

14A

14B

3

12

2

1

9

5

9 Families with Plant Members

4

12

2

1

4

Kinesin -1

Monomer = 110KDa N-terminal motor, transport of cellulose

synthase containing vesicles to specific

plasma membrane sites and transport of

secretory vesicles to the cell plate.

Kinesin1/Atkin12 Family

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Tubulin DAPI AtPAKRP2

PAKRP1 and PAKKRP2 are members of the conventional, plus-end directed Kinesins

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AtPAKRP1/2

(conventional

Kinesins)

KNOLLE

t-SNARE

DAPI

Co-Localization of KRP2 with Knolle

AtPAKRP2 is associated with the incoming vesicles,

KNOLLE is a t-SNARE associated with the cell plate

Kinesin1/Arabidopsis Atkin12 Family

Kinesin

AKRP2

4

4 Kinesin -4, Monomer = 116KDa

forms dimers, 3 isoforms in Arabidopsis

Kin4 members in animals

associate with chromosome arms

(also called chromokinesins).

Rice ortholog Bcl1 (brittle culm),

Arabidopsis ortholog AtFra1

(fragile fibre) associate with

cortical MTs.

Class 4 Kinesin in Plants izmb.uni-bonn.de izmb.uni-bonn.de

Fra1-GFP moving along a microtubule in vitro

at a speed of 0,4µm/sec

Zhu & Dixit 2011 Zhong et al. 2002

Microtubule pattern in fibre cells

C) wild type

D) fra1 mutant

Microfibril pattern in fibre cells

C) wild type

E) fra1 mutant

fra2-mutant is katanin, see lecture 17

1

9

5

4 Kinesin -5, Monomer = 120KDa

AtKRP125a/ AtKRP125b/ AtKRP125c/ AtF16L2.60

bipolar tetramer

Kin5 / Bipolar Kinesins

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Bannigan et al. 2007

1 2 3

(A) Arabidopsis seedlings of

1: wild type

2. rsw7 mutant

3. rsw7 mutant complemented with

AtKRP125c-GFP

Visualization of AtKRP125c-GFP in root (B), Hypocotyl (C) and division figures of root

cells: Preprophase (D), Metaphase (E), Phragmoplast (F).

rsw7 is a temperature-

sensitive mutant of

kinesin-5 motor protein

AtKRP125c is a member

of the bipolar

Kinesin-5/BIMC family

Spindle &

Phragmoplast

rsw7

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Failure of spindle assembly in rsw7 mutant cells after shift of temperature from 21°C to 29°C

Bipolar Kinesins

rsw7 is a temperature- sensitive mutant of kinesin-5 motor protein

Miki et al. 2005

Kinesin-7, CENP-E, NACK

AtZCF125, AtF14P13.22

N-terminal motor

CENP-E is an integral component

of the kinetochore corona fibers

that link centromeres to the

spindle microtubules from early

metaphase to anaphase

7

KIN7 / Kinetochore Structure izmb.uni-bonn.de izmb.uni-bonn.de

Kim et al. 2008

(Kin7)

CENP-E = centromer protein E

Kinesin-7 Member Interacting with the

MAP-Kinase Signalling Pathway

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NACK = NPK1-activating kinesin

NPK = Nicotiana protein kinase

NACK binds to NPK1 and transports it to the

cell plate center. At the same time it activates

its MAPKKK function and thus triggers the

MAP-cascade. As a consequence MT_turnover

is enhanced. Target of the NPK1 cascade is

MAP65. Dominant negative NPK1 inhibits MT-

turnover and prevents centrifugal maturation of

the phragmoplast. Taxol treatment has the

same effect.

Takahashi et al. 2004

NACK is another member of the kinesin-7 family

3

12

Kinesin-12 family

POK1 = At3g17360

POK2 = At3g19050

TAN - Tangled Mutant of Maize izmb.uni-bonn.de izmb.uni-bonn.de

What determines the plane of division?

wild type tangled

Smith et al. 1996

It is mostly the shift from the transversal to

the longitudinal division plane that is

affected in the „tangled“ mutant.

POK: Phragmoplast Orienting Kinesin izmb.uni-bonn.de izmb.uni-bonn.de

Is TAN a plant MAP? Approximately 85% of TAN1 is related to vertebrate adenomatous

polyposis coli (APC) proteins in a region coinciding with a basic domain, which has been

shown to bind to tubulin, promote polymerization and bundling of MTs in vitro and to

associate with MTs in vivo

Adenomatous polyposis coli is a familiary genetic disease of the large intestine. APC-potein is involved in

the so called wnt-signalling pathway controlling cell proliferation and differentiation.

