The microtubule connection:

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The microtubule connection To Zebrafish spinal
neurons from Xenopus mitotic spindles
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Project aim
Develop and apply Zebrafish as a model to
address cell biological questions
concerning neuronal wiring of the organism
Six main wiring questions (temporally ordered)
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Majority of research surface ligand-receptor acti
n
Short-range
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GTP
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(Tanaka and Kirschner, 1991)
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10 nM Vinblastine
(Tanaka and Kirschner, 1995)
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Local change of MT polymerization
SCG10 and Stathmin/Op18, CRMP2 (neg.
regul.) (pos. reg.)
rac, cdc42
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Signal bound/diffusible
Premises
Interaction?
Actin is primed for turning but needs MTs to
deliver (motors/ dynamics) components required
for polar actin assembly
The area of primed actin positively promotes MT
stability (cortex (EB1-APC)/kinetochore analogy)
Primed actin and MTs positively promote each
others polar assembly leading to turning
(positive feedback)
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Which system?
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Why shift to Zebrafish? Genetics Transparent Genom
e sequence Vertebrate Room temperature More
reproducible Beautiful
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Work in progress
M T Dynamics

• - Remove EB1 by Morpholino antisense technique
• Study growth and turning at a substrate boundary
• - Study MT polymerization (GFP-tubulin)
• - Any observable changes in vivo?
• ----------
• SCG10, Stathmin/Op18, CRMP2 (single, double)?

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Isolation of the spinal cord
1) Forceps (arrows) 2) Trypsin (15 min)
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Plated on Laminin coated glass (6 hours after
plating)
Look at bipolar neurons (DRG?) as consistently
growing
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Preparation of dissociated Zebrafish spinal
neuron cultures
Growth rate 35.8 µm/h18.2 µm/h (n22)
70 µm
(Methods in Cell Science, 2001)
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Microtubule dynamics Microtubule polarity Local
stabilization
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global changes in microtubule dynamics
?
local regulation of microtubule dynamics
?
?
organization by microtubule-based motor proteins
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I
?
M
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Xenopus laevis egg extract system
collect cytoplasm
centrifuge
spindle assembly in vitro
Xenopus sperm nucleus
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Frogs/Xenopus
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Purification of XMAP230/XMAP4
(JCB, 1994)
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14-18
0.5 µM
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Observing microtubule dynamics
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Ac
2-D gel
2-D gel
Ac
XMAP230/XMAP4 is highly phospho. during mitosis
No MT binding in M phase ext.
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DNA
Tubulin
XMAP230
Binding to M phase MTs
Gradient
XMAP230 localization during the cell cycle
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Long distance effect of chromatin on
microtubule assembly (1996, JCB)
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10 kb DNA attached to beads No kinetochores or
centrosomes
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Bead spindle formation
10
20
60
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À distance effect of chromatin on microtubule
assembly (2003, CB)
30-40 µm
Molecular explanation?
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Stathmin/Op18 (18 kDa)
Idea Local inactivation of the MT destabilizer
Stathmin/ Op18 contributes to local MT
stabilization
Without chromatin beads, no differential
phosphorylation between I and M
Stathmin/Op18 has at least two isoforms in Xenopus
(Nature, 1997)
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Mitotic beads induce Stathmin/Op18 phosphorylation
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Phosphorylation of Stathmin/Op18 induced by
chromatin beads
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Okadaic Acid and Chromatin beads induce similar
phosphorylation of Stathmin/Op18
Mitotic extractOAgt stable MTs
OA PP2A inhibitor
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Okadaic Acid and Chromatin beads induce very
similar phosphorylation of Stathmin/Op18
suggesting same signaling pathway
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Depletion of Stathmin/Op18 accelerates MT assembly
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Spindles shorten in the presence of increasing
amounts of Stathmin/Op18
Stathmin/Op18 concentraction
control
L E N G T H (µm)
Time (min)
µg/ml
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To growth cone MT stabilization
(BioEssays, 1999)
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(Guidosome)
Signal bound/diffusible
Premises
Interaction?
Actin is primed for turning but needs MTs to
deliver (motors/ dynamics) components required
for polar actin assembly
The area of primed actin positively promotes MT
stability (cortex (EB1-APC)/kinetochore analogy)
Primed actin and MTs positively promote each
others polar assembly leading to turning
(positive feedback)
To appear in the Hypothesis section of
BioEssays (in press)
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Acknowledgements
People at the EMBL Eric Karsenti Kai Simons Tony
Hyman Rebecca Heald (DNA Beads) Tony Ashford
(Op18/2D gels) James Endres (Zebrafish) James
Bamburg (Colorado) Many colleagues
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Some questions to start with

• - Remove EB1 by Morpholino antisense technique
• Study growth and turning at a substrate boundary
• - Study MT polymerization (GFP-tubulin)
• - Any observable changes in vivo?
• ----------
• SCG10, Stathmin/Op18, CRMP2 (single, double)?
• ---------------------------------------------

M T Dynamics
--------------------------------------------- -
How does synapse formation in vivo correlate
with MT polymerization and these proteins
temporal localization/presence? - How does
functional synapse formation correlate with
this? ---------- Upon nerve regeneration how do
MTs reorganize?
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XMAP310
XMAP230/XMAP4
XMAP/XMAP215
Tubulin
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(Arimura et al., 2004)
(Gordon-Weeks, 2004)
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XMAP310 localization
(JCB, 1997)
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XMAP310 localization during metaphase
Tubulin
Overlay
XMAP310
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Soma
Myocyte
Bead
Bead diameter 10 µm
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Internalization of the TrkB receptor not required
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(Saint-amant and Drapeau, 2003)
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?
MT
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Acknowledgements
Eric Karsenti Kai Simons Tony Hyman Rebecca
Heald (DNA Beads) Tony Ashford (Op18/2D
gels) Many people at the EMBL --) Mu-ming Poo
Xiaohui Zhang (BDNF beads) Jimmer Endres
(Zebrafish) Gong Tong (La Jolla) James Bamburg
(Colorado) Richard Reimer (Stanford/VGLUT1) Many
colleagues