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Transcript of Plant Cell, Development & Ultrastructure - Botanik in...
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
izmb.uni-bonn.de izmb.uni-bonn.de
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.
Subunit flux causes treadmilling
izmb.uni-bonn.de izmb.uni-bonn.de
FRAP
= Fluorescence after photobleaching
Shaw et al. 2003
„Treadmilling“ = polymerization/depolymerization at opposite ends
„Breathing“
= replacement
MT-dynamics in situ
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stabilized
stabilized
crosslinking could slow down dynamic instability
plus end stabilization could prevent catastrophe
minus end stabilization could prevent shrinking
Dynamic cortical MT-cytoskeleton
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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
izmb.uni-bonn.de izmb.uni-bonn.de
• 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)
MT-cytoskeleton affected in MOR1
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Whittington et al. 2001
Wild-Type MOR1
Sub-plasmalemma localization of MOR1
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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
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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
izmb.uni-bonn.de izmb.uni-bonn.de
• MOR1 (217kDa, member of XMAP-family)
• MAP18 (18kDa, plant specific, destabilize 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)
MAP65 associates with cortical MTs
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Overexpression of MAP65-1
Chan et al. 1999
9 MAP65 genes in Arabidopsis
MAP65
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Tubulin MAP65-1
Smertenko et al. 2004
co-localization with
major MT-arrays
in plants
Regulation of MAP65-1
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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
Regulation of MAP65-1
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Inhibition of PP2A by okadaic acid
OKA
C-terminal MAPK-phosphorylations sites of MAP65
MAPK loss of function mutants
Hamada 2007
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MAPK-double mutant Wildtype
Martina Beck et al.2010
Regulation of MAP65-1
Plant MAPs
izmb.uni-bonn.de izmb.uni-bonn.de
• 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 (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)
Plus-end binding EB1
izmb.uni-bonn.de izmb.uni-bonn.de
+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
izmb.uni-bonn.de izmb.uni-bonn.de
GFP-MCAK, Borm & Menzel GFP-EB1, Timmers & Menzel
EB1: Microtubule plus end-binding protein MCAK: Mitotic centromere associated protein
Plant MAPs
izmb.uni-bonn.de izmb.uni-bonn.de
• 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 (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)
gamma-tubulin spirals
izmb.uni-bonn.de izmb.uni-bonn.de
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
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Animal Higher Plant
intermediate forms of
spindle poles
Plant MAPs
izmb.uni-bonn.de izmb.uni-bonn.de
• 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 (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)
Plant MAPs
izmb.uni-bonn.de izmb.uni-bonn.de
• 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 (MT plus end binding)
• γ-Tubulin (MT minus end binding )
• Spc98 (98kDa MTOC-Komponente)
gamma-
complex
- +
+ -
Plant MAPs
izmb.uni-bonn.de izmb.uni-bonn.de
• 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 (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)
izmb.uni-bonn.de izmb.uni-bonn.de
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
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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
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Sedbrock & Kaloriti 2008
(EB1)
zippering (Katanin)
(MAP65)
(γ-tubulin)
(SPC98)
Murata 2005
Plant MAPs
izmb.uni-bonn.de izmb.uni-bonn.de
• 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 (MT plus end binding)
• γ-Tubulin (MT minus end binding )
• Spc98 (98kDa MTOC-Komponente)
• Katanin (60kDa microtubule severing ATPase)
• Phospholipase D (PLD)
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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
izmb.uni-bonn.de izmb.uni-bonn.de
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