First members of the wnt-signalling pathway were wg (wingless gen locus in Drosophila genome, int1

(mutation in this gene causes mamma-carcinoma in mice).

APC-protein

POKs are Kinesin 12 Members izmb.uni-bonn.de izmb.uni-bonn.de

Genetik evidence for the involvement of Kinesin12-family

members POK1 and POK2 in phragmoplast alignment

wild type POK1-1, POK2-1

double mutant

POK1-2, POK2-2

double mutant

POK = Phragmoplast orienting Kinesin

Kinesin-12 family

POK1 = At3g17360

POK2 = At3g19050

Kinesin 12 - POK-Dependent Localization of TAN1 izmb.uni-bonn.de izmb.uni-bonn.de

TAN is one of a few proteins

that marks the PPB after the

MTs have disappeared untreated

wild type

POK1-1, POK2-1

double mutant

5min Oryzalin

treatment,

wild type

untreated

POK1; 2

double mutant

POK Interacts with TAN

Kinesin 12 - POK: Phragmoplast Orienting Kinesin izmb.uni-bonn.de izmb.uni-bonn.de

TAN-binding

coild coil motor domain neck linker

POK isoforms are the largest kinesins in Arabidopsis

most other kinesins have around 1000-1200aa (110 and 130 KDa)

2771 aa

2066 aa

Plant MT-Based Motor Proteins izmb.uni-bonn.de izmb.uni-bonn.de

Rasmussen et al. 2010 MTs in PPB Disappear

TAN Stays Behind

Stabilization of TAN at the PPB site is POK1/POK2-Dependent.

Without TAN the Approaching Cell Plate does not Fuse with the

Parental Plasma Membrane.

13

8

10

11

6 Kinesin -13, MCAK mitotic centromere-associated kinesin

70-80 kDa

Kinesin-13 / MCAK MT +Pole Destabilizer izmb.uni-bonn.de izmb.uni-bonn.de

Hashimoto 2003

Kin1/MCAK - Very Unusual, MT-Destabilizing Motor Molecule, Breaks Both Ends

MCAK = mitotic centromere associated kinesin

Monomer = 70-80KDa

Kinesin-13 / MT +Pole Destabilizer Kinesin izmb.uni-bonn.de izmb.uni-bonn.de

Desai et al. 1999

KIN13

Miki et al. 2005

13

8

10

11

6

7

14A

14B

Kinesin-14

C-terminal motor, MT-minus end

directed, 18 Members of this

family in Arabidopsis

Monomers = 84-143KDa

ATP-independent

MT-binding site

Kinesin-14 / C-Terminal Motor Kinesin izmb.uni-bonn.de izmb.uni-bonn.de

GFP-KCA1 TUA2-RFP merge

Vanstraelen et al. 2006

Localization of KCA1 in Tobacco BY2 Cells

Calponin-Homology-domain

KCA = Kinesin CDK-associated

= KCH-ortholog in tobacco BY2

CDK = Cyclin-dependent kinase A

CDK-phosphorylation site

folding & dimerization

6 isoforms of C-terminal kinesins

with CH-domain in Arabidopsis

KCH, KatD are Members of the C-terminal

Motor Kinesins: Kinesin14 Family

Possible Function: crosstalk to the actin-cytoskeleton.

CH-domain binds to F-actin.

Future division site is deplete

of F-actin!

neck linker neck mimic

Summary of Kinesin Involvements

in Mitosis and Cytokinesis

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Vanstraelen, Inze, Geelen 2006

AtKRP 125c

KCA: nuclear positioning

crosstalk to actin cytoskeleton

minus-end transport?

KRP125: maintenance of

MT plus-end overlap

PAKRP: secretory vesicle

and CESA transport towards

plus-ends

NACK promotes phragmoplast

maturation

Kinesin-14

Kinesin-5

Kinesin-1

Kinesin-7

PPB-formation

MCAK: Anaphase promoting

destabilizes kinetochor fibres

Kinesin-13

POK controls CDS*-positioning Kinesin-12

*CDS = cell division site

KINUa marks PPB Ungrouped

KINUa

POK