Plant Cell, Development & Ultrastructure - Botanik in …ds9.botanik.uni-bonn.de/zellbio/AG-Baluska-Volkmann/pdf...MT polymerization izmb.uniizmb.uni--bonn.de bonn.de Isolated centrosomes:

PCDU – Lecture

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

Plant Cell, Development & Ultrastructure

Plant Cell Biology Labs

Download at:

http://goo.gl/111Tha

MT polymerization


Isolated centrosomes: seeds for MT polymerization in vitro

Nuclear fragment isolated from tobacco BY-2 cells

used as a nucleation center for microtubules in vitro.

Shibaoka 1994

basal bodies isolated from sea urchin sperm

used as a nucleation center for microtubules in vitro.

Dynamic instability of MTs


Tim Mitchison, Harvard Medical School

Subunit flux causes treadmilling


FRAP

= Fluorescence after photobleaching

Shaw et al. 2003

„Treadmilling“ = polymerization/depolymerization at opposite ends

„Breathing“

= replacement

MT-dynamics in situ


stabilized

stabilized

crosslinking could slow down dynamic instability

plus end stabilization could prevent catastrophe

minus end stabilization could prevent shrinking

Dynamic cortical MT-cytoskeleton


Timmers & Menzel 2002

1

5

4

3

1 2

1: growing

2: shrinking & gliding

3: growing

4: breaking & shrinking

5: looping

transient Expression of the GFP-MBD-MAP4 construct in Onion bulb scale cells

Plant MAPs


• MOR1 (217kDa, member of XMAP-family)

• MAP18 (18kDa, plant specific, destabilizes cortical MTs

• MAP65 (65kDa, plant specific antiparallel MT-bundling)

• MAP70 (70kDa, plant specific, decorates all 4 MT-arrays)

• EB1/Clip170 (MT plus end binding)

• γ-Tubulin (MT minus end binding )

• Spc98 (98kDa MTOC-Komponente)

• Katanin (60kDa microtubule severing ATPase)

• MAP 1-4 (not known in plants)

• Tau (not known in plants)

MOR1 mutant phenotype


Whittington et al. 2001

MT-cytoskeleton affected in MOR1


Whittington et al. 2001

Wild-Type MOR1

Sub-plasmalemma localization of MOR1


HEAT-Domain armadillo-like apha-helical motif

37- 47aa repeat named after its discovery in

huntingtin elongation factor-3,

a subunit of protein phosphatase-2A

protein kinase mammalian target of rapamycin

cortical microtubules in extracted plant cytoplasts

with bridges to plasma membrane

Polarity of cortical MTs is not uniform


Lloyd 2004

antiparallel

Hook formation assay

McIntosh & Euteneuer 1984.

Tian et al. 2004

Hook assay

hooks turn clockwise

when viewed towards

the minus end

MOR1

?

Plant MAPs



• MAP18 (18kDa, plant specific, destabilize cortical MTs



• EB1/Clip170 (MT plus end binding)






MAP65 associates with cortical MTs


Overexpression of MAP65-1

Chan et al. 1999

9 MAP65 genes in Arabidopsis

MAP65 associates with cortical MTs


dimerization domain

MAP65


Tubulin MAP65-1

Smertenko et al. 2004

co-localization with

major MT-arrays

in plants

Regulation of MAP65-1


GFP:AtMAP65-1 expression in ocadaic acid-treated cells

15min 17min 19min

Phosphorylation of MAP65-1 inhibits microtubule binding Alpha-tubulin: red

MAP65-1: green

Immunofluorescence

Model of AtMAP65-1

regulation by

phosphorylation

Smertenko et al 2006

Smertenko et al 2006

MAPK = Mitogene activated protein kinase



Inhibition of PP2A by okadaic acid

OKA

C-terminal MAPK-phosphorylations sites of MAP65

MAPK loss of function mutants

Hamada 2007


MAPK-double mutant Wildtype

Martina Beck et al.2010


Plant MAPs






• EB1 (MT plus end binding)






Plus-end binding EB1


+tip comet tail binding behavior of EB1 at growing plus ends

CH = Calponin homology domain

EBH = EB-homology domain

+ - + -

EB1 is a homodimer, the monomer has a mass of 32kDa, Arabidopsis has 3 isoforms

individual EB1-Alexa 488 comets at the indicated growth velocities in µm/min

Plus-end binding EB1


GFP-MCAK, Borm & Menzel GFP-EB1, Timmers & Menzel

EB1: Microtubule plus end-binding protein MCAK: Mitotic centromere associated protein

Plant MAPs












gamma-tubulin spirals


gamma complex proteins

(GCPs 2–6)

GCP3 = SPC98

Pastuglia & Bouchez 2007

early concept of MTOC structure current concept of MTOC structure

gamma-tubulin has a mass of 58kDa

Localization of gamma-tubulin in plants


Animal Higher Plant

intermediate forms of

spindle poles

Plant MAPs













Localization of Spc98 & gamma-tubulin in plants

Plant MAPs









gamma-

complex

- +

+ -

Plant MAPs













Katanin/fra2-mutant

wild type

wild type

wild type wild type

fra2-mutant fra2-mutant

fra2-mutant

fra2-mutant fra2-mutant

microtubules microfibrils

immunofluorescence of Arabidopsis hypocotyl cells

Burk & Ye 2002


Plant Katanin

incubation of in-vitro polymerized

microtubules with recombinant

katanin

control plus katanin

Burk & Ye 2002

Role of MAPs in the dynamics

of corticle microtubules


Sedbrock & Kaloriti 2008

(EB1)

zippering (Katanin)

(MAP65)

(γ-tubulin)

(SPC98)

Murata 2005

Plant MAPs










• Phospholipase D (PLD)


control MAS7 taxol

taxol +

n-butanol

Dhonuksche et al. 2003

MAS7 = Mastoparan, an activator of PLD

Effects of PLD-activation on the cortical MT-system

Plant MAPs


Dhonuksche et al. 2003

(1-Butanol)

Phosphatidic acid

(Phosphatidyl-butanol)

Earlier, PLD has been identified as 90kDa crosslinker by structural, immunocytochemical and

biochemicals means (Gardiner et al. 2001)

covalently bound intermediate

Model for PLD-mediated microtubular reorganization

Statistically, 50% of the PLD population is in step 1 and 50% in step 2, n-butanol shifts the

equilibrium to 100% step 2

Summary of plant MAPs


90kDa

200kDa

60kDa

110kDa

Documents

Plant Cell, Development & Ultrastructure - Botanik in …ds9.botanik.uni-bonn.de/zellbio/AG-Baluska-Volkmann/pdf...MT polymerization izmb.uniizmb.uni--bonn.de bonn.de Isolated centrosomes: